History of Arbovirology: Memories From The Field: Nikos Vasilakis Laura D. Kramer Editors

Nikos Vasilakis
Laura D. Kramer Editors
History
of Arbovirology:
Memories
from the Field
Volume I:
Personal Reflections
History of Arbovirology: Memories from the Field
Nikos Vasilakis • Laura D. Kramer
Editors
History of Arbovirology:
Memories from the Field
Volume I: Personal Reflections
Editors
Nikos Vasilakis Laura D. Kramer
Department of Pathology School of Public Health
University of Texas Medical Branch University at Albany, State University of
Galveston, TX, USA New York
Albany, NY, USA
ISBN 978-3-031-21998-6 ISBN 978-3-031-21999-3 (eBook)

https://doi.org/10.1007/978-3-031-21999-3
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Introduction
The close of WW II left the USA virtually in control of the entire world. We had the
best universities, and money seemed almost unlimited. Shortly, cell tissue culture
was perfected with the use of antibiotics. Even the word arbovirus (arthropod-borne
virus) is nearly an American idiom. Here, I should note that my wife Merle has
informed me that I never worked on an arbovirus. Neither Machupo virus nor other
arenaviruses or the hepadnaviruses involve any arthropods. They all utilize rodents
as reservoirs and vectors but are included here because they were published mainly
in the journal of the American Society of Tropical Medicine and Hygiene and were
the subject of much discussion by members of that society for many years.
There was infectious disease everywhere to confront. When other countries,
especially African ones, experienced infectious disease outbreaks, they had to
request our ambassador for help, usually from our Centers for Disease Control
(CDC), and the USA made the final decision.
I was born in 1929 and came up in that generation when the world was ours.
After graduating from Oberlin College in biology, I studied medicine at the
University of Rochester School of Medicine in Rochester, NY. The microbiology
teacher, Herbert Morgan, MD, was from John Ender's Harvard Medical School. He
was very diligent, and I took a year out of medicine in his department to get a mas-
ter’s. After 2 years of internal medicine at Presbyterian Hospital in New York City,
I was awarded a position in the Public Health Service at NIH. For 4 years I had good
luck with mild respiratory viruses in the laboratory of Dr. Robert Chanock, but
when the boss decided that I should become the director of a national campaign to
develop a vaccine for the common cold, I wanted something different. NIH’s Middle
America Research Unit (MARU) in the Panama Canal Zone needed a virologist, so
off I went. MARU, like many other laboratories, used baby white mice to recover
viruses and measure antibodies to them. I hired a cell culture expert, and before
long, we used cells and dilutions of very finite volumes of sera from candidate ani-
mals to recover and determine which critters were important in life cycles of par-
ticular agents. A particular early study was with the late Dr. Michael Young on VEE
virus, 12 years before the advent of molecular biology but still referenced fre-
quently today.
v
vi Introduction
I have not read all the chapters in this arbovirus history, but I will cite several,
either because they were parts of my own experience or more importantly because
they underscore the history of the main subject.
If one wishes to know something about arbovirology in Asia please consult the
chapter by John Mackenzie and Sam Lai Kit, a colleague I’ve not yet met. He
includes early research at least 100 years ago. Especially interesting is their atten-
tion to a form of Zika virus, and I still wonder if they have, or there exists, any data
of natural history of infection on that continent.
Perhaps there is no more succinct tale to relate than that of Dr. Robert Tesh. Now
retired, has been almost everywhere and done almost everything. My own interac-
tion with him occured at MARU where he and Dr. Byron Chaniotis proved trans-
ovarial transmission of vesicular stomatitis virus (VSV-Indiana) in a single species
of the Panamanian sandfly. When MARU closed, he migrated to Hawaii with now-
deceased Leon Rosen, one of our greats in arbovirology. After that, he moved to
Yale with Bob Shope, another of our late great arbovirology scientists, and finally
to the Galveston lab in Texas. He was always great at writing grant proposals that
were approved by NIH.
We also became interested in other countries in Latin America, principally
Bolivia, where we worked on Bolivian hemorrhagic fever (BHF). We discovered
and named the virus Machupo after the local river right there in a hastily modified
house in the town of San Joaquin and found and controlled the murine reservoir/
vector of the disease, but not before the virus claimed three of us as victims. That
detailed story still awaits publication.
Clarence J. Peter’s story of Machupo virus in Cochabamba, Bolivia, is close to
my heart because C.J. launched that trip from MARU when I was the lab director.
My then wife, Patricia Webb, and I were both immune to that virus, but she was ill
with a tumor on her thymus so could not travel, and as lab director, I had to stay
home. Despite the fact that he was not immune, I asked C.J. if he would like to go.
His work proved classic, and he did not get the disease. He even took the virus to
Fort Detrick to initiate study in a modified lab there that today would be called
Level 4. He found that this strain of Machupo virus would now be termed a variant.
Charlie Calisher tells the US portion of the initial VEE equine/human epidemic
in Central America, but at MARU we had learned more, earlier. The virus was intro-
duced in Guatemala by a Colombian company that wished to promote a cattle prod-
uct. The VEE strain was a subtype that caused disease and death in equines and
humans and moved both north and south. We had done an equine serosurvey in
Costa Rica a year earlier and found no antibodies to the virus. Dr. Gerald Eddy, an
Army officer with a recent PhD in virology from Notre Dame and was an advisor to
Panama, was a frequent visitor to MARU from his advisory post in the city. You
could plainly see where he’d rather be. One day, he reported that his job would be
cancelled, and he would be sent to a desk job at Walter Reed Hospital. I began to
think. Recalling that the current Commander of the Army Veterinary Corps had
once held that position in Panama City, I called him and asked that Gerry be trans-
ferred to our military unit at MARU. I told him why. This was done quickly and
before long Gerry found himself in northern Costa Rica among many sick horses.
Introduction vii
He approached a man who had lots of rangeland and many horses, and offered a
magnum of Johnny Walker (how, when, and where he obtained this prize remains
unknown) if the man would gather 40 of his horses in a corral and allow Gerry to
vaccinate them all and bleed them daily. The live TC-83 VEE vaccine had never
been tested in horses. Three animals got VEE encephalitis in the first two days, but
all the others were completely protected. We finally obtained virulent virus from
two that sickened, but only antibodies from the rest of the group. Gerry Eddy never
had to go to Walter Reed. He published his paper and was soon rewarded by promo-
tion to the Chief of Virology at Fort Detrick. My good friend Gerry sadly is recently
deceased.
Finally, there are five chapters about dengue viruses, led by Scott Halstead who
spent many years with the Army Unit in Thailand where the late Phillip Russell also
spent several years, came home to the USA, and was promoted to General in the
health arena. Not many people know that Phil once provided money to CDC for
overseas investigations when money was tight. Phil and I graduated from the same
medical school.
Duane Gubler, now retired, also contributes to the dengue story, and Rebecca
Rico-Hesse, Margo Brinton, and Amy Morrison also chime in. It is no surprise that
dengue viruses make up so much of this history, as the agents are found everywhere
in the tropics if mosquitoes are present (as they always are). I also recommend the
chapter by Sergey Alkhovsky. Stories about tick-borne encephalitis will be appreci-
ated by all who dream of a career in field arbovirology. I had the good fortune to
visit with Dr. Mikhail Chumakov while part of a US team that visited the Soviet
Union in the 1970s.
I also note that David Morens, an experienced worker in Africa, now Tony
Fauci’s right hand at NIH, will do a chapter on “Famous Scientists of the Golden
Age.” I cannot wait.
Placitas, NM, USA Karl M. Johnson

Preface
Into the twenty-first century, arboviruses continue to be the causes of major public
health challenges, as exemplified by new and repeated disease emergence, including
Zika, yellow fever, West Nile, chikungunya, dengue, and many others. Peaks of
arbovirus discovery have coincided with seminal paradigm shifts in knowledge and
emergent technologies. The first wave of arbovirus discovery in the 1930s was
fueled by advances in virus isolation and identification (see Kramer and Jozan chap-
ter). The second wave in the 1950s and 1960s coincided with the complimentary
support of the Rockefeller foundation (see Tesh, Jozan, and Monath chapters), the
institute Pasteur network (see Saluzzo and Gonzalez chapter), Organization de
Recherche pour la Recherche Scientifique et Technique Outre Mer (ORSTOM,
Research Organization for Scientific and Technical Research Overseas and cur-
rently known as Institute of Research for Development, IRD (see Saluzzo and
Gonzalez chapter)), and the British Colonial Medical Services (see Haddow and
Hardy chapters) and their international virus discovery programs supporting field
ecology and epidemiology studies. The third wave in the 1980s, which coincided
with the gradual decline of field studies, fundamentally transformed the field of
arbovirology through seminal discoveries in molecular biology, paving the way for
the development of polymerase chain reaction and reverse genetics. These new tools
revolutionized arbovirology, allowing studies to dissect pathogenesis and evolution
as well as contribute to the development of novel antivirals, vaccine platforms, and
teasing out the foundations of virus-host interactions. Lastly, the fourth wave coin-
cided with the advent of next-generation sequencing (NGS), which dramatically
increased the breadth and depth of the known virosphere. The expansion of the
known virosphere has also allowed the taxonomic assignment of an increasing num-
ber of viruses, with a total of 6 realms, 10 kingdoms, 17 phyla, 2 subphyla, 40
classes, 72 orders, 8 suborders, 264 families, 182 subfamilies, 2818 genera, 84 sub-
genera, and 11,273 species of viruses currently approved by the International
Committee on Taxonomy of Viruses (ICTV) (https://ictv.global/taxonomy accessed
on April 11, 2023). NGS has facilitated epidemiological studies at scales extending
from very local to global, generating a wealth of new knowledge essential for public
health policy and decision makers.
ix
x Preface
As of today, despite all the technological advances in understanding arbovirus

ecology, evolution, virus-host interactions, and control methods, it appears that the
challenges of the past, emergence, transmission, and morbidity and mortality,
remain unabated and sobering. Dengue viruses have reached hyperendemicity at a
global scale with rolling epidemics on an annual basis; West Nile, Chikungunya,
and Zika viruses have significantly expanded their geographic range into temperate
regions causing global pandemics and putting at risk of severe disease millions of
immunologically naïve populations. Spillover from zoonotic reservoirs and climate
change are affecting the landscape of pathogen ecology. Critically, our ability to
predict virus emergence and outbreaks as well as mitigate their human impact and
economic costs remain a challenge. Moreover, over the last 40 years, a generational
gap has developed between the giants of arbovirus research and discovery and the
new generation.
This apparent gap developed due to an ebbing of training and lack of investment
in passing the scepter to the next generation, leading to a lack of continuity among
the generations that threatens to derail the rich history of virus discovery, field epi-
demiology, laboratory advances, and understanding of the wealth of diversity that
surrounds us. This lack of continuity may have immediate and disastrous conse-
quences for human public health and understanding of zoonotic diseases when yet-
to-be-discovered arboviruses emerge.
The purpose of this book is to bridge the generation gap by providing continuity
through personal historical perspectives. In addition, the book addresses the role
women have played, often overlooked especially in the seminal days of arbovirol-
ogy but critical to the advancement of the field, and it speaks to the challenges they
face even today (see Kramer and Jozan chapter). We hope this book will convey the
trajectory of the field of arbovirology, and express the excitement of the research
process and the sense of the adventure of field and lab experiences of both earlier
and the current generation of scientists.
Galveston, TX, USA Nikos Vasilakis

Albany, NY, USA Laura D. Kramer
Further Readings
Frederick AM (2012) The foundations of virology: discoverers and discoveries, inventors

and inventions, developers and technologies. Infinity Publishing, West Conshohocken.
ISBN-10:0741473658; ISBN-13:978-0741473653
Vasilakis N, Tesh RB, Popov VL, Widen SG, Wood TG, Forrester NL, Gonzalez JP, Saluzzo JF,
Alkhovsky S, Lam SK, Mackenzie JS, Walker PJ (2019) Exploiting the legacy of the virus
hunters. Viruses 11(5):pii: E471
“To those trailblazers on whose shoulders
we stand”
Inspired by Bernard de Chartres
Contents
Part I Personal Reflections

The Quest for Excellence and the Challenge of Gender Equality in
Arbovirology: A Celebration of Women’s Contributions to the Field�� 3
Laura D. Kramer and Martine Jozan
Serendipity and Arboviruses�� 115
John Aaskov

The Tapestry of Life: Weaving an Arbovirologist �� 159
Duane J. Gubler
Arbovirus and Viral Hemorrhagic Fevers Research Inception
in Central and West Africa: A French Researcher’s Experiences�� 187
Jean-François Saluzzo and Jean-Paul Gonzalez
From Bwamba to the Present: The Changing Forest
of Arbovirology�� 205
Andrew D. Haddow

Fighting Dengue, Chikungunya, and Japanese Encephalitis�� 227
Scott B. Halstead
Chronicles of Hantaviruses: Foundations of Epidemiology
and Ecology�� 315
James W. Le Duc and James E. Childs

Yellow Fever and Other Viruses in West Africa�� 359
Thomas P. Monath

The “Golden Age” of Arbovirology, 1950–1969�� 381
David M. Morens
xiii
xiv Contents
Twenty-Seven Years of Field Studies on Dengue and Aedes aegypti

in Latin America�� 425
Amy C. Morrison
Years of Medical Entomology: Miscellaneous Interesting Findings�� 465
50
William K. Reisen
Remembrances of Virology Past �� 485
Sondra Schlesinger
Overview of Arbovirology in São Paulo State, Southeastern Brazil,
An
Highlighting the Virus Research Center, in Ribeirão Preto City�� 493
Luiz Tadeu Moraes Figueiredo
Unplanned Career in Arbovirology�� 513
An
Robert B. Tesh
Index�� 539
About the Editors
Nikos Vasilakis Professor Nikos Vasilakis is the vice

chair of research in the Department of Pathology and
Professor of Pathology at UTMB. He earned his BA and
MA degrees in biology from Hofstra University and his
PhD in arbovirology from the University of Texas
Medical Branch, Galveston. His research interests have
been to understand the evolution and pathogenesis of
arthropod-borne viruses as well as virus–mosquito and
virus–host interactions. He has served as chair of the
American Committee on Arthropod-Borne Viruses and
is the current chair of the Subcommittee on the
Interrelationships Among Catalogued Arboviruses. He
currently leads The Coordinating Research on Emerging
Arboviral Threats Encompassing the Neotropics
(CREATE-NEO), one of the Centers for Research In
Emerging Infectious Diseases (CREID) funded by the
National Institutes of Health.
Department of Pathology, Center for Vector-borne and

Zoonotic Diseases, Center for Biodefense and Emerging
Infectious Diseases, Center for Tropical Diseases and
Institute for Human Infections and Immunity, University
of Texas Medical Branch, Galveston, Texas, USA
xv
xvi About the Editors
Laura D. Kramer Professor Emeritus Laura Kramer

earned her BA degree from the University of
Pennsylvania and her PhD from Cornell Medical
College. As the former (now retired) director of the
Arbovirus Laboratories of the Wadsworth Center,
New York State Department of Health, she led a
research program on basic and applied field and labora-
tory studies examining the interactions of mosquito-
and tick-borne arboviruses, arthropod vectors, and
vertebrate hosts, and how these interactions impact
transmission dynamics as well as emergence, persis-
tence, and evolution of the virus. She retired from
NYSDOH in December 2020 and is currently professor
emeritus in the Department of Biomedical Sciences of
the School of Public Health, State University of
New York at Albany. She also serves as an associate edi-
tor of the International Journal of Infectious Diseases, a
virology moderator for ProMED (Program for
Monitoring Emerging Diseases) in the International
Society for Infectious Diseases, and a contributing virus
author for Merck Manuals in virology.
Former (retired) Director Arbovirus Laboratory,

Wadsworth Center, NYSDOH, Albany, NY, USA
Biomedical Sciences, School of Public Health, State
University of New York, Albany, NY, USA
Contributors: Volume I
John Aaskov Queensland University of Technology, Brisbane, QLD, Australia

Charles H. Calisher Colorado State University, Fort Collins, CO, USA
James E. Childs Yale University, New Haven, CT, USA
Luiz Tadeu Moraes Figueiredo School of Medicine of the University of São
Paulo at Ribeirão Preto, Ribeirão Preto, Brazil
Duane Gubler Duke-NUS Medical School, Singapore, Singapore
Jean-Paul Gonzalez Georgetown University, Washington, DC, USA
Centaurus Biotech. LLC, Manassas, VA, USA
Andrew D. Haddow Kennesaw State University, Kennesaw, GA, USA
Scott Halstead Uniformed University of the Health Sciences, Bethesda, MD, USA
Laura D. Kramer Albany School of Public Health at State University of New York,
Albany, NY, USA
James W. Le Duc The University of Texas Medical Branch at Galveston,
Galveston, TX, USA
Thomas P. Monath Crozet BioPharma LLC, Devens, MA, USA
David M. Morens National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD, USA
Amy C. Morrison University of California, Davis, Davis, CA, USA
William K. Reisen University of California, Davis, Davis, CA, USA
Jean-François Saluzzo Fab’entech, Lyon, France
Sondra Schlesinger Washington University of Saint Louis, Saint Louis, MI, USA
Robert B. Tesh The University of Texas Medical Branch at Galveston,
Galveston, TX, USA
xvii
Contributors: Volume II
Sergey V. Alkhovsky D.I. Ivanovsky Institute of Virology, Ministry of Health of

The Russian Federation, Moscow, Russia
Harvey Artsob Public Health Agency of Canada, Winnipeg, MB, Canada
Barry J. Beaty Colorado State University, Fort Collins, CO, USA
Carol D. Blair Colorado State University, Fort Collins, CO, USA
Margo A. Brinton Georgia State University, Atlanta, GA, USA
Ibrahima Dia Institut Pasteur Dakar, Dakar, Senegal
Diawo Diallo Institut Pasteur Dakar, Dakar, Senegal
Mawlouth Diallo Institut Pasteur Dakar, Dakar, Senegal
Gerhard Dobler Bundeswehr Institute of Microbiology, Munich, Germany
Michael A. Drebot Public Health Agency of Canada, Winnipeg, MB, Canada
Anna-Bella Failloux Institut Pasteur, Paris, France
Alioune Gaye Institut Pasteur Dakar, Dakar, Senegal
Dieter Gniel Independent TBE-consultant, Zurich, Switzerland
Diane E. Griffin John Hopkins Bloomberg School of Public Health,
Baltimore, MD, USA
Roy A. Hall The University of Queensland, St Lucia, QLD, Australia
Anne Hardy London School of Hygiene and Tropical Medicine, London, UK
Jenny C. Hesson Uppsala University, Uppsala, Sweden
Martine Jozan Orange County Mosquito and Vector Control District, Garden
Grove, CA, USA
Segenet Kelemu International Center of Insect Physiology and Ecology (ICIPE),
Nairobi, Kenya
xix
xx Contributors: Volume II
Desiree A. LaBeaud Stanford University, Stanford, CA, USA

Sai-Kit Lam University of Malaya, Kuala Lumpur, Malaysia
L. Robbin Lindsay Public Health Agency of Canada, Winnipeg, MB, Canada
Jan O. Lundström Uppsala University, Uppsala, Sweden
Joel Lutomiah Kenya Medical Research Institute (KEMRI), Nairobi, Kenya
Dmitrii K. Lvov D.I. Ivanovsky Institute of Virology, Ministry of Health of The
Russian Federation, Moscow, Russia
John S. Mackenzie Curtin University, Perth, Australia
The University of Queensland, Brisbane, QLD, Australia
Kuichi Morita Institute of Tropical Medicine (ITM), Nagasaki University,
Nagasaki City, Japan
Pat Nuttall University of Oxford, Oxford, UK
Victor Ofula Kenya Medical Research Institute (KEMRI), Nairobi, Kenya
Maria Gorreti Onyango Texas Tech University, Lubbock, TX, USA
C. J. Peters The University of Texas Medical Branch at Galveston,
Galveston, TX, USA
Rebecca Rico-Hesse Baylor College of Medicine, Houston, TX, USA
Rosemary Sang International Center of Insect Physiology and Ecology (ICIPE),
Nairobi, Kenya
Connie S. Schmaljohn National Institute of Allergy and Infectious Diseases
Integrated Research Facility, Frederick, MD, USA
Ellen G. Strauss California Institute of Technology, Pasadena, CA, USA
James H. Strauss California Institute of Technology, Pasadena, CA, USA
Michael J. Turell United States Army Medical Research Institute of Infectious
Diseases (USMRIID), Fort Detrick, MD, USA
Jandouwe Villinger International Center of Insect Physiology and Ecology
(ICIPE), Nairobi, Kenya
Scott C. Weaver The University of Texas Medical Branch at Galveston,
Galveston, TX, USA
Thomas M. Yuill University of Wisconsin, Madison, WI, USA
Part I
Personal Reflections
The Quest for Excellence
and the Challenge of Gender Equality
in Arbovirology: A Celebration
of Women’s Contributions to the Field
Abstract Women have made significant contributions to arbovirology, which

have played a valuable role in building our knowledge base of the biology, ecol-
ogy, epidemiology, pathology, diagnostics and much more of arboviruses. The
chapter begins with brief sketches of the female scientists who, while receiving
very little recognition or remuneration for their work, built the base upon which
the field of arbovirology grew. It explores their career paths and accomplishments
that moved the field forward to where it is today. It looks at our personal experi-
ences as well as a sample of accomplished women whose work continues to build
our understanding of arboviruses and introduces a few junior scientists embarking
on their careers in the field of arbovirology. This section is meant to inform and
preserve the place of these scientists in the historical backdrop of the field. It dem-
onstrates the different directions one can go while fulfilling one’s dreams of con-
ducting research that will help control the serious diseases caused by arthropod-borne
and zoonotic viruses. Finally, the chapter explores the changing status of women
in the life sciences over time. We explore some of the current hypotheses for the
continuing inequity at the highest levels of academia and possible approaches to
address the problem. While the chapter is focused mainly on gender, it is not meant
to exclude other aspects of diversity such as race and ethnicity, which also need to
be explored more in depth.
Dedication: This chapter is dedicated to all the women in science who have contributed so much
to the field and all those who have encouraged and mentored them in support of their dreams. It
honors the accomplishments of pioneer arbovirologists, scientists who are now leaders, mentors,
senior investigators, and professors, and those junior women embarking on their career paths.
L. D. Kramer (*)
School of Public Health, University at Albany, State University of New York,
Albany, NY, USA
M. Jozan
Malibu, CA, USA
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 3

N. Vasilakis, L. D. Kramer (eds.), History of Arbovirology: Memories from the
Field, https://doi.org/10.1007/978-3-031-21999-3_1
4 L. D. Kramer and M. Jozan
1 Introduction
It is now almost 60 years since I (LDK) became a doctoral student, enthusiastically

embarking on the path to a scientific career. The occasion of editing and writing a
chapter for this book, History of Arbovirology: Memories from the Field, taken
together with my very recent retirement lent itself to looking back and reflecting on
how far women in scientific pursuits have come, and looking forward to consider
where do we go from here to attain parity. Together with Martine Jozan, we have
been immersing ourselves in the original documents on the early history of arbovi-
rology, following the trajectory of women’s involvement and the advances in the
field up to the current time.
Women have made significant contributions to arbovirology that have played a
valuable role in building our knowledge base of arbovirus biology, ecology, epide-
miology, pathology, diagnostics, vaccines, and more. We begin by setting the his-
torical backdrop for the field of arbovirology as it exists today, exploring the career
paths and accomplishments of selected outstanding female pioneers in the field. We
describe our personal career paths and those of a sample of women who are leaders
in the field, highlighting how they are moving the field forward; we felt women in
arbovirology needed a separate chapter highlighting their accomplishments. There
are certainly many other women who could have been included, but space limita-
tions didn’t permit. Finally, the chapter explores the nagging lack of parity between
male and female scientists in upper-level positions. We present data on the improv-
ing status of women in the life sciences over time, exploring some of the current
hypotheses for the continuing inequity at higher levels of academia, and possible
approaches to address the problem.
This has been a labor of love that kept me (LDK) focused during the 2020
COVID-19 lockdown but at the same time was made more difficult by the demands
of the ongoing pandemic. Martine and I had many lively discussions while going
through Telford Work’s records and photos. Martine has an invaluable wealth of
knowledge on many of the “old soldiers” who played important roles in advancing
the field. We hope this chapter will help preserve the contributions of those scien-
tists whom we discuss.
Writing this chapter was inspiring and fun, and very personal; we hope it will
serve similarly to inspire young scientists entering the field today who may not real-
ize the struggle it has been for many women to gain recognition for their work. We
also hope it will inspire mentors to encourage women expressing interest in scien-
tific pursuits, as one of the most powerful determinants of whether a woman goes on
in science is whether someone she respects emboldens her to believe in her own
abilities.
The Quest for Excellence and the Challenge of Gender Equality in Arbovirology… 5
2 The Pioneers: Profiles of Selected Female Scientists (Who

Are No Longer with Us) Who Contributed
to the Development of the Field of Arbovirology
The pioneers in the field described here have lessons to teach us still. In discussing
with Karl Johnson how these women were able to be so successful, he noted that
New York was an outlier which allowed the three Rockefeller women (Drs Clarke,
Causey, Buckley) to be able to be recognized on the basis of their own work.
The earliest arboviral research begins with the saga of yellow fever. Drs. Ottis
and Calista Causey (Figs. 1 and 2), both physicians, were researchers at the
Rockefeller Foundation (RF). In 1940, the Causeys began working in the Yellow
Fever Service and the Northeast Malaria Service in Brazil. In 1954, they set up a lab
in Belem, Brazil, and became the first directors of the lab. They supervised the col-
lecting and breeding of mice, a practice used for the first time by Max Theiler for
yellow fever virus, but not often used because of the prohibitive cost. They invented
the “Causey hood,” a container that held mice and trapped mosquitoes under a hood.
Using these new techniques, the Causeys isolated 1593 viruses, 34 new to science
from 1954 to 1963. Robert Shope, the successor of the Causeys, stated that “it was
one of the greatest quests for viruses of all times” (Causey 1979). Figure 1 shows
Fig. 1 Ottis and Calista Causey recording mouse data. (Kindly provided by Martine Jozan)
Fig. 2 Ottis and Calista Causey in field. (Kindly provided by Martine Jozan)
them going over mouse records; the Causeys and Fred Soper were diligent about
keeping precise records. In 1964, the Causeys set up a lab in Ibadan, Nigeria, and in
1970 the Nigerian lab isolated 1500 airborne viruses and 266 other viruses. Calista
never received a cent of salary because of nepotism rules. She was acknowledged by
the RF but could not be paid.
• Recommended reading: Causey, Calista E. Interview. A National Medical
Audiovisual Center Production in Cooperation with the American Society of
Tropical Medicine and Hygiene. Sept. 1979. (Causey 1979)
• Recommended reading: Rômulo de Paula Andrade. “A Forest Full of Viruses!”
Science and development in the Frontiers of the Brazilian Amazon. Dossier:
Amazonian Borders • Rev. Bras. Hist. 39 (82) • Sep-Dec 2019 • https://doi.
org/10.1590/1806-93472019v39n82-02. (Andrade 2019)
The RF virus laboratory (RFVL) in NYC at the beginning of the expanded virus
program in 1951 was staffed by Theiler, Casals, Clarke, Taylor, Smithburn, and
Loring Whitman. Field laboratories were being established in collaboration with
foreign governments; the goal of RF members assigned to the labs was to detect and
investigate human disease. The search for viruses in arthropods, birds, and mam-
mals went on in parallel.
A major advance came with the use of continuous cell culture introduced by
Earle along with colleagues Katherine Sanford and G.D. Likely in 1948, as a work-
ing tool for the virologist. The RFVL established a tissue culture section and in
1957 brought in Sonja Buckley (Figs. 3, 4, and 10) who had that expertise after
Fig. 3 Jordi Casals, Sonja Buckley, Wilbur Downs at Yale. (Yale events and activities photographs
(RU 690). Manuscripts and archives, Yale University Library; Public access)
Fig. 4 Sonja Buckley and

RE Williams in Soviet
Union conference 1969.
(Kindly provided by
Martine Jozan)
having worked for the New York State Department of Health beginning in 1953 as
an Associate Medical Bacteriologist. They offered her a position as staff member
starting 1 Jan 1957 with a salary of $1000, as a probationary job for the first year of
employment. When all the scientists moved to Yale to the Dept of Epidemiology
and Public Health in 1965, Sonja Buckley writes in her autobiography that only she
was not given tenure when all the male scientists who moved there from the RF
were! (Buckley 1998).
Sonja Buckley worked initially under Max Theiler who thought cell culture was
too slow to yield results; so he relegated Sonja to a dark corner of the laboratory
where she managed to emerge as an eminent virologist. But Martine Jozan noted
that Theiler proved his point. She wrote, “When I came to the lab, Sonja asked me
to reisolate the Asibi strain of Yellow Fever from the frozen serum of a monkey
infected 40 years earlier. Theiler immediately inoculated baby mice and sure enough
isolated the virus after 7 days. Meanwhile I had to nurture my PS cells for 28 days
to just get a hint of virus infection, and it took three passages to adapt the isolate to
cell culture. Theiler was crowing and scoffing at us.” Sonja Buckley was a pioneer
in adapting cultures of mammalian and later insect cells for arbovirus isolation and
identification.
Sonja Buckley was the first to use fluorescent protein tracing techniques (immu-
nofluorescence) and she established cell culture neutralization tests. She tested sus-
ceptibility of cells to many arboviruses, which could then be classified and submitted
to the Arbovirus Catalogue [See later]. By Jan 1957, there were nearly 270 recog-
nized arboviruses, with the number of new isolates increasing each year.
She made the first isolation of the Lassa virus when two other institutions could
not isolate anything. At the time she got all the credit for it, and in fact, the story is
told very well in “Fever” by John Fuller. But as time went on, the men of the RF
forgot to mention this accomplishment, and it disappeared into the woodwork. In
her biography, she explained how women were prevented tenure and other positions
at the RF (Buckley 1998).
“I was there for 7 months. We still mouth pipetted everything including viruses
and tissue culture, and wore no gloves. To change the tubes we would empty the
racks on paper towels, but never had virus cross contamination, and no personal
infections”.
“Sonja Buckley was very quiet, tough, but ran the lab with ‘demonic efficiency.’
The techniques were rigorous and she supervised everything. She did not like very
young technicians because they wanted to be married and then leave. At 9:30 AM
and 3:00 PM every day Theiler would come up for coffee, often joined by Casals,
Clark and any visitor; we would all babble together and it was an incredible experi-
ence for me. Downs had told me to write to Telford Work and when I told Theiler
that he did not have a position available for me, Theiler replied ‘it is better. You
would not have been happy working with him.’ I learned a lot from Sonja – controls
in experiments, consistency, discipline and the importance of good records. The first
thing she had me do was titrate seven times the virus from a lyophylised ampoule to
teach me the importance of consistency and accuracy” (Oransky 2005).
The RFVL program was refining existing techniques for virus study, basically
complement fixation (CF), hemagglutination inhibition (HI), neutralization (NT)
tests, effecting improvements in preparation of antigen and antisera for the perfor-
mance of these tests, development of new techniques, application of these tech-
niques to the sorting, identification, classification of viruses isolated in the field.
Delphine Clark (Fig. 5) began working at the Rockefeller Foundation Virus
Laboratory (RFVL) prior to 1951 and moved to Yale in 1965. She helped advance
serologic identification of arboviruses, including the HI technique along with Casals
and LV Brown (Clarke and Casals 1958). She demonstrated that the Kyasanur
Forest Disease virus, isolated by Telford Work and Harold Trapido, 1957, from
man, monkeys, and ticks, could be distinguished from other members of the TBE
complex of Group B arboviruses (Clarke 1964). She defined two antigenic subtypes
of the Omsk hemorrhagic fever virus.
With Pierre Ardoin from the Pasteur Inst, they developed superior group C hem-
agglutinins using sucrose-acetone method of purification, sonication, and calcium
phosphate chromatography.
As part of the RFVL, and with the threat of WWII, Delphine Clark was assigned
the job of working with Alice Moore to develop techniques and equipment for pro-
duction of large quantities of 17D YF vaccine. [From an interview with Dr. Albert
Sabin, March 13, 1976] (Sabin 1976):
Interviewer: You know, I have never run across Alice Moore’s name before
you just mentioned it.
Fig. 5 Delphine Clark.

(Courtesy of Rockefeller
Archives; Rockefeller
Archive Center https://
rockarch.org/)
Sabin: Well, Alice Moore had worked with Tommy Francis* at the Rockefeller
and then she got her Ph.D., and then she went to Sloan Kettering. She was one of the
old soldiers so to speak. [*Thomas Francis Jr. (1900–1969) was an American physi-
cian, virologist, and epidemiologist. Francis was the first person to isolate influenza
virus in the United States, and in 1940 showed that there are other strains of influ-
enza, and took part in the development of influenza vaccine.]
Amelia P. A. Travassos da Rosa (Figs. 6 and 7) became director of a new Section
of Arbovirology (Seção de Arbovirologia e Febres Hemorrágicas – SAARB) sup-
ported by the Pan American Health Organization (PAHO) from 1979 until her
retirement in 1998.
In her Memoria on the Institute Evandro Chagas, she wrote, “From 1980–1998:
It was a very important period in the Arbovirology history, not only in Pará State,
however to the world. …This period was very productive, many research activities
were carried out in order to study arbovirus outbreaks in different Brazilian States.
Hundreds of arboviruses were antigenically characterized, comprising 94 distinct
serological types. Many important research projects were developed in collabora-
tion with national and international organizations. More than 200 papers were pre-
sented in scientific meetings, as well as published in national and international
journals in the virology field” (Travassos da Rosa 2016).
During that time, research projects in the Amazon Region investigated eco-
epidemiological aspects of the arboviruses and the impact of anthropogenic change.
For example, the “Tucuruí hydroelectric dam was built, and several other human
actions in the forest that led to the emerging of several viruses.” The research con-
tributed substantially to a better understanding about the biodiversity of zoonotic
viruses in the Amazon, creating a challenge for several future generations of arbo-
virologists (Travassos da Rosa 2016).
Fig. 6 Dr. Robert Shope

and Amelia P. A. Travassos
da Rosa conducting the CF
test in their laboratory
facility at the Belém Virus
Laboratory, Pará State,
Brazil 1959. (Kindly
provided by Martine
Jozan)
Fig. 7 From L, Jordi Casals, Robert Shope, Thomas Aitken, Calista Causey, Jack Woodall, Amelia
Travassos da Rosa, International symposium on tropical arboviruses. (Kindly provided by
Martine Jozan)
Because of the proliferation of arbovirus research, a conference was convened at

Gould house on the Hudson in 1959. Drs. William F Scherer and Edward Buscher
urged adoption of a proposal for the informal exchange among American laborato-
ries, forming the new Subcommittee on Information Exchange (Richard Taylor,
William F Scherer, Telford Work who replaced Ken Smithburn). Over dinner during
the Federation Meetings, April 1960, the first meeting of the American Committee
on Arthropod-borne Viruses (ACAV; Fig. 8) took place also including distinguished
virologists Max Theiler, Jordi Casals, Delphine Clarke, Richard Taylor, Smithburn,
Loring Whitman, and Sonja Buckley. The first issue of the Arbovirus Information
Exchange was shared during this dinner meeting, as was the first issue of the
Arbovirus Catalog. This represented “universal documentation that brought arbovi-
rology into the forefront of virological science.” The ACAV was organized into a
formal entity 1 year later at the Centers for Disease Control (CDC) in Atlanta,
Georgia. A photo of a meeting of the ACAV in 1964 shows a single female, Gladys
Sather, among 13 men.
The establishment of the Arbovirus Information Exchange became an extraordi-
nary conduit of information, linking arbovirus programs worldwide, giving more
isolated laboratories access to information, and scientific exposure for their work.
The casual format for the publication nurtured a nucleus of scientific relationships
which created a unique network of intellectual and technical resources.
Knowledge of tick-borne viruses and the diseases they caused was still in its
infancy. Neurologic tick-borne viruses, e.g., tick-borne encephalitis virus and
Fig. 8 American Committee on Arthropod-borne virus subcommittee. Left (L) to Right (R) D
Davis, CH Andrewes, CM Eklund, RM Taylor, WF Scherer, T Work, A Shelolov, P Gibbs, G
Sather, Webb, J Casals. Along the wall: Overman, RW Chamberlain, N Wiebenga, 1964. (Kindly
provided by Martine Jozan)
visceral (hemorrhagic) tick-borne viruses, e.g., Crimean-Congo hemorrhagic fever

virus (CCHFV), Kyasanur forest disease virus (KFDV), Alkhumra or Alkhurma
hemorrhagic fever virus (AHFV), and Omsk hemorrhagic fever virus (OHFV) were
emerging in the Eurasian and African continents. This fostered two “US delegations
to the USSR for Hemorhagic Fevers,” in 1965 and 1969, respectively (Casals et al.
1970; Work 1965; Figs. 9 and 10). “On these hemorrhagic fever missions to the
Soviet Union, the American scientists were accompanied by Bela Kaplan, the inter-
preter. They would never have been able to do the work without her” (From M
Jozan; Figs. 9 and 11). Helena Leshchinskaya was a physician/epidemiologist
(Figs. 9, 11, 12 and 13). Her expertise as a physician was paramount in the under-
standing of hemorrhagic fever.
Michael Chumakov, together with his colleagues, discovered the etiology of a
new transmissible neurological disease and tick-borne encephalitis (TBE) and iso-
lated the virus that causes it. He was accidentally infected with the virus and devel-
oped encephalitis that led to a permanent loss of hearing and paralysis of the
right arm.
Beginning in the 1940s, Chumakov organized multiple medical expeditions to
Siberia and other regions of the Soviet Union to investigate outbreaks caused by
new viruses. Among the viruses discovered and studied by Chumakov are Omsk
hemorrhagic fever and Kemerovo fever viruses, hantavirus causing renal syndrome,
Crimean-Congo Hemorrhagic Fever virus, and many others (Chumakov et al. 1964).
Fig. 9 Tick-borne hemorrhagic fever delegation. L to R, Ned Wiebenga, Shelokov, Bela Kaplan,
Michael Chumakov, Karl Johnson, Helena Leschinskaya, Voroshilova (virologist and wife of
Chumakov), Jordi Casals. (Kindly provided by Martine Jozan)
According to Telford Work, Helen Leshchinskaya had seen more hemorrhagic

fevers than anyone. Like all the Russian scientists she was full of zip, charming, and
very efficient and knowledgeable. During World War II, many men were killed, and
there was thus a huge rise in women doing their jobs, many scientists among them.
From an interview with Alex Shelakov, 2005: “Leshchinskaya was a superb
human being. ‘Superb clinician.’ I’ve seen her work with patients like a magician.
She was just incredibly competent in being able to relate to them, to make them feel
that they are somebody and so on. Unlike tales about Soviet medicine being cold
and indifferent, and these superiors pushing people around. She was not like that,
and I don’t think very many were.”
“I met B.E. Henderson in 1965 in Dakar. He was sent by Telford to help us deal with
the big yellow fever epidemic of ‘Diourbel,’ which greeted me as a beginner virologist.
Fig. 10 International Symposium on tick-borne arboviruses, (Excluding group B) Sonya Buckley,
R.E.Williams, Yalka Vesinjak of Yugoslavia, B. Henderson, M. Balduci, P. Verani, 1969. (Kindly
provided by Martine Jozan)
We were running out of vaccines and syringes and used the pedojet for the first time.
Children loved it and came back for more, which was problematic” (M Jozan).
Dr. Helena Libikova was a remarkable virologist who worked on tick-borne
viruses in the Institute of Virology in Bratislava, Slovakia (Fig. 14). (From Martine
Jozan remembrances): “I met Libikova when I was at the Institut Pasteur Paris, in
Claude Hannoun’s lab (influenza, hepatitis B, Arboviruses) where Gresikova
(Fig. 53; tick-borne encephalitis virus, Hantaan virus) came to visit. Later that year
(1969), I worked with Doubravka Malkova in Prague; she was studying the latency
of tick-borne viruses in the lymphatic system of small mammals, which was quite
avant garde at the time. I met Libikova at U’Flaku, a very legendary restaurant illus-
trating the work of writer Yacek, and here she was with her husband. The Russians
had come to Prague in 1968, isolating these scientists. Yet they worked hard together
with little opportunity for outside scientific communication, and the collaboration
was extraordinary.”
Fig. 11 L to R. Bela Kaplan, Helena Leschinskaya, Michael Chumakov during Delegation on
hemorrhagic fevers. (Kindly provided by Martine Jozan)
Fig. 12 Helena Leschinskaya. (Kindly provided by Martine Jozan)

Fig. 13 Helena Leschinskaya in field. (Kindly provided by Martine Jozan)
Fig. 14 Helena Libikova, Institute of Virology, Bratislava, Slovakia, with colleagues and Telford
Work, 1965. (Kindly provided by Martine Jozan)
Fig. 15 Pospelova-Strohm, Telford Work and Harry Hoogstral looking at a Russian book of tick
taxonomy, Moscow, 1965. (Kindly provided by Martine Jozan)
Dr. M.V. Pospelova-Strohm was the tick expert in the Soviet Union, the counter-
part of Harry Hoogstraal, who had immense regard for her (Figs. 15 and 16; photos
taken in Moscow during the 1965 Russian expedition to the Soviet Union).
Dr. Patricia Webb (1925–2005) earned her MD in 1950 from Tulane University.
She completed her pediatric residency in 1953 at the Kern General Hospital in
Bakersfield, CA. She started working on Rhinoviruses (USPHS NIH, 1961) and
then went to the Middle America Research Unit in Panama from 1963 to 1975.
Patricia Webb was involved in the discovery and characterization of some of the
most lethal infectious diseases to date (e.g., Ebola, Lassa, Machupo, and more). Her
work on the Machupo virus led to the creation of the family Arenaviridae. She stud-
ied Filoviruses with the Special Pathogens Branch, CDC, 1975 (Fig. 17) and made
the initial isolation of Ebola in Vero cells. She demonstrated with IFA that Ebola
was a new agent distinct from Marburg. She conducted intensive field, laboratory,
and clinical studies in West Africa on Lassa virus infection and demonstrated the
effectiveness of ribavirin for Lassa. Patricia Webb became director of the CDC
Kenema field station in Sierra Leone in 1978 (Fig. 18), and from 1981 to 1988 she
worked on virus vectors between humans and livestock at the Division of Vector-
borne Viral Diseases, CDC Fort Collins.
“Colleagues fondly remember her as smart, sharp-witted, demanding, tenacious,
detail-oriented, and crusty, but all knew that she could be counted on for support
when needed…. Most remember Patricia as a tough – even relentless – taskmaster,
but fun to be with. [Co-workers] would comment that working with her was not for
Fig. 16 Pospelova-strohm (center) and her staff. (Kindly provided by Martine Jozan)
the ‘faint of heart,’ but all appreciated the experience and remember it fondly”
(Chu 2018). I (LDK) personally can attest to this having worked with Patricia at the
Middle America Research Unit in Panama from 1974 to 1977.
“Patricia taught me the fundamentals of critical thinking and problem solving.
She taught me work ethics and gave me confidence to pursue knowledge that would
help humanity. She would tell me that my place of birth in the small and dusty little
town of Kenema should be a source of pride and not an impediment for standing tall
among international peers” (from Austin Demby, Director of the PEPFAR program
for the Department of Health and Human Services).
• Recommended reading. May C. Chu. The Legacy of Patricia Ann Webb: Broken
Vials and Urgency. Women in Microbiology. Edited by Rachel J. Whitaker and
Hazel A. Barton © 2018 American Society for Microbiology. All rights reserved.
https://doi.org/10.1128/9781555819545.ch31. Chapter 31 (Chu 2018)
Dr. Margaretha Isaacson (Figs. 19, 20 and 21) suffered internment as a child
during the Nazi occupation of the Netherlands, her country of birth. Her knowledge
of tropical and infectious diseases encompassed a breadth and depth that few could
match. She had vast clinical experience and an extensive list of publications. She
served as a member of a World Health Organization working group. She also served
on the senate of Witwatersrand University and was a member of the Biological
Fig. 17 Patricia Webb. (Photo courtesy of Fred Murphy 1976)
Weapons Working Committee of the South African Non-proliferation Council, as

well as serving as associate editor of Journal of Travel Medicine (ACTM 2008).
Isaacson was a member of the team that investigated Marburg fever in
Johannesburg, 1975; and Ebola, 1976, when she was involved in the first Ebola
outbreak in 1976 in Zaire (Fig. 20). She saw that the virus was able to spread rapidly
because of poor infection control measures and promptly instituted isolation pre-
cautions and quarantined all hospital workers; as a result, the outbreak was eventu-
ally contained. She advocated “universal precautions” in handing the patient’s blood
and body fluids as well the wearing of protective gowns, gloves, and face mask
whenever in contact with the bleeding patient (ACTM 2008).
Isaacson was a founding member of the International Medical Commission for
the Investigation and Control of Viral Haemorrhagic Fever (Ebola) in Zaire and a
founding member of the South African National Haemorrhagic Fever Emergency
Team. She was founder and editor of VHF Directory, a member of the South African
Medical Research Council advisory panel on tropical diseases, and Associate editor
of Journal of Travel Medicine. Her investigations also included other infectious
diseases such as hepatitis, cholera, sporotrichosis, babesiosis, and plague.
Fig. 18 Patricia Webb. (Photo courtesy of Joe McCormick 1978 Sierra Leone)
Fig. 19 Margaretha
Isaacson. (Jennifer Burgess
j.burgess@amaq.com.au
permission granted)
Fig. 20 Margaretha Isaacson in hospital room. (Photo in public domain provided by Dr. Lyle
Conrad (https://phil.cdc.gov/Details.aspx?pid=6921) 1976)
Fig. 21 Margaretha Isaacson in field. (Photo in public domain)
• Recommended reading. The Memoirs of Margaretha Isaacson. Annals of the

ACTM. 2008. (https://search.informit.org/doi/pdf/10.3316/informit.20664378
1669169) (ACTM 2008).
Dr. Enid Cook de Rodaniche (1906–1988) was an American virologist and bac-
teriologist. She was the Chief of the Public Health Laboratory at the Instituto
Conmemorativo Gorgas in Panama City, Panama (Fig. 22), where she was the first
Fig. 22 Pedro Galindo, Enid Rodaniche, Harold Trapido
person to isolate the yellow fever virus in Panama and, along with her physician
husband Arcadio Rodaniche, identified and characterized the viral strain responsi-
ble for an outbreak of polio in Panama in 1950–1951. She was on the founding
faculty of the University of Panama School of Medicine and its first professor of
parasitology and microbiology.
She was the first Black student to graduate from Bryn Mawr College, majoring
in chemistry and biology. The Enid Cook ‘31 Center at Bryn Mawr College is
named for her, and the Dr. Enid Cook de Rodaniche Medal is awarded by the Rotary
Club of Panama for work in virology (“Enid_Cook_de_Rodaniche” n.d.).
Enid Cook de Rodaniche was a lecturer at the University of Chicago from 1937
to 1944. During her time at the University, she published a number of journal arti-
cles on her research into St. Louis encephalitis and on herpes. Beginning in 1946,
she was the chief of the Public Health Laboratory at the Instituto Conmemorativo
Gorgas in Panama City where her work centered on viral diseases and rickettsias
(“Enid_Cook_de_Rodaniche” n.d.).
Gladys Sather (Fig. 23) was head laboratory technician for Dr. William Hammon
at Univ. of Pittsburgh. She published 82 papers, mostly with Hammon, but also with
others, on dengue diagnostics and cases, as well as other arboviruses.
During an encephalitis outbreak in Yakima Valley, CA, Dr. William Hammon
collected samples of blood from domestic animals. “He didn’t collect any wild ani-
mal bloods, because at that time we [WC Reeves] thought this was a horse disease
and also in domestic mammals and man, so he collected those bloods and found
antibodies in them. [The antibody testing] was to differentiate polio, St. Louis, and
western encephalitis [which have similar early symptoms].
Fig. 23 Gladys Sather
Fig. 24 Pauline Peralta – permission generously granted Jean-Paul Carrera, Investigador en
Salud, Department of Virology, Gorgas Memorial Institute and the Peralta family
Hammon established the diagnostic facilities and the diagnostic tests, and
recorded the clinical histories” (Reeves 1993).
Dr. Pauline Peralta (1926–2021) (Fig 24). In 1958, Alex Shelokov, then director
of the Middle America Research Unit (MARU), first hired Dr. Peralta as a microbi-
ologist/virologist, a career she pursued until she retired. Pauline worked with, and
in some cases mentored, many noted arbovirologists, medical entomologists, infec-
tious disease epidemiologists, and other past and present members of the ASTM&H,
including Alexis Shelokov, Karl M. Johnson, Patricia Webb, Pedro Galindo,
Nathanial Young, Arnold Monto, William C. Reeves, Jr., Clarence C. Peters, John
Craighead, Barnett Cline, Byron Chaniotis, Jack Peterson, Abdiel Adames, Gerald
Eddy, Margaret Grayson, Robert Tesh, David H. Martin, Thomas Walton, Laura
Kramer, Charles Seymour, Bedsy Dutary, Sandra Lopez-Verges, Evelia Quiroz,
Jean Paul Carrera, Gladys Oro, Gustavo Justines, Sunthorn Srihongse, and Yamilka
Diaz (Fig. 24).
Pauline was a very modest person by nature, preferring to let others take the
spotlight, but she was an important contributor and co-author on many significant
publications. Some of her collaborative research projects included the following:
ecologic and epidemiologic studies of vesicular stomatitis virus, ecologic investiga-
tions of encephalomyocarditis virus, studies of the seasonal occurrence of respira-
tory illness among children in Panama, epidemiology and potential hosts of eastern
equine and Venezuelan equine encephalitis viruses, as well as virologic and sero-
logic studies in Panamanian sloths and cormorants, first description and character-
ization of the Changuinola virus group, discovery of new viruses in the sandfly fever
group of phleboviruses, participation in nutritional and arbovirus prevalence sur-
veys of rural residents in Central America and Panama (Tesh and Kramer 2021).
3 My Career Path: Coming Full Circle from Southam

to Sturman
Laura D. Kramer
My pathway to becoming a professor and laboratory director began and ended with
good luck, first, having landed in the classroom of an inspiring science teacher in
high school, and later, with the good luck to have an advocate to recommend me for
a position in the right place at the right time to study an emerging virus, West Nile
virus, following its introduction into the naive environment of the western hemi-
sphere. In between these bookends of good luck, I took advantage of opportunities
that presented themselves; honed the art of patience, resilience, and perseverance;
developed a tough skin; and had amazing mentors (Figs. 33, 34, 35, 36 and 38). My
career path demanded long work hours and dedication and had many memorable
moments, too many to recount here. While my professional path was rather direct;
my hope is by sharing it; I might provide some insight for young scientists who are
just now working their way through the quagmire of choices and decisions to
be made.
Historically, I am the first research scientist in my family, which is composed of
doctors, lawyers, economists, accountants, writers, teachers/librarians, with one
very notable exception, my cousin, a renowned anthropologist May Mandelbaum
Edel, who was the first American woman anthropologist to live in an African vil-
lage. Perhaps it is from her I inherited my wanderlust. My interest in science was
sparked relatively early when I was a student at Sanford H. Calhoun High School,
Long Island, NY. It has been observed repeatedly that interest and attachment to a
career in science are formed early in life. Teachers and parents play a huge role in
attracting young people to the field. And so it was for me, my biology teacher,
Howard Crouch (Fig. 33), with whom all the girls were enamored, was a terrific role
model and my first mentor. He loved teaching and was always enthusiastic, and he
encouraged me to apply for a summer position at Sloan Kettering Institute in NYC. I
was accepted and worked in the lab of J. Spencer Munroe and Chester M. Southam,
who had experimented with using West Nile virus to treat cancer patients, but my
studies at the time focused on Rous sarcoma virus. I have little recollection of the
actual research other than cages and cages of animals we infected and studied. What
stays with me mostly is the hard-driving competitive research environment. I found
that pressured work atmosphere difficult, so I decided to look for a different type of
job in NYC the following summer. But nothing materialized. At the same time, Mr.
Crouch once again offered help, suggesting that I explore a resident summer pro-
gram for students at Brookhaven National Laboratory on Long Island. I was
accepted and hoped this opportunity would allow me to see a gentler side of science
working in the lab of Dr. Donald Baker. And what a different experience that was – a
great research program again under a wonderful PI, integrated with lots of fun activ-
ities around science – lunchtime tennis, bike rides, a Long Island, NY summer with
other like-minded students, a great work/life balance. I realized life as a scientist
could be a great life choice after all.
My high school mentor and the heads of the labs in which I worked were always
male. I never thought further about it, and I never felt uncomfortable as a female
among predominantly male faculty and students at Brookhaven. I never felt held
back, overlooked, or underappreciated as a female. Perhaps it was my naiveite or
perhaps my passion for science obscured my vision, making me rather gender-blind
in those early days, motivated by the thrill of being in a lab.
I went to college at the University of Pennsylvania where I was a science major
taking pre-med classes with the intention of going into research. At this time, in the
1960s, there were very few women at Penn, especially in the sciences. With only a
handful of women in a class of hundreds, the focus on the few females was intense.
But I worked hard and persevered and went on to graduate school at Cornell Medical
College in New York City. Here too in the late 1960s, there were very few women
and the pressure to excel was great.
Although it was clear I was leaning toward a research career, my parents tried to
persuade me to pursue an MD rather than a PhD at Cornell Med. But my passion for
international fieldwork led me to the doctoral program of Dr. William F Scherer
(Figs. 8, 26 and 34) to work on the Venezuelan equine encephalitis virus (VEEV) in
Central America. This would give me the opportunity to do field research in Mexico
and Guatemala on a mosquito-borne virus with a giant in the field – a brilliant sci-
entist, a great virologist, field worker, and mentor. The lab was balanced between
male and female students, although I have been told that Dr. Scherer at one point
would not accept females. Peter Jahrling (former Chief of the Emerging Viral
Pathogens Section of the National Institute of Allergy and Infectious Diseases)
entered the same year I did, and Priscilla Schaffer (1941–2009, Chief of the
Laboratory of Molecular Virology, Harvard Medical School at the Beth Israel
Deaconess Medical Center), graduated as we started. Later, Scott Weaver (Scientific
Director, Galveston National Laboratory; Professor, Microbiology & Immunology
and Pathology, UTMB) and Rebecca Rico-Hesse (Professor Virol and Microbiol
Baylor College of Medicine) joined Scherer’s lab at Cornell Medical College. Bill
Scherer was a demanding mentor, with a very hands-on approach to training stu-
dents (Fig. 26). He was extremely supportive and I never felt any stigma as a female
in his lab, unlike in the medical school classes. The main stigma came with pursuing
a PhD in a medical school.
My thesis was on Aedes taeniorhynchus as a competent vector of VEEV epi-
demic strains (IAB) but incompetent for endemic strains (IC), in Central America,
thereby serving as a biologic marker for epidemic strains of virus. There were
unforgettable experiences in the field in Guatemala and Mexico, in abattoirs collect-
ing blood, climbing trees collecting nestling birds, living in a village schoolyard
with scorpions and spiders and roosters crowing all night [one of which we pur-
chased and cooked for dinner in order to give us some peace and quiet], following
school children who would lead us to our hamster-baited mosquito traps when we
could not find them, harrowing experiences with lab vehicles breaking down in the
middle of nowhere, spending days stranded in a remote village in the mountains of
Chiapas en route to Guatemala (Fig. 25) patiently waiting for a car part, negotiating
our way across borders of Guatemala and Mexico, with a van full of biologic speci-
mens. None of these experiences would I replace with any other graduate program
were I to do it again.
After earning my PhD, I went to the Middle America Research Unit and the
Gorgas Memorial Lab in Panama as a post-doctoral fellow. This experience solidi-
fied my love of arboviral research and ecological questions. There were
Fig. 25 Laura Kramer, Brownsville, TX 1969 en route to Guatemala to conduct doctoral field
research, 1969–1974, under William F Scherer, Cornell Medical College. (Kramer, personal photo)
Fig. 26 William F Scherer, Cornell Medical College, PhD mentor. (Photo kindly provided by
Rebecca Rico-Hesse of our doctoral mentor at Cornell Medical College, working in the field in
Guatemala. Dr. William F Scherer loved his summer field work with students and field help and as
can be seen in the photo, was fully an active member of the team)
uncomfortable times as a female in the tropical forest study site in the Bayano forest
(Figs. 27 and 28) sleeping in a common space with field workers and their mis-
tresses and pet monkeys, fear of bush masters on the trails at night, and all the mud
and dust, biting insects and bumpy roads. But I was incredibly fortunate to have the
opportunity to work with two visionary and brilliant scientists, Pedro Galindo and
Karl Johnson. Both of these men were incredibly stimulating and exciting to work
with, and each offered unique expertise and insights. Here my research mostly
focused on yellow fever virus forest vectors Sabethes chloropterus, one of the most
beautiful mosquitoes in the world, and Haemagogus equinus, and St. Louis enceph-
alitis virus (Figs. 27, 28 and 29).
From Panama, and after a brief stint at the Univ. of Wisconsin, Madison, I was
invited to join the UC Berkeley group of Dr. James L Hardy (1932–1997) (Figs. 35
and 37), Chair of the Department of Biomedical Sciences and Dr. William C. Reeves
(1916–2004) (Figs. 36 and 37), professor and Dean Emeritus of UC Berkeley’s
School of Public Health, both world-renowned arbovirus experts and outstanding
entomologists. This was another stroke of luck. Interestingly, Jim Hardy also had
been trained by Bill Scherer. Together their labs established the natural history of
western equine encephalitis and St. Louis encephalitis viruses, among other arbovi-
ruses. They were the go-to authorities for any questions on mosquito-borne viruses.
They were devoted to their students and excellent teachers. It was at UC Berkeley
that my own interests expanded and intensified. I always felt I had the full support
Fig. 27 Laura Kramer, Bayano, Panama 1974, Middle America Research Unit/Gorgas Memorial
Laboratory – post-doctoral research on forest mosquito vectors of yellow fever virus. I am standing
together with the field team. (Kramer, personal photo)
Fig. 28 Laura Kramer, Bayano, Panama 1974, Middle America Research Unit/Gorgas Memorial
Laboratory establishing field colonies of Sabethes chloroptorus and Haemagogus equinus mosqui-
toes. (Kramer, personal photo)
Fig. 29 L to R – Gerry Eddy, Laura Kramer, Bob Tesh, Gustavo Justines, Pat Webb, John Seal,
Karl Johnson, Tom Walton, Charley Seymour, ??? Dinner with Panama group at ASTMH annual
meeting. (Kramer, personal photo)
Fig. 30 The original New York West Nile team: Kristen Bernard, Laura Kramer, Greg Ebel,
Elizabeth Kauffman at Griffin Lab, Wadsworth Center, Albany, NY, 2001. (Personal photo)
Fig. 31 Laura Kramer and Larry Sturman, 2012. Receiving the first Sturman Excellence in
Research Award, 2012 [Wadsworth Center, Albany, NY]. (Personal photo)
of Hardy and Reeves, and we had a terrific team of scientists who enjoyed working
together (Fig. 37), but my ambitions were restricted by being in research (non-ten-
ure) track and no effort was made to promote me to tenure tract (until I made the
decision to leave for the New York State Department of Health (NYSDOH) at the
time of my recruitment following WNV introduction). I had the freedom to design
and conduct my own projects and contributed to grant applications mostly to the US
Army and NIH, but the proposals could not be in my name. (Eventually, at UC
Davis, I was allowed to submit a grant to NIH on St Louis encephalitis virus, which
was my first NIH grant to be awarded.)
While in Berkeley, I was extremely fortunate to know Professor Andy Jackson, a
plant virologist, through a comparative virology course he co-taught with Dr. Loy
Volkman, an insect virologist. He generously offered me precious bench space in
his lab to learn molecular approaches to virus research. In those days, sequencing
and cloning were laborious and hands on, which students today may not appreciate,
but I accomplished a lot and moved our understanding of temperate arbovirus ecol-
ogy and genetics one step further through this opportunity. Landmark findings I
made in Berkeley included our description for the first time of the mosquito midgut
escape barrier for the alphavirus, western equine encephalitis virus (WEEV); selec-
tion of a WEEV-resistant population of Culex tarsalis with which we could better
study determinants of vector competence, and much more.
With the untimely death of Dr. Hardy, the classical virology conducted by our lab
was no longer considered a high priority for the School of Public Health, UC
Fig. 32 L to R. Nikos Vasilakis, Shannan Rossi, Gregory Ebel, Carol Blair, Laura Kramer Ann
Powers. Delegation of ACAV members invited to participate in a historic NIH-sponsored meeting
in Havana, Cuba on 28–30 November 2016. This scientific conference entitled ‘Exploring
Opportunities for Arbovirus Research Collaboration’ was convened to share information about
current arbovirus research, recent findings and future research priorities. NIH and ACAV responded
to a call for such a meeting at Cuba-U.S. leadership meetings where collaboration on arbovirus
research was identified as a priority for scientific interaction. (Photo kindly provided by
ACAV, ASTMH)
Berkeley. Fortunately, we were invited to move the lab to UC Davis where we estab-
lished the Center for Vector-borne Disease Research, which would not have been
possible without the tremendous support of Dr. Fred Murphy and Dr. Bennie Osborn
under the auspices of the UC Davis Veterinary school. But in the Vet school, there
were few women and even fewer PhDs (rather than DVMs) making for a difficult
existence. This was pre-West Nile virus and arboviruses were not highly considered
by the Department Chair as important research topics before its introduction and
spread in the USA.
When WNV was introduced into the USA in 1999 and the country, CDC and
New York State Department of Health (NYSDOH) and most other health depart-
ments in the Eastern US, were gearing up frantically to stay on top of this virus, I
was recruited to NYSDOH to set up a laboratory for surveillance and research on
medically important arboviruses in NYS, particularly WNV and eastern equine
encephalitis virus. I was brought in largely on the strong recommendation of Dr.
Fig. 33 My mentors – (1)

Howard Crouch: Founder/
President of the Damien-
Dutton Society for Leprosy
Aid, Inc., a worldwide
foundation, aiding the
victims of leprosy, with
headquarters in Bellmore,
NY. My high school AP
biology teacher
Fig. 34 (Mentor 2, PhD)

William F Scherer
(1952–1982). (Photo
kindly provided by ACAV,
ASTMH)
Fig. 35 (Mentor 3,

post-doctoral) James L
Hardy (1932–1997).
(Photo kindly provided by
ACAV, ASTMH)
Fig. 36 (Mentor 4, career)

William C Reeves
(1916–2004)
Jack Woodall (Fig. 38), another renowned arbovirologist and colleague whom I had
known well for many years, who was then Director of the Arbovirus Lab at the
NYSDOH, more good fortune. At that time, the lab was extremely diminished in
size operating on a shoestring budget and staff, and Jack wanted to retire to devote
Fig. 37 L.to R. William C Reeves, Robert E Chiles, Chuck Fulhorst, James L Hardy – The
Arbovirus group UC Berkeley. (Photo kindly provided by Tom Ksiazek)
Fig. 38 (Mentor 5, ProMED) Jack Payne Woodall (1935–2016)
himself full time to ProMED (Program for Monitoring Emerging Diseases), which
he had co-founded. This was a difficult decision for me, moving from the west coast
to upstate New York to a city, Albany, about which I knew almost nothing, and leav-
ing my family behind in Berkeley. But Wadsworth Center went all out with a won-
derful offer of basically everything I asked for, and more. Furthermore, the
opportunity to study a newly introduced virus as it adapted to a naive environment
and evolved over space and time was not to be passed up! And finally to be
considered, Wadsworth Center is a unique environment with a large number of

strong female scientists in senior positions. This was the first time I found myself in
such an environment. Dr. Lawrence Sturman, the Wadsworth Center Director (Fig.
31), was an extremely supportive and visionary scientist who made himself avail-
able to listen at almost any time; he seriously considered my ideas and extended
himself to bring them to fruition. The female scientists at Wadsworth were terrific
and talented and a joy to have as colleagues. Not only were they superb scientists
but also talented poets and artists. This was the first time, albeit late in my career, I
had this experience of more than an occasional female peer. About ten of us formed
a vibrant support group, WoW (Women of Wadsworth), which brought us emotional
and intellectual support over the most enjoyable dinners full of science and laughter
and commiseration.
I stayed at Wadsworth until I retired in late 2020 after more than 20 years. There
were certainly problems – at one rather confrontational meeting between WoW and
the Wadsworth Director, it was suggested by the Director that the men were able to
solve their problems on the golf course and why didn’t we female scientists con-
sider that approach to solving our problems, a typically male perspective! But at
Wadsworth, with the support I had, I was able to build a lab with a wonderful team
of scientists, technicians, students, and administrative help (Fig. 30). I had a score
of amazing collaborators and developed exciting national and international research
collaborations which allowed me to travel to exotic locations to collaborate or pres-
ent our findings, making lifelong friends all over the world, from Central America
to Cuba (Fig. 32), South America, Europe the Middle East, Africa, Asia, Australia,
New Zealand, and more. Adding to this, the lab hosted senior scientists and students
also from all over the world who came largely to take advantage of our expertise as
well as our wonderful high containment insectary, built following a successful facil-
ities grant from NIH that I co-wrote with terrific Wadsworth support staff almost
immediately upon arriving there in 2000.
Along the way, I was awarded a Fulbright fellowship in La Plata, Argentina to
study the ecology of dengue and chikungunya, and now have completed a second
Fulbright award in Cordoba, Argentina to teach viral ecology. The research of the
Arbovirus Lab produced seminal papers on the ecology of the West Nile virus, the
impact of temperature on vectorial capacity of Culex pipiens complex mosquitoes,
evolutionary pressures on arboviruses, as well as diagnostics and antivirals. The
overall focus of the program I established was to dissect virus-vector-vertebrate
interactions and elucidate the factors that impact virus emergence, persistence, and
spread in the environment and determine how these interactions impact virus adap-
tation and evolution. We also conducted surveillance for medically important vec-
tor-borne viruses in mosquitoes and ticks, providing us with an invaluable bank of
virus strains and allowed us to analyze patterns of virus activity with changing con-
ditions. The research/surveillance pairing is ideal. It would be hard to imagine a
more fulfilling career, and many days I would wake up and pinch myself to be sure
this life I was leading was all real, and even more, to be honored for my life’s work
with receipt of the Richard M Taylor award for outstanding contributions to
arbovirology as the third female in its history by the American Committee on

Arthropod-borne Viruses/American Society of Tropical Medicine and Hygiene.
Looking at my professional history, while it was a rather direct path, both family
and professional decisions were made every step along the way that at the time felt
agonizingly difficult. My son Benjamin was born in 1981, several years after I
started at UC Berkeley, and while challenging and exhausting of course, I learned
quickly how to work more efficiently and establish networks with other parents.
There is no best time to start a family as a scientist, and one must weigh many fac-
tors before making such a momentous decision. No doubt, it slowed down my pro-
fessional progress, but having a baby enriched my life. Raising a child with all the
love they bring out in us, or completing a composition in music or art or whatever
one’s passion, can only be positive, albeit trying at times, like a research project. In
addition, either focus can provide an escape when one or the other is causing frus-
tration. When my family expanded to include grandchildren, I was in a very differ-
ent place professionally, well established in my career with greater confidence in
my abilities and my position. Life choices become easier with maturity, a wonderful
position to be in, but we discover that science and life never cease presenting new
challenges and new rewards.
I decided to retire in 2020 to spend more time with my grandchildren in California;
Albany, New York, being 3000 miles away from Berkeley, CA, was just too far and
COVID-19 made travel much more difficult. I also felt it was time to step aside and
let the next generation take charge. I find I am working as hard as ever following in
the footsteps of Jack Woodall, moderating Ebola and COVID-19 for ProMED,
which has more than 80,000 subscribers, writing for Merck Manuals as a virus
author, and serving as an Associate Editor of the International Journal of Infectious
Diseases, among other scientific endeavors, advising, and mentoring. One day I
picture actually feeling retired and sitting down in a comfy chair to knit or play my
flute and do all those things I have always dreamt about having the time to enjoy.
4 Movers and Shakers
Our (MJ and LDK) career paths and scientific pursuits were very different from
each other and represent just two paths among endless possibilities, as will be clear
from the brief biosketches below. We have tried to highlight a selection of individu-
als who have had and continue to have a strong impact on the field, and others who
are just embarking on that path. This group of trailblazers is not at all meant to be
exclusive, as everyone working to the same end in the field or lab makes an impact,
but space constraints limit the number of women we could include.
Ellen Strauss, PhD (Figs. 39 and 40)
Senior Research Associate in Biology, Emeritus
California Institute of Technology
Fig. 39 Ellen and James Strauss, February 2008, Vienna, in the major cemetery in a garden of
tributes to musical greats beside the statue of Johann Strauss
Fig. 40 Strauss group photo from around 1983. From left: Henry Sun, James Ou, Jeff Mayne,
Charlie Rice, Jim Strauss, Caroline Vermaes, Ellen Strauss, unknown (probably Caroline’s daugh-
ter) and Edith Lenches
and
James H. Strauss, PhD (1938–2021)
Ethel Wilson Bowles and Robert Bowles Professor of Biology, Emeritus
California Institute of Technology
As was the case with some of the pioneer women described in section I, for
example, Calista Causey, there have been very accomplished husband and wife
teams. Jim and Ellen Strauss exemplify this.
James H. Strauss and Ellen G. Strauss (Fig. 39) both received their doctorate
degrees in biochemistry from Caltech; it is notable that Ellen Strauss was the “first
woman admitted to graduate study in biology through the regular admissions pro-
cess and ultimately became the 10th woman to receive a PhD from Caltech.” The
Strauss’s returned to Caltech in 1969 to begin a lifelong program in teaching and
research in animal virology. Their research has focused on togaviruses and flavivi-
ruses and has resulted in many original contributions to our understanding of these
two families of viruses. Their groundbreaking research examines the “molecular
structure and replication of these viruses and the connection between virology and
human disease” (E and J Strauss).
[Following the submission of this very brief biosketch by the Strausses, we
learned that Jim Strauss passed away on 28 December 2021. Not only was he a
wonderful scientist, but also “a quiet and thoughtful man, always thinking before he
spoke, generous with his time and expertise, kind to all, slow to anger, and pos-
sessed of a wry and gentle sense of humor. His office door was always open, but he
believed in permitting his students to develop their own projects. He believed pas-
sionately in meritocracy for selecting faculty or admitting students, and scientific
truth as a guide for many decisions; admired expertise in varied subjects; champi-
oned Free Speech even for those who disagreed with him, abhorred hypocrisy and
lies at all levels…” Truly a wonderful person. His obituary can be found at Strauss,
James Obituary. https://www.legacy.com/us/obituaries/latimes/name/james-strauss-
obituary?id=32239236].
Sondra Schlesinger, PhD (Figs. 41 and 42)
Professor Emeritus
Washington University School of Medicine
Sondra Schlesinger’s research interests first focused on microbial genetics. Later,
she redirected her research toward understanding the structure and replication of
RNA-enveloped viruses, in particular the alpha virus, Sindbis. Insight gained from
these studies has led to the development of vector systems for the expression of
heterologous genes. As such, these systems are used worldwide for many molecular
and neurobiological applications.
For almost 40 years, Sondra Schlesinger has contributed to the Washington
University School of Medicine as a teacher, investigator, and administrator. As the
first woman to hold a faculty position in her department, she recalls primarily being
ignored as she began to establish her lab. As academics woke up to the paucity of
women faculty, Schlesinger found herself taking on many local and national com-
mittee responsibilities.
Fig. 41 Sondra
Schlesinger
Fig. 42 Sondra
Schlesinger
Highly involved in efforts to racially integrate the medical school classes, she
remembers several attempts to organize women faculty prior to the inception of the
Academic Women’s Network, of which she was an active member during the initial
years. Deeply committed to encouraging trainees and young faculty, Schlesinger has
been highly instrumental in advancing their scientific careers and goals (Academic
Women’s Network of Washington University School of Medicine 2002–2009)
Fig. 43 Diane Griffin
Diane Griffin, PhD (Fig. 43)

Vice President, National Academy of Sciences
University Distinguished Service Professor
W. Harry Feinstone Department of Molecular Microbiology and Immunology
Johns Hopkins Bloomberg School of Public Health
Diane Griffin is a past president of the American Society for Virology, the
Association of Medical School Microbiology Chairs, and the American Society for
Microbiology. She was elected to the National Academies of Science in 2004 and
currently serves as vice president. Dr. Griffin is the recipient of numerous presti-
gious awards including the Rudolf Virchow Medal, FASEB Excellence in Science,
Maxwell Finland Award from the National Foundation for Infectious Diseases, and
Alice C Evans Award from the American Society for Microbiology (National
Academy of Sciences 2021).
“Dr. Griffin’s research on RNA viruses that cause two different acute diseases
(measles and alphavirus encephalitis) seeks to understand the mechanisms of dis-
ease pathogenesis, recovery, and development of protective immunity. These stud-
ies involve identification and characterization of viral determinants of virulence and
host responses to infection in animal model systems. The mechanism(s) of non-
cytolytic viral clearance from mature neurons during recovery from encephalomy-
elitis is currently under study. Viral RNA persistence may lead to unexpected late
complications of infection but during recovery, continued stimulation of the immune
response may also contribute to development of lifelong protective immunity”
(National Academy of Sciences 2021).
Fig. 44 Margot Brinton
Margo Brinton, PhD (Fig. 44)

Regents; Professor, Department of Biology
Georgia State University
Dr. Brinton’s laboratory studies molecular and cellular basis of virus-host inter-
actions and host antiviral responses to West Nile virus and other flaviviruses and
simian hemorrhagic fever virus. They also study regulation of West Nile virus rep-
lication and functional analysis of flavivirus genetic resistance. She received the
2013 Georgia State University Distinguished Professor Award, to honor her interna-
tionally recognized research accomplishments and her outstanding teaching and
student mentorship (Outstanding Alumna 2013). She has fostered the exchange of
information among scientists in her field by founding an international positive
strand RNA virus symposium in 1986; and in 1991, she started a very successful
Southeastern regional virology conference for scientists working in eight southeast-
ern states.
Martine Jozan, MD (Fig. 45)
Retired Consultant
Orange County Mosquito and Vector District
“Figure 44 was taken at the second West Nile virus conference which I organized
and monitored in 2004 for the Orange County Mosquito and Vector District where
I (MJ) was a consultant for 23 years. The first conference was held in 2000.
I was born in France, in Cannes of all places, where the bikini was introduced by
Brigitte Bardot in a big journalistic splash on the local beach. I am from four gen-
erations of military servants, and my very strict father was in the Navy. His very
brilliant and renowned career brought us to Morocco, Tunisia and Vietnam, and
from the age of 13, I developed a keen interest in the different cultures I was exposed
to. I did my pre-med year at the Vietnamese University of Ho Chi Min City (then
Fig. 45 L to R Tom Monath, Martine Jozan, Fred Murphy. (Kindly provided by Martine Jozan)
Saigon) as the Americans eager to get involved in Vietnam were undermining the
departure of the French following the miserable defeat of the French at Dien Bien
Phu. From there it was Medical School for seven more years, with a year spent as a
resident in country hospital in Algeria during the final year of the war. I became
acquainted with war injuries and exotic infectious diseases. I eventually got my
diploma in Paris in 1964, and joined my young husband in Mali, where I practiced
in the pediatric ward of the local hospital. The untimely death of my husband made
me abandon practice to do research instead. I was hired by the Pasteur Institute who
had me trained in Paris for the basics in tissue culture, virus isolations and serology.
Andre Lwoff was the principal investigator of the project (attenuation of the neurot-
ropism of the now abandoned mouse brain yellow fever vaccine). I spent 15 days in
his lab, coached by his wonderful wife, Marguerite. Upon meeting Lwoff for the
first time he made it very clear that ‘women were very useful in research but cer-
tainly did not need to get a Nobel prize’. During my 26 months contract in Dakar,
Wilbur Downs (Yale) came for a visit, and being the only one speaking English I
was asked to escort him everywhere. Back in the States, Downs asked Chambon, the
Director of the Institute to send me for a year to work with Sonya Buckley. The
Director thought that I was not ‘ready for that’ but then Downs came up with a sup-
portive WHO fellowship, which could not be ignored. Both Sonja and Edwin
Westaway became my mentors and friends. Back at the Pasteur Institute, in Paris
now, in the arbovirus laboratory (Claude Hannoun, Director) I continued working

on possible virus attenuation, especially Tahyna recently recognized as a medical
problem in Eastern Europe. Rosisky in Prague invited me to spend two months in
Rehacek’s lab, to help them prepare a Tahyna sucrose acetone antigen. Doubravka
Malkova was in charge, a remarkable virologist and one of the few who were study-
ing virus latency in the lymphatic system of rodents. At the end of my stay I was
asked to attend the Smolensk International Symposium on Tick-Borne arboviruses
(excluding group B) September 8–12 1969. There I met Telford Work, who wrote a
chapter in ‘Virus Diagnostic Procedures for Viral and Rickettsial Diseases’, 3rd edi-
tion, 1964, Lennette Edwin H and Schmidt Nathalie J, APHL, Inc. 1964, a book that
Work gave to Paul Bress director of the Dakar Pasteur Institute, that I used it all the
time. This was the first day cocktail hour, and here he was standing tall, imposing
with his glittery and smiling eyes. Richard Moreland Taylor was at his side, the
ultimate elegant silhouette of a southern gentleman. Sonja dragged me to them,
insisting on a meeting. I had written three times to Work for a position in his newly
established laboratory at the UCLA School of Public Health. Each time the answer
was ‘Thank you for your interest in our program, but we have nothing available at
this time. Please write again in six months’. The truth was as Telford put it, ‘there
were three strikes against me: I was French, a woman and I had a child’. This time
I was offered a job as an assistant researcher, and following 6 months of bureau-
cratic shenanigans, I arrived (with the three kings!) on 6 January 1970. Three days
later I was teaching epidemiology to graduate students. Eventually I became the lab
mentor of incoming doctoral students, a wonderful experience indeed. We were
married six months later, and engaged in a unique working partnership which lasted
25 years. Because of the University nepotism rules I had to work with no renumera-
tion at all. But the work was exhilarating dotted by two sabbatical years in Iran/
Australia and Argentina, with in between serological survey in three Indonesian
islands and Papua New Guinea. Telford was my third mentor, a man of immense life
appetite, doggedly pursuing his public health service to science, and totally con-
vinced that ‘if it is not hard work it is probably not worth pursuing’” (M Jozan).
Mercedes Weissenbacher (Fig. 46)
National Reference Center for AIDS,
Department of Microbiology, School of Medicine,
University of Buenos Aires, Buenos Aires, Argentina
Martine wrote (edited): “Mercedes is a remarkable woman and Telford and I owe
her a lot. Joe Held at NIH recruited us for a year at CEPANZO (now defunct) to
establish an arbovirus diagnostic program. The laboratory had just finished the
experimental evaluation of the Koprowsky rabies vaccine, and some sort of incident
had occurred which prevented anyone from any work with live virus. We could do
nothing else but cell culture and serology. They offered us an empty p3 unit at the
University. CEPANZO gave us the equipment and lab ware and the necessary mice
that we carried by taxi to the 15th floor of the University! We were assigned a doc-
toral student, Vida Hodara who is now in San Antonio, TX, and we developed all the
antigens and sera for Dengue and Yellow Fever. They had Yellow Fever in Brazil
Fig. 46 Mercedes Weissenbacher (L) and Martine Jozan Work (R) in front of the University of
Buenos Aires Medical school and laboratories, Argentina. (Kindly provided by Martine Jozan)
and Misiones, Argentina but no way to test and no one was vaccinated. At the veteri-
nary school two veterinarians assisted us to study the mosquito population along the
Parana delta. It was a fascinating year in a beautiful variegated country, among a
highly educated population.”
Carol Blair, PhD (Figs. 47 and 48)
Professor Emerita, Microbiology, Immunology, and Pathology,
Colorado State University, Ft. Collins, CO
Dr. Blair was the first female department head in the College of Veterinary
Medicine and Biomedical Sciences, CSU. She spent 39 years teaching and conduct-
ing research at CSU.
After she had changed her major from chemistry to the brand new (at the time)
field of microbiology, she says, “I was given the opportunity to work as a lab assis-
tant with John Stanton and Joel Dalrymple. (My duties were to capture snakes in the
freshwater marshes east of Great Salt Lake, care for and conduct experiments with
the snakes in the lab, prepare primary chick embryo cell cultures, assay infectious
Fig. 47 Carol Blair
Fig. 48 Carol Blair and Patrick Brennan at Bolder Boulder 10 k race in 2017
virus, etc.) John and Joel taught me so much and they enjoyed their work so much,
and I enjoyed working with them so much, that I decided I wanted to pursue the
academic life for the rest of my career” (Pace n.d.).
Quote from Carol Blair during the American Society of Tropical Medicine meet-
ing in Denver years ago: “I remember talking to two female friends in my age group
Fig. 49 Patricia Nuttall speaking on how changing conditions are increasing the risk of tick-borne
viruses and how these viruses benefit from the peculiar properties of tick saliva
[Connie Schmaljohn and Laura Kramer], and we looked around and we couldn’t see
any other women there. We said, ‘This has got to change.’” And so it has!
Patricia Nuttall, PhD (Figs. 49 and 50)
Emeritus Professor of Arbovirology & Fellow of Wolfson College, Department of
Zoology, University of Oxford, UK
“Trained as a virologist, Pat became interested in tick-borne viruses and was the
first to describe ‘non-viremic transmission’, a phenomenon that challenged the
accepted mode of arbovirus transmission. Her subsequent research helped demon-
strate the essential role of tick saliva in tick-borne virus transmission, and she con-
tinues her interests in exploiting tick saliva proteins and peptides for vaccine and
therapeutic drug development.
Now that she is ‘retired,’ she enjoys helping others by sharing her knowledge,
giving advice, editing, reviewing, and writing a few articles. She is just finalising a
book entitled Climate, Ticks, and Disease, which is due for publication by CABI in
November 2021” (P Nuttall).
Polly Roy, PhD (Fig. 51)
Professor of Virology
Department of Pathogen Molecular Biology within the Infectious and Tropical
Diseases Faculty London School of Hygiene and Tropical Medicine
“The situation with arthropod bluetongue virus today is one of being tantaliz-
ingly close to a complete molecular understanding of its replication cycle. In the
30 years of research leading to this position, the fundamental structural and molecu-
lar aspects of the virus, including its sequence and its functional coding capacity,
have been painstakingly mapped, dissected and validated. It is, today, a very well
Fig. 50 Patricia Nuttall in front of tick painting
Fig. 51 Polly Roy
understood virus that has contributed to fundamental discoveries in molecular and

cell biology as well as providing new opportunities for pathogen control. The most
recent developments have related to the mechanism of replication including (i) virus
attachment and entry in two diverse host cells, (ii) the sorting of the multi-segment
RNA genome into the newly-formed virus particle and its engagement by the
resident RNA-dependent RNA polymerase. These findings are wholly novel and
present a fascinating picture of a previously obscure pathway in unprecedented
detail; a few angstroms of movement leading to a complete switch in a key biologi-
cal process. Further, these understandings have led to develop more efficacious vac-
cines and alternate therapeutic possibilities” (P Roy).
Many vector biologists don’t recognize that BTV is arthropod-borne. Roy’s con-
tributions to virology, in particular to virus structure and assembly, have been rec-
ognized by her peers worldwide through numerous awards and prizes especially as
BTV itself has become more widely distributed.
Connie Schmaljohn, PhD (Figs. 52, 53 and 54)
Chief Scientist and Director
Integrated Research Facility
NIAID, Frederick, MD
Dr. Connie Schmaljohn’s research background is in molecular virology and
molecular vaccine development. Dr. Schmaljohn was selected to become director of
the NIAID IRF-Frederick in November 2019. Prior to that time, she was the senior
research scientist for medical defenses against infectious disease threats for the US
Army and directed a research program at the U.S. Army Medical Research Institute
of Infectious Diseases (USAMRIID) (“Integrated Research Facility” 2022).
Fig. 52 Connie
Schmaljohn named
Director of NIAD
Integrated Research
Facility – Frederick in
November 2019. (Photo
credit NIAID. 30 April
2022. (4/30/2022 IRF))
Fig. 53 Connie Schmaljohn accepts an award for honorable mention from MG Barbara Holcomb,
Commanding General, U.S. Army Medical Research and Materiel Command, 28 August 2017, which
was given to USAMRIID for the successful development and human clinical testing of a state-of-the-
art DNA vaccine to prevent hemorrhagic fever with renal syndrome. (“Military Health” n.d.)
Fig. 54 Connie Schmaljohn and M Gresikova. Dr. Gresikova, from Bratislava, Slovak Republic
conducted research on tick-borne encephalitis virus, eastern equine encephalitis virus, Sindbis
virus, among others
Fig. 55 Mary Jane

Cardosa
• Recommended reading. (Schrage 2019) Q&A: Husker alumna talks small-town

Nebraska, football, women in virology. University Communication. https://
news.unl.edu/newsrooms/today/article/qa-h usker-a lumna-t alks-s mall-
town-nebraska-football-women-in-virology/
Mary Jane Cardosa, PhD (Fig. 55)
Director, Chief Scientific Officer & Co-Founder at Sentinext Therapeutics Sdn. Bhd.
Director, Chief Scientific Officer & Co-Founder MAb Explorations, Penang,
Malaysia.
Presently Chief Technical Officer of Integrated Research Associates, LLC, San
Rafael, CA, USA.
“Mary Jane Cardosa earned a Masters from Columbia University in 1975, then
returned to Malaysia to begin a family. From 1980 to 1984 she continued her post-
graduate training at the Sir William Dunn School of Pathology in England under Dr.
James Porterfield, where she completed a DPhil thesis on the ‘Immunology of
Dengue Haemorrhagic Fever.’ Following a brief postdoctoral training period at the
Scripps Institute in California in 1985, she subsequently returned to Malaysia as a
lecturer at the School of Medical Sciences at Universiti Sains Malaysia where she
set up a laboratory to continue her research in dengue haemorrhagic fever (Das
2001). ‘I have been a pioneer in setting up a research laboratory and leading a group
of young local scientists in the investigation and control of emerging viral infections
in challenging resource-limited environments. Since my retirement from University
service in 2010, I took on a different challenge in product development setting up a
start up biotechnology company to work on VLP vaccines for infectious diseases of
importance to developing countries. I also set up a startup biotech company to gen-
erate monoclonal antibodies for use in diagnostics and possibly therapeutics.’ She
has successfully demonstrated that ‘solutions for local and regional problems can be
effectively researched and developed in the region.’ Her studies resulted in the
development and commercialization of several diagnostic kits for dengue and
Japanese encephalitis” (Das 2001).
Elizabeth Ann McGraw, PhD (Fig. 56)
Professor and Head of Biology Department
Huck Scholar in Entomology
Center for Infectious Disease Dynamics
The Pennsylvania State University, PA
“Elizabeth obtained her undergraduate and PhD degrees in Biology, from the
University of Michigan and The Pennsylvania State University, respectively. She
did two postdocs, one at Yale University School of Medicine, Department of
Epidemiology & Public Health and another at The University of Queensland in
Australia. In 2002 she accepted an assistant professor position at The University of
Queensland. In 2011 she moved to Monash University. In 2017 she returned to the
U.S. as a Professor at The Pennsylvania State University in the Entomology
Department. Elizabeth is trained as an evolutionary biologist and has spent her
career studying the genetics and evolution of mosquito x virus x Wolbachia interac-
tions. While in Australia her team helped to prepare Wolbachia for field release and
testing for dengue virus biocontrol. Her major contributions include discovering
somatic tissue infection associated effects of Wolbachia and the mechanistic bases
of Wolbachia-mediated pathogen blocking.
She has been awarded the Ross Crozier Medal in Genetics from the Genetics
Society of AustralAsia and The Australian Eureka Prize as part of the ‘Eliminate
Dengue’ team. She has been named a ‘Huck Scholar’ in Entomology by Penn State.
She has held multiple leadership roles including Director of the Center for Infectious
Disease Dynamics (CIDD) and the Head of the Department of Biology at Penn
State” (E McGraw).
Fig. 56 Elizabeth Ann McGraw

Andrea Gamarnik, PhD (Figs. 57 and 58)

Principal Investigator, Institute Leloir
Director of the Biochemistry Institute of Buenos Aires, IIBBA, at the National
Research Council of Science and Technology of Argentina (CONICET)
In 2001, Andrea “created the first laboratory of Molecular Virology in the Leloir
Institute, one of few in Latin America. Her laboratory has made seminal contribu-
tions on our understanding of the mechanism of dengue virus replication. Dr.
Gamarnik received a number of national and international awards such as the
L’Oreal UNESCO for Women in Science in representation of Latin America in 2016
and the Konex award for her trajectory in Microbiology 2003–2013. She became a
Howard Hughes International Scholar in the Infectious Diseases Program, fellow of
Fig. 57 Andrea Gamarnik
Fig. 58 Andrea Gamarnik

the American Academy of Microbiology in 2014 (the only woman from Argentina
in this Academy) and fellow of the American Academy of Arts and Sciences in 2020.
Her team discovered the mechanism by which the viral polymerase discriminates
between the viral RNA and cellular mRNAs, identifying the promoter for viral
genome amplification. Unexpectedly, this promoter was found 11,000 nucleotides
away from the initiation site. Using elegant in vivo an in vitro systems, together with
atomic force microscopy, in a series of landmark manuscripts, her lab demonstrated
that cyclization of the viral genome places the promoter and initiation sites in close
proximity, allowing relocation of the polymerase and viral RNA replication. This
discovery, using dengue virus as a model, was more recently extrapolated to other
flaviviruses, including about 50 human pathogens such as yellow fever and Zika
virus. Studies from her laboratory also presented the first connection between den-
gue virus replication and the metabolism of host lipid droplets, and presented
molecular mechanisms of how the virus manipulates the cellular splicing machin-
ery, providing novel targets for antiviral intervention. Her laboratory also made a
remarkable findings about dengue virus transmission from mosquito to humans.
These studies identified a novel mechanism of viral adaptation to different species.
These findings provided new concepts on viral RNA evolution” (A Gamarnik).
Ana Fernandez-Sesma, PhD (Fig. 59)
Professor, Department of Microbiology
Department of Medicine, Division of Infectious Diseases
Graduate School of Biomedical Sciences
Icahn School of Medicine at Mount Sinai
“Dr. Fernandez-Sesma is currently a professor with tenure in the department of
Microbiology at Icahn School of Medicine at Mount Sinai (ISMMS) in New York,
NY, USA. She is interested on the modulation of innate immunity by viruses of
human health interests, such as dengue (DENV), influenza (IAV), chikungunya
Fig. 59 Ana Fernandez

Sesma
virus (CHIKV), human immunodeficiency virus (HIV) and Zika (ZIKV) and more
recently, also SARS-CoV-2. Her group uses mainly primary human systems, such
as dendritic cells (DCs) and macrophages as well as primary lung epithelial cells
and human tonsils for these studies combining molecular and immunological tech-
niques. Their main goal is to understand the mechanisms of immune evasion used
by these viruses to establish infection in humans. Using these primary human cells
her group identified the interferon (IFN) antagonist for DENV, namely the NS2B3
protease complex, that is able to inhibit the production of this important antiviral
cytokine in primary human dendritic cells by cleaving the adaptor molecule
STING. Her group subsequently showed that DENV and CHIKV can degrade the
DNA sensor cGAS in infected cells, which is triggered by leakage of mitochondrial
DNA during infection.
She currently participates in several multi-investigator projects on DENV and
IAV that use OMICS technologies to study immune responses to infection. She has
also participated in several study sections for the National Institutes of Health (NIH/
NIAD) and is currently a member of the Scientific Council of the Division of
Microbiology and Infectious Diseases (DMID) at NIH/NIAID. Dr. Fernandez-
Sesma is also the Chair of the NIAID Human Immunology Project Consortium
(HIPC) Steering Committee.
Dr. Fernandez-Sesma is very dedicated to graduate education, mentoring and
advocacy for women in science. She co-directed the Microbiology Main Training
Area of the Graduate School of Biomedical Sciences at ISMMS from 2010 to
2020. She has co-authored numerous publications in Virology and Immunology
journals and is on the editorial board of PLoS Pathogens and mSphere and. In 2021
she was elected Fellow of the American Academy of Microbiology (AAM) and
was also awarded the Jacobi Medallion from ISMMS and elected as Honorary
Member of the Alumni Association of the University of Salamanca, Spain” (A
Fernandez-Sesma).
Rebecca Rico-Hesse, PhD (Figs. 60 and 61)
Professor, Baylor College of Medicine, Department of Molecular Virology &
Microbiology
Houston, TX
“Rebecca Rico-Hesse’s laboratory uses animal models of disease to (a) under-
stand the basics of dengue pathogenesis, especially the mechanisms that lead to the
more severe DHF, and whether vaccination might actually enhance disease, and (b)
to develop therapeutics for treatment after infection, or for persons at high risk of
developing severe disease. The humanized mouse model of dengue developed by
the Rico-Hesse laboratory is the only one that shows consistent signs of disease as
in humans, after infection with low-passage, or “clinical” isolates of the virus, via
mosquito bite. We have streamlined the production of these mice, using NOD/
SCID/IL2gnull mice and human hematopoietic stem cells from normal birth cord
blood and we also infect these mice by mosquito bite, to mimic natural transmission.
We currently pursue two lines of research: the effect of mosquito saliva on den-
gue virus pathogenesis and testing genetically-modified, neutralizing monoclonal
Fig. 60 Rebecca
Rico-Hesse
Fig. 61 Rebecca Rico-Hesse netting birds
antibodies, for efficacy in treating the clinical signs of DF or DHF in humanized

mice” (R Rico-Hesse).
Eva Harris, PhD (Figs. 62 and 63)
Professor, Division of Infectious Diseases and Vaccinology; Director, Center for
Global Public Health; Chair, Infectious Diseases and Immunity Graduate Group,
School of Public Health, University of California, Berkeley, Berkeley, CA
“Dr. Harris has developed a multidisciplinary approach to study the molecular
virology, pathogenesis, immunology, epidemiology, diagnostics, clinical aspects
and control of dengue, Zika and chikungunya, the most prevalent mosquito-borne
viral diseases in humans. Her work addresses immune correlates of protection and
pathogenesis and viral and host factors that modulate disease severity, using in vitro
approaches, animal models, and research involving human populations. One
Fig. 62 Eva Harris
Fig. 63 Eva Harris, collecting blood samples in house in Nicaragua, 2005
research major focus is on studies of arboviral disease in humans, including anti-

body and B cell responses and correlates of protection, diagnostics and seropreva-
lence studies, and viral evolution. Another focus is viral pathogenesis, specifically
the role of flavivirus NS1 protein as a “viral toxin” leading to vascular leak and
promoting viral dissemination. Her international work includes laboratory-based
and epidemiological studies of arboviral diseases in endemic Latin American coun-

tries, particularly in Nicaragua through close collaborations for over 30 years.
Ongoing long-term projects in Nicaragua include a 23-year clinical and biological
hospital-based studies of severe arboviral disease and a long-term pediatric cohort
study (currently in its 18th year) and household transmission studies of dengue,
Zika, and chikungunya, as well as a cluster randomized controlled trial of evidence-
based community-derived interventions to prevent and control arboviral diseases.
She is deeply committed to scientific capacity building globally; in 1997, she
received a MacArthur Award for work over the previous ten years developing pro-
grams to build scientific capacity in developing countries to address public health
and infectious disease issues. This enabled her to found a non-profit organization in
1998, Sustainable Sciences Institute, with offices in San Francisco and Nicaragua,
to continue and expand this work worldwide” (E Harris).
Desiree LaBeaud, MD, MS (Figs. 64 and 65)
Professor Pediatric Infectious Diseases, Stanford University
Bechtel Faculty Scholar, Stanford Child Health Research Institute, Stanford, CA
“Since the early 2000s, I have devoted my efforts to better understanding the risk
factors and long-term health consequences of arboviral infections, including Rift
Valley fever, chikungunya, and dengue viruses. Currently, I have two large field
projects ongoing in Kenya. As a physician-scientist, I split my time between research
(75%) and clinical practice (25%), including travel clinic experience. In my current
position, I balance a productive research career, a stimulating hospital-based clini-
cal practice, and a rewarding family life” (LaBeaud n.d.).
Fig. 64 Desiree LaBeaud

Fig. 65 Desiree LaBeaud

in a field of plastic refuse
where mosquitoes can
breed profusely, as part of
HERI program,
Kenya 2021
I am currently fulfilling a decade-long dream to launch a new nonprofit, the

Health and Environmental Research Institute (HERI). HERI aims to inspire com-
munity education, new research, policy change and grassroots activism in environ-
mental health issues in Kenya. “We will empower communities to change cultural
norms, destigmatize trash, safely clean up local environments, and improve health.
Importantly, we will provide training and support for local scientists whose research
aligns with these aims.
In short, our goal is to harness the knowledge and power created in science,
translate it to actionable community messages, and then change the trajectories of
health for our communities and our planet” (LaBeaud 2021).
Rosemary Sang, PhD (Figs. 66 and 67)
Advisor and Chief Research Officer
Centre for Virus Research, Kenya Medical Research Institute (KEMRI), Kenya
“Dr. Sang is a biomedical research scientist with specialization in arbovirology,
a background in medical entomology and medical virology. She has more than
30 years’ experience conducting field and laboratory studies on emerging arbovirus
disease epidemiology, ecology, and emergency and outbreak response. She has
served in various research capacities at the Kenya Ministry of Health (MOH), Kenya
Medical Research institute (KEMRI) and, through collaboration, the U.S. Medical
Research Directorate and the International Center of Insect Physiology and Ecology
(ICIPE), leading and mentoring strong arbovirus research teams to conduct
Fig. 66 Rosemary Sang
Fig. 67 Rosemary Sang
surveillance, ecological studies, and outbreak investigations and contributing to

much of what is documented about distribution, ecology, and epidemiology of
disease-associated arboviruses in Kenya. Her studies have focused on Rift Valley
Fever, for which she demonstrated evidence of interepidemic low level transmission
in hot spot areas of Kenya, and dengue. Dr. Sang also demonstrated circulation of
other known and novel arboviruses of public health importance in Kenya and the
growing threat of such arboviruses in the region. In 2021, she was selected to serve
on the Scientific Advisory Group for the Origins of Novel Pathogens (SAGO).
Capacity building has been an integral part of her research including teaching
medical virology at the Institute of Tropical Medicine and Infectious Disease
ITROMID, (a collaboration between KEMRI and Jomo Kenyatta University of
Agriculture and Technology, JKUAT) where she has served as adjunct professor at
the College of Health Sciences” (R Sang).
Ann Powers, PhD (Figs. 68 and 69)
CDC Ft. Collins, Vector-borne Disease Branch
Associate Director for Science
Division of Vector-Borne Diseases
CDC
“As a research microbiologist for CDC’s National Center for Emerging Zoonotic
and Infectious Diseases and now Associate Director for Science, my job is to better
understand and occasionally chase (literally) an often overlooked, mosquito-borne,
threat to public health; one that holds the potential to spread sickness and misery in
the United States. Much of CDC’s work is with regional partners. Since 2010 for
example, CDC has joined with the Pan American Health Organization to craft a
regional surveillance and response plan for the Americas. CDC has also developed,
evaluated, and published diagnostic testing protocols, produced and distributed
diagnostic test reagents and positive controls and developed notices with specific
information for health departments, health care providers, and travelers, among
other acts” (A Powers 2014).
Tamara Segeevna Gritsun (1956–2015) (Fig. 70)
(Oxford University)
Obituary (Gould, 2015; edited)
Fig. 68 Ann Powers

Fig. 69 Ann Powers in

Comoros 2005
Fig. 70 Tamara Segeevna

Gritsun
“Tamara Gritsun graduated with an honour’s degree from Moscow State

University in 1980. Her career as a virologist then began with postgraduate studies
in the Medical Academy of Sciences Institute of Poliomyelitis and Viral
Encephalitides, Moscow, Russia. She obtained her PhD in 1987, focusing primarily
on biochemical studies of West Nile virus and tick-borne encephalitis virus (TBEV),
the latter becoming her speciality for the remainder of her scientific career.
Her studies on the non-structural NS1 protein of TBEV were years ahead of
western scientists when she described oligomeric molecules, one of which was hex-
americ (with a doughnut-like appearance) and membrane associated. This was sub-
sequently ‘re-discovered’ by western scientists years later!
In 1990 Tamara moved to Oxford where she worked in Dr. Gould’s research
team, having to learn to speak English whilst teaching and supervising D Phil and
other students, many of whom have since progressed to senior career positions.
Within two years of commencing research in Oxford, Tamara embarked on a
[very risky] project to develop an infectious clone of TBEV using high fidelity
reverse transcription to synthesise full-length genomic cDNA which was then
in vitro transcribed to produce full-length RNA. With perseverance and using her
strong organic chemistry background, Tamara achieved her first goal, ie to produce
the full length genome in two halves which were ligated together. This led to the
first infectious virus being recovered directly using RT PCR methodology. Within
another year full-length genomes were being synthesised by RT PCR and the
in vitro transcribed RNA proved to be infectious. This technology reduced the time
to produce an infectious clone from years to weeks.
Tamara was also determined to demonstrate that chronic tick-borne encephalitis
was a real disease in Russia. Using molecular methods she demonstrated that the
virus persistence in brains of healthy animals and humans is responsible for a range
of slowly progressive neurological sequelae.
Her research on the evolution of structure and function of the untranslated
regions (UTRs) of viral RNA, was innovative and has wide implications in our
understanding of viral replication mechanisms in association with virus virulence,
evolution, innate immunity. It also has practical applications for the development of
live attenuated vaccines and antiviral therapeutics.
Tamara developed her own unique methods of comparing RNA sequences that
were disparate (particularly amongst UTR sequences). This led to the discovery of
‘ancestral building blocks’ that were in fact repeated sequence elements. Her man-
ual sequence alignment technique then demonstrated very clearly the evolutionary
process of insertion and deletion which when associated with polymerase infidelity
illustrated the simple but effective mechanism by which evolution could progress
rapidly, ultimately being refined by conventional single base substitutions.
Tamara also applied her imaginative mind to understanding the role of ticks in
TBEV pathogenesis. Finally, during her last two years Tamara was developing a
novel approach to understanding the molecular basis of viral neuropathogenesis.
Her papers will leave a lasting legacy of her amazing contribution to
arbovirology.”
Kathryn A. Hanley, PhD (Figs. 71 and 72)
Regents’ Professor of Biology
New Mexico State University
Las Cruces, New Mexico
Fig. 71 Kathryn Hanley, (~2014). Left to right. Kathy Hanley, Katie Young, Nikos Vasilakis.
Mosquito identification in Sarawak (Malaysian Borneo), in a makeshift station within a traditional
longhouse. Katie Young was at the time Kathy’s student at New Mexico State University
Fig. 72 Kathryn Hanley
“In the Hanley lab, we investigate the molecular biology, evolution and ecology
of emerging RNA viruses and use our discoveries to design better methods to pre-
vent and treat infection with these dangerous pathogens. We focus on the flavivi-
ruses, a group that includes dengue and Zika virus, as well as vesicular stomatitis
virus and, most recently, the novel coronavirus SARS-CoV-2.”
Much of Kathryn Hanley’s time is currently taken up by her role as co-PI along
with Nikolaos Vasilakis (UTMB) on the NIH-/NIAID-funded project, Coordinating
Research on Emerging Arboviral Threats Encompassing the Neotropics (CREATE-
NEO), which will “provide a flexible network of surveillance sites in Central and
South America coupled to cutting-edge modeling approaches in order to anticipate
and counter emerging arboviruses. Importantly, once established, CREATE-NEO
can quickly redirect to address any emerging zoonotic or vector-borne disease.”
This program is one of the Centers for Research in Emerging Infectious Disease
(CREID), a coordinated network with centers in regions around the globe where
emerging and re-emerging infectious disease outbreaks are likely to occur (“Create
Neo” n.d.).
Anna-Bella Failloux, PhD (Figs. 73 and 74)
Director of Research Institut Pasteur Paris
Head of the Arboviruses and Insect Vectors Unit
Institut Pasteur
Fig. 73 Anna-Bella Failloux, Institut Pasteur, 2016
Fig. 74 Anna-Bella Failloux, Institut Luis Mallarde, 1993

“Since 1996 at the Institut Pasteur, I have developed a research on transmission

of arboviruses by Aedes/Culex mosquitoes and set my objectives of research with
the support of the Pasteur Network. I contributed to uncover the complex genetic
structure of the mosquito Aedes aegypti in relation with its competence to transmit
dengue viruses. Capitalizing on our expertise on vectorial transmission, my team is
at the front line to decipher the factors leading to viral emergence: Chikungunya
virus in 2005, Zika virus in 2015, and Yellow fever virus in 2016” (Failloux n.d.).
Laura Harrington, PhD (Figs. 75 and 76)
Professor of Entomology
Cornell University, Ithaca, NY
Fig. 75 Laura Harrington
Fig. 76 Laura Harrington in field

“Professor Harrington became interested in global health issues and vector-borne

diseases after living and working for several years in rural Thailand. She contracted
both dengue and malaria while living abroad and realized the impact these infec-
tions have on children and adults in resource poor nations.
Her research focuses on the biology, ecology and behavior of mosquitoes that
transmit human diseases. Current research projects in her laboratory address the
blood feeding and mating behavior of mosquito vectors of dengue, Zika,
Chikungunya, and West Nile viruses, and malaria. She also studies human and
animal-mosquito interactions and the role of climate change and globalization on
emerging vector-borne diseases. Dr. Harrington studies mosquito biology in the
field locally as well as abroad, with past or present field sites in Thailand, Tanzania,
and Mexico” (Harrington n.d.).
Sonja Best, PhD (Fig. 77)
Chief, Laboratory of Neurologic Infections and Immunity
Rocky Mountain Laboratories
National Institute of Allergy and Infectious Diseases
Fig. 77 Sonja Best

Sonja Best’s research group, the Laboratory of Neurologic Infections and

Immunity, focuses on “understanding the host-pathogen interface associated with
the intrinsic antiviral response of cells to emerging RNA viruses, with a primary
focus on flaviviruses. Dr. Best earned her Ph.D. from Australian National University
in Australia where she examined pathogenesis of the poxvirus, myxoma virus. She
then joined the RML, NIAID, where she conducted postdoctoral research focusing
on the role of host immune factors in flavivirus pathogenesis prior to establishing
her independent laboratory. Key findings have included the identification of flavivi-
rus NS5 as the major flavivirus-encoded antagonist of interferon-dependent JAK-
STAT signaling, the early identification of tripartite motif (TRIM) proteins as
restriction factors with specificity for certain flavivirus species, and identification of
specific mechanisms encoded by flaviviruses to manipulate mitochondrial dynam-
ics. She has been awarded the Presidential Early Career Award for Scientists and
Engineers (PECASE) for her work on flaviviruses” (S Best).
Carla Saleh, PhD (Figs. 78 and 79)
Principal Investigator, Viruses and RNAi Unit, Department of Virology
Pasteur Institute, Paris
“Carla Saleh was born in Argentina, where she obtained her Master degree in
Biology at the National University of Cordoba. She earned her PhD in Cellular and
Molecular Physiopathology at the University of Paris 6, and then moved to UCSF,
where she trained as a postdoctoral researcher. In 2008, she obtained a tenure-track
position at the Institut Pasteur, where she was appointed head of the ‘Viruses and
Fig. 78 Carla Saleh

Fig. 79 Carla Saleh
RNAi’ research group, which was converted to a full unit in 2013 (equivalent to
tenure in the USA). Since 2020, she is Full Professor at Institut Pasteur.
As a post-doc, Dr. Saleh became acquainted with, and fascinated by RNA inter-
ference (RNAi). At this time, it was widely accepted that insects lack a specific
antiviral response and that their only defense against viral infections is an off-shoot
of antibacterial pathways. In 2006, she provided key evidence that RNAi is the
major antiviral defense in the fruit fly and that the viral immune signal enters the
RNAi pathway via a specific pathway involving clathrin-mediated endocytosis and
pattern-recognition receptors. These findings highlighted that antiviral defense in
arthropods is achieved via a ‘nucleic-acid based immune system.’ In 2008, she
established her ‘Viruses and RNAi’ laboratory at Institut Pasteur and, with her team,
exploited the powerful combination of basic science and bioinformatics to unravel
this new immune system.
Carla’s research has increased our understanding of the invertebrate immune sys-
tem and antiviral response. Her work embodies the success of a multidisciplinary
approach, combining genetics and cell biology in the context of immunity and
infection. With the use of next generation sequencing, it has a profound impact on
both fundamental biological and evolutionary questions and medically-related
research in epidemiology, infection biology, and immunology. Carla’s work not
only pioneered the field of antiviral immunity in insects but set a new standard in
entomology and infectious diseases that she keeps raising” (C Saleh).
Katharine Bossart, PhD (Figs. 80 and 81)
Chief Scientific Officer, Chief Executive Officer
Integrated Research Associates, LLC
San Rafael, California
Fig. 80 Katharine Bossart
Fig. 81 Katharine Bossart in BSL-4
“I did my post-doctoral research at CSIRO and took a faculty position at Boston

University where I was actively involved in trying to get the National Emerging
Infectious Diseases Laboratory (NEIDL) open. After 5 years, the community was
still against the laboratory and I started looking for a change. I wasn’t sure what I
wanted to do but I made the decision to leave the BSL4 field, and I moved back to
San Francisco (my home town) which I never thought possible when studying so
many BSL4 viruses (as there was no BSL4 facility in California). As funding in
academia was bleak, I decided to venture into the biotech arena by launching a
small company focused on early discovery and development of viral vaccines and
therapeutics. That was 7 years ago now and I haven’t looked back! In a super nut-
shell, the highlights are having more freedom and time to pursue your scientific
research and interests and the hardships include the costs and responsibilities of
doing something new on your own. I have forged collaborations with academic and
government labs, benefitted from relationships with other small businesses and I
still review manuscripts for top tier journals and sit on important NIH study sec-
tions. Just last month the USPTO granted our patent related to a novel vaccine
platform for flaviviruses”(K Bossart).
Lisa Ng, PhD (Fig. 82)
A*STAR Infectious Diseases Labs
Executive Director
“Lisa joined A*STAR’s Genome Institute of Singapore (GIS) in 2002 as a post-
doctoral fellow where she provided major contributions in the containment, preven-
tion and treatment of epidemic viral infections including SARS (Severe Acute
Respiratory Syndrome) and avian influenza H5N1 (bird flu). Lisa then joined
A*STAR’s Singapore Immunology Network (SIgN) in 2007 as a Principal
Investigator, where she expanded her focus on arboviral immunity. These include
chikungunya virus, dengue virus, Zika virus and other related arboviruses. Her team
has published in top tier scientific journals and made several key important findings
Fig. 82 Lisa Ng

in understanding infection and immunity in controlling arboviruses. While Lisa

continues her research in arboviral immunity, she was appointed Executive Director
of A*STAR’s Infectious Diseases Labs (ID Labs), a new entity positioned to drive
and translate mission-inspired basic research on infectious diseases for better health
and economic outcomes” (L Ng).
Felicity Burt, PhD (Figs. 83 and 84)
Professor/Medical Scientist
Research Pathogen Laboratory,
South African Research Chair (SARChI): Vector borne and zoonotic pathogens
research
Division of Virology, Faculty of Health Sciences
Republic of South Africa
“I have worked my entire career in the field of viral haemorrhagic fevers (VHF)
and arboviruses focusing on detection, pathogenesis and molecular epidemiology. I
started my virology career at the Special Pathogens Unit at the National Institute for
Communicable Diseases in Johannesburg (1988–2006) where I worked for Bob
Swanepoel and completed my PhD on the Diagnosis, Pathogenesis and Epidemiology
of Crimean-Congo haemorrhagic fever virus. While working in the laboratory I
participated in the diagnosis and investigation of outbreaks of VHF and arboviruses
Fig. 83 Felicity Burt, Field trip to collect mosquitoes at the Mokala National Park, in the Northern
Cape Province of South Africa with Dr. Mduduzi Ndlovu (2021)
Fig. 84 Felicity Burt
including, outbreaks of Crimean-Congo haemorrhagic fever virus (CCHFV) in

South Africa; outbreak response in other regions of Africa (Ebola DRC1995,
Uganda2000, Marburg DRC1999); Rift Valley fever virus in Saudi Arabia 2000 to
name a few. The facility had biosafety level 2, 3 and 4 laboratories and at the time
was the only maximum containment level 4 laboratory with VHF diagnostic capa-
bility in Africa and hence we were involved in confirming outbreaks of VHF in
Africa. Frequently working through the night to get an answer. I relocated to
Bloemfontein (from 2006 to current) and established a research group to primarily
focus on host immune responses to arboviral infections specifically characterizing
humoral and cellular immune responses in patients with infections such as CCHFV
and other arboviruses; epitope discovery for development of diagnostic tools; devel-
opment of molecular and serological assays and establishment of surveillance pro-
grams; virus discovery; and development of vaccines. I am currently responsible for
the Research Pathogen Laboratory in the Division of Virology, University of the
Free State, and National Health Laboratory Service (NHLS), Bloemfontein, South
Africa and I hold a NRF-DST South African Research Chair in Vector-Borne and
Zoonotic Pathogens Research. I have recently established a new biosafety level 2
and 3 facility at our Institution which has broadened our research scope. My career
has provided me with the opportunity to travel as a member of outbreak response
teams and to participate in field trips for collection of mosquitoes and ticks in our
quest to further understand viruses and their vectors especially in South Africa and
neighbouring countries” (F Burt).
Amy Morrison, PhD (Fig. 85)
Project Scientist
Department of Pathology, Microbiology, and Immunology
School of Veterinary Medicine
Fig. 85 Amy Morrison, Community meeting Iquitos, Peru, to recruit people into a cohort study
University of California, Davis

Amy Morrison has been stationed in Iquitos full-time while directing dengue
research activities there for the past 15 years. She joined the UC Davis laboratory of
Thomas Scott (now professor emeritus) in 1996 and is currently a project scientist
and scientific director of the Naval Medical Research Unit No. 6 (NAMRU-6)
Iquitos Laboratory.
Dr. Morrison’s “research focus has been on the epidemiology of dengue fever,
the role of its primary vector Aedes aegypti in virus transmission dynamics, as well
as using vector control interventions to control transmission measured through epi-
demiological endpoints. She has worked onsite in Iquitos, Peru since 1998”
(Garvey 2018).
[Be sure to read the chapter by Amy in this book for a fun read!]
Sandra Lopez, PhD (Fig. 86)
Gorgas Memorial Institute of Health Studies, Senior Health Researcher
Chief, Department of Research in Virology and Biotechnology, Gorgas Memorial
Laboratory.
Sandra heads the molecular diagnostics lab at Instituto Conmemorativo Gorgas
de Estudios de la Salud, Panama City, Panama. She also heads human surveillance
cohorts in three sites in Panama as part of an NIH project led by N Vasilakis and K
Hanley. She trained in the Vasilakis lab in virology and molecular genetics as part
Fig. 86 Sandra Lopez
of her L’ Oreal “International Fellowships for Young Women in Life Sciences” fel-
lowship (2014–2016).
“My work focuses on two main subjects: the first, in participating in collabora-
tive projects on molecular epidemiology and discovery of emergent and reemergent
viruses, with a special focus on arboviruses. The second, in characterizing the
immune responses in patients infected with arboviruses or other emergent viruses,
as SARS-CoV-2.”
“She was the second Panamanian to obtain the Young Women in Science 2014
scholarship, awarded by L’Oréal and UNESCO in recognition of her work as a sci-
entist. In 2018, she was among the group of scientists awarded for the excellence of
their research by the Open Forum of Sciences of Latin America and the Caribbean
(CILAC) of UNESCO” (S Lopez).
Sandra Junglen, PhD (Fig. 87)
Group Leader
Charité Universitätsmedizin Berlin: Berlin, DE
“Our research focuses on the ecology and evolution of arthropod-associated
viruses. We are particularly interested in the genetic diversity of these viruses and in
ecological mechanisms that influence the geographic spread and emergence of
arboviruses. We conduct fieldwork in pristine ecosystems and adjacent landscapes
in Africa and Central America to investigate initial emergence processes from enzo-
otic to epidemic transmission cycles” (S Junglen).
Lark L. Coffey, PhD (Fig. 88)
Associate Professor
University of California, Davis
Lark obtained her Ph.D. from the University of Texas Medical Branch. Before
joining UC Davis in 2013, she worked at Institut Pasteur, Paris and the Blood
Systems Research Institute at the University of California, San Francisco. Lark‘s
research program focuses on studying the transmission dynamics, pathogenesis,
Fig. 87 Sandra Junglen
Fig. 88 Lark Coffey, University of Texas Medical Branch Ph.D. student pinning mosquitoes to
learn identification in Iquitos, Peru, 2002
and evolution of arboviruses with a goal of interrupting transmission to reduce dis-

ease. “Our research focuses on several central themes with a common goal of reduc-
ing the burden of disease caused by arboviruses. These include: understanding viral
genetic factors that promote arbovirus outbreaks, predicting viral mutations that
enhance arbovirus transmissibility by mosquitoes and disease in humans or ani-
mals, increasing safety of candidate live-attenuated vaccines, improving arbovirus
surveillance in mosquitoes (Coffey n.d.).
Patricia Aguilar, PhD (Figs. 89 and 90)
Associate Professor, Department of Pathology
Associate Director, Center for Tropical Diseases
University of Texas Medical Branch
Galveston, TX
Fig. 89 Patricia Aguilar
Fig. 90 Patricia Aguilar with Dr. Robert Shope taken during one of Dr. Shope’s trips to
NAMRU-6 in Lima, Peru, with Dr. Laura Chandler from the Shope lab, front center, when they
came to train personnel in arbovirus diagnostics
Dr. Aguilar’s primary research interest has “focused on the epidemiology, genet-
ics, and understanding of human diseases caused by arboviruses. I am also heavily
involved in global capacity-building efforts and training the next generation of sci-
entists to provide them with the skills and tools to recognize and study the next virus
outbreaks.”
Dr. Aguilar provide them with the skills and tools to recognize and study the next
virus host immune responses and cause disease, and to identify cellular factors that
contribute to viral infection.
“As a co-investigator of the World Reference Center for Arboviruses and
Emerging Viruses (WRCEVA), I am also involved in the identification and charac-
terization of novel pathogens affecting humans in Tropical regions” (Aguilar n.d.).
Naomi Forrester, PhD (Fig. 91)
Reader in Vector Biology
Keele University, UK
“Dr. Forrester-Soto’s primary research focus is how arboviruses persist in
endemic cycles and the role that minority variants play in sustaining endemic trans-
mission. Her research has demonstrated that minority variants are likely generated
de novo in mosquitoes and, if this is so, are likely beneficial to the virus in overcom-
ing bottlenecks and facilitating endemic virus transmission.
With the emergence of SARS-CoV-2, Dr. Forrester-Soto took on a second career.
She was asked to participate in a phone-in in the early days of COVID, in March
2020 with a local radio station. Since then she has been answering questions about
the Coronavirus pandemic every week on Radio Stoke, as well as national Radio
and TV appearances to discuss the pandemic and the ways in which viruses repli-
cate and how this impacts the pandemic. Over the course of the pandemic she has
Fig. 91 Naomi Forrester

done more than 300 live interviews about the pandemic, in both TV and Radio” (N
Forrester).
Anna K Överby, PhD (Fig. 92)
Associate Professor
Umeå University, Sweden
“Anna Överby graduated with a Master of Science in Engineering Biology at
Umeå University in 2003, and in 2007 she completed her PhD at the Karolinska
Institute, Stockholm where she studied the assembly of bunyaviruses. Thereafter,
Anna moved to Freiburg, Germany for a post doc in the laboratory of Friedemann
Weber were the focus was tick-borne encephalitis virus (TBEV) interaction with the
innate immune system. In 2011 she started her own research group within the
Laboratory for Molecular Infection Medicine Sweden, one of the Nordic EMBL
nodes at Umeå University. Her research team has characterized different aspects of
what determines TBEV pathogenicity and tropism in mice, in particular how the
local type I interferon response within the central nervous system restricts viral
replication. To pursue this research, she was awarded several competitive grants and
prestigious awards, the latest being the Eric K. Fernström prize, to a young success-
ful researcher showing particular promise. Throughout her research career, she has
established a national and international network with collaborators, and she has
successfully supervised numerous postdocs, PhD students, and master studies,
underlying her excellent teaching and supervision skills” (AK Overby).
Fig. 92 Anna Overby

Erin Mordecai, PhD (Fig. 93)

Associate Professor of Biology
Senior Fellow in the Woods
Inst for the Environment at Stanford University
“Erin Mordecai is a disease ecologist who uses fundamental knowledge of spe-
cies responses to climate and environment to better understand the transmission of
vector-borne diseases. Her work has demonstrated that temperature has distinct,
nonlinear effects on multiple traits of mosquitoes and arboviruses, resulting in
hump-shaped relationships between temperature and transmission that differ across
systems in their optimal and limiting temperatures. For example, Aedes aegypti
transmission of dengue, chikungunya, and Zika viruses peaks at 29 °C while Culex
spp. transmission of West Nile and other viruses peaks between 23–25 °C, and these
nonlinear relationships determine patterns of disease incidence in the field. Her
research has also shown that ecological models that combine physiological traits,
species interactions, and biogeographic information can accurately predict arbovi-
rus dynamics, ranging from the rate of yellow fever spillover to humans in Brazil to
the competence of different host and vector species for Ross River virus.
She received her B.S. in Mathematical Biology at the University of Georgia in
2007 and her PhD in Ecology, Evolution, and Marine Biology at the University of
California in 2012. She was a National Science Foundation Postdoctoral Research
Fellow in Biology at the University of North Carolina at Chapel Hill and North
Carolina State University from 2013–2014, where she studied the intersections of
mathematics and biology in infectious disease dynamics. She has been on the fac-
ulty at Stanford since 2015” (E Mordecai).
Fig. 93 Erin Mordecai.

(Photo of field work in
Costa Rica, 2018, by
Chuck Katz)
Courtney Murdock, PhD (Figs. 94 and 95)

Associate Professor
Cornell University Dept. of Entomology
Fig. 94 Courtney
Murdock
Fig. 95 Courtney
Murdock
“One of the main drivers of vector-borne disease transmission is the ecology of

the insect vector. Changes in climate and land use alter ecological relationships vec-
tors have with their hosts and pathogens, resulting in shifts in transmission. Some of
the most recent emerging infectious diseases that have impacted the human popula-
tion have been mosquito-borne viruses (dengue, chikungunya, and Zika). Our
research applies ecological and evolutionary theory to better understand: 1) the
host-vector-virus interaction, 2) key environmental drivers of transmission, and 3)
how environmental change (e.g., climate and land use change) will affect arbovirus
disease transmission and control” (C Murdock).
Joan (Joanie) Kenney, PhD, MPH (Figs. 96 and 97)
Biologist
Centers For Disease Control and Prevention
Division of Vector-Borne Diseases
Arboviral Disease Branch
Fort Collins, CO 80521
Joanie Kenney completed her undergraduate degree in the department of Ecology
and Evolution at Tulane University, Louisiana before acquiring her MPH from the
Yale School of Public Health while working with Dr. Durland Fish in 2001. She was
awarded a CDC/APHL fellowship in Emerging Infectious Diseases under the direc-
tion of Dr. Pascale Leonard and the New Mexico Department of Health working to
improve molecular detection and surveillance for vector-borne pathogens including
Yersinia pestis and Francisella tularensis. Continuing to follow her interest regard-
ing dynamics of pathogen-vector interactions, Joanie pursued her Ph.D. at the
Fig. 96 Joan Kenney,
Kidepo Valley National
Park in Uganda, 2014
Fig. 97 Joan Kenney Zika

Forest, Uganda, 2017
University of Texas Medical Branch studying under Dr. Scott Weaver. Her research
primarily focused on genetic determinants of vector competence of multiple alpha-
viruses, though she also examined the stability and safety of several chimeric vac-
cine candidates.
Joanie became a CDC/ASM postdoctoral fellow with Dr. Aaron Brault in the
Virology section of the Arboviral Disease Branch at the Centers for Disease Control
and Prevention, Ft. Collins, CO, where she examined the genetic determinants of vec-
tor competence between tick-borne and mosquito-borne flaviviruses. She accepted a
full-time position in the Fort Collins CDC’s Entomology and Ecology section under
the direction Dr. Harry Savage and Dr. Roxanne Connelly. In this position, her respon-
sibilities shifted from primarily research to outbreak response and arbovirus surveil-
lance. Joanie’s outbreak response and surveillance work have given her the opportunity
to work throughout the USA and territories as well as collaborations with the Uganda
Viral Research Institute based in Entebbe, Uganda. She continues to work at CDC,
currently focusing on improving molecular methods of arbovirus detection.
Shannan L. Rossi, PhD (Figs. 98 and 99)
Associate Professor (Non-tenure track)
Departments of Microbiology & Immunology and Pathology
University of Texas Medical Branch, Galveston, TX
“Dr. Rossi attended Cornell University and graduated in 2001 with a Bachelor’s
of Science. Her dissertation was completed in 2008 with a focus on the role of point
mutations on persistent West Nile virus infection. Following a brief 2-year postdoc
Fig. 98 Shannan Rossi
Fig. 99 Shannan Rossi with Scott Weaver, 2016

at the University of Pittsburgh where she helped move forward potential West Nile
virus vaccines, she returned to UTMB to continue her postdoctoral education. At
UTMB, she continued to not only work on alphaviral and flaviviral vaccines but
developed the first useful animal model for Zika virus and refined small animal
models for chikungunya disease.
Her current research centers around understanding how arboviruses, which typi-
cally cause acute infection, can persist in the host. Understanding these infections
can enter and be maintained in immune-privileged sites like the testes and eye is
critical to controlling infection and understanding how to combat long-term
sequelae” (SL Rossi).
Hilda Guzman, BS (Figs. 100 and 101)
University of Texas Medical Branch, Galveston, TX
Fig. 100 Hilda Guzman
Fig. 101 Hilda Guzman

“Hilda Guzman was born in Mariquita, Tolima Dept., Colombia in 1956. She
graduated with a B.S. in Biology in 1985 from the Universidad Nacional in Bogota.
From 1985–1988, she worked as a biologist in the Parasitology Section, Colombian
National Institute of Health, Bogota, involved primarily in field and laboratory stud-
ies on the ecology of phlebotomine sandflies and leishmaniasis. In 1989, she moved
to the Yale Arbovirus Research Unit (YARU) in the Dept. of Epidemiology and
Public Health, Yale University School of Medicine, New Haven, CT., where she
remained for 6 years as a Research Associate. Her work at YARU included mainte-
nance of laboratory colonies of sandflies and mosquitoes (photo 1), research on
protozoan and viral pathogens transmitted by these insects, and processing clinical
and arthropod samples for virus isolation, in collaboration with Drs. Robert Shope
and Robert Tesh. In 1995, Ms. Guzman moved with Drs. Shope and Tesh to the
Dept. of Pathology, University of Texas Medical Branch (UTMB), Galveston. Ms.
Guzman was responsible for packing up the extensive WRCA virus collection and
reestablishing and curating it at UTMB. At UTMB, she was involved in the isola-
tion, identification and characterization of viruses from arthropods, animals and
human clinical samples using classical cell culture and serologic techniques
(Figs. 95 and 96) in collaboration with Dr. Tesh and other investigators. She also
developed the first computer-based inventory of all viruses in the WRCA collection
and where they were physically located along with detailed information on the geo-
graphic and host origin, date of collection, passage history, and source if they were
received from another investigator or institution. She also trained graduate students
and visiting scientists in her laboratory at UTMB. Ms. Guzman is author or coau-
thor of 116 scientific publications. She retired from UTMB as a Senior Research
Scientist in 2017. Throughout her scientific career, her interests and research were
focused in three areas: (1) ecology of insect vectors of disease; (2) pathogenesis of
arboviruses in their vertebrate and invertebrate hosts; and (3) isolation and charac-
terization of novel arboviruses and insect-specific viruses” (H Guzman).
Nisha Duggal, PhD (Figs. 102 and 103)
Assistant Professor, Department of Biomedical Sciences and Pathobiology
Virginia-Maryland College of Veterinary Medicine at Virginia Tech
“Nisha Duggal received her PhD in the laboratory of Dr. Michael Emerman at
the University of Washington in Seattle, where she studied the evolution of the
intrinsic immune response to HIV-1 and other retroviruses in primates. She com-
pleted her postdoctoral training in the laboratory of Dr. Aaron Brault at the CDC in
Fort Collins, CO where she studied the impact of West Nile virus evolution on
transmission potential and established systems for studying Zika virus sexual
transmission.
Her current work investigates the viral determinants and host immune correlates
of Usutu virus disease, avian and vector species important for Usutu virus transmis-
sion, and mechanisms of Zika virus sexual transmission” (N Duggal).
Rebekah Kading (Figs. 104 and 105)
Assistant Professor
Colorado State University
Fig. 102 Nisha Duggal
Fig. 103 Nisha Duggal,

2015 responding to
outbreak of St. Louis
encephalitis virus in
Arizona
“Dr. Kading’s research focus is on the ecology and transmission of emerging

arboviruses. Her contributions to the field include novel insights into mosquito
blood feeding patterns and arbovirus circulation, entomological risk factors for the
potential for introduction and establishment of Rift Valley fever virus to the United
States, and viruses associated with bats” (R Kading).
Fig. 104 Rebekah Kading

with Dr. Robert Kityo of
Makerere University taking
an oral swab from a bat in
the genus Rhinolophus;
Kaptum cave, Uganda,
May 2021. (With kind
permission of Anna Fagre,
photographer)
Fig. 105 Rebekah Kading. (With kind permission of John Eisele/Colorado State University
Photography)
Helen Lazaer, PhD (Fig. 106)

Assistant Professor Microbiology and Immunology
Univ North Carolina School of Medicine
Fig. 106 Helen Lazaer
“Helen Lazear grew up in Calgary, Alberta, Canada. She received her B.Sc. in
2003 from the University of Alberta and her Ph.D. in 2009 from the University of
Pennsylvania. Although her graduate research was on herpes simplex virus neuronal
transport, during this time she developed an interest in arboviruses, and pursued
postdoctoral training with Mike Diamond at Washington University in St. Louis.
Her postdoctoral research focused on innate immune control of West Nile virus
pathogenesis but she always maintained an interest in obscure flaviviruses, particu-
larly ones with potential for wider emergence. This led her to pursue early work on
Zika virus, which proved useful when that virus caused a pandemic in 2015–2016.
She started her lab at UNC Chapel Hill in 2015 where she continues to investigate
viral and host mechanisms that control flavivirus tissue and species tropism” (H
Lazaer).
Jenny Hesson, PhD (Fig. 107)
Uppsala University
Uppsala, Sweden
“In 2014, I received my PhD in Biology studying Culex vectors and Sindbis virus
under Dr. Jan Lundström, with specialisation in Population Biology, at Uppsala
University in Sweden. I did my post-doc in arbovirology with Professor Matthew
Baylis at University of Liverpool (UOL) in the United Kingdom. This position gave
me experience infecting mosquitoes and performing vector competence experi-
ments in a Biosafety level (BSL) 3 laboratory. The experiments were performed at
the Liverpool School of Tropical Medicine where I enjoyed contact with other vec-
tor biologists, which was a rare specialisation where I came from in Sweden. Around
this time, I also did a short term post-doc granted by the Japan Society for the
Fig. 107 Jenny Hesson
Promotion of Science with Professor Noboru Minakawa at Nagasaki University,

Japan. In his lab, I performed life traits experiments on different populations of
Aedes aegypti. After my time in Japan I returned to working half time at UOL and
half time at the Zoonosis Science Center (ZSC) at Uppsala University, commuting
between the two countries. This allowed me to continue my work with mosquito-
virus experiments at UOL at the same time as I began to build up my research base
in Sweden. working on Sindbis virus that is highly endemic in Sweden. With time,
this funding also allowed me to move, I am currently working full time at ZSC
building up my own research o program on vector biology and arbovirology. I com-
bine field and laboratory studies and use Sindbis virus as a model to study adapta-
tion of arboviral transmission to northern climates” (J Hesson).
Gillian Eastwood, PhD (Figs. 108 and 109)
Assistant Professor of Entomology
Fralin Life Sciences Institute of Virginia Tech
“The Eastwood lab focuses on vector-borne disease ecology with a One Health
perspective. We are interested in enzootic transmission dynamics and the potential
for disease emergence of arboviral vector-borne pathogens, such as mosquito-borne
viruses (e.g. La Crosse virus, or novel agents) and tick-borne viruses (Powassan,
Heartland, Bourbon viruses); as well as the general prevalence of co-infection
in ticks.
Fig. 108 Gillian Eastwood
Fig. 109 Gillian Eastwood

Considering the impact of ecosystem land-use is a theme throughout our proj-

ects, in particular the influence of tropical forest degradation on vector species
diversity as a driver for emerging infectious disease” (Eastwood n.d.).
Maria Onyango, PhD (Figs. 110 and 111)
Assistant Professor
Texas Tech University, Lubbock, TX
“I am interested in understanding mosquito and tick biology and their interaction
with viruses. I study the tripartite interactions of arthropods, their microbiome and
the pathogens that they transmit and how these interactions may influence disease
Fig. 110 Maria Onyango
Fig. 111 Maria Onyango

emergence. Further, I endeavor to uncover the mechanisms that govern these inter-
actions with the aim of identifying novel ways to block transmission of pathogens
as well as discover novel therapeutics” (M Onyango).
Eili Huhtamo, PhD (Figs. 112 and 113)
Docent in Virology
Medicine and Veterinary Medicine University of Helsinki, Finland
Fig. 112 Eili Huhtamo
Fig. 113 Eili Huhtamo.

(With kind permission of
photographer Joonas
Kolehmainen/University of
Helsinki)
“I am a university researcher and a principal investigator affiliated to faculties of

Medicine and Veterinary Medicine University of Helsinki, Finland. My research
focuses on mosquito-borne viruses, especially flaviviruses. I have been involved in
developing diagnostics and characterizing mosquito-borne viruses from patients
and mosquitoes. The work has resulted in identification of atypical flaviviruses from
Finnish mosquitoes, and various mosquito-borne viruses from patients. Recently,
my work has been focused on virus discovery from mosquitoes and patient samples
collected from Finland, Italy and Kenya. The ongoing work is linked to collabora-
tive consortium projects such as EU funded Versatile Emerging infectious disease
Observatory (VEO) and Academy of Finland funded VECLIMIT” (E Huhtamo).
Meghan Hermance, PhD (Fig. 114)
Assistant Professor
University of South Alabama
“Meghan Hermance earned her Ph.D. from the University of Texas Medical
Branch under the mentorship of Saravanan Thangamani. Her training provided sig-
nificant experience in conducting BSL3 research with Powassan virus-infected
ticks. She is now faculty at the University of South Alabama where her lab uses
biologically relevant tick-virus-host systems to investigate the determinants of tick-
borne virus transmission and pathogenesis. Within this framework, the research
team seeks to discover biological weak links in arbovirus transmission cycles that
can be targeted for novel disease control strategies” (M Hermance).
Fig. 114 Meghan
Hermance
Betania Drumond Ph.D. (Fig. 115)

Biologist
Federal University of Minas Gerais/Brazil
“Betânia Drumond is a Biologist, BS, M.Sc and Ph.D in Microbiology (Federal
University of Minas Gerais, Brazil), and postdoctoral in genomics (Oswaldo Cruz
Foundation/FIOCRUZ, Brazil). She has a broad background in virology/microbiol-
ogy and molecular biology applied to viral infectious diseases. She started her
career as an associate professor in 2010 and since 2016 she has a permanent posi-
tion at Federal University of Minas Gerais/Brazil. She is involved with academic
activities, teaching Virology and Microbiology for undergrad and graduate students,
and research activities coordinating projects and mentoring graduate students. Her
early studies/publications focused on the origins, biological and genetic properties
of Vaccinia virus causing a significant occupational zoonotic disease in Brazil. She
also has other studies in veterinary virology, and studies of giant viruses, focusing
mainly on evolutive aspects of these viruses. Her major research interest is the study
of arboviruses (arthropod-borne viruses). She has been focusing on virus surveil-
lance, molecular and biological characterization, viral evolution and dynamics,
including arboviral investigation in vectors and vertebrate hosts. Her studies dem-
onstrated important aspects of viral emergence, circulation and epidemiology of
Dengue virus, Zika virus, Saint Louis encephalitis virus and more recently Yellow
fever virus, among others viruses in Brazil. Her team has a record of collaborative
work with public local health authorities as illustrated by the responses to yellow
fever epidemics and epizootics and COVID-19 pandemic in Brazil. During the YF
outbreaks that took place in Brazil from 2016–2018, she developed collaborative
Fig. 115 Betania Drumond

research that has elucidated the origin, ecological aspects and dynamics of yellow
fever outbreaks, and also important aspects of yellow fever disease course including
new virological, epidemiological and immunological findings in humans”
(B. Drumond).
5 Challenges in Pursuing a Professional Career

in the Life Sciences
Laura D. Kramer
This last section addresses the question of why women do not receive the same
recognition as men in the sciences in general, focusing on the life sciences in par-
ticular. As will be discussed, some of the barriers that existed historically are no
longer deterrents, leading to parity between males and females in graduate school,
as post-doctoral fellows, and near parity as assistant research scientists. Yet females
who venture into academic science do not rise to become Associate and Full
Professors, Chairs, Deans, and Department Heads in a proportion equal to men.
They are selected for awards (e.g., Wetzel 2021; ACAV n.d.) less often than men,
sometimes referred to as the Matilda effect (Lincoln et al. 2012), and invited less
often to serve on expert panels. There are certainly exceptions, and we have pre-
sented a selection of such outstanding women earlier in this chapter. But we felt that
starting a dialog on the causes for the lack of parity at the upper echelons of aca-
demia, which may lie partially in the hands of the women themselves, is an impor-
tant part of any discussion on recognition of women in science.
Goal 5 of the 17 Sustainable Development Goals of the 2030 Agenda for
Sustainable Development adopted by the UN in September 2015 is to achieve gen-
der equality and empower women and girls (United Nations 2015). Women com-
prise a mean of ~30% of researchers around the world, with the gap greater at
senior-level positions, according to the most recent UNESCO Science Report
(UNESCO 2019; Fig. 116). If one compares the proportion of women researchers
by region of the world (Fig. 116), it varies from 18.5% (South and West Asia) to
48.2% (Central Asia) of the global researcher pool (UNESCO 2019; Fig. 116).
Economic prosperity does not guarantee parity, as can be noted (Fig. 116; UNESCO
2019). South and West Asia, East Asia and the Pacific, North America and Western
Europe, among other developed regions lag in gender equity, while less prosperous
regions such as Central Asia, Latin America and the Caribbean, and the Arab states
are closer to achieving parity in the sciences.
Because of space limitations, this section of the chapter will predominantly
address scientists in the USA, although a comparison of the status of women in the
different global regions would be very informative and would be a study in and of
itself. Similarly, this chapter does not address race, ethnicity, or age, all of which are
being measured assiduously by many institutions. Underrepresented minority
Fig. 116 Share of female researchers by country, 2016 (%). (Figure 3.2 (UNESCO 2019); World
map from Shutterfly)
faculty members make up only 12.9% of full-time faculty members across the USA,
despite making up 32.6% of the US population. These differences also need to be
addressed (Colby and Fowler 2020).
Women have been gaining parity over time since 1993 in occupations in the life
sciences, including biological, agricultural, and environmental fields, more than in
computer and mathematical sciences, physical sciences, and engineers [(National
Science Foundation 2018a, b, c); Fig. 117]. Social scientists fare best, being the
only group to have achieved equity, 2013–2015.
5.1 Graduate Degrees
Veterinary schools now enroll 70–80% female students (Lincoln 2010), and as
of 2020, according to the Association of American Medical Colleges, women
accounted for 53.7% of medical school matriculants (Association of American
Medical Colleges 2020), basically the same proportion of women earning doc-
torates in the biological sciences in 2020 (American Physical Society 2021).
But disparities remain. While the proportion of women faculty members in aca-
demia is increasing, far more students and postdoctoral fellows are trained than
the number of available faculty positions, making for a sharply competitive
environment for grant dollars, upper rank faculty positions, and stature [Fig.
118; (Leeming et al. 2016; Kelly 2019)]. This presents a dilemma for institu-
tions seeking to achieve parity in academic positions. The question has been put
forth whether we should stop actively encouraging women to study life
Fig. 117 National Science Foundation. National Science Board. Women in Science and
Engineering. (National Center for Science and Engineering Statistics, SESTAT (1993–2013),
Fig. 3–27 (NSF 2018a, b, c). Available at: https://www.nsf.gov/statistics/2018/sestat [Accessed
10/5/2021]; and the National Survey of College Graduates (NSCG) (2015), Available at: https://
www.nsf.gov/statistics/srvygrads/ [accesses 10/5/2021])
sciences – a field in which they face unique challenges with few positions avail-
able [Fig. 118; (Xue and Larson 2015; Feder 2012)]. To counter this concern,
the Bureau of Labor Statistics indicates that we need at least a million more
STEM [Science, Technology, Engineering, Math] professionals right now and
this number is only expected to increase over the coming decades. (U.S. Bureau
of Labor Statistics 2020) STEM includes many subdisciplines in addition to
biology, and academia is not the only career path for biologists. Alternative
scientific careers other than academia need to be discussed with students and
explored more thoroughly.
The proportion of women going into the life sciences has increased steadily
since 1995 from 49.75% (BA), 47.73% (MS), and 37.40% (PhD) to 58.01%
(BA), 56.56% (MA), and 52.66% (PhD) reported in 2018 (Catalyst 2020).
Thus, women ostensibly have achieved parity with men, if not a slight advan-
tage, in award of graduate degrees in the biological sciences. Unfortunately,
this apparent achievement of parity has two caveats. First, it only applies to
white women. Among students enrolled in graduate school in Science and
Engineering (S&E fields) in 2018, white US citizens were the largest group
(38.3%). Proportion of female students who are Black or African American
Fig. 118 Challenging trajectory in academic careers. Academic positions in biological sciences;
design by Vanessa Czerwenka; Guest contributors Philipp Gramlich and Karen Bodewits. (Women
in science: Clogging the leaky pipeline. 23 Mar 2016 Posted by Jack Leeming. (Leeming et al.
2016). Available at: http://blogs.nature.com/naturejobs/2016/03/23/women-in-science-clogging-
the-leaky-pipeline/ [Accessed 09/10/2021])
among S&E [Science and Engineering] was 4.6% of doctoral students and
7.7% of S&E masters, an unacceptably low proportion but larger share than
that found for Black or African American male students (2.4% of all male S&E
doctoral students, 4.7% of all male S&E masters) (NSF 21-321. 29 Apr 2021).
Second, while parity at the doctoral degree level is present in the untenured
academic positions, i.e., adjunct faculty, post-doctoral fellows and research
assistants, a lack of parity becomes evident in the tenured academic positions.
Of the 53% of the PhDs awarded to women in 2015, 50% continued on to post-
docs, 45% assistant professors and 33% full professors [Fig. 119; (Catalyst
2020; Colby and Fowler 2020)]. Analysis by the American Association of
University Professors confirms the NSF data and found that women made up
42.5% of all tenured faculty at 4-year institutions (Colby and Fowler 2020).
The transition from graduate programs to assistant professorships shows more
attrition in the fields in which women are already very prevalent (e.g., life sci-
ences, social science) than in the math-intensive fields in which they are under-
represented (Ceci et al. 2014).
Fig. 119 The leaky pipeline, share of women in higher education and research worldwide, 2013.
((Huyer 2015) Fig. 3.3; Updated data from Catalyst 2020; Colby and Fowler 2020; NSF 2021a, b).
Source: IPEDS HR survey component 2018–2019. Some data compiled by AAUP research dept)
5.2 Tenure Track
The tenure process in academia is the first critical gatekeeping event where signifi-
cant faculty attrition takes place (Ceci et al. 2014). There has been an overall reduc-
tion the past 15–20 years in the proportion of US-trained S&E doctorate holders who
have achieved tenure, but interestingly, the reduction occurred predominantly in
male doctorate holders even though fewer women than men held tenured positions in
both 1995 and 2015. In the life sciences, 52.0% (males) and 30.4% (females) doctor-
ate holders were tenured in 1995. This proportion remained basically the same for
women through 2015 (29.6%), but for men decreased from 52% to 44.8% (National
Science Foundation 2018a). Thus, the process of advancing through tenure (and cor-
respondingly becoming an Associate Professor) appeared to be stagnant for women,
and by some accounts, ascending the professional ladder is now harder than ever.
5.3 Academic Faculty Positions
Overall, female full-time faculty increased from 31% to 45% from 2003 to 2019,
but as stated earlier, this increase is deceptive because it is the result of more female
faculty hired in the untenured roles of instructors, lecturers, and assistant professors.
When one compares the proportion of males and females in the different aca-
demic positions at 4-year colleges, males have a distinct advantage in upper echelon
Teaching assistant 350.00

200.00
Research assistant 1,050.00

650.00
Postdoc 12,050.00
8,95.00
Academic position
Adjunct faculty 10.950.00

8,150.00
Teaching faculty 134,150.00

77,900.00
Research faculty 113,650.00

62,650.00
Dean, department head, chair 23,450.00

13,150.00
President, provost, chancellor 2,200.00

1,550.00
0 20,000 40,000 60,000 80,000 100,000 120,000 140,000

Number
Men Women
Fig. 120 Doctoral scientists and engineers employed in universities and 4-year colleges. Women,
Minorities, and Persons with Disabilities in Science and Engineering. National Center for Science
and Engineering Statistics (NCSES). (Figure 44. Directorate for Social, Behavioral and Economic
Sciences. National Science Foundation. Alexandria, VA | NSF 21–321 | April 29, 2021 (NSF
2021a) Available at: https://ncses.nsf.gov/pubs/nsf21321/report/academic-careers [Accessed
09/15/2021])
positions such as deans, department heads, research, and teaching faculty; but males
and females show greater equivalency in the number of adjunct faculty, post-
doctoral fellows and research assistants, i.e., all untenured positions [Fig. 120; (NSF
2021a)]. Science and engineering male doctorate holders outnumbered their female
counterparts in all major positions in 2019 in 4-year colleges and universities.
Women made up about 36% of both research faculty and teaching faculty when all
academic levels were combined. This represents an increase of only 5% over the
previous 75 years while over the same time period, the number of women enrolling
in college and earning college degrees has tripled (Kelly 2019).
Interestingly, women are still more poorly represented, but less so, at the very
highest levels of president, provost, chancellor, comprising 43.1% of the total, still
lower than men but an improvement over lower level position discrepancies [Fig.
120; (NSF 2021a)].
5.4 Academic Rank
The percentage of S&E female doctorate holders in academia in the USA has
increased gradually and consistently at all academic ranks, i.e., Assistant, Associate,
Full Professor, over time; see 1973–2015 displayed in Fig. 121 (NSF 2018b). Yet it
is remarkable that women still make up less than 50% of the total at each level of
Fig. 121 Women as a percentage of S&E doctorate holders employed full time in academia, by
academic rank: Selected years, 1973–2015. National Science Board. Science and engineering
Indicators 2018. Alexandria, VA: National Science Foundation (NSB-2018-1). (Figure 5–12.
Women in S&E occupations. (NSF 2018b) Available at https://www.nsf.gov/statistics/indicators/.
https://www.nsf.gov/statistics/2018/nsb20181/figures/fig 05-12)
academic achievement, with female Full Professors comprising the lowest percent-
age at under 30% (NSF 2018b).
As implied by the lower proportion of women as one moves up the academic
ladder, appointments at the associate and full professor ranks, which are tenured,
disproportionately went to men. And women experienced disproportionately high
rates of leaving academia before tenure (Kelly 2019).
More than 40% of women with full-time jobs in science leave or go part-time
after having their first child, according to a study of how parenthood affects career
trajectories in the USA. By contrast, only 23% of new fathers leave or cut their
working hours. And this compares with 16% of child-free men and 24% of child-
free women (Cech and Blair-Loy 2019). According to the AAUW, approximately
70% of male professors with tenure have children compared to 44% of female ten-
ured professors. Even earlier in the career track, it is likely family considerations vs.
career that lead to attrition of females after earning a PhD and completing a post-
doctoral fellowship; it is at this time when they are weighing whether to they really
want to compete for an assistant professorship or higher grade [Fig. 122; (Goulden
et al. 2009)].
Yet it was found in an experimental study that faculty members of both genders
in four S&E fields exhibited approximately a 2:1 preference for hiring female assis-
tant professors over males with identical qualifications and lifestyles (n = 339: 171
women and 168 men; χ2 = 40.38; P < 0.0001) (Williams and Ceci 2015). This was
true regardless of gender of the faculty member making the decision. In contrast, in
a real-life examination of top institutions, it was found that elite male faculty, e.g.,
those whose research was funded by the Howard Hughes Medical Institute, who had
been elected to the National Academy of Sciences, or who had won a major career
award, were less likely to select female scientists than male scientists to work in
their labs (Sheltzer and Smith 2014).
No children, no future plans

19%
Men Women
20%
No children, future plans to have children

17%
28%
Children previous to postdoc

19%
32%
New children since postdoc

20%
41%
Fig. 122 Percentage of University of California postdoctoral scholars who shifted away from
professor with research emphasis as a career goal, broken down by gender and family status/future
plans. Staying competitive: Patching America’s leaky pipeline in the sciences. (The University of
California, Berkeley, Berkeley Center on Health, Economic, & Family Security and The Center for
American Progress (Goulden et al. 2009). Available at: http://cdn.americanprogress.org/wp-
content/uploads/issues/2009/11/pdf/women_and_sciences.pdf [Accessed 08/15/2021]. Permission
generously provided by Marc Goulden)
5.5 Wadsworth Center
Because the number of women in senior research positions was one aspect that
attracted me (LDK) to Wadsworth Center, New York State Department of Health,
we examined composition of the staff at different grade levels in this institution.
Even though Wadsworth is a state health lab, it is the exception among state health
laboratories in that the staff at Wadsworth also serve as faculty in the School of
Public Health, State University of New York at Albany, and grant-funded research
plays a major role in most scientist’s programs and their evaluations for promotion.
An Assistant Professor is equivalent to a G25–27, Associate G31–35, and Full G38.
The proportion of females/males (Table 1; kindly provided by Carleen VanPatten,
Associate Director for Administration, Nov 2018) follows the pattern seen in the
biological sciences in academic institutions, that is, females predominate in lower-
level positions, reach parity at G25, and then are more poorly represented in upper-
level positions.
Table 1 Research staff by grade and gender at the Wadsworth Center, New York State Department
of Health in 2018
Grade level Male Female % Female
Postdoctoral 17 15 47%
G14 7 29 85%
G18 20 52 72%
G22 34 54 61%
G25 23 26 53%
G27 20 16 44%
G31 18 8 31%
G35 15 10 40%
G38 4 1 20%
Proportion of females at Wadsworth Center, New York state department of Health, Albany, NY,
2018. Data graciously provided by Carlene VanPatten, Wadsworth Center, Nov 2018
5.6 Professional Societies: The American Society for Tropical

Medicine and Hygiene (ASTMH)
The American Committee on Arthropod-borne Viruses (ACAV), a subcommittee

under the umbrella of ASTMH, established the groundwork for arbovirology as a
field of study [see section 1]. A photo of a meeting of the ACAV in 1964 shows a
single female, Gladys Sather, among 13 men (photo 8 section 1). Being the single
or one of a few females reflects my experience for most of my training and profes-
sional life until I (LDK) joined Wadsworth Center in 2000!! Recent data for mem-
bership in ASTMH and ACAV (Table 2; data kindly provided by Buffy Finn, Oct
2019) demonstrate the same problem with parity among members of the larger par-
ent organization (ASTMH) and the subcommittee (ACAV), respectively. Here too
the proportion of postdocs and graduate students is approximately balanced between
males and females, with females having slightly higher numbers, but an imbalance
remains among members that yet needs to be addressed. No change is evident
between years 2015 and 2019. An examination of ACAV awards indicates males
greatly surpass females as recipients: e.g., Dalrymple/Young award – 2F/11M, RM
Taylor Award 3F/28M; Scherer/Hardy 4F/10M (ACAV n.d.).
The slightly greater parity with the Scherer/Hardy award is a consequence of it
being given to scientists early in their careers, reflecting a stage where there is a
greater balance in gender ratio.
5.7 Causes and Solutions
Why is there greater attrition of women contributing to lack of parity between

women and men in upper echelon academic positions? Women faculty members
continue to face unique challenges in academia with respect to employment,
Table 2 Number of ASTMH and ACAV members in 2015 and 2019 by membership status
and gender
ASTMHa ACAVb
Male Female % Female Male Female % Female
2015
Member 1000 593 37 84 40 32
Emeritus 48 12 20 8 0 0
Lifetime 23 2 8 3 0 0
Postdoc 125 161 56 18 30 63
Student 86 128 60 9 10 53
2019
Member 1018 604 37 102 51 33
Emeritus 48 12 20 8 0 0
Lifetime 23 2 8 3 0 0
Postdoc 158 197 55 51 66 56
Student 140 201 59 63 83 57
Proportion of females American Society for Tropical Medicine and Hygiene (ASTMH) and sub-
committee American Committee on Arthropod-borne Viruses (ACAV), 2019. Data graciously pro-
vided by Buffy Finn, Manager, Membership ASTMH
a
American Society for Tropical Medicine and Hygiene
b
American Committee on Arthropod-borne Viruses
advancement, salary, and job security. Addressing these gaps is a major challenge
that must be discussed, not only by universities and society but also among the
women themselves, searching for innovative solutions and organizing sustained
effort to correct the inequity. It is to the benefit of all that there is diversity in gender,
race, ethnicity, and age, since individuals from different backgrounds bring different
perspectives, make unique contributions, and offer different approaches to problem-
solving. With diversity comes a broader point of view, and greater sensitivity and
respect for different perspectives (Rosser 2012).
5.8 Funding Longevity
The gradual loss of female academics moving up the academic ladder has been
attributed to such factors as child rearing responsibilities, committee and teaching
obligations, misperception that men are better at science and math, insecurity among
females, lack of mentors and role models, fewer networking opportunities, and more.
Because scientists in academia must maintain a funding stream to support their
labs, students, and post-doctoral fellows who will conduct the research that will lead
to publications and visibility, a study was conducted to determine whether women
remain funded at the same rate as men after receiving their first major research grant
(e.g., NIH RO1, PO1, UO1) as independent investigators (Hechtman et al. 2018).
This time point was selected because results indicated that women are as successful
as men in being awarded their first major grant. Their success in receiving major
grants beyond the first grant is a key indicator of their ability to conduct research,
build their laboratories, qualify for tenure, and be promoted to Associate Professor.
As discussed earlier, men maintain an advantage even as their numbers are dropping.
The investigators found that women submitted fewer grant applications (approx-
imately one third fewer), and their review scores were slightly worse than males
with score percentiles averaging 23.76 compared with 23.00 in men, but this differ-
ence is not statistically significant. In regard to renewal applications, women on
average submitted 42.45% of their eligible projects for renewal, compared with
45.44% for men. And women had a funding rate of 35.98% for project renewals,
while men had a renewal rate of 39.28%, with the exception of the most recent
cohort (2006–2010).
It was concluded male and female academic researchers have generally compa-
rable funding longevity, contradicting the basic concept of accelerated attrition
compared to men across the consecutive career ranks. Instead, women are under-
represented among NIH grantees largely because of lower rates of new and renewal
application submissions, representing approximately 30.66% of the investigator
pool. This is significant because what was determined to be most predictive of an
investigator’s survival was the rate the investigator submitted new and renewal
applications (Hechtman et al. 2018).
Similar to grant funding, manuscript reviewing has been found to be gender neu-
tral, i.e., manuscripts by male and female authors are equally likely to be accepted
by journal editors just as they are to have their grants funded, with only very occa-
sional exceptions [(Ceci et al. 2014) and see below]. Another factor contributing to
professional advancement is citation index. How often academic articles are cited is
a key measure of scholarly impact. In one study looking at high-impact medical
journals [Annals of Internal Medicine, British Medical Journal, JAMA, JAMA
Internal Medicine, and The New England Journal of Medicine], articles written by
women had fewer citations than those written by men, particularly when both pri-
mary and senior authors were women (Chatterjee and Werner 2021). This may
partly be a consequence of men’s increased productivity (NSF 2018a, b, c, Fig. 5-G;
Ceci et al. 2014); publications by women represent <35% of total. It also may reflect
the greater tendency men have to cite their own work than women do.
However, a large study of 1,523,002 scientists (412,808 female and 1,110,194
male) whose publishing careers ended between 1955 and 2010 found that “differ-
ences in publishing career lengths and dropout rates explain a large portion of the
reported career-wise differences in productivity and impact, although productivity
differences still remain” (Huang et al. 2020). Thus retention of women in academic
career tracks again presents a significant problem.
Publication and funding differences will certainly impact chances for profes-
sional success of women in academia and attainment of gender parity. With fewer
grants to support their research, women may not have the same ability to attract the
top students. Added to that, women receive less recognition for their accomplish-
ments [Matilda effect (Lincoln et al. 2012)]. Decreased recognition will lead to their
being less likely to be called upon to serve as experts and leaders, fewer speaking
invitations at national conferences, fewer prestigious awards, https://jamanetwork.
com/journals/jamanetworkopen/fullarticle/2781617 – zoi210439r4 and diminished

likelihood to be promoted.
How do we address this problem? An AAUP analysis confirms that women fac-
ulty members continue to face unique challenges in academia with respect to
employment, advancement, salary, and job security. Pay and opportunity gaps are
the result of many factors beyond gender, race, and ethnicity, and closing them will
require innovative and sustained efforts. Attention must be paid to find ways to cre-
ate more free time, child care at a reasonable price, and free after-school programs
for new female faculty to allow them to increase productivity in grant submission
and papers. Family-responsive policies have been recommended in (Powell 2021),
among other publications. Such policies as paid parental leave and tenure deferment
have contributed to decreasing the female productivity gap (Morgan et al. 2021).
Tenured female faculty often feel internal pressure to take on more mentorship
and service, which is generally uncompensated and undervalued. Teaching, com-
mittees, and students all eat up “free” time. Further, women are held to a higher bar
than men [Fig. 123; data from (Wennerås and Wold 1997)]. Scientists in Sweden
were assigned a peer review evaluation (competence) score based on scientific com-
petence (number and quality of scientific publications), relevance of the submitted
research proposal, and quality of proposed methodology. It revealed that women
with the greatest scientific impact (>99) are considered equal to men with the mid
to lowest (<19) productivity; the women had to be 2.5 times more productive than
men in order to get the same peer review ratings. And Sweden has been named by
the UN as the world leader in sexual equality! A contributing factor to this discrep-
ancy may be the paucity of women serving on the peer review committee. As noted
in the NIH study discussed earlier (Hechtman et al. 2018), peer review scores are
basically equivalent between males and females, but the MRC and NIH systems are
Fig. 123 Peer review evaluation scores. The mean competence score given to male (Blue bars)
and female (Red bars) applicants by the MRC (Medical Research Council, Sweden) reviewers as
a function of their scientific productivity, measured as total impact. One impact point equals one
paper published in a journal with an impact factor of 1. (Data from Wennerås and Wold 1997)
different and the MRC study is from 1997. Thus this may serve to demonstrate that
advances in equity have been made. However, in controlled studies in 2012, the
male applicant was rated significantly more competent and hirable than the (identi-
cal) female applicant. Women in academia received less favorable evaluations,
received lower salary offers, and were mentored less frequently than men (Moss-
Racusin et al. 2012). Nonetheless, we need to reappraise methods of teaching, hir-
ing, and promotion to allow greater flexibility for women. Some approaches follow
to address these problems among others not discussed. Women need to hone their
networking skills for support and mentoring, just as men do on campus, in bars and
on golf courses, but with their own touch, e.g., dinners, scientific salons.
The attrition of females pursuing scientific careers can begin early in a student’s
education when peer pressure has a strong impact on the choices females make. It is
the same time as stereotypes of boys being better in science and math prevail, and
perceptions persist that science and math are masculine pursuits not suited for
women (Carlana 2019). Parents and educators must encourage children from an
early age and teach them not to stereotype people, e.g., considering women as being
too emotional to hold leadership positions. Gender biases may be conscious and
unconscious; when identified, they must be addressed. Parents and educators can
also help women build self-confidence, crucial in advancing and enjoying a research
career. To change this situation, encouragement and inspiration early in the educa-
tional process is critical to engage young girls.
Women in upper echelon positions must recognize their importance as role mod-
els and make time to serve as mentors. To accomplish this, the support structure for
graduate students needs to be strengthened with institution of a mechanism for
matching student with mentor, who does not have to be the actual advisor. There
needs to be proactive recruitment for women for tenure-eligible positions to provide
a balanced faculty where the percentage of female tenured professors is an appropri-
ate measure of the percentage of female PhD in the discipline. This balance facili-
tates mentoring relationships. Universities and other institutions should allow
flexible working hours for women (and men) to devote time to child-rearing, elder
care, and other household activities so they can balance career and family responsi-
bilities. However, flexible hours may be difficult to manage with scientific experi-
mentation. Even in homes where men assist in household activities, women in the
USA generally bear the greater burden [Fig. 124; (Hess et al. 2021)] regardless of
age group but particularly among 25–44 yo, the time women are starting families,
are more involved with elder care, and, at the same time, are going for tenure. There
is a gender gap in effort of 54% in 25–34 yo group and 51% in 35–44 age group
(Hess et al. 2021).
It is interesting that when the hours spent in the workplace were compared, they
were equal for tenure and tenure-track male and female academics (Ceci et al.
2014). One might conclude that it is in the home environment where work effort
differs due to women taking on more of the familial responsibilities. It is here that
writing grants and manuscripts may take place. Another not exclusive explanation
is men are able to publish more papers because they have greater funding (more
8.8
8.0
Average hours per day
5.6
5.2
4.2 4.4
3.9 4.1
3.7 3.5
3.3
1.7
15 to 24 25 to 34 35 to 44 45 to 54 55 to 64 61 and older
Age Group
Female Male
Fig. 124 Average Hours per Day Spent on Unpaid Household and Care Work by Gender (Hess
et al. 2020). (Source: IWPR analysis of America Time Use Survey microdata [US Bureau of Labor
statistics]. Notes from the authors: For aged 15 and older – Care work includes secondary child
care as well as primary child and elder care. Secondary child care is considered as a separate activ-
ity and is counted independently even though it may be performed while doing housework or pri-
mary care work. This “double counting” (counting both secondary and primary care performed at
the same time) makes our estimates of the hours spent in unpaid care work higher than many oth-
ers. While the convention in the field is to only count secondary care if not performed simultane-
ously with primary care, our method captures the intensity of unpaid care work and the multitasking
it often requires. This may provide a better measure of the actual time spent on unpaid care work
activities since it gives insight into the caretaker’s quality of life or well-being. Permission gener-
ously provided by Jodi Narde narde@iwpr.org)
resources, more students and fellows), and larger labs producing more papers. Thus
collaboration becomes even more important for female scientists.
The competitiveness of the system forces PIs and their fellows who want to
advance, to work long hours. When women have families, career advancement is
likely impeded. It is difficult to take time off from work and return to the same
career trajectory. But there is no easy solution since funding depends on a peer
review process that counts publications and evaluates recent progress. Clearly, more
policies supportive of families are needed.
One solution to the rigid academic track women face is to reshape our perception
of the career path. Rather than a nonstop straightforward progression from college
to PhD to Professor, students should recognize that careers can be redirected or
reshaped in the dynamic environment in which we live. Furthermore, the standard
model does not reflect the full range of career opportunities available to STEM
degree holders and ignores the many factors that influence career choices over a
lifetime.
We end this chapter with a brief description of an informative book, The
Autobiography of a Transgender Scientist by Ben Barres (1954–2017) (Barres
2020). In 1997, when Ben Barres was a Stanford University neuroscientist, he wrote
a letter to colleagues. He signed the letter with his birth name, Barbara Barres, but
made it clear that from now on he wished to be known as Ben. “I hope that despite
my trans-sexuality you will allow me to continue with the work that, as you all
know, I love,” he concluded in his letter. His fellow scientists responded with
unwavering support.
S/He had attended MIT in Biology, earned an MD at Dartmouth, a PhD at
Harvard, and then conducted research at Stanford. He wrote “By far the main differ-
ence that I have noticed is that people who don’t know I am transgender treat me
with much more respect [as a male]. I can even complete a whole sentence without
being interrupted by a man.” How better to understand the different life experiences
of a female and male scientist having experienced both the female and male sides of
the picture! (Barres 2020).
The academic landscape is changing, and a number of the historical barriers
impeding advancement of women in the life sciences have been overcome. There is
parity in graduate programs, post-doctoral fellowships, and near parity in assistant
professors. There is no difference in acceptance of manuscripts by journal reviewers
and editors (except perhaps top tier medical journals), grants are nearly equally
funded, hours worked outside the home are equivalent, but males demonstrate sig-
nificantly increased productivity in manuscript and grant submission. Problems
remain with women’s sense of self, ability to gain recognition and credit for their
ideas, networking skills, and probably most significantly, women experience per-
ceived and real difficulty managing both a family and a demanding science career.
New interventions are needed addressing faculty gender bias. In addition, although
not discussed in this chapter in depth, racial inequities are large as is diversity in
general. These are the issues on which attention must be focused now to achieve
further equity.
Acknowledgments We are indebted to the large collection of Rockefeller diaries and pictures
kept by Telford Work during his extraordinary career from working in Fiji (on elephantiasis), to
Egypt (on West Nile, Chenuda, Quaranfil), India (on Kyasanur Forest Disease, Jamshedpur Fever),
CDC and the UCLA School of Public Health. Many quotes come from Telford Work’s personal
recollections continuously and generously shared with his wife Martine for 25 years. We also
thank Elizabeth Kauffman for her help with figures, tables, and editing; Shibani Karovalia with
help with references; and all those who so generously shared photos and bios with us.
References
Academic Women’s Network of Washington University School of Medicine (2002–2009) Sondra

Schlesinger – We’ve come a long way – maybe [Online]. Bernard Becker Medical Library.
Available: https://beckerexhibits.wustl.edu/women/schlesinger.htm Accessed 3 Nov 2021
ACTM AOT (2008) The memoirs of Margaretha Issacson [Online]. Available: https://search.infor-
mit.org/doi/pdf/10.3316/informit.206643781669169. Accessed 3 Nov 2021
Aguilar PV (n.d.) UTMB health. Department of Pathology. Available: https://www.utmb.edu/
pathology/faculty/patricia-v-aguilar-phd. Accessed 20 Oct 2021
American Committee on Arthropod-Borne and Zoonotic Viruses (ACAV) (n.d.) [Online]. American
Society of Tropical Medicine and Hygiene. Available: https://www.astmh.org/subgroups/acav.
Accessed 10 Nov 2021
American Physical Society (2021) Doctoral degrees earned by women, by major [Online].
American Physical Society. Available: https://www.aps.org/programs/education/statistics/
fraction-phd.cfm. Accessed 8 Nov 2021
Andrade RDP (2019) “A forest full of viruses!” science and development in the fron-
tiers of the Brazilian Amazon. Dossier: Amazonian Borders 39(82). https://doi.
org/10.1590/1806-93472019v39n82-02
Association Of American Medical Colleges (2020) 2020 Fall applicant, matriculant, and enroll-
ment data tables [Online]. Associate of American Medical Colleges. Available: https://www.
aamc.org/media/49911/download. Accessed 8 Nov 2021
Barres B (2020) The autobiography of a transgender scientist. The MIT Press, Cambridge,
MA/London
Buckley S (1998) The late bloomer: an autobiography of a female medical scientist
Carlana M (2019) Implicit stereotypes: evidence from teachers’ gender bias*. Q J Econ
134:1163–1224
Casals J, Henderson BE, Hoogstraal H, Johnson KM, Shelokov A (1970) A review of soviet viral
Hemorrhagic fevers, 1969. J Infect Dis 122:437–453
Catalyst (2020) Women in science technology, engineering, and mathematics (STEM) [Online].
Available: https://www.catalyst.org/research/women-in-science-technology-engineering-and-
mathematics-stem/. Accessed 8 Nov 2021
Causey CE (1979) Workers in tropical medicine. American Society of Tropical Medicine
and Hygiene
Cech EA, Blair-Loy M (2019) The changing career trajectories of new parents in STEM. Proc Natl
Acad Sci 116:4182–4187
Ceci SJ, Ginther DK, Kahn S, Williams WM (2014) Women in academic science: a changing
landscape. Psychol Sci Public Interest 15:75–141
Chatterjee P, Werner RM (2021) Gender disparity in citations in high-impact journal articles.
JAMA Netw Open 4:e2114509–e2114509
Chu MC (2018) The legacy of Patricia Ann Webb: broken vials and urgency. In: Whitaker RJ,
Barton HA (eds) Women in microbiology. https://doi.org/10.1128/9781555819545.ch31
Chumakov MP, Sarmanova ES, Bychkova MV, Bannova GG, Pivanova GP, Karpovich LG, Izotov
VK, Rzhakhova OE (1964) Identification of Kemerovo tick-borne fever virus and its antigenic
independence. Fed Proc Transl Suppl 23:852–854
Clarke DH (1964) Further studies on antigenic relationships among the viruses of the group B
tick-borne complex. Bull World Health Organ 31:45–56
Clarke DH, Casals J (1958) Techniques for hemagglutination and hemagglutination-inhibition
with arthropod-borne viruses. Am J Trop Med Hyg 7:561–573
Coffey L (n.d.) Coffey Laboratory – mosquito-borne virus evolution and the viral genetic factors
that promote emergence and disease [Online]. WordPress. Available https://coffeylab.ucdavis.
edu/. Accessed 5 Nov 2021
Colby G, Fowler C (2020) Data snapshot looks at full-time women faculty and faculty of color.
Academic Blog [Online]. Available from https://academeblog.org/2020/12/09/data-snapshot-
looks-at-full-time-women-faculty-and-faculty-of-color/. Accessed 8 Nov 2021
Coordinating Research on Emerging Arboviral Threats Encompassing the Neotropics CREATE-
NEO, UTMB Health. Available from https://www.utmb.edu/createneo/specific-aims
Das P (2001) Mary Jane Cardosa – bringing local solutions to Sarawak, Malaysia. Lancet
1:128–132
Eastwood G (n.d.) Eastwood Lab at Virginia Tech [Online]. Weebly. Available https://eastwoodlab.
weebly.com/. Accessed 4 Nov 2021
Enid Cook De Rodaniche (n.d.) [Online]. In Wikipedia, 22 September 2021. Available https://
en.wikipedia.org/wiki/Enid_Cook_de_Rodaniche. Accessed 3 Nov 2021
Failloux A-B (n.d.) [Online]. The Conversation. Available https://theconversation.com/profiles/

anna-bella-failloux-869702. Accessed 3 Nov 2021
Feder M (2012) One decade, one million more STEM graduates. Available from https://obam-
awhitehouse.archives.gov/blog/2012/12/18/one-decade-one-million-more-stem-graduates.
Accessed 8 Nov 2021
Garvey KK (2018) Battling dengue at a Field Station in Iquitos, Peru. Bug Squad. Available https://
ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=26056. Accessed 8 Sept 2021
Gould EA (2015) In memory of two scientific friends and colleagues who lost their fight against
cancer [Online]. EVA-GLOBAL. Available https://www.european-virus-archive.com/evag-
news/tribute. Accessed 3 Nov 2021
Goulden M, Frasch K, Mason MA (2009) Staying competitive: patching America’s leaky pipeline
in the sciences. Berkeley Center on Health, Economic, & Family Security and The Center for
American Progress
Harrington L (n.d.) [Online]. Cornell cals: 2021 Cornell University. Department of entomology.
Available https://entomology.cals.cornell.edu/people/laura-harrington/. Accessed 3 Nov 2021
Hechtman LA, Moore NP, Schulkey CE, Miklos AC, Calcagno AM, Aragon R, Greenberg JH
(2018) NIH funding longevity by gender. Proc Natl Acad Sci 115:7943–7948
Hess C, Ahmed T, Hayes J (2021) Providing unpaid household and care work in the
United States: uncovering inequality, Brief, IWPR #C487. Institute for Women’s
Policy Research, Washington, DC. https://iwpr.org/iwpr-publications/report/
providing-unpaid-household-and-care-work-in-the-united-states-uncovering-inequality/
Huang J, Gates AJ, Sinatra R, Barabási A-L (2020) Historical comparison of gender inequality in
scientific careers across countries and disciplines. Proc Natl Acad Sci 117:4609–4616
Huyer S (2015) Is the gender gap narrowing in science and engineering? UNESCO Science
Report: towards 2030. Fig 3.1 Available http://uis.unesco.org/sites/default/files/documents/
unesco-science-report-towards-2030-part1.pd
Integrated Research Facility Leadership and Scientists (2022) [Online]. National Institute of
Allergy and Infectious Diseases, 30 April 2022. Available https://www.niaid.nih.gov/research/
integrated-research-facility-leadership-scientists. Accessed 3 Nov 2021
Kelly BT (2019) Though more women are on college campuses, climbing the professor ladder
reamins a challenge. [Online]. Brookings. Available https://www.brookings.edu/blog/brown-
center-chalkboard/2019/03/29/though-more-women-are-on-college-campuses-climbing-the-
professor-ladder-remains-a-challenge/. Accessed 10 Nov 2021
Labeaud AD (n.d.) [Online]. Stanford profiles. Available https://profiles.stanford.edu/angelle-
labeaud. Accessed 3 Nov 2021
Labeaud AD, HERI-Kenya (2021) Career, the plastic problem, solutions and HERI-Kenya!
https://www.oneblueearth.com/post/the-health-consequences-of-plastic-pollution. Accessed 3
Nov 2021
Leeming J, and guest contributors Gramlich P, Bodewits K (2016) Women in science: clogging
the leaky pipeline. Available from http://blogs.nature.com/naturejobs/2016/03/23/women-in-
science-clogging-the-leaky-pipeline/. Accessed 8 Nov 2021
Lincoln AE (2010) The shifting supply of men and women to occupations: feminization in veteri-
nary education. Soc Forces 88:1969–1998
Lincoln AE, Pincus S, Koster JB, Leboy PS (2012) The matilda effect in science: awards and prizes
in the US, 1990s and 2000s. Soc Stud Sci 42:307–320
Military Health System (n.d.). Available https://health.mil/News/Gallery/Photos/2017/08/29/2017-
Team-Research-Accomplishment-Military-Honorable-Mention-2. Accessed 3 Nov 2021
Morgan AC, Way SF, Hoefer MJD, Larremore DB, Galesic M, Clauset A (2021) The unequal
impact of parenthood in academia. Sci Adv 7:eabd1996
Moss-Racusin CA, Dovidio JF, Brescoll VL, Graham MJ, Handelsman J (2012) Science faculty’s
subtle gender biases favor male students. Proc Natl Acad Sci 109:201211286
National Academy Of Sciences (2021) Member directory. Diane E. Griffin. Available http://www.
nasonline.org/member-directory/members/3007695.html. Accessed 10 Oct 2021
National Science Foundation (2018a) National Science Board. Science and engineering indicators
2018 [Online]. National Center for Science and Engineering Statistics (NCSES), Alexandria.
Available https://www.nsf.gov/statistics/2018/nsb20181/figures/. Accessed 8 Nov 2021
National Science Foundation (2018b) National Center for Science and Engineering Statistics, spe-
cial tabulations (2016) of the Survey of Doctorate Recipients (SDR) [Online]. National Science
Foundation. Available https://www.nsf.gov/statistics/2018/nsb20181/figures. Accessed 10
Nov 2021
National Science Foundation (2018c) Women as a percentage of S&E doctorate holders employed
full time in academia, by academic rank: selected years, 1973–2015. National Science
Foundation, Alexandria. NSB-2018-1
National Science Foundation (2021a) Doctorate recipients from U.S. Universities: 2020. National
Center for Science and Engineering Statistics (NCSES). Directorate for Social, Behavioral
and Economic Sciences. Alexandria, VA | NSF 22–300 | November 30, 2021. Available https://
ncses.nsf.gov/pubs/nsf22300/report/fields-of-study. Accessed 3 Oct 2021
National Science Foundation (2021b) Women, minorities, and persons with disabilities in science
and engineering. National Center for Science and Engineering Statistics (NCSES). Directorate
for Social, Behavioral and Economic Sciences. Fig 44. Alexandria, VA | NSF 21–321 | April
29, 2021. Available https://ncses.nsf.gov/pubs/nsf21321/report/academic-careers. Accessed 3
Oct 2021
Oransky I (2005) Sonja Buckley obituary. Lancet 365:932
Outstanding Alumna (2013) Dr. Margo Brinton. [Online]. Available http://www.prooftoprint.
com/host/cvsd/Alumni%20Relations/2013AlumniAwardMargoBrintonhtml.html. Accessed 3
Nov 2021
Pace D (n.d.) Carol Blair, BA’64 [Online]. University of Utah. College of Science. Available
https://science.utah.edu/alumni/carol-blair-ba64/. Accessed 3 Nov 2021
Powell K (2021) The parenting penalties faced by scientist mothers. Nature 595:611–613
Powers A (2014) CDC scientist fights Chikungunya. Available from https://blogs.cdc.gov/
global/2014/07/14/cdc-scientist-fights-chikungunya/. Accessed 3 Nov 2021
Reeves WC (1993) “Arbovirologist and professor, UC Berkeley School of Public Health,” an oral
history conducted in 1990 and 1991 by Sally Smith Hughes. In: University Of California, B
(ed) Online archive of California: regional oral history office, The Bancroft Library. University
of California, Berkeley
Rosser SV (2012) Breaking into the lab: engineering progress for women in science. New York
University Press, New York
Sabin A (1976) Interview with Dr. Albert Sabin
Schrage S (2019) Q&A: Husker alumna talks small-town Nebraska, football, women in virol-
ogy [Online]. University Communication, University of Nebraska – Lincoln. Available https://
news.unl.edu/newsrooms/today/article/qa-husker-alumna-talks-small-town-nebraska-football-
women-in-virology/. Accessed 3 Nov 2021
Sheltzer JM, Smith JC (2014) Elite male faculty in the life sciences employ fewer women. Proc
Natl Acad Sci 111:10107–10112
Strauss, James Obituary [Online]. Los Angeles Times. Jan. 14 to Jan. 16, 2022. Available https://
www.legacy.com/us/obituaries/latimes/name/james-strauss-obituary?id=32239236. Accessed
1 July 2022
Tesh RB, Kramer LD (2021) Pauline Huntington Peralta, PhD (1926–2021). Microbiologist, virol-
ogist, and mentor [Online]. American Society of Tropical Medicine and Hygiene. Available
https://www.astmh.org/blog/may-2 021/in-m emoriam-p auline-h untington-p eralta,-p hd.
Accessed 3 Nov 2021
Travassos de Rosa APA (2016) The history of Arbovirology at Instituto Evandro Chagas, Belém,
Pará, Brazil, from 1954 to 1998. Revista Pan-Amazônica de Saúde 7:61–70
U.S. Bureau Of Labor Statistics (2020) Employment in STEM occupations [Online]. United
States Department of Labor Available https://www.bls.gov/emp/tables/stem-employment.htm.
Accessed 8 Nov 2021
UNESCO (2019) Sustainable Development Goals UIS Fact sheet no. 55 [Online]. UNESCO
Institute for Statistics. Available http://uis.unesco.org/sites/default/files/documents/fs55-
women-in-science-2019-en.pdf. Accessed 5 Nov 2021
United Nations (2015) United Nations Sustainable Development Goals [Online]. United Nations.
Available https://www.un.org/sustainabledevelopment/sustainable-development-goals/.
Accessed 5 Nov 2021
Wennerås C, Wold A (1997) Nepotism and sexism in peer-review. Nat 387:341–343. https://doi.
org/10.1038/387341a0
Wetzel C (2021) No Nobel Prizes in Science Went to Women This Year, Widening the Awards’
Gender Gap. Smithsonian Magazine. Oct 8, https://www.smithsonianmag.com/smart-news/
the-nobel-gender-gap-widens-as-no-women-awarded-science-prizes-180978835/
Williams WM, Ceci SJ (2015) STEM faculty prefer hiring women professors 2:1. Proceedings of the
National Academy of Sciences 112(17):5360–5365. https://doi.org/10.1073/pnas.1418878112
Work TH (1965) Letter of transmittal and recommendation from US delegation on Hemorrhagic
Fevers to the USSR.
Xue Y, Larson RC (2015) STEM crisis or STEM surplus? Yes and yes. Mon Labor Rev. https://doi.
org/10.21916/mlr.2015.14. Epub May 26. PMID: 29422698; PMCID: PMC5800410.
Serendipity and Arboviruses
John Aaskov
Abstract In the latter half of the twentieth century, an enormous number of “new”
arboviruses were discovered, and some of these were found to be the causative
agents of well-recognised diseases. Making the connection between the causative
agent and a disease, developing appropriate diagnostics and then attempting to miti-
gate the effect of the disease have provided meaningful employment for an army of
arbovirologists. That many of these diseases occur in exotic settings has added fas-
cination to the field. While the next frontier will be discovering and understanding
uncultivatable viruses, or those for which only nucleotide sequences are available,
it is important that future generations of arbovirologists appreciate the excitement
and satisfaction that come from working with colleagues in the settings where these
viruses/diseases are most likely to be found – often in the developing world.
These things have never yet been seen,
But Scientists who aught to know, assure us that they must be so,
So never, never, doubt,
What no one is sure about.
(Hillaire Belloc)
I became an arbovirologist by default. As a young post-doctoral scientist, I applied

for a job at the Queensland Institute of Medical Research and was offered two proj-
ects. The first was with the Epstein-Barr virus group but that project had a strong
smell of cancer to it (Burkitt’s lymphoma, nasopharyngeal carcinoma, etc.). Having
just completed a Ph.D. in a Department of Cancer Research in the U.K., I was com-
pletely disillusioned with the cancer field and so opted for a project with the Institute
Director on Ross River virus, which he had recently discovered, although I knew
nothing about arboviruses. It turned out to be a great choice, because the Director,
Ralph Doherty, was the perfect boss for a newly minted post-doc. He was a com-
plete renaissance man – an accomplished musician, widely read, had a keen interest
in the visual arts and was a scientist who had a clear view of the big picture. A few
lines from the abstract of his M.D. thesis illustrate his scientific reach –
Murray Valley encephalitis virus was isolated from mosquitoes for the first time; Ross River
virus, a group A arbovirus distinct from any known, was isolated and evidence accumulated
that it was the causative agent of epidemic polyarthritis. Three viruses (Sindbis, Getah and
J. Aaskov (*)
Queensland University of Technology, Brisbane, Australia
116 J. Aaskov
Akabane) previously known in Africa or Asia were isolated in Australia; 24 other viruses
apparently new to science were also isolated. (Doherty 1972)
Doherty’s search for “new” arboviruses was supported, in part, by the Rockefeller
Foundation, and this included establishment of a research station at Mitchell River
Mission, now Kowanyama, in north-east Australia. This site yielded an enormous
number of these viruses as evidenced by the MRM prefix on so many Australian
isolates. It was during a visit to this site in 1966, on behalf of the Foundation, that
Harald Johnson made a side trip to a Tern colony at Upolu Cay on the Great Barrier
Reef where he recovered a number of ticks from which Doherty and colleagues
isolated Johnston Atoll virus and Upolu virus. Upolu virus was the prototype of a
new antigenic group in the Bunyaviridae (Doherty et al. 1969) (Fig. 1).
After he left the Queensland Institute of Medical Research, Doherty moved
through the upper ranks of the Medical Faculty of the University of Queensland
eventually becoming Pro-Vice Chancellor and also chairing an inquiry, for the
Australian Government, into the education of medical students in Australia. The
“Doherty Report” revolutionised how Australian medical students are selected and
educated.
These were interesting times for a young arbovirologist because of the “new”
arboviruses that were being identified and investigated in Australia. It was not pos-
sible to bring antisera into the country because of our quarantine restrictions so a
number of the “unknown” viruses were sent to Bob Shope at Yale for identification.
Unless these were associated with an outbreak of disease, they sat in a freezer until
someone had time to characterise them. One example was CSIRO 19 which had
been isolated by Toby St. George and his group at the Commonwealth Scientific and
Industrial Research Organisation. It turned out to be Bluetongue virus, believed to
be exotic to Australia, and possibly a major threat to our livestock industry. The
telegram from Yale was read at our regular Friday afternoon laboratory meeting and
triggered a multimillion dollar response by the Australian Government because of
the threat, not only to our own livestock but also to our export trade. Eventually, it
was shown that this strain of Bluetongue virus was relatively avirulent (Fig. 2).
Fig. 1 Ralph Doherty performing haemagglutination inhibition assays in his laboratory in the
original Queensland Institute of Medical Research building
Serendipity and Arboviruses 117
Fig. 2 Telegram from Bob Shope at Yale informing Ralph Doherty that CSIRO 19 was
Bluetongue virus
Another example of difficulties associated with the identification of “new”

viruses was those surrounding Barmah Forest virus. Ian Marshall, from the
Australian National University, suggested it was “bunyavirus-like” and unrelated to
known alphaviruses (Marshall et al. 1982), but several years later, Doherty showed
Marshall’s virus was indistinguishable from a “new” alphavirus he had called
Murweh virus after the region from where his isolate was made (Doherty et al.
1979). Although Marshall had misidentified his isolate, Doherty felt that the virus
should be called Barmah Forest because Marshall had made the first isolate.
Following Marshall’s retirement from the John Curtin School of Medicine, in
Canberra, his entire arbovirus collection was discarded because “someone” thought
“no one would be interested in it”.
My first day at the Queensland Institute of Medical Research, in 1976, was a bit
of a shock. My laboratory was in a 30-year-old accommodation block, on two lev-
els, built by the U.S. Army during World War 2 and located on a golf course.
Ventilation was through a window containing Perspex rather than glass and held
open by a piece of wood. My laboratory was above the institute animal house, and
there was a strong smell of mice percolating through the floor boards. When I asked
the Institute Storeman for some stationary, he told me to “f… off” and then arrived
at my office later in the day with enough writing material to last a lifetime and asked
me what I was doing at the Institute. Being somewhat vexed, I told him I was going
to cure cancer, and he went away happy with the reply, for more than a decade.
Some months later, I realised that the Institute had a cat. In the pre-tissue culture
days, arboviruses were isolated in suckling mice, and neutralisation tests also were
performed in newborn mice. At the conclusion of these experiments, there were a
large number of “mother” mice that had to be euthanised and then incinerated. The
animal attendants would euthanise 6–12 of these by cervical dislocation, so there
were no unpleasant anaesthetic odours associated with them, and then lie them out,
118 J. Aaskov
side by side, outside the animal house, for the cat to eat. This the cat did, in small
meals, throughout each morning (Fig. 3).
When I commenced work with Doherty, he gave me a brief typed note suggest-
ing I might like to consider a possible role for antibody-mediated enhancement of
Ross River virus infection in the pathogenesis of epidemic polyarthritis and referred
me to Scott Halstead’s 1973 paper in Nature New Biology (Halstead et al. 1973)
discussing antibody-mediated enhancement of dengue virus infection. The first
report of antibody-mediated enhancement of flavivirus infection was by Royle
Hawkes (1964) when he was a PhD student at the Australian National University.
He observed an increase in the number of plaques produced by Murray Valley
encephalitis, West Nile, and Japanese encephalitis viruses when he added antisera
from fowls infected with these viruses to his virus stocks. He also showed that anti-
sera against one flavivirus could enhance the infection of chick embryo fibroblasts
by a second Group B (flavi) virus. He and Kevin Lafferty showed, subsequently, that
this enhancement was due to IgG antibody and not the IgM (19 S antibody) origi-
nally suspected by Hawkes (Hawkes and Lafferty 1967) (Fig. 4).
The primary chick embryo fibroblast cultures these investigators used for their
plaque assays contained Fc receptor-bearing cells to which antibody-virus com-
plexes could attach, resulting in the enhancement of infection observed (Kliks and
Halstead 1983). Interestingly, Hawkes’ Ph.D. supervisor, Ian Marshall, refused to
have his name on these two papers.
I received a bit of sage advice from the Deputy Director of the Institute, John
Pope, at this time “Never do an experiment in a mouse if you can ask the question
Fig. 3 The Queensland Institute of Medical Research in the 1950s although nothing much, apart
from the car, had changed when I commenced work in 1976
Fig. 4 Royle Hawkes (left) and Kevin Lafferty (right)
in a patient”. While I tried to find a way to get actual epidemic polyarthritis patients
to study, I began a series of, entirely fruitless, attempts to induce polyarthritis in
mice of several strains, and of all ages, by injecting them with an extensive range of
doses of Ross River virus, by any route I could imagine. If mice survived the infec-
tion, they showed no sign of stiffness or swelling in any joints. A new avenue of
research emerged when the animal house supplied me with several cages of female
CBA mice that, inadvertently, contained a few males. I was unaware of this until,
halfway through some experiments, a mouse aborted and we recovered Ross River
virus from the foetal tissues. In the subsequent, planned, experiments, we showed
that Ross River virus readily crossed the placenta and caused foetal infection and
death in mice (Aaskov et al. 1981a). An unresolved conundrum from these studies
was why doses of virus that would kill newborn mice did not kill foetuses. The
“controls” in these experiments were Getah (a serologically related alphavirus) and
Murray Valley encephalitis virus (flavivirus). Murray Valley encephalitis virus
infected, but did not cross, the placenta in mice. Nonetheless, the placental infection
and damage following the infection with Murray Valley encephalitis virus was
enough to cause significant post-partum mortality in the newborns.
Serendipity stepped in again with an enormous outbreak of Ross River virus
infection in the Pacific in 1979–1980 during which we, and Fijian colleagues, were
able to obtain maternal and umbilical cord blood from more than 800 women and
their newborn babies in Fiji. About 4% of the cord bloods contained anti-Ross River
virus IgM antibodies suggesting the babies had been infected in utero but all were
apparently normal at delivery (Aaskov et al. 1981b). A subsequent analysis of data
for miscarriages and dilations and curettages at several hospitals in Fiji in the year
of the outbreak and for the previous year revealed no evidence of foetal deaths due
120 J. Aaskov
to Ross River virus infection. Leon Rosen did not believe this data and organised a
retrospective study in the Cook Islands with a small cohort of mothers and children.
In the subsequent report, he and colleagues concluded that our results were due to
“laboratory or other error” (Aleck et al. 1983). The American Journal of Tropical
Medicine and Hygiene very generously broke with tradition and published my
rebuttal of the claim by Leon and his colleagues (Aaskov 1984) and then their reply
to our rebuttal. There was no significant difference between the Cook Island results
and ours from Fiji. Furthermore, the Cook Island study population was drawn from
women infected in their first trimester of pregnancy when the foetus is not immuno-
competent and so Leon and colleagues would have been unlikely to detect anti-Ross
River virus antibodies produced by a foetus infected at this stage.
It was during this outbreak that Jona Mataika, in the Wellcome Virus Laboratory
in Suva, Fiji, made the first isolation of Ross River virus from an epidemic polyar-
thritis patient – using suckling mice (Aaskov et al. 1981c). Doherty had isolated this
virus from a febrile child, who did not have polyarthritis, by this method in Australia
in 1971 (Doherty et al. 1972), but we had been unable to make any isolates from
patients by this method, or using a range of vertebrate cell lines. Once we began
using Akira Igarashi’s C6–36 cells, we recovered virus from 10% to 20% of acute
phase, seronegative, sera from epidemic polyarthritis patients. I was surprised to
learn several years later that there are two variants of this cell line. The one we, and
may others, use adheres to the surface of culture vessels, but the C6–36 cells we
obtained directly from Igarashi, and which also are in use in many laboratories, are
non-adherent (Fig. 5).
Jona Mataika was one of Fiji’s first medical graduates, and he played a pivotal
role, for decades, in the diagnosis of vector-borne diseases in Fiji, particularly fila-
riasis, and provided invaluable assistance to anyone wanting to undertake virus
research in Fiji or neighbouring islands. He worked well into his retirement as the
Fijian Ministry of Health struggled to find a replacement.
The problems associated with identifying epidemic polyarthritis patients for
study seemed endless. Most Australian General Practitioners were unaware of Ross
River virus and so didn’t request diagnostic serology, and the responsibility for
diagnostic serology for Ross River virus infections had moved from the Queensland
Institute of Medical Research to the Queensland Health Department Laboratory of
Microbiology and Pathology. They relied on detecting a fourfold or greater rise in
haemagglutination inhibiting (H.I.) antibody titres in paired sera or detecting anti-
Ross River virus IgM antibody in an acute phase serum sample. It was uncommon
to get a convalescent sample to look for diagnostic rises in titre, and the IgM assay
involved separating IgM from IgG on a sucrose gradient spun overnight in an ultra-
centrifuge, recovering gradient fractions and then comparing the H.I. titres in each
fraction before and after treatment with 2-mercapto-ethanol which denatured the
IgM but had no effect on IgG at the concentrations employed (Desmyter et al. 1971).
As the laboratory didn’t operate on the weekend and only six samples could be
centrifuged each evening, only 24 sera could be tested each week. It was not uncom-
mon for testing to take 6–8 weeks by which time many patients were beginning to
recover. The next obstacle was that this laboratory felt that forwarding letters, which
Fig. 5 Dr. Jona Mataika at the Wellcome Virus Laboratory in Tamavua Hospital, Suva, Fiji
I had prepared in advance, to General Practitioners whose patients had been diag-
nosed as having epidemic polyarthritis, placed too great a demand on their time.
This led us to develop an indirect immunofluorescence assay to detect anti-Ross
River virus IgM antibodies ourselves (Aaskov and Davies 1979) based on that
Schmitz and Haas (1972) had developed to detect anti-cytomegalovirus IgM.
There was an interesting diversion years later when Chiron began patenting their
hepatitis C discoveries. Unrecognised by them at the time, one of the peptides they
proposed to use in their ELISAs to diagnose hepatitis C infection had a similar
amino acid sequence to that in the Ross River virus non-structural protein nsP2 and
our Ross River virus immunofluorescence assay could detect anti-hepatitis C anti-
bodies reliably. The pattern of immunofluorescence when anti-hepatitis C antibod-
ies reacted with Ross River virus-infected cells was different to that with sera from
epidemic polyarthritis patients. As part of the challenge to the Chiron patents, I was
asked to test a panel of coded sera using our Ross River virus immunofluorescence
assay before an audience of about a dozen lawyers and scientists in a Microbiology
Department laboratory at the University of Reading in the U.K. I managed to iden-
tify all sera from hepatitis C patients, and I think all the non-hepatitis C samples
were non-reactive. Chiron managed to have the results disregarded in the High
122 J. Aaskov
Court challenge in London because I had used Ross River virus-infected Vero cells
in Reading when our published method employed L929 cells – I had not been well
briefed. At the subsequent hearing at the European Patent Office in Munich, Chiron
managed to have the results of new testing using L929 cells dismissed because they
hadn’t received the data 28 days before the hearing.
With the immunofluorescence assay for anti-Ross River virus IgM in hand, a
private pathologist in rural Queensland agreed to send me acute phase sera from
suspected epidemic polyarthritis patients for testing and to raise awareness of this
infection among the medical practitioners for whom he provided diagnostic ser-
vices. We reported results within 1 or 2 days of receiving sera and soon had signifi-
cant numbers of samples coming to us for testing. These samples also were being
sent to the Queensland Health Department Laboratory of Microbiology and
Pathology for testing using their traditional serological methods, and, within a cou-
ple of months, it became apparent that almost all our results were being confirmed
by them. About 1% of our results were false positives due to the presence of rheu-
matoid factor in the sera. By reporting results promptly, we often were able to obtain
blood from patients for our research within a week of onset of symptoms, and with
Igarashi’s C6–36 cells, we began recovering Ross River virus from acute phase,
IgM non-reactive, sera (Aaskov et al. 1985). Many of the patients lived in isolated
rural settings but, despite this, and in order to support our research efforts, would
drive 60 or 70 kilometres over dirt roads to be bled in the late afternoon so samples
could come to Brisbane on an overnight bus, and they would do this every month
until they had fully recovered – sometimes eight or nine trips.
Lymphocytes were recovered from these blood samples for HLA typing and for
lymphoproliferation assays on the premise that the underlying pathology of epi-
demic polyarthritis might have a cell-mediated basis and that there might be some
genetic association with clinical infections (Aaskov et al. 1981d). We had been
unable to find any relationship between disease severity and anti-Ross River virus
antibody titres. In order to perform these assays, we developed procedures for mak-
ing, and U.V.- inactivating, large amounts of Ross River virus, usually from about 2
litres of infectious tissue culture supernatant, from 20 large roller bottles, and puri-
fied on tartrate gradients. This material also allowed us to move from indirect immu-
nofluorescence assays to an indirect ELISA to detect anti-Ross River virus IgM
antibodies (Oseni et al. 1983).
With the ELISA to detect anti-Ross River virus IgM antibodies, we were able to
identify even more patients through our connections with General Practitioners and
Commonwealth Health Department Pathology Laboratories in regional centres in
Australia. In a bureaucratic power play in the 1920s and 1930s, the Australian
Government had established diagnostic Pathology laboratories around the country,
ostensibly to support quarantine measures for which the national Government was
responsible. Over time, these laboratories had come to assume responsibility for all
diagnostic pathology in the large regional centres in Australia in which they were
located. In general, state governments maintained a large central laboratory in the
state capitals and only small, hospital-based, laboratories in centres not covered by
the Commonwealth laboratories.
The formal and informal collaboration with public and private pathology labora-
tories played an important part in our research programme for decades. Apart from
the first recovery of Ross River virus from epidemic polyarthritis patients in
Australia, it alerted us to the first clinical infection with Edge Hill virus (Aaskov
et al. 1993) and enabled us to recover Barmah Forest (Phillips et al. 1990) and
Kunjin viruses (Phillips et al. 1992) for the first time from patients infected in
nature. It also enabled us to identify 10–12 patients with clinical infections with
Kokobera virus. More recently, it enabled us to recover the most primitive strain of
dengue virus identified to date (DENV 2 QML 22, Liu et al. 2016). Because of the
ethical constraints under which we worked, the Queensland Health Department
Laboratory, unknowingly, subsequently isolated the same dengue type 2 virus from
the same patient and gave it another name (Sab 2015).
A Research Assistant working with me in the 1980s was diagnosed with simul-
taneous infections with Ross River and Epstein-Barr viruses by a local Pathology
Laboratory. This was a significant concern given her potential exposure to Ross
River virus in the laboratory, but her symptoms matched those of an Epstein-Barr
virus infection more closely than those of epidemic polyarthritis – and she had
someone new in her social life. After a thorough investigation by the Epstein-Barr
virus group at the Queensland Institute of Medical Research, we concluded that
Epstein-Barr virus was the causative agent and that she had not been infected
recently with Ross River virus in the laboratory or anywhere else. In subsequent
years, Debbie Phillips, by then in charge of the Arbovirus Laboratory in the
Queensland Health Laboratory of Microbiology and Pathology, began to notice a
pattern of, apparent, dual infections with Ross River or Barmah Forest viruses and
agents like Epstein-Barr virus, cytomegalovirus, malaria, Leptospira spp. and
Coxiella burnetii (Q fever), i.e., sera from these patients contained IgM antibodies
against both Ross River and Barmah Forest virus and one of these other agents but
with symptoms unlike those of Ross River or Barmah Forest virus infections. Many
of these patients had significant amounts of anti-Ross River or Barmah Forest virus
IgG in their acute phase serum samples. We concluded that infection of people, who
had prior infections with Ross River or Barmah Forest virus, with agents like
Epstein-Barr or cytomegalovirus resulted in some sort of polyclonal activation of
the immune system and transient production of IgM antibodies against agents which
had caused earlier infections. These observations reinforced the importance of good
communication between clinicians and the laboratory and the importance of relat-
ing serological data to clinical signs and symptoms (Fig. 6).
Diagnosis of large numbers of epidemic polyarthritis patients provided material
for our research program and cast new light on the epidemiology of Ross River
virus infection. Prior to this, colleagues had expended an enormous effort trying to
find the reservoir for this virus in the periods when no patients were being detected.
With an expanded capacity to diagnose Ross River virus infections nationally, it was
recognised that people were being infected all year round and many of them were
being infected in urban settings far from the presumed native marsupial resevoirs
(Aaskov et al. 1981d). The epidemiology of the outbreak of Ross River virus
124 J. Aaskov
Fig. 6 Debbie Phillips cycling to report the results of her recent arbovirus serological tests
infection in the Pacific in 1979–1980 also demonstrated that transmission of this

virus was not dependent on intermediate vertebrate hosts.
Some fundamental questions that arose very early in the study of Ross River
virus were “How many people are being infected with this virus in Australia each
year” and “How many of those infected develop symptoms”? We approached this in
two ways. The first was to bleed a panel of subjects at six monthly intervals and to
look for seroconversions, and the second was to measure the prevalence of anti-
Ross River virus antibodies in an age and gender-stratified cross section of the com-
munity. As with much of science, we took the most difficult approach first, although
we didn’t appreciate this at the time, and set off to bleed military personnel and their
partners and a group of agricultural workers and their partners every 6 months.
Contrary to popular opinion, even in the 1970s, the Australian Army didn’t give up
volunteers by nominating “you, you, and you” and I had to make a pitch to the sol-
diers to be bled. Interestingly, women in the broader Australian community were far
less “needle shy” than men and were more likely to participate in a study of this
kind and to be re-bled. Both approaches suggested that approximately 1.5% of peo-
ple in northern Australia were being infected with Ross River virus each year
(Aaskov et al. 1981d). There appeared to be an enormous difference between the
numbers being infected and the numbers being diagnosed with clinical infections.
At a time when there was a bit more altruism in science, I offered our ELISA to
detect anti - Ross River virus IgM to the Queensland Health Laboratory of
Microbiology and Pathology, but the offer was refused because the head of the
serology laboratory felt the assay was too sophisticated and he preferred to stay with
the density gradient fractionation of serum followed by haemagglutination inhibi-
tion tests. The new Director at the Queensland Institute of Medical Research, Chev
Kidson, then instructed me to stop performing routine diagnostic serology for Ross
River virus infections. After prolonged negotiations, the Commonwealth Serum
Laboratories (now CSL) agreed to manufacture virus for those laboratories wishing
to perform in-house Ross River virus IgM ELISAs, based on our protocols, and I
signed a “no compete” agreement. It was shortly after this that I accepted a position
as an academic at the Queensland Institute of Technology (Q.I.T.), now the
Queensland University of Technology, (Q.U.T.), and there was extensive feedback
that the Ross River virus being produced by CSL could not be used to detect anti-
Ross River virus IgM antibodies. CSL had decided to develop their own protocol for
the preparation of virus, and it resulted in partial denaturation of the virions. CSL
then announced that they would no longer produce virus and they released me from
the “no compete” agreement so we began manufacturing Ross River virus again for
anyone interested, in particular, for David Wyatt, who I had replaced at the
Queensland Institute of Technology and who now was Head of Serology at a private
Pathology Laboratory. This was the beginning of a long and very productive col-
laboration with him in the development of commercial assays for the diagnosis of
arboviral infections (Fig. 7).
David Wyatt eventually left the pathology system and formed a very successful
biotechnology company, PanBio. One of the company’s first products was an ELISA
to detect anti-Ross River virus IgM based on our assay. I was offered a royalty
stream from this but couldn’t see how it would ever make money so settled for sell-
ing him purified Ross River virus for the kits – poor decision! The widespread avail-
ability of a commercial kit to diagnose Ross River virus infections had a profound
effect on the number of cases diagnosed (i.e., from ~50 in the 1970s to ~5500 annu-
ally after 1992, Fig. 8) and resulted in the Australian Government making epidemic
polyarthritis a notifiable disease in 1992. With an appreciation of the disease burden
due to Ross River virus infection, discussions began about the possibility of a vac-
cine – see below. In the following year, Barmah Forest virus infection also was
made a notifiable disease because PanBio’s development of a commercial ELISA
for the detection of anti-Barmah Forest virus IgM had resulted in 500–1000 cases of
Barmah Forest virus infection being detected annually in Australia. We had never
been able to stabilise the Barmah Forest virions sufficiently to be confident in our
prototype Barmah Forest virus IgM ELISAs and PanBio also experienced difficulty
with their Barmah Forest IgM ELISA kits from time to time.
Subsequently, David Wyatt and I obtained a grant from the Australian Government
to develop IgM capture ELISAs to diagnose dengue, Japanese encephalitis, and yel-
low fever. The yellow fever kit never eventuated because we couldn’t obtain the
necessary IgM reactive sera to validate the assay or for internal controls. When
this grant came to an end, the project was supported by an Australian
126 J. Aaskov
Fig. 7 David Wyatt
10,000
RRV infection
notifiable
8,000
Cases notified
6,000 Commercial
lgM ELISA
4,000
Indirect lgM IFA
2,000
1970 1980 1990 2000 2010 2018

Year
Fig. 8 Number of cases of epidemic polyarthritis diagnosed each year in Australia. Notification
only became compulsory after 1992
Government-funded Co-operative Research Centre for Medical Diagnostics. There

were three critical aspects to the development of these assays, in particular the den-
gue ELISA. The first was the availability of recombinant dengue virus envelope
protein from Hawaii Biotechnology (I doubt that we could have produced pure den-
gue virus in the quantities required, and at a realistic cost), the second was the
enormous amount of time and money spent on panels of sera to validate the assay,
and the third were the modifications made to the procedure and reagents so the test
could be performed in hours rather than days. An important lesson from this project
was that research scientists often don’t know much about the commercial world.
The “word on the street” from the arbovirology research community was that no
one would buy a dengue test that cost more than 50 cents. The reality was that there
was an enormous global market for a reliable, relatively rapid, dengue test that cost
from US$ 5 to 10.
There was significant resistance to the commercial Ross River ELISAs from
some Australian State Health Department Laboratories and to the dengue ELISA
within Australia and from some U.S. government laboratories as well. This stemmed,
principally, from the democratising of diagnostic serology for infection with these
agents – knowledge is power and those doing the testing generate the knowledge
and publish the papers. Eventually, the World Health Organisation arranged a multi-
country evaluation of the PanBio dengue ELISA and a number of other kits that had
been produced more recently. Remarkably, the gold standard for this evaluation was
two incompletely validated in-house assays, so it was impossible for any of the
commercial assays to be found to perform better than the in-house assays (Peeling
et al. 2010). Like the ocean and King Canute, the commercial assays have gone
round this opposition, and the pathology community is approaching diagnosis of
arboviral infections in the same way they approach that of most other infectious
diseases – by employing commercial kits.
As I gained greater exposure to the field of arboviral diagnostics, and to com-
municable disease diagnostics in general, in Australia and throughout the Asia
Pacific region, I began to appreciate that even the best diagnostic tests could be
misused to produce erroneous results and how critical quality assurance processes
were. I had organised a very basic Quality Assurance Program for Ross River virus
serology in Australia in the early 1980s, and this now is part of the routine, and far
more professional, Quality Assurance Programs conducted by the Royal College of
Pathologists of Australia. I also had a rudimentary Quality Assurance Program in
our AusAID-funded dengue project in Vietnam (below) and eventually interested
Frank Konings at the World Health Organisation regional office in Manila in a
Quality Assurance Program for dengue diagnostics in the Western Pacific Region of
W.H.O. Frank put together a team and made this happen. Eventually, the World
Health Organisation’s Health Emergencies Programme in Lyon became involved,
and the programme became global and was expanded to include diagnostics for
Chikungunya and Zika as well as for dengue. Consideration is being given to includ-
ing yellow fever in this programme in the future.
By the time I moved to the Queensland University of Technology in 1986, my
former Research Assistant, Debbie Phillips, had moved to the Queensland Health
Laboratory of Microbiology and Pathology to head the Arbovirus Serology
Laboratory and concern about the loss of material from Doherty’s arbovirus collec-
tion at the Queensland Institute of Medical Research resulted in remaining material,
in their freezers, being moved to the Laboratory of Microbiology and Pathology
where 24-h monitoring was available. As my laboratory at the Queensland University
128 J. Aaskov
of Technology was only 100 metres up the road from the Health Department
Laboratory, I prepared a proposal for both institutions, the Australian Government
and W.H.O. that they designate a joint Queensland Health–Queensland University
of Technology Collaborating Centre for Arbovirus Reference and Research. The
W.H.O. designation came in January 1993. While the designation didn’t change the
reach or extent of our existing collaboration with colleagues in the Asia Pacific
region, the W.H.O. imprimatur greatly facilitated the administration around these
activities. It also led to some very productive interactions with W.H.O.
By the late 1980s, Ross River virus had emerged from being an obscure Australian
virus of little interest to the remainder of the world. Thousands of cases of epidemic
polyarthritis were being reported each year, and it was realised that many of these
were incapacitated for 6 months or more (Fraser 1986); the Medical Journal of
Australia used a number of Ross River–related images on a September 1983 cover;
the outbreak of Ross River virus infection in the Pacific in 1979–1980 gained inter-
national attention (Aaskov et al. 1981c); tourists and military personnel were return-
ing to the U.S.A. and Europe suffering from epidemic polyarthritis and scientists
wanting to investigate an alphavirus began employing Ross River virus rather than
the old standby, Sindbis virus.
In 1989, I was invited by Dmitry Lvov from the D.I. Ivanovsky Institute of
Virology to give two talks in Moscow at a symposium entitled “Arboviruses and
arboviral infections”, one of which was about Ross River virus. During this meet-
ing, Tom Monath came to lunch one day and said “the f……g Russians have called
off the meeting. I bet they are trying to hide something”. At the time, this comment
made little sense to me. Tom had been steeped in bilateral Biological Weapons
meetings up to Perestroika and then worked with the U.S. National Academy of
Science on verification issues through the 1990s. The head of the Soviet Union’s
bioweapons program at that time was Ken Alibek. In his book Biohazard, he
recounts that the U.S.A. and the U.K. had presented a diplomatic demarche to
President Gorbachev’s foreign policy advisor in late 1989 claiming that Russia was
violating the 1972 Biological Weapons Convention, probably as a result of informa-
tion provided by Vladimir Pasechnik the Director of the Leningrad Institute of
Ultrapure Biopreparations before he defected to the U.K. Alibek goes on to describe
the steps the Soviet Union took in 1989, and afterwards, to conceal their program
before the mutual inspection program began several years later.
As we and others, particularly Dr. Bob Fraser, a Rheumatologist at the University
of Melbourne, took a more detailed look at Ross River virus infection and epidemic
polyarthritis patients, it became obvious that this disease placed a significant finan-
cial and social burden on Australians. The number of mosquitoes likely to act as
vectors for Ross River virus and their varied habitats (Claflin and Webb 2015) made
vector control an unlikely mechanism to prevent the disease. In a quiet bar, many
Entomologists will agree that a significant component of Australia’s mosquito con-
trol programs is intended as nuisance control rather than disease control. With the
encouragement of the Australian Cattlemen’s Union and the proceeds of their
annual Cattle Queen contest, we began attempts to develop an inactivated Ross
River virus vaccine. The Australian National Health and Medical Research Council
had consistently refused to fund development of this vaccine. The rationale for an
inactivated vaccine was that licensing a live-attenuated one against a disease for
which the pathogenesis was not understood would be almost impossible. However,
we could grow and purify enormous amounts of Ross River virus, and there was
extensive expertise in the vaccine community in inactivating viruses for use in vac-
cines. I was aware of the Cutter Incident with polio vaccine (Nathanson and
Langmuir 1963a, b) and so chose not to use formalin as an inactivating agent. I also
was aware that beta-propiolactone destroyed key conformational epitopes on some
viruses. The choice of binary ethylenimine as an inactivating agent was based on its
target being the viral genome rather than proteins that might be important for immu-
nogenicity, and the Veterinarians appeared to have used it successfully as an inacti-
vating agent for some of their vaccines (Bahnemann 1976). The second tranche of
money for vaccine development came from a health insurance company, the Medical
Benefits Fund of Australia, which, unfortunately, no longer exists. We then attracted
funds from a local Rotary Club, and the final tranche of non-commercial funding
came from a benefactor who had made their fortune framing pictures. At this point,
we had an inactivated vaccine that was immunogenic and protected mice against
infection – developed without any government or industry support (Aaskov
et al. 1997).
After toying with the idea of trying to develop the vaccine ourselves, common
sense prevailed, and I approached a range of vaccine manufacturers with experience
in preparing inactivated vaccines against human pathogens. The only response was
from Immuno, in Vienna, who were making a formalin-inactivated vaccine against
tick-borne encephalitis. The last contract they signed before being taken over by
Baxter was to develop the Ross River virus vaccine. The first hurdle encountered at
this stage of the development of the vaccine was the absence of an approved proto-
col to confirm that all binary ethyleneimine and its derivatives had been removed
once the virus had been inactivated. The default position was to use formalin for
inactivation and to add a U.V. irradiation step to damage viral RNA, i.e., “double”
inactivation targeting both viral proteins and the genome. Baxter had confirmed our
mouse data using the new vaccine (Kistner et al. 2007) and began pilot production,
when two aircraft flew into the twin towers in New York on September 11, 2001,
and President George Bush decided the U.S.A. needed one dose of smallpox vac-
cine for every American in case there was an attack with biological weapons. Tom
Monath and Acambis, who already had a contract from the U.S. Centres for Disease
Control to work on smallpox vaccine, partnered with Baxter in Vienna to produce
180 million doses of smallpox vaccine in Vero cells. This was a far more profitable
exercise than a Ross River virus vaccine, and Baxter didn’t return to the Ross River
virus vaccine until about 2005. By the time the phase III clinical trials of the vaccine
had been completed (Wressnigg et al. 2015), Baxter had sold its vaccine division,
and the Ross River virus vaccine sits in limbo until someone is prepared to complete
the licensing trials. Ross River virus doesn’t kill anyone, and almost none of the
costs, financial and social, associated with infection with the virus are borne by the
Government so there is little incentive for them to support a vaccine like this to
become part of Australia’s free immunisation program. It might help if this virus
130 J. Aaskov
was introduced, by tourists or returning military personnel, to the southern states of

the U.S.A., where there are mosquitoes competent to transmit it (Mitchell et al.
1987), and if it became endemic there! Alternatively, global warming might see
Ross River virus transmission begin around the Mediterranean and in southern
Europe in the future.
Following a warning of the threat in 1980 (Marks et al. 1980), dengue reappeared
in Australia in 1981 (Kay et al. 1984). Barry Gorman, the elder statesman of arbo-
virology at the Queensland Institute of Medical Research after Doherty left, sug-
gested that we might begin to focus on dengue which was of far greater significance,
internationally, than many of the viruses with which we had been working. Barry
also became aware that Australia was providing funding for the dengue vaccine
efforts at Mahidol University in Thailand. This information may have come through
Peter Doherty who, in his pre-Nobel Prize days, acted as a Peer Review Panel mem-
ber for the S.E.A.R.O./W.H.O. Dengue Vaccine Development Programme.
Australia had made some significant contributions to the early understanding of
dengue with Dr. Hare in Charters Towers, in 1897 (Hare 1898), recording the first
cases of dengue haemorrhagic fever and clearly delineating it from earlier outbreaks
labelled as dengue in south-east Asia and the Caribbean, which almost certainly
were outbreaks of Chikungunya (Carey 1971). Most of the death certificates for
patients in the Charters Towers outbreak were signed by Drs Forrest, Huxtable and
Vores. None were signed by Hare, a renowned surgeon, suggesting that he may have
drawn together the data for the subsequent paper, on which he was sole author,
rather than having cared for any of the patients himself (Fig. 9).
The first two fatalities in the dengue outbreak in Charters Towers in 1897 were
Christina Fardon and Harold Aspinall. Christina Fardon was my great-great
Grandfather’s sister-in-law (Fig. 10).
In 1906, Bancroft demonstrated that Aedes aegypti, and not Culex fatigans as
claimed by Graham (1903), was a vector of dengue viruses (Bancroft 1906). After
allowing his Aedes aegypti mosquitoes to feed on a dengue patient “the mosquitoes
were kept in boxes and fed on dates” for 10–20 days, Bancroft allowed them to bite
five normal healthy residents of Brisbane – two developed dengue (Fig. 11).
Both Hare (1898) and doctors in Brisbane (Anon 1905) noted the clustering of
dengue cases. Seven of the first eight cases identified in the 1905 outbreak in
Brisbane occurred within a radius of 100 metres in the suburb of Spring Hill and
Hare recorded that following the first case of dengue in someone newly arrived and
staying in a hotel in central Charters Towers, cases radiated out thereafter. These
observations are compatible with Aedes aegypti mosquitoes being a vector for den-
gue viruses (Fig. 12).
Cleland et al. (1918) and Cleland and Bradley (1919) determined the incubation
period, duration of infectivity, and the source of dengue viruses in blood by passing
material from subject to subject among the Sydney medical fraternity and, eventu-
ally, volunteers from the community. “Further experiments were abandoned….
chiefly due to the unexpected difficulty in obtaining volunteers even with a consid-
erable monetary inducement. …. in two experiments, the finding of a positive com-
plement fixation reaction for syphilis in the volunteer prevented further utilisation
of the virus in his blood”.
Fig. 9 Staff at the Charters Towers Hospital in 1895. Front row, left to right. Dr. Hare (Resident
Surgeon), Dr. Vores, Mr. Millican (Honorary Secretary), Dr. Huxtable (Assistant Resident
Surgeon), Mr. Moore (Honorary Dentist), Dr. Forrest (Honorary Surgeon). Standing, left to right.
Nurses Pratt, West, Mr. Fraser (Wardsman and Dispenser), Campbell, Fraser, Duncan, Hindmarsh,
England (Head Nurse), McKenzie, Earl. It was the custom in Australia, well into the twentieth
century, for Medical Practitioners with a private practice to work free of charge, as Honoraries, in
public hospitals
In 1981, I obtained a World Health Organisation Fellowship to go to the

Department of Medical Microbiology in Mainz, Germany, to explore the possible
role of serum complement in the innate immune response to Ross River virus infec-
tion. The Mainz group was at the cutting edge of the complement world, and several
members had trained in the Muller-Eberhard laboratory at Scripps Clinic. My
review of the complement literature, in preparation for Mainz, alerted me to the
reports from Phil Russell (Russell et al. 1969) and from the Muller-Eberhard labora-
tory (Bokish et al. 1973) of the possible role of complement in the pathogenesis of
dengue, and this seemed to be an interesting facet to explore in our expansion into
dengue research in general. W.H.O. very generously allowed me to modify my
Fellowship itinerary to spend a week in Burma to see some dengue first-hand and,
after my time in Mainz, to visit Scott Halstead in Hawaii to learn more about
antibody-dependent enhancement. The rationale for visiting Burma was that they
had a lot of dengue and it seemed a more interesting place to visit than most other
dengue-endemic areas.
132 J. Aaskov
Fig. 10 Renowned tennis player and dengue researcher, Scott Halstead, at Christina Fardon’s
grave in Charters Towers, Australia
Fig. 11 Thomas Bancroft
Dengue was reported first from Rangoon, in Burma, in 1824. It is a coastal city
that straddles the Irrawaddy River, and in 1865, the Irrawaddy Flotilla Company
began to turn the river into the major transport link between the capital and
upper Burma.
Fig. 12 Clustering of the initial cases of dengue in the 1905 outbreak in Brisbane, Australia.
Seven of the first eight cases occurred within 200 metres of each other
The Road to Mandalay

Come you back to Mandalay,
Where the old Flotilla lay,
Can’t you ‘ear their paddles chunkin’ from Rangoon to Mandalay?
(Rudyard Kipling)
Unlike earlier outbreaks which appear to have been confined to coastal regions, the
1902 dengue outbreak began in Rangoon and quickly spread along the Irrawaddy
River as far as Bhamo, which is past Mandalay and just a few bends in the river past
Katha where, in 1927, Eric Blair, an officer in the Indian Imperial Police had a den-
gue infection severe enough to justify his return to England on sick leave. In
England, he resigned from the police and then fought in the Spanish Civil War with
the International Brigades, changed his name to George Orwell, and wrote novels
like “Burmese Days”, “Homage to Catalonia”, “1984”, “Animal Farm”, and others.
Burma was only the second country in Asia I had visited, after Singapore in
1972, and the flight from Bangkok to Rangoon on Burma Airways should have
alerted me that things were going to be different – lunch on the flight was a manda-
rin, a cheese sandwich, and a cup of sweet milky tea. At Customs, on arrival, I had
to declare everything of value including my watch and pen and, as I left the airport,
an official with a large pot of glue slopped a dollop on my suitcase and attached a
piece of paper stamped “passed”. The floor of the taxi I took into the city had rusted
134 J. Aaskov
Fig. 13 Staff at the Virology Department of the National Health Laboratory in 1981. Than Swe is
second from the left in the centre row and Soe Thein on the right of the centre row. Than Swe
would become the Director General of the Department of Medical Research and Soe Thein,
Deputy Director General
away, and the road below was clearly visible at all times. The next day, I caught a
three-wheel taxi (passengers in the utility tray at the back and the driver in the cab
at the front chewing and spitting betel) to the National Health Laboratory where I
was met by Than Swe and Soe Thein in the Virology Department. Both would come
to play significant roles in my subsequent studies in Myanmar (Fig. 13).
The Virology Laboratory was relatively new, and attached to what had been a
Pasteur Institute, but was perpetually short of funds to undertake research or even
to perform systematic diagnostic serology. Despite this, Soe Thein had undertaken
significant studies of dengue, Chikungunya and Japanese encephalitis in Burma.
The Strand Hotel in Rangoon, where I was staying, served local lobster for $1.50,
and it took only 24 h before I became acutely ill from eating these lightly cooked
crustaceans. While visiting the Rangoon Children’s Hospital to see my first dengue
shock patients, I vomited over a hospital trolley, the wall and the floor in my rush to
a toilet. It being 29 July, 1981, the Australian Embassy and all the British
Commonwealth Embassies were closed for the wedding of Prince Charles to Lady
Diana Spencer, and my colleagues were uncertain what to do with me for medical
care. I was dispatched to the Rangoon General Hospital, and after several conflict-
ing sphygmomanometer readings in the Casualty Department, I was admitted for
observation. Food and medication were the responsibility of the patient, but Soe
Thein soon took this in hand for me. Within a day or so, I had recovered and was
Fig. 14 Rangoon General Hospital in 1981. Hospitals of the same design can be seen throughout
British Colonial Asia
discharged. Two days later, I was at the International Virology Congress in

Strasbourg, totally culture shocked (Fig. 14).
In Strasbourg, I met Leon Rosen and Scott Halstead for the first time – in that
order. After chatting for some time, Leon suggested we go and hear Scott “deliver
the party line”. During question time, Leon stood up and asked “Scott, do you really
believe that stuff”? This wasn’t the sort of scientific debate with which I was famil-
iar, and at that stage of my career, I hadn’t read Leon’s Presidential address to the
American Society of Tropical Medicine and Hygiene “The emperor’s new clothes
revisited, or reflections on the pathogenesis of dengue haemorrhagic fever” (Rosen
1977), and I had yet to cross swords with Leon over Ross River virus infection in
pregnancy. I witnessed another astonishing dengue event at the International
Congress of Tropical Medicine and Malaria in Calgary in 1984. Don Burke, in
U.S. military uniform, was chairing a session in which Gustavo Kouri was the first
speaker and was to discuss the 1981 dengue outbreak in Cuba (Guzman et al. 1984).
Gustavo began “Following the introduction of dengue into Cuba by the American
Central Intelligence Agency, we experienced….….”. Don didn’t bat an eyelid and at
the end of the presentation thanked Dr. Kouri and invited questions. There was a
feeling that the opening statement might have been the price Gustavo had to pay to
come to the meeting.
During the time in Mainz, I learnt a lot about the serum complement system and
scientific leadership. The Director of the Department of Medical Microbiology in
Johannes Gutenberg University, Paul Klein, saw it as his role to advance the careers
of his staff rather than his own. Most of the laboratory heads, when I visited in 1981,
about seven I think, went on to other German or Austrian Universities to head their
own Departments. One also became President of a German University and another
136 J. Aaskov
President of the Robert Koch Institute in Berlin – an astonishing progression from a

small Institute in the Rhineland, or any Institute for that matter.
On return to Australia, I attempted to find funds to begin collaborative studies
with Burma – without success. In the following years, Soe Thein was transferred to
the Department of Medical Research to work with Mi Mi Khin in the Virology
Research Division, and she managed to get funds through W.H.O. for me to make
two, 1 month, visits in 1985 and 1986. Among her other Virological and
Entomological achievements, Mi Mi Khin was the first person to demonstrate trans-
ovarial transmission of dengue virus in nature (Khin and Than 1983). Her subse-
quent involvement in the politics of the post-1988 turmoil in Burma resulted in her
transfer from the Department of Medical Research to the National Health Laboratory
and Than Swe being moved from the National Health Laboratory to the Department
of Medical Research where he subsequently became Director General (Figs. 15
and 16).
In 1984, almost two-thirds of the children admitted to the Rangoon Children’s
Hospital with a clinical diagnosis of dengue were found, after serology was per-
formed, not to have dengue and Soe Thein suspected Chikungunya. We modified
our Ross River virus IgM ELISA (Oseni et al. 1983) to detect anti-Chikungunya
virus IgM by using serum-free supernatant from cultures of Chikungunya virus-
infected C6–36 cells as a source of antigen, performing all plate washing with a
wash bottle of buffer and reading the colour change after addition of substrate and
chromogen, by eye. This was a time of endless, unpredictable, shortages in Burma,
and each roll of toilet paper (only pink available) we used to dry the ELISA plates
cost almost as much as a week’s salary for a Technician. After some preliminary
assay validations, we found 103 of a sub-sample of 163 of the non-dengue cases had
Chikungunya virus infections (Thein et al. 1992). Shortly after this, I was evaluating
an early prototype of an indirect ELISA to detect anti-dengue virus IgM when we
received sera from two grandchildren of the President, General Ne Win, to be tested.
I didn’t want to do the test because the assay was completely unvalidated, and I felt
an incorrect result would go badly for us all. Under duress, I performed the assay,
and the result was “non-reactive.” I guess my report would have been something
Fig. 15 Department of Medical Research, Rangoon (left). Soe Thein, Mi Mi Khin and May La
Linn. Virology Research Division, Department of Medical Research (right)
Fig. 16 The view from the Virology laboratories in the Department of Medical Research in
Yangon. Shwe Dagon pagoda
like “No anti-dengue virus IgM was detected and if this sample was collected within
7 days of onset of symptoms another sample should be collected for testing in
7–10 days”. The children were found not to have dengue, we were off the hook, and
I presume everyone thought the test was marvellous!!
In 1984, Soe Thein began a prospective study of dengue in two suburbs of
Rangoon, funded by the World Health Organisation and the Wellcome Trust, to
determine whether anamnestic dengue virus infections were a risk factor for severe
dengue. Soe Thein had established an excellent collaboration with the clinical staff
at the Rangoon Children’s Hospital that continued for decades and enabled our
subsequent studies of dengue in Burma/Myanmar. This probably was assisted by his
wife, Than Nu Shwe, being a paediatrician at the Children’s Hospital. She went on
to become Rector (Head) of one of the two Medical Schools in Yangon. For decades,
the Government of Burma/Myanmar would not permit invitations to conferences or
workshops to be extended to named individuals – they had to be made to be a skill
description. On one occasion, I wanted Soe Thien to present some of his data at an
Australian Arbovirus Symposium and so extended an invitation for a Clinical
Virologist with extensive research experience in dengue. The Government sent his
wife!! We got an excellent presentation on the clinical management of dengue but
we didn’t get the talk for which we had planned. On one occasion when we were
purchasing supplies for the Children’s Hospital to support our research, I noted
there were orders for needles but no syringes. The Hospital staff preferred to collect
blood by inserting a needle into a vein and simply allowing the blood to drain into
a tube. They felt the children weren’t as frightened as being confronted by a needle
and a syringe. Apart from some occasional fungal and bacterial contamination, the
procedure appeared to work well.
138 J. Aaskov
We managed to assimilate this prospective dengue study by Soe Thein into a

Ph.D. program for him as an external student of the University of Queensland.
Throughout the Ph.D. program, I organised to spend a month each year at the
Department of Medical Research in Rangoon and Soe Thein came to my laboratory
in Australia for visits of several months at the beginning and conclusion of the proj-
ect. The annual visits were an important way to get supplies to the Department of
Medical Research without having to use the labyrinthine ordering system of the
Myanmar Ministry of Health. This study was the first, truly independent, confirma-
tion of the data generated by Scott Halstead, and those who followed him at the
U.S. Armed Forces Research Institute of Medical Sciences in Bangkok, that sec-
ondary dengue virus infections were associated with a significantly increased risk of
severe dengue (Thein et al. 1997). A lot of the heat in the early Halstead-Rosen
debates around this issue seemed to focus on whether all secondary dengue virus
infections resulted in severe dengue and an implied corollary that severe dengue
didn’t occur in primary dengue infections. Soe Thein’s study seemed to provide a
much-needed nuance to this. There was another wonderful example of the focal
nature of dengue virus infections, when in 1 year of the study, one of the study sites
in Rangoon city, for no obvious reason, had significantly more dengue than the
second. This was an issue that came back to concern us in the Mesocyclops dengue
control project in Vietnam – could we be certain that differences observed between
a treated and an untreated, control, Commune were due to our intervention or were
they just the random, focal, variations in dengue virus transmission?
One of Soe Thein’s visits to Brisbane coincided with a visit to my lab by Susumu
Hotta who was searching for material on dengue in Australia for one of his forth-
coming books (Figs. 17 and 18).
Hotta was the first person to isolate dengue virus from a patient, a Mrs. Mochizuki,
and he made the isolate using suckling mice – a procedure that rarely works with
tissues from dengue patients. When I asked him how he knew the isolate was den-
gue virus, he hedged a bit and then told me he injected it into his Mother, she devel-
oped classical dengue, and he recovered dengue virus from her blood – Ah! What a
Mother will do for her Son’s career. In Table 2 of Hotta’s 1952 paper in the Journal
of Infectious Diseases (Hotta 1952), he described the symptoms in human volun-
teers injected with different passage levels of the Mochizuki strain of dengue virus.
The first subject is a female, 55 years of age, who was injected with fifth passage
virus in October 1943 and who developed classical dengue fever. Could this have
been his Mother?
Soe Thein’s Ph.D. program overlapped with demonetizations of the currency in
Burma in 1985 in which 100, 50 and 20 Kyat notes were replaced with 75, 35 and
25 Kyat ones and in 1987 in which these new notes were replaced by 45 and 90 Kyat
notes. The official exchange rate at this time was about 6 Kyat to the $US, while the
black market rate was 600–900 Kyats to the $US. This made purchases with exter-
nal grant funds quite a challenge because they were supposed to be made at the
official rate while, in practise, everything cost the black market rate. Following the
political turmoil which began in 1988 and coincided with Burma changing its name
to Myanmar in 1989, I travelled on a W.H.O./U.N passport to avoid the difficulty of
Fig. 17 Left to right. John Aaskov, Susuma Hotta and Soe Thein. Late 1980s
Fig. 18 Mrs. Mochizuki.

(From Susumu Hotta)
getting a Myanmar visa in my Australian passport. On occasions, there were cur-

fews and armed soldiers at the gate to the Department of Medical Research and on
its verandas, but I never felt at any personal risk. A Director General of the
Department of Medical Research in this period, Dr. Khin Maung Tin, recounted
several interesting experiences. On one occasion, two students being pursued by
police/military ran into the administration block of the Department of Medical
Research. When the authorities arrived, Khin Maung Tin explained that the students
couldn’t be the ones being sought because he had been discussing their studies with
them for ages. He said the authorities didn’t believe him, but they weren’t inclined
to question the word of a Director General in the Ministry of Health. On another
140 J. Aaskov
occasion, he intervened to try to prevent a body of students and their supporters

from beheading three individuals who they claimed were Army spies. He rescued
the woman, but the two men were killed. When I suggested that this seemed a fairly
risky activity on his part, he assured me that, as a good Buddhist, he felt the quality
of his next life would have been assured!! Some years later, on a long ferry crossing
returning from a visit to Bassein (Pathein), I was approached by a policeman whose
opening question wasn’t “Can I see your passport/permit”? but “Have you been
saved”? Noticing my bewilderment at the question, he then asked “Have you taken
the Lord Jesus Christ into your heart”? The policeman turned out to be a very
friendly Baptist Chin who assumed that all westerners were Christians and he was
simply making a friendly enquiry about my soul.
After Soe Thein completed his Ph.D. and became Head of the Virology Research
Division at the Department of Medical Research, he asked me to consider taking on
one of his new Medical Officers as a Ph.D. student. After chatting with several, I
made an offer to Hlaing Myat Thu (Fig. 19).
Our interest by this time had extended to dengue virus evolution and to whether
genetic variation between isolates at any single locality was due to local evolution
or to the introduction of new strains. As I had done previously with new research
interests, I searched for someone who knew infinitely more about the subject than I
did and a quick search on PubMed led me to Eddie Holmes who was still at Oxford.
This was the beginning of a very pleasant and productive collaboration that went on
for years – despite Eddie’s fascination with Elvis Presley. I applied to the Wellcome
Trust for support for Myat Thu’s PhD project, but the application was rejected. As
some of the Reviewer’s comments were wrong and there were publications to
Fig. 19 Myanmar New Year (Thingyan) at the Department of Medical Research. Left to
right. Future Director General, Kyaw Zin Thant, Hlaing Myat Thu, John Aaskov, Deputy Director
General Soe Thein. Note the wet figures, of all ranks, and the bucket and hose on the left
demonstrate this, I appealed the decision. Perhaps only the Trust would (a) consider
such an appeal and (b) then make the funding available when my objections had
been sustained. We adopted a similar approach with Myat Thu’s Ph.D. to that we
had taken with Soe Thein – she spent the dengue “season” (June–November) in
Myanmar and the other 6 months in my lab in Brisbane. Myat Thu’s Ph.D. coin-
cided with the largest outbreak of dengue on record in Myanmar, in 2001, due to a
“new” strain of dengue type 1 that almost completely displaced the other three sero-
types, and this provided a wealth of interesting grist for her Ph.D. mill. In the course
of these studies, we identified an intragenic stop codon in the envelope protein gene
of dengue type 1 that, with Edie Holmes insights, we were able to demonstrate was
being transmitted in nature for years (Aaskov et al. 2006). On completion of her
Ph.D., we managed to obtain a World Health Organisation/Tropical Diseases
Research Re-entry Grant to support Myat Thu to continue her research when she
returned home. In later years, we also were able to get an AusAID Australian
Leadership Award Fellowship for her to gain experience in administration in a med-
ical research environment to prepare her for her subsequent roles on the Board of
Directors and as Deputy Director General of the Department of Medical Research.
Soon after Myat Thu began her Ph.D., outbreaks of dengue virus type 1 infection
were reported from throughout south-east Asia and the Pacific. With $5000,000
from a very generous philanthropist in Singapore, we set out to investigate these
outbreaks. The plan was to undertake a detailed study inside Myanmar and also to
sequence as many dengue type 1 viruses from as many countries in the region as we
could. The approach for countries outside Myanmar was for me to fund a local sci-
entist to bring their viruses to my lab in Australia and to sequence them there.
Alternatively, I would pay for sequencing in-country or would sequence viruses sent
to us if no one was interested in coming here to do it themselves. For most countries,
this was straightforward, and we had a wonderful group of young scientists from the
region in my laboratory over several years, but it took more than a year to get an
agreement with the Thai Ministry of Public Health. In the end, they agreed someone
could come to Brisbane and do the sequencing of the Thai viruses, but at the conclu-
sion of the sequencing, we had to destroy all viral material and derivatives. These
studies suggested that the outbreaks of dengue type 1 infection in the Pacific had
been due to three discrete introductions of virus from Myanmar/Thailand, from the
Philippines, and from Malaysia (A-Nuegoonpipat et al. 2004). Serendipitously, we
also identified recombinant and parental dengue viruses in a mosquito and in a
dengue patient during these studies (Craig et al. 2003; Aaskov et al. 2007).
In an effort to capture strains of dengue virus coming into Myanmar from neigh-
bouring countries, we had established study sites at Moulmein (Mawlamyine,
viruses from Thailand), Lashio (viruses from China), Mandalay (movement of
viruses within Myanmar), and Sittwe on the west coast of Myanmar. Sittwe was of
particular interest because there was no dengue being reported from Rakhine State
in the west of Myanmar, and we wondered if the Arakan Yoma mountains were act-
ing as some sort of biological barrier restricting the movement of viruses from the
remainder of Myanmar where dengue was endemic. Once we had the surveillance
program working in Sittwe, cases of dengue were diagnosed, and dengue viruses
142 J. Aaskov
were recovered for analysis – there was no barrier. The need to be well briefed also
was brought home to me when visiting the Hospital in Sittwe for the first time. In
return for their assistance in our surveillance program, I offered to provide dengue
point-of-care tests free of charge. Throughout the morning, our discussions went
nowhere, but at lunch, one of my colleagues took me aside and pointed out that the
Hospital Pathologist was using point-of-care dengue tests in his private clinic, and
if I provided the tests free of charge, I would wipe out much of his dengue business.
After lunch, I provided “clarification” that our tests were intended only for Hospital
in-patients and all went swimmingly after that.
Sittwe has become the centre of the current Rohingya crisis, but at the time I was
working with Hospital staff there, it was safe and peaceful – so much so that the
authorities allowed me to travel by road to Mrauk-U to see its magnificent 500-year-
old pagodas when, officially, tourists were supposed to travel only by boat. The
antipathy between the Arakanese, particularly Muslim Arakanese, and Burmans
goes back for a century or more. General Slim described the problems he faced try-
ing to limit the bloodshed between these groups during the return of the British
Army into Burma at the end of World War 2. The Arakanese, and many other non-
Burman ethnic groups, had supported the British in the hope of some sort of inde-
pendence after the war, while Aung San (Aung San Suu Kyi’s Father) and many
Burmans had supported the Japanese invasion and fought with them against the
British until it became obvious the Japanese would be defeated.
In 2000 and 2001, there had been large outbreaks of infection with dengue virus
type 3 in Bangladesh, and, interestingly, in a country bordering others where dengue
is endemic, many of the Bangladesh patients, including adults, were experiencing a
primary dengue infection. Phylogenetic analyses suggested the viruses had come
from Myanmar, but when I drafted a manuscript suggesting that the viruses may
have been introduced with formal and informal cross border human traffic, both my
Myanmar and Bangladeshi colleagues asked for the phrase to be removed (Podder
et al. 2006). During a visit to Sittwe, I discussed the issue of cross border traffic with
various authorities and was told that officials on both sides of the border provided
unofficial 24- and 48-h “visas” to allow ongoing cross border commerce.
Our early dengue focus was on vaccinology and whether it would be possible to
make some sort of hybrid dengue virus envelope protein or virus that elicited pro-
tective immunity against all four dengue virus serotypes. This adventure was
severely constrained by a dearth of amino acid sequence data for dengue envelope
proteins, and the absence of a three-dimensional model of the envelope protein or
the virion. Ph.D. students in my laboratory and visiting Scientists prepared large
panels of mouse anti-DENV IgM monoclonal antibodies to map neutralising epit-
opes on the dengue virus envelope protein. We decided to employ IgM antibodies
because they neutralise but don’t enhance infection of cells by DENV in vitro
(Halstead and O’Rourke 1977), and anti-dengue virus IgM antibody is the predomi-
nant class of antibody in primary dengue infections where severe disease is uncom-
mon. However, none of these IgM monoclonal antibodies reacted with linear
serological epitopes so we were unable to map the epitopes they recognised using
PepScan technology. However, we did manage to identify a large number of linear
epitopes in dengue virus envelope proteins recognised by IgG antibodies using this
technology (Aaskov et al. 1989), and we met a modicum of success in identifying
epitopes recognised by the IgM monoclonal antibodies by using them to select neu-
tralising escape mutants (Beasley and Aaskov 2001; Serafin and Aaskov 2001; Lok
et al. 2001). About this time, Franz Heinz and his colleagues (Mandl et al. 1989)
developed an antigenic model of the Tick-borne encephalitis virus envelope protein,
and they and Felix Rey (Rey et al. 1995) eventually solved the structure to a 2 ang-
strom resolution. This allowed other flavivirologists to make informed guesses
about the structure of the envelope protein of their viruses.
In 1990, I took myself off to Ernie Gould’s laboratory at the Institute of Virology
in Oxford to learn the finer points of the Baculovirus expression system as a way of
producing our proposed hybrid dengue virus envelope protein. On return to Q.U.T.,
the initial dengue 2–dengue 3 hybrid envelope protein that we prepared contained
putative epitopes involved in neutralisation, but the subsequent response by mice
immunised with this material was underwhelming (Bielefeldt-Ohmann et al. 1997),
and, for a variety of reasons, this approach was discontinued. One of the reasons for
discontinuing this work was the enormous genetic and, potentially, antigenic diver-
sity being observed in dengue viruses as improved technology increased the rate at
which they could be sequenced. The other was the growing realisation, from our
work with the Ross River virus vaccine, that vaccine development was not a cottage
industry to be undertaken by small groups of enthusiastic amateurs.
An issue that arose very early in the days of dengue vaccine development was
that of an in vitro correlate of protection in humans. There are two basic mecha-
nisms for performing a serological test – varying amounts of antibody added to a
constant amount of antigen to determine an antibody titre (Ramon 1922, e.g. plaque
reduction neutralisation test [P.R.N.T.]) or adding varying amounts of antigen to a
constant amount of antibody (Dean and Webb 1926, e.g. neutralisation index).
These two tests can have different endpoints even with the same antigen and anti-
sera. The P.R.N.T. is unlikely to provide a good correlate of protection because, by
diluting out the sera to determine a titre, the test measures only the activity of the
most abundant antibody. The dilution also abrogates the cooperative interactions
between antibodies recognising different epitopes (Beasley and Aaskov 2001). The
weakness of the P.R.N.T. became clear in early epidemiological and vaccine studies
where P.R.N.T. titres of 100–300 were not associated with protection against infec-
tion. I was Rapporteur at a Dengue Vaccine Peer Review Meeting in Bangkok in the
early 1990s and advocated strongly for the use of the Neutralisation Index in place
of the P.R.N.T., but the committee decided that since less sera was required to deter-
mine the P.R.N.T. titre than the Neutralisation Index, it would recommend continu-
ing use of the P.R.N.T. I was so concerned that logistics had trumped science that I
minuted the decision and the rationale and felt that one day the dengue vaccine
community would appreciate that the P.R.N.T. is a poor correlate of protection. It
would be interesting to know what the interpretation of the early dengue vaccine
clinical trial data would have been if it had been based on Neutralisation Indices
rather than P.R.N.T. data. The courage of my convictions about P.R.N.Ts. was on
show when we used Neutralisation Indices to screen participants for the licensing
144 J. Aaskov
trial of the Sanofi Pasteur Japanese encephalitis vaccine (Nasveld et al. 2010) and
for the phase I Ross River virus vaccine trial (Achinger et al. 2011).
The political turmoil in Burma that began in 1988 was continuing – the name of
the country was changed to Myanmar in 1989 – so, as an insurance policy, I looked
for an alternate location for dengue investigations that wasn’t already firmly in the
U.S. or Japanese spheres of influence and settled on Vietnam. In 1993, I visited the
Pasteur Institute in Ho Chi Minh City and the National Institute of Hygiene and
Epidemiology (N.I.H.E.) in Hanoi to try to get a feel for what the arboviral interests
of those Institutes were and then returned in 1995 for Pasteur celebrations and
organised for a Scientist from N.I.H.E. to come to my laboratory for 6 months. The
Director of N.I.H.E., Hoang Thuy Nguyen, suggested I interview three people and I
chose Nguyen Thi Hong Hanh because she had the best English language skills. I
learnt much later that she also was the Director’s daughter-in-law. Hoang Thuy
Nguyen’s Father was the first Minister of Health in Ho Chi Minh’s government
(from 1945 to 1956), and he had wonderful tales about the bureaucracy surrounding
the departure of the French after their defeat at Dien Bien Phu. One of the other
interviewees was Truong Uyen Ninh who I had met first at a workshop in Malaysia
in 1985 and with whom I, subsequently, worked for more than a decade on AusAID-
funded dengue control projects (Figs. 20 and 21).
In 1996, W.H.O. asked Dr. Brian Kay, the Head of the Entomology Unit at the
Queensland Institute of Medical Research, to undertake a review of the vector-borne
Fig. 20 Participants in the Toxorhynchites workshop in Malaysia in 1985. (Left to right) Dr. Joe
Korovueta, Fiji; Professor Ramalingam, Malaysia; Unidentified; Dr. Truong Uyen Ninh, fourth
from left
Fig. 21 Brian Kay
disease control programme in Vietnam. Brian suggested he needed a Virologist to

accompany him and proposed me. Two critical issues that became apparent during
this Consultancy, and which I have encountered repeatedly in the Asia Pacific
region, was the amount of effort put into counting Aedes aegypti mosquitoes but
without any meaningful analysis of the data or response to it. The second was the
disconnect between the reporting of cases of dengue and the results of any serologi-
cal testing performed on the patients, i.e. even when serological testing reveals that
a suspected patient does not have a dengue infection, the number of reported cases
may not be altered accordingly.
Remarkably, our subsequent report to W.H.O. and the Vietnam Ministry of
Health was the catalyst for the development of Vietnam’s National Dengue Control
Program. I think an important concept in the report was the acknowledgement that,
for public health purposes, particularly in less developed settings, it is not necessary,
nor cost-effective, to try to provide comprehensive laboratory testing of all sus-
pected dengue patients. I proposed a target of 10% of suspected cases to undergo
laboratory testing and the Vietnamese Ministry of Health provided the funding for
this to happen. Shortly thereafter, Brian Kay teamed with the Australian Foundation
for Peoples from Asia and the Pacific (A.F.A.P.) and obtained funds from the
Australian Government aid agency AusAID to evaluate Mesocyclops as a control
agent to prevent breeding of Aedes aegypti mosquitoes in water containers and
invited me to participate as Senior Virology Adviser. The project began in Hai
Phong, Nam Dinh and Yen Bai Provinces near Hanoi in north Vietnam. The initial
study sites had been chosen by the Entomologists as ideal for Entomological studies
but without determining whether they had significant numbers of dengue cases –
they didn’t, and Nam Dinh Province was added because it reported cases of dengue
on a regular basis. The project very quickly floundered over control of its financial
resources, and, after months of delay, it was resumed with new administrative pro-
cesses and with the Australian side having to replace its Project Manager. The sec-
ond hurdle, which lasted for almost a decade, was our inability to get any freight
through Hanoi airport without officials demanding bribes. The “work around” was
146 J. Aaskov
that Qantas, very generously, allowed me to carry enormous amounts of excess

personal baggage free of charge – 100 kg on one occasion.
An important feature of this project was that we wanted to be able to say that the
intervention undertaken prevented dengue, not just that numbers of Aedes aegypti
mosquitoes were reduced. While the Entomologists counted Aedes aegypti, Truong
Uyen Ninh and I focussed on cases of dengue. The approach was to provide free
diagnostic serology for any patient presenting at Commune Health Centres with a
fever. The first hurdle was to get blood/serum samples from the patient to a Provincial
Health Centre, so serology could be performed with the reagents provided by the
project. The ambit bid by the local health authorities was for a Toyota Land Cruiser
to transport the occasional sample from the Commune Health Centre to the
Provincial capital. The compromise was a refrigerator at the clinic and for the sam-
ples to be transported on the Entomology motorcycle if they couldn’t be shipped
with one of the regular Ministry of Health visitors in their Toyota Land Cruisers.
Initial obstacles included the records in the Commune not being completed or main-
tained and the Provincial Laboratories not sending results back to the Commune.
We promised results would be returned in a week, and I put a notice to this effect on
the clinic wall also explaining that the test was free and providing the contact details
of the Director of the Provincial Health Laboratory. Record keeping became very
good when it was realised that I was there for the long haul and would return at
regular intervals to collect data from the Clinic Record Book.
I also was interested to capture data for unapparent infections or for patients who
might not seek formal health care – there was a strong element of traditional medi-
cine in some areas. I proposed sampling 1200 healthy residents at six monthly inter-
vals and testing the samples for seroconversions or a rise in anti-dengue virus
antibody titres. My Vietnamese counterparts were shocked at the numbers sug-
gested, and we settled for 300, and they insisted I should have nothing to do with the
collections. These samples eventually provided a useful measure of dengue virus
infection rates. Almost a decade later, my Vietnamese colleagues described how this
all nearly fell at the first hurdle. On their way back to Hanoi with the first collection
of sera, they received a call that those bled were collapsing and were being admitted
to the District Hospital. It being late evening, my colleagues decided to return to
Hanoi and go back to the Commune the following morning. By the time they
reached Hanoi, the calls had escalated to the Deputy Director at N.I.H.E. He decided
that everyone, and the samples, should turn around and return immediately. Shortly
after dawn, the N.I.H.E. folk arrived at the District Hospital to inspect some of the
collapsed participants, and one of the N.I.H.E. people immediately realised that
some of the “patients” didn’t appear to be people who had been bled. When he
inspected the arm of the first “patient”, with no sign of a venepuncture, the child
leapt out of bed and ran away and by good fortune, this was a relative of a senior
local official. The Commune had become aware that this was an internationally
funded project and had decided to try for some “compensation” in addition to the
school materials and vitamins that had been provided. When this failed, they com-
plained that someone had told them we planned to sell the blood to China. My col-
leagues explained that this was completely untrue, showed them the samples, still in
the jeep, and invited them to come to N.I.H.E. and see the samples whenever they
were in Hanoi – no one ever did.
This project eventually involved Quang Nam, Quang Nghai and Khanh Hoa
Provinces in central Vietnam and then Provinces in the Mekong Delta. We did man-
age to prevent dengue using Mesocyclops and associated public health interventions
but only in rural communities that had a cohesive social structure (Vu et al. 2005).
Dengue was not prevented in urban areas with their associated distractions and lack
of “community”. It was reassuring that the measures introduced in a number of the
Communes were still in play 5–7 years later despite the project and the associated
funding having moved on (Sinh Nam et al. 2012). Another innovation was the use
of a Quality Assurance Program covering all participating Provincial Laboratories
to ensure that dengue serology was being undertaken in a reliable manner. This was
very basic and worked well. Twice each year, all our laboratories received four
serum samples that they had to test on 3 different days and then forward the ELISA
absorbance values for all tests to our counterparts at N.I.H.E. in Hanoi. One sample
contained strongly reactive anti-dengue virus IgM antibody, one contained no
detectable anti-dengue virus IgM antibody and the other two were the same serum
sample from a patient and were borderline reactive. Most laboratories obtained the
correct result for the reactive and non-reactive samples, and the split, borderline
reactive, samples provided some information about reproducibility of testing and
the sensitivity of the testing. If problems arose, one of my counterparts, Truong
Uyen Ninh, Le Quynh Mai or later Nguyen Thuy, would go immediately to the
laboratory to identify the problem and initiate corrective action.
In 1999, Australia led a U.N. Peace Keeping Force into East Timor, but many of
the lessons learned from previous conflicts about preventing vector-borne diseases
had been forgotten, and in 2000, the Australian Defence Force had hundreds of
cases of malaria and dengue among personnel in Timor. I was invited to join the
Australian Army Medical Corps as a Reservist and to establish an arbovirus research
laboratory at the Australian Army Malaria Institute in Brisbane. An immediate chal-
lenge, while I still was going through the recruiting process, was how to diagnose
the dengue cases in Timor in the absence of any real laboratory facilities. I sug-
gested colleagues take some PanBio dengue IgG and IgM capture ELISA kits and
perform the rinse steps with wash buffer from a plastic wash bottle and to hold the
plates up to the light to read the final colour change. When the first batch of samples
was retested under proper laboratory conditions in Australia, only one or two results
obtained in Timor were found to be incorrect – improvisation was trumps again.
My first major project on becoming a Reservist in 2000 was to look for an alter-
native to the inactivated, mouse brain-derived, Japanese encephalitis vaccine. There
was a history of adverse reactions to the vaccine, and an immunisation schedule of
6 months wasn’t very convenient when soldiers were to be deployed at short notice,
e.g. to East Timor in our U.N. peace keeping role, or for tourists planning a hiking
holiday in south-east Asia. There also was the possibility that Australia might need
a Japanese encephalitis vaccine one day.
Australia’s first outbreak of Japanese encephalitis, in 1995, almost went unde-
tected. Sera were sent to the Queensland Department of Health Arbovirology
148 J. Aaskov
Laboratory for Murray Valley encephalitis serology – a flavivirus known to cause

encephalitis in Australia and Papua New Guinea. The head of the laboratory, Debbie
Phillips, included Japanese encephalitis virus antigen in the serological testing
because a local Medical Practitioner felt there was something unusual about the
patients. The first tests showed more than fourfold higher IgM antibody titres against
Japanese encephalitis virus than against Murray Valley encephalitis virus. Debbie
repeated the tests, got the same results and asked me to review the reactions. The
results were clear, and reproducible, so we mulled over the possible effects on our
careers if Debbie called Australia’s first outbreak of Japanese encephalitis, and
some subsequent testing showed it was something else. The outbreak was Japanese
encephalitis, and Debbie’s second achievement was to recover Japanese encephali-
tis virus from sera from two healthy residents of Saibai Island, where the outbreak
was occurring. These individuals had been bled as part of a serological survey for
anti-Japanese encephalitis virus antibodies. These isolations of Japanese encephali-
tis virus were met with quite a bit of scepticism until both people seroconverted a
short time later. They are, to my knowledge, the only isolates of Japanese encepha-
litis virus from asymptomatic infections (Fig. 22).
Tom Monath agreed to let us trial the cell-derived, live-attenuated, Chimerivax
Japanese encephalitis virus vaccine he and colleagues had developed, but it took
some effort to interest the Medical Officers at the Army Malaria Institute in this
project. The real challenge was that this was to be the first genetically modified
organism for intentional release in humans in Australia, and approvals were needed
from three regulatory bodies, the Australian Quarantine Service, the Office of the
Gene Technology Regulator and the Australian Standards, Safety in Laboratories
Committee – and often with conflicting or contradictory regulations. The Chair of
the Australian Standards, Safety in Laboratories Committee threw up the first
obstacle by designating the vaccine a BSL 3 agent. Months of correspondence and
support from Tom Monath got this changed to BSL 2. A remarkable young Captain
Fig. 22 Tom Monath

Fig. 23 Captain Mark

Reid, Arbovirology
Department, Australian
Army Malaria Institute
in my laboratory at the Army Malaria Institute, Mark Reid, spent more than a year
of his life negotiating with the Safety in Laboratories Committee and the other two
regulatory bodies in order for the trial of the vaccine to go ahead. It was successful,
and Australia was the first country to license the vaccine (Nasveld et al. 2010).
Defence personnel and tourists can now deploy/travel 2 weeks after a single dose of
vaccine, although two doses are recommended (Fig. 23). More importantly, in
2022, it began use in response to Japanese encephalitis becoming endemic in
Australia.
Although Australia had recognised North Vietnam at the end of the Vietnam War/
American War, Australia’s political engagement with the post-1975 Government of
Vietnam only really began in 1991. In 2000, Australia and Vietnam exchanged
Defence Attaches, and as part of a soft diplomatic approach, the Vietnam People’s
Army and the Australian Defence Force began two, long-term, health-related, pro-
grams – first malaria and then dengue. The dengue program, which began in 2004,
reflected, in part, that the Australian Defence Force now had a Reservist with almost
a decade of experience working on dengue projects with the Vietnam Ministry of
Health. My Vietnamese military counterparts indicated that there would be no
restrictions to working in Military Hospitals in any area where dengue was a signifi-
cant problem, but I could not wear an Australian military uniform while in country.
The project began with the refurbishment and re-equipping of a laboratory in the
Military Institute of Hygiene and Epidemiology in Hanoi and the training of coun-
terparts in basic dengue virology and serology. The initial study sites were in Da
Nang (Army Hospital 17), Nha Trang (Army Hospital 87) and Can Tho (Army
Hospital 121). Army Hospital 13 at Quy Nhon was added several years into the
project.
150 J. Aaskov
An irony of this project was that, if I had a different birthday, I would have been
required to undertake National Service in the Australian Defence Force and would,
most likely, have served in Vietnam during the Vietnam War/American War. A
Vietnamese People’s Army counterpart who acted as my translator for most of this
project had served with the North Vietnamese Army, in South Vietnam; the
Grandfather of one of the Vietnamese People’s Army Scientists working on this
project had been killed fighting the Americans; the Head of the Vietnam People’s
Army Medical Corps during the project, Lieutenant General Cuong, had served as
a Doctor in the North Vietnamese Army, including a period, post-1975, in the
Mekong Delta and retired General Bui Dai, who took an interest in most of what we
did, had travelled to South Vietnam twice during the war to provide oversight of the
North Vietnamese Army’s medical activities and claimed to have recognised the
first recorded cases of dengue in Laos (Fig. 24).
Because military hospitals in Vietnam often treat civilians living nearby, it also
was possible to generate epidemiological data for the broader community from the
military hospitals. With viruses we recovered during this project, we and civilian
collaborators were able to produce quite a detailed map of the movement of dengue
viruses into and within Vietnam (Rabaa et al. 2013). It also was possible to generate
data for the rate of misdiagnosis of dengue patients based on clinical signs and
symptoms alone and to demonstrate the ineffectiveness of a single round of fogging
in controlling outbreaks of dengue. Between 20% and 90% of reported dengue
cases in areas where we worked in Vietnam did not have dengue when diagnostic
serology was performed. I have seen similar figures in other countries in the Asia
Fig. 24 Counterparts at the final workshop of the Vietnam Australia Defence Dengue project.
Front row, L to R. – Colonel Anh, Director of Hospital 87, Senior Colonel Thanh (Director,
Military Institute of Hygiene and Epidemiology), LT. General Cuong (Director, Vietnamese
People’s Army Medical Services), author, Retired General Bui Dai, Dr. Trieu Nguyen Trung
(Director of the Institute of Malaria, Parisitology and Epidemiology, Quy Nhon)
Pacific region as well. It is much more difficult to determine how many people with
dengue are not reported because they do not seek medical care.
There are significant issues that arise from this misreporting, and I assume it is
not a problem particular to dengue. The first is the waste of resources responding to
presumed dengue outbreaks that may not even be due to a mosquito-borne virus.
The second is the failure to identify what agent(s) is/are causing an outbreak. For
example, measles vaccine is notoriously unstable if the cold chain is not maintained,
and there have been enormous sums spent on both immunising against measles and
conducting “catch up” immunisation programs. There is an assumption that if a
child has received the measles vaccine, they will not get measles. This assumes the
vaccine was viable (I have seen unpublished data that up to 50% of children in some
areas who have been immunised have no detectable anti-measles virus antibodies)
and that the herd immunity is sufficient (~85% required) to prevent transmission. I
suspect that an unquantified number of reported dengue cases are children with
fever and rash due to measles.
A challenge with studies in settings like the ones in which I have been involved
is to find incentives/rewards for national counterparts. In general, there is no system
for rewarding those who go out of their way to make a project successful – or for
avoiding those who take the resources on offer and then do nothing. Having spent
more than a year trying, unsuccessfully, to get a key player on board a project, I was
greatly relieved when he retired. When I was invited to his farewell, I discovered he
was an accomplished poet and didn’t want to be an Army Pathologist in the
first place!
I made a conscious decision not to get on the Chikungunya or Zika bandwagons
when they began to roll because I felt there was a high risk of falling off or being
run over by them. However, this caution did Dr. Van-Mai Lao-Cormeau from the
Institute Louis Malarde in Tahiti a significant dis-service. When she presented her
preliminary data linking Zika infection to Guillian Barre syndrome at a meeting in
Tahiti in 2014, I and several well-known Arbovirologists were very sceptical and
suggested that what she was seeing was probably due to the comorbidities she had
identified. Subsequent events have shown I was wrong.
In the late 1990s, I began to take a greater interest in the broad issue of arbovirol-
ogy/disease surveillance, and in 1996 I was fortunate to obtain a W.H.O. Fellowship
to travel throughout India, Thailand and Indonesia looking at their communicable
disease surveillance and reporting systems. Several issues caught my attention at
that time, and they are not exclusively issues for these three countries. There was
very little quality assurance – how do you know that the laboratory test has pro-
duced the correct result? There was a significant disconnect between clinicians car-
ing for patients and the scientists performing diagnostic tests, so the results of
laboratory tests were, often, not being interpreted correctly. While data for a range
of communicable diseases were being reported to Ministries of Health, the report-
ing often was not timely, and it was rarely analysed in any detail. There was little
interest in the patients for whom laboratory tests were non-reactive in the assays
performed. This was an issue that I encountered repeatedly in years to come. If 90%
of patients during a suspected outbreak of dengue don’t have dengue, then it is
152 J. Aaskov
important to determine what they do have (see the Burma Chikungunya story
above). However, I was impressed with the efforts the Thai Ministry of Public
Health were making to link case reporting and laboratory testing and to change data
accordingly.
The next adventure in this space was participation in John Ehrenberg’s efforts at
W.H.O. to develop a bi-regional (Western Pacific and South-East Asia WHO
regions), 5-year, Dengue Plan, and it was a real tribute to his organisational skills
that this happened. Unfortunately, no one could identify a source of the estimated
US$800 million required to fund the plan. I proposed an international fund-raising
committee chaired by someone with the regional profile of the retired Singapore
Prime Minister, Lee Kuan Yew, who had the social and political clout to approach
corporations and ultra-high-net-worth individuals in the region for support. This
was unacceptable to W.H.O. because they felt they would have no control over the
process and, in the end, I am sure no more than 10% of the necessary funds ever
eventuated. Unfortunately, when this bi-regional plan came to be renewed, it
reverted to regional plans. This was a tragedy. Post World War 2 geopolitics put
Thailand and Indonesia in the South-East Asia Region of W.H.O., and China and
Malaysia in the Western Pacific Region – nations in the Asia Pacific region have
common borders, enormous cross-border migration and shared interests in reducing
the burden of dengue.
While attending a W.H.O. Regional Dengue Conference in Hanoi, I heard that
the Asia Development Bank had funded extensive refurbishment of a large number
of Provincial Health Laboratories in Vietnam and was astonished at the amount and
relative sophistication of the equipment involved. Following some informal discus-
sions with Bank staff, I was invited to participate in the mid-term review of the
project. There had been a significant disconnect between a centrally planned social-
ist economy and modern diagnostic Pathology. The Vietnamese Ministry of Health
policies, at that time, were more concerned with floor space and the number of
chairs and desks than with what was needed to diagnose the most pressing health
problems facing each Province. In addition, there was equipment that was still in its
boxes because it wouldn’t fit through doors, there was equipment that couldn’t fit in
the space available inside laboratories, there were enormously expensive pieces of
equipment that would be used to perform only a few tests each month, there were
no plans to test class 2 biohazard hoods after delivery to determine if they were safe
to use, and there were almost no quality processes in place in the laboratories. Plans
for waste disposal also were novel, rudimentary or non-existent. The procedure for
the disposal of cadmium and mercury waste at one laboratory involved putting it in
a plastic bucket, adding cement and, when solid, discarding the concrete block in a
local garbage tip.
When I was invited to act as a Consultant for the expansion of the Greater
Mekong Communicable Disease Control Project to Laos and Cambodia (CDC II),
the Asia Development Bank agreed to give me a car, a driver and a national counter-
part for about 3 weeks in each country to visit as many project sites as possible
before I got started, and this was invaluable. I felt that before large amounts of
equipment were purchased, it was important to have a clear view of the situation on
the ground. Did the laboratory staff have shoes? Most staff preferred slip-on plastic
sandals. Did they take their laboratory coats/clothes home to wash or was there a
laundry in the Hospital? Did they have an autoclave? Did it work? Could all the
windows and doors be closed? Were the micropipettes clean, operational and cali-
brated? Were disposable micropipette tips being washed and re-used? Were all diag-
nostic kits legal and in date? Could staff read the instructions on kits? Were there
any standard operating procedures? Was there a safe waste disposal system? Sadly,
all too often, the answer to many of these questions was no. Taking laboratory coats/
gowns/clothes home for laundry was a particular concern because of the likelihood
of spreading agents like influenza, measles or sudden acute respiratory syndrome
(S.A.R.S.) viruses on these garments, and so I arranged the purchase of washing
machines for a number of laboratories. How many people reading this know that
only front-loading washing machines are able to heat water? Top loading washing
machines require an external source of hot water.
These Hospital/Laboratory visits also gave me the opportunity to discuss the
issues and problems that most concerned the people on the spot. When a Doctor at
one site told me he saw about 10 cases of encephalitis each month, I was taken
aback when he then told me he didn’t know how many of these died. Families at this
locality take moribund patients home to die, and deaths at home are rarely reported.
The consequence of this is that the Ministry of Health was unaware that they may
have had a focus of Japanese encephalitis – that is vaccine preventable. As was the
case in Australia until relatively recently, there was almost no communication
between doctors treating patients and the laboratory performing diagnostic tests. As
a result, it was common for laboratory results to be misinterpreted. Another signifi-
cant issue is what, euphemistically, is called co-payment by patients for their health
care. In practise, it means that patients pay almost the entire cost of their treatment,
and if the patient can no longer pay, treatment usually ceases. The patient on the
back of the motorcycle in Fig. 25 has paid the motorcycle owner to take him home
from hospital, still in his pyjamas and with a drip in his arm, because he has run out
of money. The patient keeps the drip because he has paid for it.
The training workshops I organised reflected the needs identified when I had
visited the Provincial laboratories. On the first morning, we either provided shoes or
went to the market and bought shoes for everyone – I had organised two laboratory
gowns to be made for each workshop participant beforehand (Fig. 26).
Each student was given a set of new micropipettes that they could take back to
their laboratory and each morning of the workshop they dismantled, cleaned and
calibrated them. Students prepared standard operating procedures (S.O.Ps.) using a
standard template provided and then gave them to their colleagues to perform basic
tasks like autoclaving waste or using, interpreting and reporting the results of a
point-of-care test for dengue. Invariably, operators were unable to perform these
tasks until the S.O.Ps. had gone through several rounds of editing. The practical
component was supported with lectures on the signs, symptoms, diagnosis and pre-
vention/treatment of the sorts of communicable diseases participants were likely to
encounter in their laboratories. Wherever the Ministry of Health had policies under-
pinning the role and activities of diagnostic laboratories, this was used to underpin
154 J. Aaskov
Fig. 25 Patient, in pyjamas and with a drip in their arm, returning home because they can no
longer afford to stay in hospital
workshop lectures and practical exercises. At the conclusion of the workshops, there
was a written theory exam and competency-based assessment of participant’s labo-
ratory skills. In a break with tradition, participants had to pass the assessments in
order to get a workshop certificate. However, they could repeat assessments until
they passed.
I also was involved, subsequently, in the planning for the Greater Mekong
Communicable Disease Control Project III which was extended to include Myanmar.
This project, involving Vietnam, Laos, Cambodia and Myanmar, had an indicative
budget of ~ US$140 million so had the potential to catapult these countries into
compliance with their obligations under the International Health Regulations.
However, as these funds would come as loans to some of these Governments, there
was very little leverage other than reasoned argument, to ensure the funds weren’t
going to be spent buying large numbers of Toyota Land Cruisers or something simi-
lar. In 2016, I found myself back in the National Health Laboratory in Yangon where
my Burma/Myanmar adventure had begun 25 years earlier, and there had been pro-
found changes at the Myanmar Ministry of Health. The health budget had increased
tenfold between 2010 and 2017, but the health system was struggling to absorb the
funds – doctors, nurses and paramedical staff can’t be trained in a year, even if the
training facilities have the capacity to do so. Nonetheless, hospitals and laboratories
were being expanded, built or renovated, and the National Health Laboratory people
were equipping State and Township laboratories with key, new, and appropriate,
equipment. However, the private Pathology laboratories in large cities like Yangon
and Mandalay were soaking up the newly graduating Medical Scientists leaving the
more distant Ministry of Health laboratories almost without staff.
Fig. 26 Workshop at the Centre for Laboratory and Epidemiology in Vientiane, Lao P.D.R. Note
the purple laboratory gowns provided for each student and the empty drink bottle into which
pipette tips are discarded. The bottles are free, and they and their contents can be autoclaved when
the bottle is full or at the end of each day
There is enormous waste and duplication in programs to help these, and many
other countries in the region, improve their diagnostic and disease surveillance sys-
tems and to meet their obligations under the International Health Regulations. One
country had at least five donor countries each providing slightly different biosafety
training and “study visits” for their nationals to the donor country. The Deputy
Minister of Health in another of the countries told me that he doesn’t know what
equipment is being brought into his hospitals by donors and so it is impossible to
develop a meaningful equipment maintenance or replacement program. There is
very little follow-up to determine whether workshops and training have any impact
and there are wonderful cartoons circulating highlighting the failures of some work-
shops. For many donors, to have conducted a workshop is sufficient outcome. Many
international players are unaware of, or ignore, national policies and strategies and
don’t train to these. There is no systematic mentoring of key players in the health
systems in many/most developing countries. There is a strong whiff of politics to
almost all aid programs, and so the outcome desired by the donor can be unrelated
to the needs of the recipient.
For much of my early career, I was captivated by the minutia of what I was doing and
paid only lip service, usually in the first paragraphs of grant applications or of sci-
entific papers, to the “big picture” to which Doherty had exposed me at the beginning
of my career. There is a pressing need for people with solid basic science training to
go to the field and to get a feel for the context of the issues they are addressing - and
it can be great fun and life changing!!
156 J. Aaskov
References
Aaskov J (1984) Absence of intrauterine infection following Ross River virus infection during
pregnancy. Am J Trop Med Hyg 33:739–740
Aaskov JG, Davies CE (1979) An immunofluorescence assay for human antibodies to Ross River
virus. J Immunol Methods 25:37–41
Aaskov JG, Davies CE, Tucker M et al (1981a) Effect on mice of infection during pregnancy with
three Australian arboviruses. Am J Trop Med Hyg 30:198–203
Aaskov JG, Nair K, Lawrence GW et al (1981b) Evidence for transplacental transmission of Ross
River virus in humans. Med J Aust 2:20–21
Aaskov JG, Mataika JU, Lawrence GW et al (1981c) An epidemic of Ross River virus infection in
Fiji, 1979. Am J Trop Med Hyg 30:1053–1059
Aaskov JG, Ross P, Davies CEA et al (1981d) Epidemic polyarthritis in northeastern Australia,
1978-1979. Med J Aust 2:17–19
Aaskov JG, Ross PV, Harper JJ et al (1985) Isolation of Ross River virus from epidemic polyar-
thritis patients in Australia. Aust J Exp Biol Med Sci 63:587–597
Aaskov JG, Geysen HM, Mason TJ (1989) Serologically defined linear epitopes in the envelope
protein of dengue 2 (Jamaica strain 1409). Arch Virol 105:209–221
Aaskov JG, Phillips DA, Wiemers MA (1993) Possible clinical infection with Edge Hill virus.
Trans R Soc Trop Med Hyg 87:452–453
Aaskov J, Williams L, Yu S (1997) A candidate Ross River virus vaccine: preclinical evaluation.
Vaccine 15:1396–1404
Aaskov J, Buzacott K, Thu HM et al (2006) Long-term transmission of defective RNA viruses in
humans and Aedes mosquitoes. Science 311:236–238
Aaskov J, Buzacott K, Field E et al (2007) Multiple recombinant dengue type 1 viruses in an iso-
late from a dengue patient. J Gen Virol 88:3334–3340
Aichinger G, Ehrlich HJ, Aaskov JG et al (2011) Safety and immunogenicity of an inactivated whole
virus Vero cell-derived Ross River virus vaccine: a randomized trial. Vaccine 29:9376–9384
Aleck KA, Rosen L, Pettitt DJ et al (1983) Absence of intrauterine infection following Ross River
virus infection during pregnancy. Am J Trop Med Hyg 32:618–620
Anon (1905) Report on the dengue epidemic in Brisbane in 1905. Australasian Med Gaz:1–8
Bahnemann HG (1976) Inactivation of viruses in serum with binary ethyleneimine. J Clin
Microbiol 3:209–210
Bancroft TL (1906) On the etiology of dengue fever. Australasian Med Gaz 25:17–18
Beasley DW, Aaskov JG (2001) Epitopes on the dengue 1 virus envelope protein recognized by
neutralizing IgM monoclonal antibodies. Virology 279:447–458
Bielefeldt-Ohmann H, Beasley DW, Fitzpatrick DR et al (1997) Analysis of a recombinant den-
gue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus sys-
tem. J Gen Virol 78:2723–2733
Bokisch VA, Top FH Jr, Russell PK et al (1973) The potential pathogenic role of complement in
dengue hemorrhagic shock syndrome. N Engl J Med 289:996–1000
Carey DE (1971) Chikungunya and dengue: a case of mistaken identity? J Hist Med Allied Sci
26:243–262
Claflin SB, Webb CE (2015) Ross River virus: many vectors and unusual hosts make for an unpre-
dictable pathogen. PLoS Pathog 11:e1005070
Cleland JB, Bradley B (1919) Further experiments in the etiology of dengue fever. J Hyg
18:217–254
Cleland JB, Bradley B, McDonald W (1918) Dengue fever in Australia. J Hyg 16:317–442
Craig S, Thu HM, Lowry K et al (2003) Diverse dengue type 2 virus populations contain recombi-
nant and both parental viruses in a single mosquito host. J Virol 77:4463–4467
Dean HR, Webb RA (1926) The influence of optimal proportions of antigen and antibody in the
serum precipitation reaction. J Pathol 9:473–492
Desmyter J, South MA, Rawls WE (1971) The IgM antibody response in rubella during pregnancy.
J Med Microbiol 4:107–114
Doherty RL (1972) Arthropod-borne viruses in Australia and their relation to infection and dis-
ease. MD thesis, University of Queensland
Doherty RL, Whitehead RH, Wetters EJ et al (1969) Isolation of viruses from Ornithodoros capen-
sis Neumann from a tern colony on the Great Barrier Reef, North Queensland. Aust J Sci
31:363–364
Doherty RL, Carley JG, Best JC (1972) Isolation of Ross River virus from man. Med J Aust
1:1083–1084
Doherty RL, Carley JG, Kay BH et al (1979) Isolation of virus strains from mosquitoes collected
in Queensland, 1972-1976. Aust J Exp Biol Med Sci 57:509–520
Fraser JR (1986) Epidemic polyarthritis and Ross River virus disease. Clin Rheum Dis 12:369–388
Graham H (1903) The dengue: a study of its pathology and mode of propagation. J Trop Med
6:209–214
Guzman MG, Kouri GP, Bravo J et al (1984) Dengue haemorrhagic fever in Cuba. I. Serological
confirmation of clinical diagnosis. Trans R Soc Trop Med Hyg 78:235–238
Halstead SB, O’Rourke EJ (1977) Dengue viruses and mononuclear phagocytes. I. Infection
enhancement by non-neutralising antibody. J Exp Med 146:201–217
Halstead SB, Chow JS, Marchette NJ (1973) Immunological enhancement of dengue virus replica-
tion. Nat New Biol 243:24–26
Hare FE (1898) The 1897 epidemic of dengue in North Queensland. Australasian Med Gaz
17:98–107
Hawkes RA (1964) Enhancement of the infectivity of arboviruses by specific antisera produced in
domestic fowls. Aust J Exp Biol Med Sci 42:465–482
Hawkes RA, Lafferty KJ (1967) The enhancement of virus infectivity by antibody. Virology
33:250–261
Hotta S (1952) Experimental studies on dengue. I. Isolation, identification and modification of the
virus. J Infect Dis 90:1–9
Kay BH, Barker-Hudson P, Stallman ND et al (1984) Dengue fever. Reappearance in northern
Queensland after 26 years. Med J Aust 140:264–268
Khin MM, Than KA (1983) Transovarial transmission of dengue 2 virus by Aedes aegypti in
nature. Am J Trop Med Hyg 32:590–594
Kistner O, Barrett N, Brühmann A, Reiter M et al (2007) The preclinical testing of a formaldehyde
inactivated Ross River virus vaccine designed for use in humans. Vaccine 25:4845–4852
Kliks SC, Halstead SB (1983) Role of antibodies and host cells in plaque enhancement of Murray
Valley encephalitis virus. J Virol 46:394–404
Liu W, Pickering P, Duchêne S, Holmes EC et al (2016) Highly divergent dengue virus type 2 in
traveler returning from Borneo to Australia. Emerg Infect Dis 22:2146–2148
Lok SM, Ng ML, Aaskov J (2001) Amino acid and phenotypic changes in dengue 2 virus associ-
ated with escape from neutralisation by IgM antibody. J Med Virol 65:315–323
Mandl CW, Guirakhoo F, Holzmann H et al (1989) Antigenic structure of the flavivirus enve-
lope protein E at the molecular level, using tick-borne encephalitis virus as a model. J Virol
63:564–571
Marks EN, Kay BH, Elson-Harris MM (1980) Potential vectors of malaria and dengue at
Townsville, Queensland. Med J Aust 2:676–677
Marshall ID, Woodroofe GM, Hirsch S (1982) Viruses recovered from mosquitoes and wildlife
serum collected in the Murray Valley of South-Eastern Australia, February 1974, during an
epidemic of encephalitis. Aust J Exp Biol Med Sci 60:457–470
Mitchell CJ, Miller BR, Gubler DJ (1987) Vector competence of Aedes albopictus from Houston,
Texas, for dengue serotypes 1 to 4, yellow fever and Ross River viruses. J Am Mosq Control
Assoc 3:460–465
158 J. Aaskov
Nasveld PE, Ebringer A, Elmes N et al (2010) Long term immunity to live attenuated Japanese
encephalitis chimeric virus vaccine: randomized, double-blind, 5-year phase II study in healthy
adults. Hum Vaccin 6:1038–1046
Nathanson N, Langmuir AD (1963a) The Cutter Incident. Poliomyelitis following formalde-
hyde inactivated polio virus vaccination in the United States during the spring of 1955.
I. Background. Am J Hyg 78:16–28
Nathanson N, Langmuir AD (1963b) The Cutter Incident. Poliomyelitis following formalde-
hyde inactivated polio virus vaccination in the United States during the spring of 1955.
II. Relationship of poliomyelitis to Cutter vaccine. Am J Hyg 78:29–60
Nuegoonpipat A, Berlioz-Arthaud A, Chow V et al (2004) Sustained transmission of dengue
virus type 1 in the Pacific due to repeated introductions of different Asian strains. Virology
329:505–512
Oseni RA, Donaldson MD, Dalglish DA et al (1983) Detection by ELISA of IgM antibodies to
Ross River virus in serum from patients with suspected epidemic polyarthritis. Bull World
Health Organ 61:703–708
Peeling RW, Artsob H, Pelegrino JL et al (2010) Evaluation of diagnostic tests: dengue. Nat Rev
Microbiol 8(Suppl):S30–S38
Phillips DA, Murray JR, Aaskov JG et al (1990) Clinical and subclinical Barmah Forest virus
infection in Queensland. Med J Aust 152:463–466
Phillips DA, Aaskov JG, Atkin C et al (1992) Isolation of Kunjin virus from a patient with a natu-
rally acquired infection. Med J Aust 157:190–191
Podder G, Breiman RF, Azim T et al (2006) Origin of dengue type 3 viruses associated with the
dengue outbreak in Dhaka, Bangladesh, in 2000 and 2001. Am J Trop Med Hyg 74:263–265
Rabaa MA, Simmons CP, Fox A et al (2013) Dengue virus in sub-tropical northern and central
Viet Nam: population immunity and climate shape patterns of viral invasion and maintenance.
PLoS Negl Trop Dis 7:e2581
Ramon G (1922) Compt Rend Soc Biol 1922, 86, 661, 711, 813
Rey FA, Heinz FX, Mandl C et al (1995) The envelope glycoprotein from tick-borne encephalitis
virus at 2 A resolution. Nature 375:291–298
Rosen L (1977) The Emperor’s New Clothes revisited, or reflections on the pathogenesis of dengue
haemorrhagic fever. Am J Trop Med Hyg 26:337–343
Russell PK, Intavivat A, Kanchanapilant S (1969) Anti-dengue immunoglobulins and serum beta 1
c-a globulin levels in dengue shock syndrome. J Immunol 102:412–420
Schmitz H, Haas R (1972) Determination of different cytomegalovirus immunoglobulins (IgG,
IgA, IgM) by immunofluorescence. Arch Gesamte Virusforsch 37:131–140
Serafin IL, Aaskov JG (2001) Identification of epitopes on the envelope (E) protein of dengue 2
and dengue 3 viruses using monoclonal antibodies. Arch Virol 146:2469–2479
Sinh Nam V, Thi Yen N, Minh Duc H et al (2012) Community-based control of Aedes aegypti by
using Mesocyclops in southern Vietnam. Am J Trop Med Hyg 86:850–859
Thein S, La Linn M, Aaskov J et al (1992) Development of a simple indirect enzyme-linked immu-
nosorbent assay for the detection of immunoglobulin M antibody in serum from patients fol-
lowing an outbreak of chikungunya virus infection in Yangon, Myanmar. Trans R Soc Trop
Med Hyg 86:438–442
Thein S, Aung MM, Shwe TN et al (1997) Risk factors in dengue shock syndrome. Am J Trop
Med Hyg 56:566–572
Vu SN, Nguyen TY, Tran VP et al (2005) Elimination of dengue by community programs using
Mesocyclops(Copepoda) against Aedes aegypti in central Vietnam. Am J Trop Med Hyg
72:67–73
Wressnigg N, van der Velden MV, Portsmouth D et al (2015) An inactivated Ross River virus vac-
cine is well tolerated and immunogenic in an adult population in a randomized phase 3 trial.
Clin Vaccine Immunol 22:267–273
The Tapestry of Life: Weaving
an Arbovirologist
Duane J. Gubler
Life is a tapestry woven by the decisions we make.

Sherrilyn Kenyon
Abstract How does one become an abovirologist? There are many paths leading to
this end, as varied as the unique characters that make up the field of arbovirology.
Here I describe the rather unusual path I followed, from a humble rural beginning to
international recognition. I focus on my early academic and work experiences and
how they influenced the decisions that drove my career path. My hope is that this
paper will encourage students and young scientists to follow their dream and make
it happen regardless of adverse events and challenges.
1 Background
In recent years, I find myself being repeatedly asked the question “how did you get
started in arbovirology”? I usually chuckle to myself and, with the paucity of time,
give a short answer to a question that requires a much longer explanation if it is to
have any meaning. For this book, I want to provide a more complete and thoughtful
response. As I turned my mind and my memories loose to free range over the paths
chosen and the roads traveled in the 55 years of my professional life, I realized how
uncertainty and indecision dominated the early years of my career and how a few
critical decisions and luck influenced and helped crystalize my transition to arbovi-
rology. Behind the question lies a story that forms the first threads woven into the
tapestry of my life, a weave that provided the foundation on which my personal and
professional life was built.
I am writing this short history of my formative years in public health and arbovi-
rology with students and young scientists in mind, remembering my own experi-
ence of being at the same point searching for direction and inspiration. I recall my
D. J. Gubler (*)
Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
e-mail: duane.gubler@duke-nus.edu.sg
160 D. J. Gubler
early uncertainty about my future in science and the direction of my professional

career. I am guessing that students and young scientists today have many of the
same doubts and uncertainties. My hope is that by recounting the rather tortuous
road, I traveled from farmer/rancher to entomology to parasitology to arbovirology,
and the experiences I describe herein will allay some of the uncertainties they may
have about the future direction of their careers. I also hope to reinforce the virtues
of patience and persistence to explore new roads until you find the one that leads to
an area of endeavor you love. Finally, I hope to ignite their sense of adventure and
underscore the rewards and joys of working in the field where studying pathogens
in their natural habitats has greater relevance to the real world.
2 The Beginning
I was raised in a small farm/ranch community in southern Utah where I completed

my primary (three grades per room) and secondary education. I spent my childhood
and teen years working on my father’s farm/ranch, never expecting or dreaming that
I would ever leave the rather closed rural Mormon community of southern Utah. I
played sandlot and high school sports (baseball, basketball, track, and football) and
excelled in football, being selected as First String on the Utah All State High School
team in 1957. I started college on a football scholarship, but soon realized that I
would never be good enough to make the big league and that studying was a more
productive use of my time. But I had no idea what I wanted to study, so I dropped
out of college after football season and began driving a produce truck. I married my
high school sweetheart and planned to be a farmer/rancher in the tradition of south-
ern Utah like so many generations before me and to drive truck to supplement the
family income. But it did not take long before I realized that driving truck was not
what I wanted to do the rest of my life. Working part-time, and with my wife work-
ing to help support us, I went back to school at the College of Southern Utah (now
Southern Utah University), a community college that specialized in training school
teachers. My plan this time was to teach school to supplement the farm/ranch
income. However, in my 3rd year when I began taking upper division education
classes, I realized that teaching primary/secondary school was also not for me. So,
I changed my major from education to biological sciences. I wasn’t sure what I was
going to do with a degree in biological sciences, but I did well in those classes and
had an excellent mentor (Dr Wesley P Larsen). Under his tutelage, I experienced my
first taste of research and had my first publication (Larsen and Gubler 1962). With
his support and advice, I applied for and was awarded an undergraduate research
fellowship at the University of North Dakota Medical School Anatomy Department
in the summer of 1961. I did well in that assignment and was invited back again in
the summer of 1962, which resulted in my second publication (Yeager and Gubler
1963). By then I knew I enjoyed biomedical research and transferred to Utah State
University where I earned my BS in Entomology, with minors in Zoology and
Chemistry, in 1963.
The Tapestry of Life: Weaving an Arbovirologist 161
3 Graduate School
Having experienced the university research environment, I knew I would not be

satisfied as a research technician working for someone else. I had an opportunity to
go to the University of North Dakota Medical School for graduate studies in anat-
omy under my mentor, Dr. Vernon Yeager, but by that time I had developed a strong
interest in mosquito-borne diseases. I applied for and was awarded a fellowship to
do a Master’s degree with Elmo Hardy at the University of Hawaii Department of
Entomology. Elmo had a grant in collaboration with some of the best drosophila
geneticists in the USA at the time (Drs Wilson Stone, Marshal Wheeler, Hampton
Carson, William Heed, Herman Speith and Lynn Throckmorton) to study the evolu-
tion of the Hawaiian Drosophila and wanted me to do my graduate work in genetics
and Drosophila evolution. I worked in that program as a field entomologist and even
have a new species of fly named after me (Drosophila gubleri). However, I was not
really into genetics, and with Elmo’s approval, I chose to do my thesis on mosquito-
borne canine filariasis in Hawaii, earning an MS in Parasitology and Entomology in
1965, and my third publication (Gubler 1966).
I was lucky to have two Johns Hopkins University School of Public Health (now
the Bloomberg School of Public Health) alumni (Drs George W. Chu and Leon
Rosen) on my MS committee. With their encouragement and help, I was accepted
into the Johns Hopkins School of Public Health (JHUSPH) doctoral program in
1965. By this time, my wife and I had started our family consisting of two boys, one
aged 18 months and a newborn. The decision to go to Johns Hopkins was very dif-
ficult because they offered me financial support for only 6 months, after which I
needed to get my own grant support to continue my studies; this was how Johns
Hopkins separated the wheat from the chaff. I also had an offer to go to Nepal to
study filariasis, but that was a 2-year nondegree program. With my wife’s encour-
agement, therefore, I accepted the JHUSPH offer and wrote a successful training
grant proposal to the NIH to develop mosquito tissue culture, which was a new field
at that time. That gave me the financial support to complete my doctoral studies and
my fourth publication (Gubler 1968). I earned an ScD in Pathobiology from the
JHUSPH in 1969. The training at Johns Hopkins was very broad with an emphasis
on tropical medicine, parasitology, medical entomology, epidemiology, disease
ecology, and even a class in social science. One could say I was by this time, a “jack
of all trades,” comfortable working in both the laboratory and the field, especially in
parasitology and entomology. It was a time before specialization and postdoctoral
fellowships became the norm in graduate training.
4 Out on My Own
When I received my ScD, I was overwhelmed with doubt and lack of confidence.
This was a scary time in my career development, with the uncertainty of the times
when the war on infectious diseases had been declared won. I was uncertain of my
162 D. J. Gubler
ability to go out on my own and compete in the cutthroat world of academic medical
research. Many JHUSPH graduates in the late 1960s were hired by pharmaceutical
companies to do drug discovery research. I visited one large company in Pennsylvania
and decided that was not for me. Luckily, JHUSPH needed help in teaching and
offered me a job as a lecturer teaching diagnostic parasitology and medical ento-
mology. My annual salary was $9500, considerably less than the salaries paid by
pharmaceutical companies, but, nevertheless, a major increase in salary from my
student stipend.
I go into this detail of my early years of work, education, and training to empha-
size that not everyone knows what they want to do in life without experiencing
doubts, uncertainties, a variety of work experiences and fields of study, and the
importance of following your instincts and taking chances to find a field that you
love. The road I followed led to a number of dead ends because I did not know
where I was going. It was also probably longer than most because of my beginnings
in rural southern Utah as a farmer/rancher, although that experience taught me the
value of honesty and a good work ethic. The take-home message is that if you are
not fully satisfied and are unsure of where you are going in your career, do not be
afraid to change the road you travel and try a new one.
I was lucky to have a wife who believed in and supported me during this arduous
journey and who also had a spirit of adventure. I was anxious to work in the field
and study mosquito-borne diseases in their natural setting, so in the summer of
1969, with my wife’s blessing, I accepted a position working for JHUSPH at the
Calcutta School of Tropical Medicine in Calcutta (now Kolkata), India, to conduct
field research on bancroftian filariasis. My research was to prospectively study the
interactions between the parasite, Wuchereria bancrofti, the mosquito vector, Culex
quinquefasciatus, and the human host in the natural endemic setting of Calcutta.
The results of this field research resulted in my first important publication, which
underscored the importance of transmission dynamics and immune tolerance in dis-
ease expression and severity of human filariasis, and of how human and mosquito
behavior influence the epidemiology of mosquito-borne diseases (Gubler and
Bhattacharya 1974; Dondero et al. 1976). These papers were used by Eli Chernin at
Harvard as a case study for field research for a number of years.
However, little did we know what we were getting into in Calcutta! The late
1960s and early 1970s were a chaotic period in Asia and especially India. The
Vietnam war was raging; China, although it had failed in its coup attempt in
Indonesia in 1965, was flexing its muscles in Asia; it was at the height of the cold
war, and the Soviet Union was very influential in India at the time with tensions high
following the 1965 war between India and Pakistan. The state government of West
Bengal had been taken over by a communist front in 1967, and as a result, chaos
reigned in Calcutta. Things became so bad in 1970, the Prime Minister of India,
Indira Gandhi, declared “President’s Rule” in West Bengal, removed the communist
front government and sent in the Indian Army to restore calm. She then used that
vantage point to send the Indian Army into East Pakistan (East Bengal), resulting in
the removal of Pakistan from that part of the Indian subcontinent and the formation
of a new country (Bangladesh). That created a mass migration of 2 million refugees
flooding into Calcutta. It was a time when Americans were not very welcome in
West Bengal and the government was in the process of forcing American institu-
tions such as the Peace Corps and Johns Hopkins University to leave Calcutta.
In short, Calcutta in 1969 was a total culture shock for my wife, our two young
sons, and me! It was like going back in time, with the overcrowding, poverty, lack
of hygiene and waste disposal, and, in general, the rigors of life in that setting. In
addition, the political situation was very disruptive to everyday life, with frequent
Hartals (general strikes), demonstrations and processions involving tens of thou-
sands of people, making it difficult to get anything done, especially if it was work
related. The following is a quote from my journal: “31 July,1969. This morning it
took me 45 minutes to go from the Johns Hopkins University administrative office
to the Calcutta School of Tropical Medicine, a short trip that usually takes
10–15 minutes. We ran into a procession that was protesting President Nixon’s visit
to India (Fig. 1). There were thousands of demonstrators waving their communist
flags with the hammer and sickle and shouting anti-American slogans.” These kinds
of demonstrations were common, and you just had to wait them out or more wisely,
turn around, and go another way. Although life in Calcutta was challenging and
sometimes dangerous, it was an exciting and uncertain time to live in that part of
the world!
Fig. 1 Communist procession, Calcutta, India, 1969. (Picture taken from inside car as taking
photos of such events was dangerous)
164 D. J. Gubler
5 Hawaii
In the fall of 1970, Dr. Scott Halstead came through Calcutta. He had been hired to
start a tropical medicine program at the new school of medicine at the University of
Hawaii. His program was co-located at Leahi Hospital with the Pacific Research
Section of NIAID, NIH, headed by Dr. Leon Rosen. It was a natural partnership
between the University of Hawaii and NIH to work on tropical diseases of the Asia-
Pacific region. Leon Rosen had been instrumental in my acceptance to Johns
Hopkins for my doctoral studies, and he had recommended me to Scott, who offered
me a position as assistant professor. The agreement was that I would teach at the
university but do my research in the NIH laboratory with Leon Rosen, who was
working primarily on parasitic diseases at the time. The JHUSPH had also offered
me a position as assistant professor of pathobiology in Baltimore, but because of the
political turmoil in Calcutta, our program there was phasing out, and I wanted to
continue work in the Asia-Pacific region. Moreover, dengue, in which I had a strong
interest, was becoming increasingly important in Southeast Asia, and both Scott and
Leon were good virologists, both interested in dengue. So I left Johns Hopkins and
joined Scott and Leon in Hawaii in July 1971. Although it seemed like a natural
transition for me at the time, I had many doubts and uncertainties about leaving
JHUSPH, a well-established world-class university for a startup program in a new
medical school. In retrospect, however, it was one of the best decisions I ever made,
one that completely changed the direction my career from parasitology to arbovirol-
ogy. Of note, Johns Hopkins again offered me a position at JHUSPH in 1973; I don’t
regret not returning to Baltimore, but have always wondered how my career would
have turned out had I taken that road. In hindsight, this decision to remain focused
on the Asia-pacific Region set the tapestry weave for my lifetime engagement
in Asia.
The move to Hawaii was timely since major epidemics of dengue were occurring
in Asia and for the first time since World War II, in the South Pacific islands.
Epidemics first occurred in Tahiti and Fiji in 1971, spreading to New Caledonia,
Niue, and Samoa in 1972, and to most of the other islands over the next few years.
There was no good virological assay for dengue viruses at that time as the viruses
did not grow well in sucking mice nor mammalian cell cultures (mosquito cell cul-
tures were not available at that time). My first dengue research project was to help
develop the mosquito inoculation method to isolate and quantitate dengue viruses
(Rosen and Gubler 1974). Mosquito inoculation not only provided the first highly
sensitive method to isolate dengue viruses from human clinical samples; it allowed
us to accurately quantitate and measure virus levels in human blood and mosquito
tissues. Since Aedes aegypti did not occur in Hawaii at that time, we used Aedes
albopictus to develop mosquito inoculation. All inoculation was done under a dis-
secting microscope and required good hand/eye coordination. I became very profi-
cient at it, being able to accurately inoculate upward of 200 mosquitoes per hour. In
the laboratory, we showed that the virus disseminates throughout the body of the
mosquito, infecting various tissues, including the brain. Tim Kuberski in Leon
Rosen’s laboratory developed a simple direct immunofluorescent assay (DFA) to

detect dengue virus antigen, making it easy and rapid to identify infected mosqui-
toes by doing DFA on a brain squash (Kuberski and Rosen 1977). We made our own
conjugate using pooled human sera with high antiflavivirus IgG titers (HI >5120)
and used these methods to study the dengue virus-mosquito interactions, variation
in vector competence, biological differences in virus strains, virus isolation from
human clinical samples, and the epidemiology of dengue as the disease spread
through the Pacific islands (Gubler and Rosen 1976a, b, 1977; Gubler, et al., 1978;
Tesh and Gubler 1975; Tesh et al. 1976; Kuberski et al. 1977).
With the mosquito inoculation method in hand, Leon and I began to study den-
gue epidemics in the South Pacific. DENV-2 was introduced to Tahiti in 1971 from
the French West Indies, causing an explosive epidemic with severe disease and sev-
eral deaths. A similar DENV-2 epidemic occurred in Fiji that same year, as did oth-
ers in New Caledonia, American Samoa, and Niue Island in 1972. The Tahiti, New
Caledonia, and Fiji epidemics were studied by French and New Zealand scientists,
although we worked more closely with the French because of Leon’s connections
(Moreau et al. 1975; Maguire et al. 1974; Loison et al. 1973). Because the French
had no laboratory capability to diagnose dengue, they sent the samples from both
the Tahiti and New Caledonia epidemics to Leon. We isolated DENV-2 from over
75% of serologically confirmed cases in both epidemics using the mosquito inocu-
lation method, a dengue virus isolation rate unheard of at that time (Gubler 1997). I
investigated epidemics in American Samoa, Manua Islands, Kingdom of Tonga,
Pohnpei, Raratonga, and Palau. With the exception of Raratonga and Palau, I trav-
eled to investigate these epidemics alone, having to do everything from taking clini-
cal histories and bleeding patients to laboratory and entomology work. A brief
description of these investigations follows.
6 American Samoa
The Samoa, Manua, and Niue island epidemics occurred almost simultaneously in
the spring of 1972. I had gone to American Samoa to investigate that epidemic in
June 1972. While there, I heard about epidemics in Niue and in the Manua Islands.
The information I had was limited, and I had no information on the severity of the
epidemics. Communication and travel between islands in the Pacific at that time
were difficult and limited, being mainly by copra boats that infrequently visited
islands to collect coconut meat (copra). My only communication with Hawaii was a
weekly Ham radio call with Leon. I checked on boats to both Niue and Manua but
was not able to confirm a time to catch one to Niue. I was able to catch a supply boat
to the Manua Islands so I went there. It was a bad decision because there was only
sporadic transmission in the Manua Islands, whereas we found out later that the
Nuie Island epidemic was explosive, infecting a high percentage of the population
and with deaths in children experiencing primary infections that were clinically
compatible with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS).
166 D. J. Gubler
As it turns out, this was the first evidence that called into question the secondary
infection (antibodydependent enhancement) hypothesis to explain DHF/
DSS. Unfortunately, because I was not able to go to Niue, we did not have acute
serum samples to confirm DENV-2 by virus isolation (IgM and PCR assays were
not available at that time). Leon did visit Niue Island in January 1973 and conducted
a clinical review of cases and a serosurvey, confirming by plaque reduction neutral-
ization test (PRNT), that it was a primary DENV-2 epidemic (Barnes and
Rosen 1974).
Interestingly, the Samoa epidemic was also not explosive like Tahiti, Fiji, New
Caledonia, and Niue, and severe and fatal disease was not observed. Rather, it was
what might be called sustained sporadic transmission (Gubler and Rosen 1972). I
conducted an entomological survey of most villages on the main island of Tutuila
and found Aedes aegypti and Ae. polynesiensis in all of them. I set up clinical sur-
veillance at the LBJ Tropical Medicine Center, the main hospital on the island, and
confirmed DENV-2 cases from most villages. All illness was mild or classical den-
gue fever. I fed uninfected Ae. polynesiensis mosquitoes on 12 patients to investi-
gate the relationship between viremia level in patients and infection rates in
mosquitoes (Fig. 2). Of the 12 patients, 6 were confirmed as dengue serologically,
but virus was isolated from only 2 of these patients by mosquito inoculation. Viremia
levels were 9 × 106 and 3 × 106 MID50/ml in those two patients. The mosquitoes
that took a blood meal from these two patients were held for 14 days after feeding
in our laboratory room at the hospital where the nonadjustable temperature was only
Fig. 2 Feeding mosquitoes on patients with suspected dengue infection, American Samoa, 1972
24 C. Twenty mosquitoes that fed on the patient with the highest viremia were dis-
sected, and 15/20 (75%) of the midguts from these mosquitoes were infected, while
none (0/19) had infected salivary glands. We concluded that the incubation tempera-
ture of 24 C was too low for efficient virus replication and dissemination.
Surprisingly, only 8% of the mosquitoes that fed on the patient with the lower vire-
mia were infected. Moreover, the overall virus isolation rate from serologically con-
firmed patients in Samoa was only 57% compared to over 75% in Tahiti and New
Caledonia (Gubler 1997). The human population under the age of 25 was mostly
naïve, and the mosquito populations were large in all villages so there was no good
explanation for why the Samoan epidemic was muted with less severe disease and a
significantly lower virus isolation rate than Tahiti and New Caledonia. We were
puzzled but didn’t have any good answers at the time.
The trip to the Manua Islands was interesting but disappointing. They are iso-
lated high islands with no passage through the reef, so that supply boats had to
anchor offshore outside the reef and transport supplies to the island in long boats
that could shoot the reef during high tide. Fuel for the island was supplied in 55 gal-
lon barrels that were thrown overboard and swimmers then floated them over the
reef (Fig. 3); once emptied, they made perfect water storage containers and mos-
quito breeding habitats (Fig. 4). I had to load all my gear into a long boat that was
already full of people who had come out to meet the supply boat. I was afraid we
would capsize, but we made it ashore. I did a mosquito survey and documented both
Ae. aegypti and Ae. polynesiensis in the main village. As I walked through the vil-
lage, I noticed that most of the water storage containers were empty with wet spots
Fig. 3 Floating barrels of fuel over the reef, Manua Islands, 1972
168 D. J. Gubler
Fig. 4 Fuel barrel used for water storage made ideal mosquito breeding sites, Tonga, 1974
on the ground; word had spread, and many villagers had emptied the containers
before I arrived at their house. There was only sporadic dengue transmission on
the island.
As an aside, a patient on one of the outer islands had reported a dengue-like ill-
ness, so we radioed ahead to the dispensary on that Island to have the patient come
out to the supply boat so I could take a blood sample. The patient arrived in an
outrigger canoe, and I took the sample, but with difficulty; I never again tried to take
a venous blood sample on a rocking boat on the ocean. There was no hotel on
Manua, so I stayed in the dispensary. The attending nurse made me dinner when I
arrived; dinner consisted of several small ocean fish boiled in seawater without
cleaning them (she just threw them in the hot water). I had to peel the skin off and
eat the meat without disturbing the stomach contents. It wasn’t too bad after I got
the hang of it. The fish were accompanied by roasted breadfruit, roasted green
bananas, and a Samoan sweet made from young coconut ground into a watery paste.
It looked like the perfect medium for bacterial growth, but I ate it so as to not insult
the nurse. It must have been safe as I didn’t get sick.
7 Kingdom of Tonga
Another puzzle was why the Kingdom of Tonga, which was right in the middle of
all of this dengue activity, reported no cases of dengue. The Tongan islands report-
edly had Ae. aegypti and another Ae. scutellaris species called Ae. tabu. In January
1974, a colleague of mine from Johns Hopkins, Jim Hitchcock, came through
Hawaii on his way to Tonga to study filariasis. I explained the dilemma of no dengue
in Tonga to him and asked if he would look out for cases or reports of dengue-like
illness in Tonga. Two months later, I received eight paired serum samples from Jim,
taken from patients with viral syndrome. Four of them seroconverted to DENV-2 by
PRNT. Thinking it was the beginning of an epidemic, I packed all my gear and
headed for Tonga, only to find a larger puzzle. Although I found large populations
of Ae. aegypti and Ae. tabu, especially in the main city of Nuku’alofa on the main
island of Tongatapu, there were no reported dengue cases. In an effort to find
dengue-like illness, I went to the hospital at 6–7 am every morning to check for viral
syndrome cases that had come into the outpatient clinic the night before. I had a
Minimoke (a small English car, Fig. 5) to get around the island and a nurse to inter-
pret for me. Our routine for several weeks was to identify potential patients from the
outpatient log, visit them at their home, check the premises for mosquito breeding
(Fig. 4), take a clinical history and blood sample (Fig. 6), and feed uninfected mos-
quitoes on the patient (Fig. 2) if I thought they might have dengue. (Note: I started
colonies of Ae. aegypti and Ae. tabu when I first arrived in Tonga to provide clean
mosquitoes to feed on patients to study the relationship between viremia levels and
Fig. 5 Minimoke, transportation in Tonga, 1974

170 D. J. Gubler
Fig. 6 Taking blood sample from patient with suspected dengue, Tonga, 1974
mosquito infection rates.) On many days, there were no potential cases identified,
and we spent a lot of time trying to find the address of patients, sometimes to no
avail. Acute blood samples were usually taken from suspected cases within 24 h of
illness onset, and the serum separated and stored in liquid nitrogen the same day the
samples were taken. Some patients were visited multiple days and blood samples
taken if I thought they had a greater chance of having dengue. Blooded mosquitoes
that had fed on suspected case patients were held in double cages for 14 days at
ambient temperature and then stored in liquid nitrogen. All samples were taken back
to the laboratory in Hawaii where serology and virus isolation were done. Very dis-
appointing was that in 6 weeks, only 20 patients were shown to have dengue by
seroconversion of paired blood samples, and only 6 (30%) of those had detectable
viremia by virus isolation, usually only on the day of onset of illness. Of nine
patients on whom mosquitoes were fed, six had confirmed dengue infection (sero-
converted), but only one had viremia adequate to infect mosquitoes (Gubler et al.
1978). With the exception of one patient, all clinical illness associated with DENV-2
infection in Tonga presented as very mild viral syndrome. The exception was a
15-year-old female who developed severe hemorrhage in association with primary
dengue infection (Gubler and Rosen 1974).
After all of that work, I almost lost all of the clinical and mosquito samples. I had
taken a full 31-liter liquid nitrogen dewar with me, and I was very careful to not
waste it, but after 6 weeks, the nitrogen was very low, and I had to get the samples
back to Hawaii. When I arrived in Pago Pago to catch a Pan American flight to
Hawaii, the agent informed me that I could not take the nitrogen container on the
plane. I showed him the International Air Regulations, where it said properly pack-
aged liquid nitrogen could be safely shipped via air. He still refused so when the
plane landed from Fiji, I asked to talk to the pilot. Luckily, the pilot was experienced
and, after reviewing the regulations, agreed to take the container on board. I had less
than 2 inches on my measuring stick when I arrived at the laboratory in Hawaii.
After returning to Hawaii, I learned of a filariasis survey that was conducted in
Tonga by Bob Desowitz in 1973, a year before I arrived. We tested those blood
samples and found about 20% PRNT positive for DENV-2. These results confirmed
that dengue had been introduced to Tonga as early as 1973 and had been maintained
on the island for over a year in silent transmission. In contrast, DENV-1 was intro-
duced to Tonga in 1975 and caused an explosive epidemic with severe and fatal
disease (Gubler et al. 1978). Of special interest was a 26-year-old female experienc-
ing a primary infection who died with a classical DSS clinical picture. DENV-1 was
isolated from her acute serum, making her the first virologically confirmed primary
fatal DSS case reported (Gubler et al. 1978), thus supporting the observations in
Niue Island.
Of historical/archeological interest in Tonga is a large trilithon called Haʻamonga
ʻa Maui, located in the northern part of the main island of Tongatapu (Fig. 7). Made
of three coral limestone slabs, it is massive, each vertical supporting pillar being 5.2
meters (17 ft) high and weighing 20–30 tons (40–60,000 pounds). The vertical slabs
have a notch carved in the top into which the top slab (lintel) is inserted to hold it in
place. The origin and purpose of the Haʻamonga ʻa Maui are uncertain and contro-
versial. Some believe it was built in the thirteenth century by the Tongan King
Fig. 7 Haʻamonga ʻa Maui, Tonga, 1974

172 D. J. Gubler
Tuʻitātui. However, Tongan lore holds that it was built in the sixth century by the
God Maui as an astronomical observatory. A mark shaped like a V on the top of the
lintel aligns perfectly with the position of sunrise at solstices and equinoxes.
8 Pohnpei, Caroline Islands
In the fall of 1974, we received a serum sample from a suspected dengue case in
Pohnpei, Federated States of Micronesia, an island in the Eastern Caroline group in
the Western Pacific. Although the serum was highly contaminated with bacteria, we
were able to isolate DENV-1. Since Ae. aegypti was not known to occur in that part
of the Pacific, and there had been no dengue reported there since World War II, I was
asked by the South Pacific Commission to conduct an entomological survey to see
if Ae. aegypti was present in Pohnpei. In February 1975, I once again headed out to
a remote Pacific island expecting to find an epidemic of dengue in progress. But as
with Samoa, Manua, and Tonga, I was disappointed to find little or no dengue-like
illness on the island (Gubler 1975). Pohnpei was difficult to get to and undeveloped
in 1975, and I had to island-hop on a Micronesian Air plane that had a few seats in
the front for people and a large cargo space in the back for cargo and animals. There
was no hotel on the island so I stayed in a guest house that had small individual
screened cottages, where I set up a makeshift laboratory to process blood and mos-
quito samples. There was no modern transportation on Pohnpei, so I had to travel to
sites around the island by boat, making it difficult to get around. There was an old
railroad track built by the Japanese during World War II that circled the island, but
it was not functional.
Kolonia, on the northern tip of the island, was the largest village and the main
population center on Pohnpei in 1975. The rest of the population was scattered in
smaller villages and individual houses around the island plain. Kolonia was the
most urbanized and ecologically suitable for Ae. aegypti, which had been reported
there by the Japanese in the 1940s. By 1949, it was difficult to find and was absent
in a 1974 WHO survey. I conducted a detailed mosquito survey in Kolonia but also
surveyed the villages of Non Mol, Awak, Mant, and the Pohnpei Agricultural and
Trade School on the Eastern and Southeastern parts of the island. I found five spe-
cies of mosquitoes, Ae. (Stegomyia) hakanssoni, Culex pipiens quinquefasciatus,
Culex maplei, Culex annulirostris, and Ae. (Aegomorphus) senyainensis. I found no
Ae. aegypti in 140 positive containers. Ae. hakanssoni, a member of the Scutellaris
complex closely related to Ae. polynesiensis, was the most common species observed
in all villages. Clinical surveillance produced only a few cases of dengue-like ill-
ness, none of which were confirmed as dengue. To determine if there had been an
outbreak of dengue in the summer-fall of 1974, I conducted a serosurvey of school
children and young adults under the age of 25 years in Kolonia and at the Pohnpei
Agricultural and Trade School on the southeastern side of the island, the rationale
being that since dengue had not been reported since 1945, persons under the age of
25 should be seronegative. I also included 12 Peace Corps volunteers in the serosur-

vey. Interestingly, 51.2% of the Pohnpei school children had DENV-1 or DENV-2
neutralizing antibodies. We thus concluded that transmission of both DENV-1
and -2, mostly silent, had occurred in Pohnpei in recent years (Gubler 1975). Of the
12 Peace Corps volunteers, only one (8.3%) had dengue antibody. Unfortunately, I
was not able to get a DENV-2 isolate from Pohnpei to see if it was the same lineage
as the Tonga DENV-2. Although we could not prove it at the time, epidemiological
and entomological data suggested that Ae. hakanssoni was the principal vector for
dengue on Pohnpei (Gubler 1975).
An interesting side story about Pohnpei is that on the southeastern side of the
island, there are ruins of a mysterious civilization that thrived during the eleventh
century AD. They had built a Venice-like city on the coral reef called Nan Madol.
With only one entrance passable only during high tide, the city was constructed on
the inner reef almost exclusively with large basalt Pillars (stone logs) of various
sizes, but many 12–15 feet in length and 8–10 inches in diameter (Fig. 8). It is not
known how the basalt logs were made or from where and how they got them to the
site of construction. The civilization and people who lived there are steeped in mys-
tery, legend, and theory, but the area is still sacred to Pohnpeians today. When I
visited the area, the site was flooded with sea water and overgrown with mangrove.
The social makeup of Nan Madol is not well understood, but apparently, the leader
was considered a King (Royalty), under whom there were two more social strata,
nobility, and commoner. The person who accompanied me to Nan Madol told me
the legend of Bwusen Mallak. On the plain southwest of Kolonia is a large volcanic
Fig. 8 Basalt logs used to construct the city of Nan Madol, Pohnpei, 1975
174 D. J. Gubler
Fig. 9 Volcanic plug known as Bwusen Mallak, Pohnpei, 1975
plug, a conical mound rising above the countryside, called Bwusen Mallak, inter-
preted literally as “the shit of chicken.” Legend has it that the King had a son who
was disrespectful of the King and of all the rules and traditions of Non Madol.
Because of his bad behavior, the Gods turned him into a chicken and banished him
from Non Madol. The chicken (son) flew off and cursed Non Madol by defecating
on the island (Fig. 9), thus, the origin of Bwusen Mallak. The curse may have been
effective since there has been little subsequent development on Pohnpei in the sub-
sequent 900 years.
On most of these trips, I was working alone, often gone to some remote island or
area for weeks at a time with no communication with Hawaii. My wife was very
understanding and supportive and just trusted me to stay well and come home. I was
fortunate and rarely had health issues while in the field. However, I thought my luck
had run out in Pohnpei. About a week into my visit, I developed a severe bout of
diarrhea that lasted for days, and I became severely dehydrated, as potable water
was very limited. I was laid up alone in my cottage and could have died without
anyone knowing. However, I hired a young boy to bring me green coconuts every
day; I drank the sterile coconut water and scraped out the young coconut meat to eat
along with some fruit. I am convinced that the coconut water saved my life (Fig. 10).
Fig. 10 Surviving on coconut water, Pohnpei, 1975
9 Raratonga, Cook Islands
Prior to 1979, epidemic polyarthritis, caused by Ross River virus (RRV), an alpha-
virus, was known to occur only in Australia, Papua New Guinea, and the Salomon
Islands in the Western Pacific basin. At that time, RRV had only rarely been isolated
from humans during outbreaks in Australia, and the dogma was that humans did not
contribute to the transmission cycle because the virus complexed with antibody and
infectious viremia was thus too low to infect mosquitoes. It was also hypothesized
that the arthritis observed in humans was associated with the immune complexes of
RRV and antibodies produced during infection. In 1979, RRV was introduced into
the Pacific islands for the first time, causing outbreaks in Samoa and Fiji, but those
outbreaks were not adequately studied. I had just joined CDC in Fort Collins,
Colorado, and was at a World Health Organization meeting on dengue in New
Delhi, India, in February 1980. While in New Delhi, I received a message from my
boss, Tom Monath, asking if I could detour to Hawaii on my way home from India.
Leon Rosen had word of an epidemic of RRV in Raratonga, Cook Islands, and
needed someone to go to investigate. I agreed and flew to Honolulu to meet with
Leon. We felt this was a good opportunity since it allowed the first investigation of
epidemic polyarthritis during the early phases of a RRV epidemic that would allow
us to answer important clinical, epidemiologic, and immunologic questions relating
to RRV infections in humans.
So once again I headed out to a Pacific island to study a potential epidemic. I was
joined by Peter Bennett of the South Pacific Commission. We set up clinical
176 D. J. Gubler
Fig. 11 Paddling to an offshore island to collect uninfected mosquitoes, Raratonga, Cook
Islands, 1980
surveillance at the hospital in Raratonga and asked physicians to collect acute and
convalescent blood samples from suspected cases. Blood samples were processed
on the day of collection, and sera were stored in ampules on dry ice. Physicians
were asked to fill out case investigation forms that included epidemiological and
clinical data on the patient. We conducted an entomological survey, fed clean unin-
fected Ae. polynesiensis mosquitoes on suspected patients (note the uninfected mos-
quitoes were collected on an uninhabited island about a kilometer offshore, reached
by outrigger canoe, Fig. 11), collected mosquitoes from inside patients’ houses, and
subsequently tested them for virus infection. A serological survey of selected
domestic animals was conducted to determine if they were being infected with RRV
and possibly playing a role in transmission. We investigated the incubation period
of RRV by monitoring visitors to the island who became ill after arriving from New
Zealand.
The clinical picture observed in Raratonga RRV-infected patients was similar to
that previously described in Australia. However, virus isolation from human patients
in Australia was rare. By contrast, we isolated virus from 50% of patients with sero-
logically confirmed RRV infection in Raratonga, documenting high viremia in acute
blood samples of humans. We established that the incubation period for RRV in
humans could be as short as 3 days. Entomological studies found only three species
of mosquitoes on Raratonga, Culex annulirostris, a known vector of RRV in
Australia; Culex quinquefasciatus; and Ae. polynesiensis. All three species were
collected inside houses of patients, but RRV was isolated from only Ae.
polynesiensis. This species was clearly the most abundant and was found breeding
in water containers in the domestic environment as well as in crab holes. We con-
firmed that Ae. polynesiensis became infected with RRV by feeding on humans,
documenting human-to-human transmission by mosquitoes, and that Ae. polyne-
siensis was the most likely vector of RRV in Raratonga (Rosen et al. 1981;
Gubler 1981).
10 Taiaro, Tuamotu Islands
In January 1972, I traveled to the remote island of Taiaro, a small uninhabited coral
atoll in the Tuamotu Archipelago of French Polynesia about 300 miles northeast of
Tahiti. The purpose of this trip was to monitor the progress of a field experiment on
competitive displacement to control bancroftian filariasis. In the way of background,
Ae. polynesiensis is the principal species of Stegomyia mosquito of the Scutellaris
complex in the South Pacific. It is a vector of dengue, Zika, chikungunya and Ross
River viruses, and of Wuchereria bancrofti in those islands. Ae. albopictus, which is
closely related to Ae. polynesiensis, also belonging to the Scutellaris complex and
has a very similar ecology, does not transmit bancroftian filariasis and was not
found naturally in the South Pacific. As part of my doctoral research at Johns
Hopkins, we showed that Ae. albopictus was an aggressive competitor in both larval
and adult stages, and when reared with Ae. polynesiensis in cages varying in size
from small to large room size, it would eliminate Ae. polynesiensis from the popula-
tion. In addition to the larval competition, an important component of the species
interaction was that Ae. albopictus males would forcibly mate with Ae. polynesien-
sis females. The resulting eggs laid by these cross-inseminated females were sterile.
Thus, competitive displacement of Ae. polynesiensis by Ae. albopictus had good
potential as a control method for bancroftian filariasis in the Pacific islands (Gubler
1970a, b, c, 1971; Ali and Rozeboom 1971; Lowrie 1973). Taiaro, an isolated pri-
vately owned island, was selected to test this hypothesis.
In 1970, when I was still in India, my major professor at Johns Hopkins, Lloyd
Rozeboom, teamed up with Leon Rosen and Bill Reeves to follow up on our research
to conduct a field trial of competitive displacement by introducing Ae. albopictus to
Taiaro to see if it could displace the native Ae. polynesiensis population under natu-
ral conditions (Rosen et al. 1976). In January 1972, Leon Rosen, Lloyd Rozeboom,
Jacques Saugrain, Remy Thirel, and I traveled to Taiaro to check the progress of that
field trial. To get there, we sailed for 3 days from Tahiti on a small French govern-
ment boat. Arrangements had been made to have a copra boat pick us up in 3 weeks.
Taiaro was an uninhabited circular coral atoll, about 5 kilometers in diameter with
a closed lagoon. The lagoon was full of fish; there were many coconut crabs on the
island, and crabs and rock lobster were plentiful in the coastal reef waters, so we ate
well (Fig. 12). Leon took his ham radio, so we could communicate with Hawaii;
otherwise, we were cut off from civilization. We also took an experimental military
178 D. J. Gubler
Fig. 12 Collecting coconut crabs, Taiaro, Tuamatu Islands, French Polynesia, 1972
satellite telephone with us to test. It was a large heavy telephone in a box about a
foot long as I recall. We also had to take an antenna along. On prearranged nights,
we would unfold the antenna and point it at a star constellation in the vicinity of the
satellite at that time of day and then try to hook up with the base in Hawaii. We were
successful in talking to Hawaii; to my knowledge, this was one to the first successful
field trials of the first military satellite telephone. It was an exciting technological
breakthrough, but of course it was classified.
We spent the next 3 weeks studying the mosquito populations, but the experi-
ment was a disappointment because Ae. albopictus did not do the job we had hoped.
It was Ae. albopictus that had disappeared from the island, not Ae. polynesiensis
(Rosen et al. 1976). A discussion of the reasons for the failure is beyond the scope
of this paper, but most likely, the main reason was that the strains of Ae. albopictus
introduced had lost the genetic variability needed to adapt to the harsh field ecology
and that there was only a single introduction as opposed to a sustained release over
a period of weeks or months.
At 3 weeks, we were ready to go back to Tahiti, but the Copra boat never showed
up. Every day we would scan the horizon for a boat, to no avail. Leon finally called
Hawaii on his ham radio and asked them to call Tahiti. We figured the French would
get a boat to pick us up because Dr. Saugrain was the director of the Institut de
Recherches Medicales Louis Malarde in Tahiti. We were correct, but it was still
another week before a large French Navy ship showed up off shore. After nearly
5 weeks, we were happy to see it! The Navy ship had to make a political call in
Fig. 13 Partying on Fakarava, French Polynesia, 1972
Fakarava, another island in the Tuamotus. The French threw a big party, with roasted
goat, red wine, and, as with all pacific island parties, singing and dancing (Fig. 13).
We were all ready to party but mainly just happy to be on our way back to
civilization.
11 Palau, Caroline Islands
It was some years later when I was with CDC in Puerto Rico, late March 1988, that
we received a report of a 46-year-old female from Palau, Caroline Islands, in the
Western Pacific, who died following an illness that was clinically compatible with
DHF/DSS. We subsequently received samples from other patients with dengue-like
illness and confirmed DENV-2 infection. As with the other Pacific islands, Palau
had not reported dengue since WWII. Neither Ae. aegypti nor Ae. albopictus were
known to occur in Palau, which at that time had not yet been developed as a tourist
destination. Scott Deitchman, a CDC EIS officer, Mike O’Leary from the National
Health Service Corps, USPHS, and I headed to Palau in early May to investigate the
epidemic. We set up a makeshift laboratory in the hospital, initiated clinical disease
surveillance, and began entomological investigations. Review of hospital and outpa-
tient records suggested the epidemic had begun in January and peaked in mid-April.
Most cases were from the main island of Koror, but transmission was also con-
firmed on the northernmost and southernmost islands of Kayangel and Peleliu,
180 D. J. Gubler
respectively. Most patients presented as classical dengue fever or mild viral syn-
drome, but there were four fatal cases. Random population-based serosurveys were
conducted along with the entomological surveys on the main island of Koror and on
Peleliu.
We stayed at a little motel called the D&W Motel on Koror. The first evening
there I was looking around the motel grounds and found Ae. aegypti breeding in
used tires and boat hulls. Entomological surveys were conducted around each house
included in the serological survey on Koror and Peleliu. Widespread distribution of
both Ae. aegypti and Ae. albopictus was documented on Koror, but Ae. albopictus
was much more common than Ae. aegypti. Nearly every house checked had Ae.
albopictus, whereas Ae. aegypti was found in only about 50% of houses. Ae. scutel-
laris complex species were also present in every house. On Peleliu, both Ae. aegypti
and Ae. albopictus were present, with house indices of 13% and 87%, respectively.
Other species of the scutellaris complex of the subgenus Stegomyia were present in
high numbers on both islands, including Ae. hensilli, Ae. palauensis, Ae. scutellaris,
and Ae. dybasi; Ae. hensilli was dominant. The attack rate as measured by the pres-
ence of IgM antibody was 51% on Koror compared to 45% on Peleliu. While we are
not certain, it appears that Ae. albopictus and/or Ae. hensilli were the principal vec-
tors on Peleliu. This was the first report of Ae. aegypti and Ae. albopictus in Palau.
Age-specific IgG antibody rates suggested that an unreported outbreak had also
occurred in Palau in the early 1970s (Epidemic Dengue 2 in Palau 1988).
Peleliu is the southernmost island of the Palau group and could only be accessed
by a 1-h boat ride from Koror. Peleliu was a Japanese stronghold during WWII,
being the main communication center for their Pacific Command. Reinforced con-
crete bunkers that housed massive generators can still be seen but were overgrown
with jungle. It is an island with over 500 natural and man-made caves that were used
by the Japanese during the war. The battle for Peleliu was one of the bloodiest cam-
paigns of WWII in the Pacific, with approximately 12–15,000 Japanese and
American soldiers killed in the effort by Americans to dislodge the Japanese. Many
Japanese soldiers who refused to surrender were entombed in the caves and tunnels.
The island had been kept as a natural museum for WWII, with tanks, landing craft
carriers, machine guns, and tunnels left as they were when the war ended. The
approximately 500 people who lived on Peleliu occupied a single village.
12 Lessons Learned
When I graduated from Johns Hopkins in 1969, it was the end of a golden era for
field work in tropical medicine, and especially arbovirology. Most of the important
arboviruses had been discovered during the 1940s–1970s time period. Moreover,
great success in controlling and reducing the global disease burden of infectious
diseases also occurred during that time period. In vector-borne diseases, the global
malaria eradication program had greatly reduced malaria incidence in the tropics,
especially in Asia and Tropical America, and had eliminated the disease from North
America and Europe. The American regional Ae. aegypti eradication program had
eliminated this mosquito from 23 countries and had successfully prevented epi-
demic yellow fever and dengue for many years. Other arboviruses and vector-borne
diseases such as yellow fever in Africa, leishmaniasis, trypanosomiasis, as well as
non-vector-borne infectious diseases were on their way to being controlled as we
had new insecticides, antibiotics, and vaccines. Smallpox eradication was on the
horizon. The Surgeon General of the United States, Sir MacFarlane Burnett in
Australia, and other infectious disease experts declared the war on infectious dis-
eases won. Public health officials and policy makers became complacent and over-
confident. As a result, funding support for infectious disease research, especially for
field research, was being redirected to other public health priorities. When I was in
my final year at Johns Hopkins, Lloyd Rozeboom actually suggested that I consider
changing my field and not focus on vector-borne diseases. Despite the projected
bright future of controlling infectious diseases, and the bleak funding future, how-
ever, I had romantic notions of working in the tropics and controlling mosquito-
borne diseases that had been the scourge of mankind from the beginning of time. I
ignored his recommendation, went to India, and persisted in my dream of working
on these diseases in their natural setting. My experiences in India and the South
Pacific were both exciting and sometimes dangerous, but they fulfilled that dream.
At this point, however, I clearly knew where I was going with my career and that the
weave of my tapestry was established!
As noted, it was a different time when you had to do everything yourself; you had
to be a “jack of all trades” to successfully work in the field. Leon Rosen had advised
me “that if you are going to work in the field, you better learn the disease you are
working on from A to Z because you can’t rely on others.” I took that advice to heart
and learned everything I could about dengue. As a result, I felt very comfortable
discussing entomological, epidemiological, clinical, and laboratory diagnostic
issues with the colleagues I met and worked with in the field. That knowledge also
gave me the confidence to successfully work alone in the field. My experience in the
South Pacific investigating epidemic dengue set the tone for the rest of my career. I
frequently had to work alone, doing everything from taking clinical histories, bleed-
ing patients, setting up and conducting laboratory tests, conducting interviews of
community members, conducting entomological surveys and mosquito taxonomy,
etc. I learned to depend on myself and have confidence in my decisions, although as
you might guess, I made many mistakes along the way. But it was during this period
that I became an arbovirologist and learned the value of working in the field and the
need to make experimental laboratory data as relevant as possible to what happens
in nature.
An important lesson I learned early and repeatedly working in foreign lands with
different cultures and languages and with public health officials and scientists with
different training and backgrounds was to try to place myself in the local colleagues’
position and look at the situation from their eyes. I had the challenge of going into
many of those early engagements alone without a support team. On arrival I would
often have to find the right contacts and identify persons with whom I could best
work with and/or work through. That was usually a mix of local government
182 D. J. Gubler
officials, local scientists, and people who I encountered as I tried to learn the lay of
the land and get established. There was often skepticism and sometimes outright
distrust due to bad experiences with earlier scientists who had approached them.
There were often significant language and cultural barriers that separated us but I
found if I was open approached them as equals and tried to understand their culture
and our differences, trusting working relationships could be built. The language bar-
rier was often intimidating but being respectful, listening and being able to laugh at
myself made the process far less intimidating.
In the process, I gained valuable insights that allowed me to be sensitive to the
many cultural differences that could adversely or positively impact my work. I
learned that respect begot respect and that differences in education, training, and
experience were easily overcome. Moreover, my local colleagues often had practi-
cal knowledge and “street smarts” that facilitated my work. Today many of those
local counterparts have moved to top leadership positions as cabinet ministers or
heading esteemed national institutions in their country. Most importantly, they have
become lifetime friends, and we remain in communications and visit each other
whenever the occasion permits.
Our work in the South Pacific led to several seminal scientific contributions.
First, we developed the first sensitive assay for dengue viruses that allowed for the
first time routine isolation and quantitation of dengue viruses from clinical samples.
Second, we showed that different geographic strains of the mosquito vector varied
in their susceptibility to infection with dengue and chikungunya viruses and that this
variability was genetically controlled. Third, that different strains of dengue virus
varied in their infectivity to and replication in the mosquito vector. Fourth, that sec-
ondary infection (antibodydependent enhancement) was not the only factor deter-
mining whether a patient experienced severe dengue disease. The Niue Island
epidemic (Barnes and Rosen 1974) and the 26-year-old Tongan female who died
with a primary DENV-1 infection (Gubler et al. 1978) reinforced the evidence that
DHF/DSS can occur in adults and patients experiencing their first or primary den-
gue infection, calling into question the importance of the antibody-dependent
enhancement hypothesis. Fifth, that the strain of virus is an important risk factor for
severe dengue disease. Thus, dengue viruses, like most animal viruses, mutate and
change genetically via drift giving rise to viruses with greater or lesser fitness and
epidemic potential. This was proven many years later because I had stored the
DENV-2 viruses from the Tahiti, Fiji, New Caledonia, Samoa, and Tonga epidemics
in a low-temperature freezer for over 40 years until they could be studied using
modern molecular methods. I did this because I was convinced that variation in the
viral strain was the only logical explanation for the epidemiological and clinical
differences we observed during these epidemics. As new technology became avail-
able, I had asked Dennis Trent to look at the virus strains by oligonucleotide finger-
printing in the 1980s and Jeff Chang to sequence them in the 1990s, but both studies
showed that all of these early South Pacific epidemics were caused by the American
genotype of DENV-2 and that the viruses were all the same lineage. However, they
only focused on the envelope gene. It was only after full-length genome sequencing
became available that genetic differences in these viruses that made
epidemiological sense were seen in the nonstructural genes. The Tonga virus was a
different lineage that had three amino acid changes in the PrM, NS2A, and NS4
genes that apparently attenuated the virus allowing it to cycle silently with mild,
undetected illness (Steel et al. 2010). These South Pacific studies were the first of a
series of investigations in Indonesia, Sri Lanka, Puerto Rico, and Singapore show-
ing that mutations in dengue viruses, mainly in the nonstructural proteins and the 3′
untranslated region, can influence phenotypic expression, including virus infectiv-
ity, replication, virulence, and epidemic potential. Thus, the strain of dengue virus
is a critical risk factor influencing disease severity and epidemic potential. Finally,
we showed for the first time that humans infected with RRV contributed to the trans-
mission cycle by infecting mosquitoes that feed on them, that Ae. polynesiensis was
the principal mosquito vector in Raratonga, and the incubation period of RRV in
humans could be as short as 3 days.
An important lesson to glean from this discourse is that one should publish the
results of your work as soon as possible after completing the investigation. I have
numerous manuscripts and reports in my files that were not published because I
moved to a new job, location, or project before writing the paper. I suspect this is the
case with many colleagues. Finally, you should establish authorship and responsi-
bilities for a research project before you begin the work and definitely before you
leave to begin a new phase of your career. I learned this the hard way with my men-
tor, Leon Rosen. In 1972, I was invited to present my Calcutta filariasis data at a
meeting at UCLA in Los Angeles, CA. At that filariasis meeting, a paper was pre-
sented by Henry Terwedow showing that if you inoculate dog heartworm microfi-
lariae into the thorax of Toxorhynchites sp., a large nonbiting mosquito, the
microfilariae developed to third stage larvae in a normal manner. Leon and I decided
to inoculate Toxorhynchites with dengue viruses to see if it would also support viral
replication. I collected Toxorhynchites larvae and pupae at Manoa Falls in Hawaii,
reared them to adults, and inoculated them with dengue viruses. The viruses repli-
cated and disseminated in Toxorhynchites just like they did in Ae. albopictus.
Toxorhynchites were larger, had better survival after inoculation, and supported
virus replication to higher titer. One of several disagreements I had with Leon over
the years was the publication of this finding. After I did the initial experiments on
Toxorhynchites, we put the project on the back burner because of all the epidemics
in the South Pacific. Unfortunately, it was left unfinished when I moved to Indonesia
in the summer of 1975. Leon subsequently colonized Toxorhynchites and conducted
more extensive experiments, resulting in a publication with him as sole author, and
not even mentioning my involvement (Rosen 1981). I only learned of this after he
had published the paper; he had forgotten my initial experiments even though the
results were in our log books. Needless to say, we had a serious knockdown fight
over this issue, which was never resolved.
184 D. J. Gubler
13 Conclusions
The road I traveled in my early career, although long and sometimes difficult, was a
wonderful journey despite uncertainties and a few wrong turns. Some things become
clearer with the passing of time, others foggier. I tried to keep good notes, but other
tasks frequently took precedence over keeping a journal. The twists and turns, the
slippery slopes, and the uphill climbs somehow form an intricate tapestry of our life
where the weave is formed with the careful integration of the personal, professional,
family, and spiritual threads. That tapestry represents our time and effort within the
environment and surroundings where we find ourselves. A fine tapestry or a life
fully lived generally requires an abundance of hard work, persistence, patience, and
learning from mistakes. Success in life and career requires not being deterred by
failures nor distracted by successes. In my life, I consider one of the factors that
carried me forward was my willingness to embrace the challenges in surroundings
where there was considerable hardship and uncertainty but also an abundance of
opportunity. The key is to look past the former and take advantage of the latter.
Acknowledgments The author thanks Frank Carlin for comments and suggestions on the paper.
References
Ali SR, Rozeboom LE (1971) Cross-mating between Aedes (S.) polynesiensis Marks and Aedes
(S.) albopictus Skuse in a large cage. Mosq News 31:80–84
Barnes WJS, Rosen L (1974) Fatal hemorrhagic disease and shock a associated with primary den-
gue infection on a Pacific Island. Am J Trop Med Hyg 23:495–506
Dondero TJ, Bhattacharya NC, Black HR, Chowdhury AB, Gubler DJ, Inui TS, Mukerjee M
(1976) Clinical manifestations of bancroftian filariasis in a suburb of Calcutta, India. Am J
Trop Med Hyg 25:64–73
Epidemic Dengue 2 in Palau. Dengue Surveillance Summary No. 54, July, 1988. Dengue Branch,
Division of Vector-Borne Viral Diseases, CDC, San Juan, Puerto Rico
Gubler DJ (1966) A comparative study on the distribution, incidence and periodicity of the canine
filarial worms, Dirofilaria immitis Leidy and Dipetalonema reconditum Grassi, in Hawaii. J
Med Entomol 3:159–167
Gubler DJ (1968) A method for the in vitro cultivation of ovarian and midgut cells from the adult
mosquito. Am J Epidemiol 87:592–598
Gubler DJ (1970a) Competitive displacement of Aedes (Stegomyia) polynesiensis Marks by Aedes
(Stegomyia) albopictus Skuse in laboratory populations. J Med Entomol 7:229–235
Gubler DJ (1970b) Induced sterility in Aedes (Stegomyia) polynesiensis Marks by cross-
insemination with Aedes (Stegomyia) albopictus Skuse. J Med Entomol 7:65–70
Gubler DJ (1970c) Comparisons on the reproductive potentials of Aedes (Stegomyia) albopictus
Skuse and Aedes (Stegomyia) polynesiensis Marks. Mosq News 30:201–209
Gubler DJ (1971) Studies on the comparative oviposition behavior of Aedes (Stegomyia) albopic-
tus Skuse and Aedes (Stegomyia) polynesiensis Marks. J Med Entomol 9:675–682
Gubler DJ (1975) Trip report, Pohnpei, Eastern Caroline Islands, February-March. South Pacific
Commission
Gubler DJ (1981) Transmission of Ross River virus by Aedes polynesiensis and Aedes aegypti. Am
J Trop Med Hyg 30:1303–1306
Gubler DJ (1997) Dengue and dengue hemorrhagic fever: its history and resurgence as a global
public health problem. In: Gubler DJ, Kuno G (eds) Dengue and dengue hemorrhagic fever.
CAB International, London, pp 1–22
Gubler DJ, Bhattacharya NC (1974) A quantitative approach to the study of bancroftian filariasis.
Am J Trop Med Hyg 23:1027–1036
Gubler DJ, Rosen L (1972) A mild outbreak of dengue type 2 in American Samoa. Unpublished
manuscript
Gubler DJ, Rosen L (1974) Primary dengue infection and gastrointestinal hemorrhagic. Dengue
Newsl Am 3:8
Gubler DJ, Rosen L (1976a) A simple technique for demonstrating transmission of dengue virus
by mosquitoes without the use of vertebrate hosts. Am J Trop Med Hyg 25:146–150
Gubler DJ, Rosen L (1976b) Variation among geographic strains of Aedes albopictus in suscepti-
bility to infection with dengue viruses. Am J Trop Med Hyg 25:318–325
Gubler DJ, Rosen L (1977) Quantitative aspects of replication of dengue viruses in Aedes albopic-
tus after oral and parenteral infection. J Med Entomol 13:469–472
Gubler DJ, Reed D, Rosen L, Hitchcock JC (1978) Epidemiologic, clinical and virologic observa-
tions on dengue in the Kingdom of Tonga. Am J Trop Med Hyg 27:581–589
Kuberski TT, Rosen L (1977) A simple technique for the detection of dengue antigen in mosqui-
toes by immunofluorescence. Am J Trop Med Hyg 26:533–537
Kuberski T, Rosen L, Reed D, Mataika J (1977) Clinical and laboratory observations on patients
with primary and secondary dengue type 1 infections with hemorrhagic manifestations in Fiji.
Larsen WP, Gubler DJ (1962) Romalea in the embryology laboratory. Turtox News 40:263
Loison G, Rosen L, Papillaud J, Tomasini J, Vauyany J, Chanalet G (1973) La Dengue en Nouvelle-
Caledonie (1971–1972). Bull Soc Pathol Exot 66:511–519
Lowrie RC (1973) Displacement of Aedes (S) polynesiensis Marks by A (S>) albopictus through
competition in the larval stage under laboratory conditions. J Med Entomol 10:131–136
Maguire T, Miles JAR, MacNamara FN, Wilkinson PJ, Austin FJ, Mataika JU (1974) Mosquito-
borne infections in Fiji. The 1971–73 dengue epidemic. J Hyg (Camb) 73:263–270
Moreau JP, Rosen L, Saugrain J, Lagraulet J (1975) An epidemic of dengue on Tahiti associated
with hemorrhagic manifestations. Am J Trop Med Hyg 22:237–241
Rosen L (1981) The use of Toxorhynchites mosquitoes to detect and propagate dengue and other
arboviruses. Am J Trop Med Hyg 30:177–183
Rosen L, Gubler DJ (1974) The use of mosquitoes to detect and propagate dengue viruses. Am J
Rosen L, Rozeboom LE, Reeves WC, Saugrain J, Gubler DJ (1976) A field trial of competitive
displacement of Aedes polynesiensis by Aedes albopictus on a Pacific atoll. Am J Trop Med
Hyg 25:906–913
Rosen L, Gubler DJ, Bennett PH (1981) Epidemic polyarthritis (Ross River) virus infection in the
Cook Islands. Am J Trop Med Hyg 30:1294–1302
Steel A, Gubler DJ, Bennett SN (2010) Natural attenuation of dengue virus type-2 after a series of
island outbreaks: a retrospective phylogenetic study of events in the South Pacific three decades
ago. Virology 405(2):505–512
Tesh RB, Gubler DJ (1975) Laboratory studies of transovarial transmission of La Crosse and other
arboviruses by Aedes albopictus and Culex fatigans. Am J Trop Med Hyg 24:876–880
Tesh RB, Gubler DJ, Rosen L (1976) Variation among geographic strains of Aedes albopictus in
susceptibility to infection with chikungunya virus. Am J Trop Med Hyg 25:326–335
Yeager VL, Gubler DJ (1963) Protein histochemistry of lathyric periosteum. Arch Pathol Lab Med
76:158–165
Arbovirus and Viral Hemorrhagic Fevers
Research Inception in Central and West
Africa: A French Researcher’s Experiences
Jean-François Saluzzo and Jean-Paul Gonzalez
Abstract The French experience in tropical diseases and, specifically in arbovi-

ral diseases, comes from the historical propensity of the French population to
extensively move around the world. The death toll to the new diseases encoun-
tered in the tropical zone led the French government for the good of development
and mankind to conduct in situ studies on tropical vector-transmitted diseases.
Therefore, since the establishment of the Pasteur Institute in Paris in 1888, Louis
Pasteur sent collaborators oversea in a variety of tropical areas to develop research
and support national public health on the matter. After World War II, mandated by
the Republic, the Research Institute for Development send, primarily to former
colonies and French oversea territories, his multidisciplinary teams of “develop-
ment soldiers” to engage the fight against vector-transmitted diseases. This chap-
ter focuses on the Pasteur Institute International Network and the Research
Institute for Development in their commitments in the field and research on arbo-
viral diseases and viral hemorrhagic fever in Central and West Africa. In partner-
ship with their national colleagues, emphasis is made on their finding, including
among others, the first yellow fever vaccine; the discovery of new arboviruses; the
demonstration of an unexpected geographical extension of the arbovirus, as well
as for the virus of Crimean-Congo, Ebola, Lassa, Hantavirus, Marburg, and Rift
Valley hemorrhagic fevers.
J.-F. Saluzzo (*)

Institut Pasteur/ORSTOM (Alias IRD), Dakar, Senegal
Fab’entech, Lyon, France
J.-P. Gonzalez
Institut Pasteur/ORSTOM (Alias IRD), Dakar, Senegal
Department of Microbiology & Immunology, Division of Biomedical Graduate Research
Organization, School of Medicine, Georgetown University, Washington, DC, USA
Centaurus Biotech LLC, Alexandria, VA, USA
e-mail: Jean.Paul.Gonzalez@georgetown.edu
188 J.-F. Saluzzo and J.-P. Gonzalez
1 Introduction
In historical times, but not so old as the Napoleonic era in the early 1800s, the
French mobilized themselves to outer-sea for a variety of reasons, including polit-
ical, social, economic, and military campaign or scientists in search of knowledge
and discovery. These adventurers were immediately and severely exposed, espe-
cially in the intertropical zone, to several infectious diseases inherent to the cli-
mates and environmental conditions. Thus, the civil and military authorities of the
new republics of France engaged several institutions in the understanding, fight,
and prevention of these exotic diseases to protect French migrants and the local
population of the tropical zone targeting the countries and territories of North,
West, and Central Africa; Southeast Asia; Polynesia; and South America.
Therefore, insect-borne diseases, as a new encounter for this traveler, appeared to
be a permanent threat and an exceptional obstacle to any tentative of expansion on
the march and in search of unknown territories. As a list of these emerging threats
to the health of these unprotected populations, malaria, yellow fever, and typhus
were on the top of it as well as other insect-borne and rodent-borne diseases as a
major factor of morbidity and mortality of these new arrivals. Despite the recent
concept of arbovirology that emerged in the middle of the twentieth century, the
health threats suffered by the French in the intertropical zone, outside of continen-
tal France, prompted the metropolitan government to invest in research and pre-
vention against insect-borne diseases in particular because of the severity of losses
due to malaria and yellow fever. This unique framework of vector-borne disease
research, and more specifically on arthropod-borne viral diseases (arbovirus dis-
eases), was naturally falling to the respected Pasteur Institute network already
located in the tropical zone from the Indochinese Peninsula to North, Central, and
West Africa. Other nascent French institutions of the twentieth century also join
this effort and were established to protect the health of humans and animals such
as the Organization de Recherche pour la Recherche Scientifique et Technique
Outre-Mer (ORSTOM, Research Organization for Scientific and Technical
Research Overseas), the Institut de Médecine Tropical des Armées du Paro (the
Pharo Army Institute of Tropical Medicine), or the Institut d’Élevage et de
Médecine Vétérinaire des pays Tropicaux (IEMVT, the Institute of Tropical
Veterinary Medicine and Stock-Keeping). The present chapter will focus on arbo-
virus research in Central and West Africa carried out by the Pasteur institute
Network and ORSTOM (alias Institute of Research for Development, IRD).
Indeed, the Institut Pasteur has a longue tradition in virology and medical ento-
mology, especially research on the transmission of many arboviruses of the tropi-
cal zone, but not restricted to, while the IRD French multidisciplinary worldwide
institution offers an extensive knowledge on fundamental medical entomology,
environmental sciences, and eco-epidemiology and joined in the early 1950s, the
Pasteur Institute network efforts in Africa on the fight against vector transmitted
tropical infectious diseases (Vasilakis et al. 2019).
Arbovirus and Viral Hemorrhagic Fevers Research Inception in Central and West… 189
2 The Pasteur Institute of Dakar, Senegal
Since the establishment of the Pasteur Institute in Paris in 1888, Louis Pasteur sent
collaborators to various countries, mainly in the French colonies of Indochina and
Africa. In that early time, Pasteur wanted to set up rabies centers where the disease
was highly and dramatically prevalent; naturally, all these centers rapidly extended
their research potential on tropical infectious diseases (Vasilakis et al. 2019).
Currently, there are 22 institutions, 19 of which bear the name of “Institut Pasteur,”
altogether these institutions constitute a complex long called the “Institut Pasteur
d’Outre-mer,” which in 1988, becomes the International Network of Pasteur
Institutes (In French. Réseau International de l’Institut Pasteur, RIIP) and Associated
Institutes. From this emerging network, the first African laboratory of microbiology
was created in 1896 in Saint Louis of Senegal, then transferred to Dakar to become
the Pasteur Institute of Dakar (In French. Institut Pasteur de Dakar, IPD).
From its inception in the modern virology, yellow fever was to play a key role in the
research of this institute in Senegal. In the 1930s, the IPD will develop the vaccine against
yellow fever French Neurotropic Vaccine (FNV) and produced it industrially (Canon et al.
1953). In the 1960s, IPD research will focus on arboviruses with the creation of a World
Health Organization Reference Center (Collaborating Center for Reference and Research
on Arboviruses, CRORA). At the end of the 1970s, following the outbreak of the Ebola
fever epidemic in Zaire (alias, Democratic Republic of Congo) in 1976, the Pasteur
Institute of Bangui set up a research program on viral hemorrhagic fevers (Digoutte et al.
1985). A permanent program on Ebola virus that will last at the IPD until 1983 followed
by other specific research (Saluzzo et al. 1980, 1981, 1982; Ivanoff et al. 1982; Morvan
et al. 1999; Gonzalez et al. 1983, 1989, 2000, 2005). This research on arboviruses and viral
hemorrhagic fevers is the result of a critical collaboration between all Pasteur Institutes in
Africa and the researchers of the French Institute of Research for Development (alias,
ORSTOM). This research was made possible, thanks to the permanent interaction with the
major research centers on transmitted diseases in the United States, which provided per-
manent support to the laboratories located in Africa, including starting from the late 1970s:
Harvard University for the initial study done on yellow fever and FNV vaccine; Yale
Arbovirus Unit (YARU) at Yale University for its permanent interaction and support of the
CRORA; the Centers for Diseases Control and Prevention (CDC) at Fort Collins Colorado,
for the study of arboviruses; the CDC in Atlanta (Special Pathogen Branch); and the US
Army Medical Research Institute of Infectious Diseases (USAMRIID) for the initiation of
viral hemorrhagic fever research in West and Central Africa. Also, all this effort has bene-
fited from ongoing WHO coordination.
3 The Institute for Scientific Research Over Sea (ORSTOM)
The French “Office de la Recherche Scientifique et Technique Outre-Mer”

(ORSTOM) was created in 1946 from a previously existing Office of Colonial
Research (French. Office de la Recherche Scientifique Coloniale, ORSC). Given
by Charles de Gaulle in 1943, ORSC had a mission to constitute a body of

researchers, create a high-level scientific training specialized in the tropical world,
and set up a network of research centers, in addition to French oversea territories
research entities. It was from that time a multidisciplinary structure of research in
environmental (e.g., oceanography, atmospheric science, soil science, agrology,
geology, chemistry, etc.), medical (human and animal), and human and social sci-
ences. In 1960, when African countries take their independence, the office was
placed under the joint supervision of the French Ministry of National Education
and the Ministry of Foreign Affairs with as new vocation to undertake fundamen-
tal research for the development of low- and medium-income tropical countries
(LMIC). In the medical sciences, ORSTOM will intensively develop expertise in
parasitology and nutrition by carrying research, training, and mentoring overseas.
In 1998, it became the Institute of Research for Development (IRD. Eng. French
Institute of Research for Development) functioning by thematic departments
including environment, living resources, societies and health, expertise, and train-
ing for capacity building.
ORSTOM (alias IRD), which has been working for years, alongside with the
RIIP with partner institutions of LMIC, all engaged in that time in the fight against
parasitic diseases, will take, in 1977, a step forward into medical virology research
in tropical areas. Indeed, ORSTOM, thanks to its experts in the fields of parasitol-
ogy and medical entomology, who formerly participated in the fight against
onchocerciasis and malaria, both vectors transmitted diseases, will naturally pro-
vide their expertise, to these IP-ORSTOM research tandems, settled within the
RIIP, to the emerging field of West and Central African arbovirus research. Dr.
Max Germain, as an ORSTOM’ medical entomologist, was already extremely
committed to the understanding of yellow fever epidemiology and ultimately
described the principle of “yellow fever zone of emergence” (Germain et al. 1980,
1986). Indeed, in its position at the IPD and then at the Pasteur Institute of Abidjan
(Côte d’Ivoire), he ardently wished to see the recruitment of a medical virologist
in his team and strongly advocates ORSTOM for it. Therefore, in November 1977,
the first ORSTOM medical virologist position for a candidate will shortly join the
team at the Pasteur Institute of Bangui (IPB) and then at the IPD. Jean Mouchet,
also an ORSTOM medical entomologist, already engaged in the main field of yel-
low fever research with M. Germain, the later having recently participate to the
recent international team discovering on site the emergence of Ebola virus in
Congo (alias Zaire) (WHO 1978), both were instrumental to develop ORSTOM’s
medical virology. Indeed, the first task of such new ORSTOM virologist (viz., J-P
Gonzalez) was, with the team at the IPB, to follow up on Ebola virus along the
Ubangi River and participate to the understanding of “yellow fever emergence
zone” (Germain et al. 1982). Ultimately, the IPB team sets up an active and pas-
sive biosurveillance on the Ebola hemorrhagic fever in these potentially Ebola
virus endemic zone of Central Africa in order to produce more insight of an even-
tual zone of filovirus emergence in the forest-savannah ecotone of the Central
African Republic (Saluzzo et al. 1980, 1982; Gonzalez et al. 1983, 1987). In the
meantime, the research in virology will extend using the expertise of the medical
entomologist and the RIIP partnership to open a new field of research in Africa, as
an example the experimental vertical transmission of arbovirus in mosquitoes
including the Zika virus among others (Cornet et al. 1979) or the major role played
by the environment (climate and latitude) in the arbovirus transmission from
arthropods to vertebrates hosts using the models of yellow fever virus (YF), or
dengue virus (Cordellier et al. 1983; Traore-Lamizana et al. 1996). Moreover,
always teaming with the virologist of the Pasteur Institute in Senegal, West Nile
virus was identified for the first time from livestock ticks along with the descrip-
tion of unique livestock-birds’ natural cycle overpassing the dry season
(Molez 2006).
4 Yellow Fever Vaccine: The French Neurotropic

Vaccine, FNV
The discovery and manufacture of the French neurotropic vaccine (FNV) was
the result of an extraordinary collaboration between researchers from Harvard
University and those of the Institut Pasteur of Dakar. On November 24, 1927,
Dr. Andrews Sellards from Harvard met the director of the Institut Pasteur of
Dakar, a physician and the French army General, Constantine Mathis. On
December 21, 1927, a young Syrian, Francis Mayali, suspected of yellow fever
infection, went to the IPD for consultation and diagnostic, and from its blood
samples, yellow fever virus was isolated and transmitted to the Rhesus macaque
monkeys (Macaca mulatta). Thus, the isolated viral strain was transferred to
Harvard University where Sellards continued its investigations. Then in Harvard
laboratory, Max Theiler will adapt this yellow fever virus strain, by intracere-
bral multiplication and passages on mice. After a several number of passages,
the virus is modified virus that was no longer able to produce the disease in
monkeys. This viral strain became the first candidate for the production of yel-
low fever vaccine (Theiler and Sellards 1928). Later on, such live-attenuated
vaccine will be called the French neurotropic vaccine (FNV). This candidate
vaccine will then be validated by clinical studies by Sellards and Jean Laigret at
the Pasteur Institute of Tunis, led time by the Nobel Prize in Medicine, Charles
Nicolle. In 1931, large-scale vaccinations were carried out by Laigret, Mathis,
and Camille Durieux in Senegal. Various modifications will be made to this vac-
cine, whose ultimate adaptation will be its administration by scarification
(Cannon and Dewhurst 1953).
In only 1 year, from 1940 to 1941, a total of 1,910,000 people will be vaccinated
in West Africa. At the end of the 1950s, yellow fever disappeared from West Africa,
thanks to the mass vaccination. However, the significant side effects, particularly
encephalitis cases, which were relatively frequent during the immunization cam-
paign following the reemergence of yellow fever in Diourbel (Senegal) in 1964 will
gradually lead to the abandonment of this vaccine in favor of the yellow fever
vaccine 17D, developed by Max Theiler at the Rockefeller Foundation. The IPD
will definitively abandon the production of the FNV vaccine in 1981, in favor of the
17D vaccine. Today, the IPD produces several million doses of this vaccine for the
people of Africa (Monath et al. 1983).
5 Inventories of Arboviruses at Pasteur Institutes in Africa
In the early 1960s, under the impetus of Dr. Louis Chambon, the research of the IPD
will focus on the study of arboviruses. The CRORA was opened at the IPD in 1963
under the leadership of Dr. Paul Brès. In Africa, research laboratories on arbovi-
ruses were created, at the Institut Pasteur of Yaoundé (IPY, former Pasteur Center of
Cameroon), the Pasteur Institute of Bangui (IPB, Central African Republic), the
Institut Pasteur of Abidjan (IPA, Côte d’Ivoire), and the Institut Pasteur of Tananarive
(IPT, Madagascar). One of the major activities of these institutes was to establish an
inventory of arbovirus circulating in different ecosystems and environments of
Africa. Therefore, from these studies of “Arbovirus discovery,” a total of 64 new
arboviruses for science were described. Most of them were isolated from vectors
(arthropods, murine) and other vertebrate animal reservoirs (e.g., cattle, rodents,
birds) as part of a broad collaboration with the ORSTOM’s medical entomologists
and mammologists and RIIP researchers. Altogether, arbovirus isolations were
made by the teams of different centers (Dakar, Bangui, Abidjan, Yaoundé,
Antananarivo) of the RIIP in Africa. This included also virus characterization and
identification final assessment carried out at the CRORA reference center of IPD,
then confirmed by YARU, to finally be registered in the International Arbovirus
Catalog (Karabatsos 1985).
6 The Incidence of Arboviruses in Human Pathology

in Central Africa
The first study of the IPB was to uncover the etiology of exanthematous fevers
coined as “Congolese red fevers,” long attributed to rickettsia alone, showed that
only 15% of these purple fevers were of rickettsia origin (Georges et al. 1983;
Gonzalez et al. 1985, 1987). A total of 16 arboviruses were associated with these
syndromes (chikungunya, Igbo-Ora, O’Nyong Nyong, Sindbis, Bouboui, yellow
fever, Wesselsbron, West Nile, Zika, Ilesha, Bwamba, Dugbe, Tatagine, Nyando,
Bangui, and Rift Valley Fever), as well as 72 human cases of arboviruses reported,
with an accompanying symptomatology consistently associating fever and diffuse
pain, and an exanthem present in more than 60% of the cases. The viral etiology was
dominated by four viruses including Chikungunya, Ilesha, Bwamba, and Tataguine
(Saluzzo et al. 2017).
7 Epidemiological Surveillance of Sylvatic Yellow Fever

in West Africa
The reemergence of yellow fever in Africa in the 1960s occurred mainly in the
savannah zone and revealed the lack of knowledge about the natural maintenance
cycle of the virus during the inter-epidemic period (and over the tropical and equa-
torial dry seasons). A large study to understand the emergence and reemergence of
sylvatic yellow fever will be established between the IPD, the Abidjan Pasteur
Institute, and IPB to detect the circulation of the yellow fever virus, apart from epi-
demics, and map its emergence in the different phytogeographic zones. Permanent
research stations will be developed in the ecotone of rain forests and savannas to
study the yellow fever virus circulation in mosquitoes and natural host from the tree
canopies to the savanna ground. For more than 10 years, thanks to a collaboration
with ORSTOM’s medical entomologists, for periodic mosquito monitoring through-
out the year, it will make possible to detect the low circulation of the YFV among
monkeys and several mosquito species (Aedes africanus, A. opok, A. furcifer-taylori,
and A. luteocephalus). This particular study will help elucidate the mechanism of
YFV maintenance in nature. In pre-forest areas and savannah areas, the circulation
of yellow fever virus is fluctuating, giving rise to epizootics of varying intensity,
which favors sporadic contamination of humans, especially in forest galleries (for-
est residue), where human activity is concentrated toward streams flow. These very
particular ecosystems were named by Max Germain, as “yellow fever zone of emer-
gence” in West and Central Africa (Germain et al. 1982). Beyond this zone is the
epidemic zone, in which yellow fever is occasionally introduced by the displace-
ment of infected individuals. In these savanna zones, the virus is maintained from
1 year to the next by ovarian transmission in selvatic vectors. This discovery was
established experimentally and by virus isolation in male mosquitoes (A. furcifer-
taylori). This maintenance of the virus in eggs allows the passage of dry season and
reappearance from the first rains, giving rise to epizootics whose intensity depends
on the turnover of nonimmune monkeys (Cornet et al. 1978).
The “yellow fever zone of emergence” clearly appeared as the main source of
epidemics in West Africa (Germain 1978). Indeed, all rural yellow fever epidemics
in recent years occurred in this “yellow fever zone of emergence,” which ultimately
represents the specific ecosystem that constitutes the privileged place of vaccination
campaigns and strategies for eradication (Salaun et al. 1981; Robertson et al. 1996).
Finally, this area of yellow fever emergence, well characterized from the spatial
point of view, was going to be clarified in its temporality. Indeed, the development
at the IPD (Coz et al. 1977) and IPB (Gonzalez et al. 1981) of the amplification of
natural weak viral loads by intrathoracic inoculation to mosquitoes colonized at the
IP laboratories by the entomological teams of the IRD, following such excellent
partnership. Another important contribution to the YF epidemiology was made by
Vincent Deubel when, during his stays at the Pasteur Institute in Dakar, he made
enormous progress in understanding the epidemiology of YF in Africa by using the
molecular analysis of viral strains, and he created the notion of YF virus topotypes.
Indeed, this discovery allowed to distinguish YFV strains origin from West or
Central African and thus demonstrated the in situ persistence of viral strains associ-
ated with a given ecosystem.
8 Arbovirus Isolated from Anthropophilic Mosquitoes at

Bangui Pasteur Institute (IPB)
This study was carried out between 1976 and 1986, among the two research stations
including gone located in pre-forest eco-zone of Bouboui (Senegal) and the other in
forest gallery savanna eco-zone Bozo (Central African Republic). The difficulty of
the fieldwork lays in the capture of non-engorged hematophagous arthropod vectors
(i.e., true vectors) and by immediately identifying the specimen collected in the
field and followed by quick deep freezing to preserve living infectious viruses. The
Bozo station was created during this study period, and more than 420,000 mosquito
species identified constituted 14,591 mosquito’s pools that were preserved for virus
isolation by intracerebral inoculation to newborn mice (Fig. 1). The most common
Fig. 1 Bozo scientific station

The Bangui Pasteur Institute and IRD-Bangui permanent field station dedicated to the study of
arbovirus transmission including monthly mosquito trapping and human population survey (serol-
ogy and clinic) located in a Sub-Sudanian domain of the central African forested area including
forest galleries (insert top left) and shrub savanna biome (i.e., yellow fever virus zone of emer-
gence) (Ombella-Mpoko province, Central African Republic, lat. 5°15 N; long. 18° 48E)
anthropophilic species caught were Aedes (Stegomyia) africanus and A. (st.) opok
inside the forest gallery and Aedes (Aedimorphus) vittatus in savannah and
Anopheles gambiae and An. funestus in the houses of the village of Bozo. A total of
321 viruses were isolated belonging to 24 different species: Chikungunya; Bagaza;
Bouboui; Bwamba; Ilesha; Kamese; Middleburg; Mossuril; M’Poko; Nyando;
Orungo; Pata; Pongola; Simbu; Sindbis; Tataguine; Wesselsbron; West Nile; yellow
fever; Zika; Bozo; Kedougou; as well as several unidentified ArD28542 Bunyavirus
and Flavivirus (Georges et al. 1980, 1983; Saluzzo et al. 2017).
Although these results were carried out within limited phytogeographic zones
and using a sole technique of viral isolation (sucking mice inoculation) with a
limited sensitivity, an important arbovirus diversity was revealed including an
active virus circulation among anthropophilic mosquitoes and potentially trans-
mitted to local populations. While the chikungunya, Bozo, and Zika viruses
showed a similar natural cycle that the one for yellow fever virus, several of these
viruses identified in a remote environment recently emerged and spread from such
natural homes, including Chikungunya, Sindbis, West Nile, and recently Zika
(Fig. 2).
Fig. 2 After a long journey on the tracks of central Africa comes the time of another long and
fastidious sample organizing and inventory (Insert bottom left) of the precious arbovirus strain
collection at the Bangui Pasteur Institute: “Bozo virus (ArB 7343); Bouboui virus (ArB 490),
M’Poko virus (BA 365); Petevo virus (ArTB 2032)…”
ArB Arbovirus Bangui, BA Bangui Arbovirus, ArTB Arbovirus Tick Bangui
9 Surveillance of the Arbovirus Sylvatic Circulation

in Senegal and Ivory Coast
Continuous surveillance by land-catch capture of anthropophilic mosquitoes,

mainly Aedes luteocephalus and A. furcifer-taylori, revealed mostly a mosquito-
monkeys’ natural cycles of the arbovirus in the savannah ecosystem, showing also
patterns like that previously described for YFV. Moreover, a new flavivirus, the
Kedougou virus, was described, and a unique dengue 2 virus sylvatic cycle was
identified for the first time in Africa (Saluzzo et al. 1986; Cordellier et al. 1983).
Incidentally, by sharing samples, technics, and reagents with our partners and col-
leagues, dengue virus was isolated during the first dengue virus epidemic described
in Africa within the semi-urban area of Bobo-Dioulasso, Burkina Faso (Gonzalez
et al. 1985).
Eventually, in 1976, this surveillance of YF epizootics (monkeys) in Eastern
Senegal (Kedougou) made possible to anticipate an epidemic with a rapid imple-
mentation of a large-scale vaccination campaign. Sadly, Gambia did not benefit
from these campaigns (Germain et al. 1980; Salaun et al. 1981). Finally, this type of
monitoring YF emergence (infected mosquitoes from monkey hosts) appears as an
early marker of epidemics and reveals the natural active circulation of the virus at a
low level.
10 Surveillance of Epidemics at the Dakar Arbovirus

Reference Center
Numerous arbovirus epidemics have been described in recent years in sub-Saharan

Africa and viruses isolated and characterized at the IPD. A large outbreak of YF
occurred in 1983, in Burkina Faso and Ghana, and this was the beginning of several
outbreaks in West Africa, and the virus which was going to spread in the neighbor-
ing countries and particularly in Nigeria, where epidemics would succeed each
other for several years (Robertson et al. 1996). Eventually, IPD played a decisive
role in the fight against such epidemic by providing tens of millions of doses of yel-
low fever vaccine 17D.
In 1987, a major outbreak of rift valley fever virus (RVFV) occurred in Mauritania,
along the Senegal River, following a survey conducted by IPD’ virologists who
alerted the health authorities about the risk of an epidemic spread in relation with
the ongoing development of the Diama dam of the Senegal River spanning the bor-
der of Senegal and Mauritania (Saluzzo et al. 1985b). This event will sign the emer-
gence of RVF in West Africa which has since continued to expand and this region
and the need of extended survey until today.
Epidemics and isolated cases of dengue viruses were reported, particularly in
Senegal and Burkina Faso (Roche 1983; Gonzalez et al. 1985; Saluzzo et al. 1986).
Finally, a major epidemic of chikungunya was described in 1982 near Dakar
(Saluzzo et al. 1983). Subsequently, these viruses continued to actively circulate,

while the rapid diagnostic tools set up by the IPD allow an appropriate response
(Digoutte et al. 1985; Saluzzo et al. 1985a).
11 Unveiling the Extensive and Active Circulation of Viral

Hemorrhagic Fever Viruses in Central and Ouest Africa
The concept of viral hemorrhagic fever (VHF) arose in the wake of the emergence
of the Ebola virus in Central Africa in 1976 [WHO, 1978]. Thus the “VHF noso-
logical envelope” was made to group these viral diseases accompanied by a severe
and anxiety-provoking clinical presentation for both health professionals and pub-
lic. Today, most of these agents enter the new framework of “high consequences
pathogens,” accordingly to their clinical and public dimensions of severity and,
therefore, showing the need of preparedness as well as awareness. Indeed, as for the
concept of emerging viral diseases, that will be established a few years later by the
scientific community (Morse and Schluederbe 1990), VHF will occupy these two
decades of the end of the century, the epoch at which the IPB will engage an exten-
sive research and understanding of VHF viruses at the regional level. At the begin-
ning, 1979, 3 years after Ebola virus emergence, a wide survey of VHF was set up
at the IPB in close collaboration with the Special Pathogen Branch (SPB) of the
CDC in Atlanta and the USAMRIID’ Special Pathogens Laboratory (SPL), Fort
Detrick. At first, the CDC, thanks to Doctor Karl Johnson, provided the basic mate-
rial for tracking the VHF viruses’ potential circulation in Central Africa. Such were
used for tracking these viruses, the historical and famous CREML slides (CREML
for Congo-Crimean hemorrhagic fever virus, Rift valley fever, Ebola virus, Marburg
virus, and Lassa virus, and later CREML-H with H for Hantavirus). These slides
made at the SPB-CDC then SPL-USAMRIID presented CHF virus infected and
inactivated cells on slide to be used for an indirect fluorescent antibody test. This
test was extremely sensitive with a limited specificity for some of the virus families.
Nevertheless, doing this pioneering research of VHF virus surveillance in Central
Africa, we were able to clearly, and later confirmed with other more specific sero-
logical test and molecular biology, the active circulation of all these viruses in many
areas and zones where then were never suspected (Gonzalez et al. 1983, 1989). The
success of these original and unique finding invites the other Institutes and partners
to continue such surveillance across all sub-Saharan Africa which continues until
today (Saluzzo et al. 1980, 1981; Ivanoff et al. 1982; Gonzalez et al. 1983; Rhodain
et al. 1989; Marrama et al. 2005).
Again, the strong partnerships between medical entomologists and virologists,
between RIIP and ORSTOM (alias IRD), were instrumental for the discovery and
beginning the understanding of VHF virus circulation and ecology in West and
Central Africa (Gonzalez et al. 1983, 1986). Let’s briefly go over these conclusive
and major findings of the RIIP bivalent teams and their partners. From these pioneer
studies on serology and passive surveillance of VHF several fundamentals, hypoth-

esis and dogma emerged.
The Crimean Congo Hemorrhagic fever virus (CCHFV), since its discovery in
the 1960s, was known to essentially circulate in Eastern Europe and the Middle East
with some sporadic cases observed in Congo (alias Zaire) and Uganda. The studies
done at the IPD and the IPB demonstrated a large distribution of CHFV in West and
Central Africa (Wilson et al. 1991) followed by the repetitive isolation of the virus
(Gonzalez et al. 1990) and a clear understanding of its eco-epidemiology in the
region, including the migration north-south of the infected ticks within the cattle
transhumance corridor and the tick vectors fleeing the desertification of the Sahel
and today the climate change lowering the rainfall and moving infected ticks to
lower latitudes (Saluzzo et al. 1985b; Camicas et al. 1994; Trape et al. 1996).
Likewise, an active circulation of Rift Valley Fever virus (RVFV) in West and
Central Africa was also demonstrated showing a consistent prevalence southward,
where it was never observed, close to the edge of the rain forest in Central Africa,
and following the herder (cattle, sheep, and camels), southward, in the Senegal river
basin, to reach out water holes and grassland (Gonzalez et al. 1983). The 1987
RVFV outbreak in Mauritania confirmed such preliminary findings (Saluzzo et al.
1985a, b; Gonzalez et al. 1987; Guillaud et al. 1988), while RVFV was identified,
carrying on, the IPD biosurveillance for arboviruses revealed unexpected host (Gora
et al. 2000; Marrama et al. 2005) or untouched areas of Upper Volta (alias Burkina
Faso) (Akakpo et al. 1989), Central Africa, or Madagascar (Morvan et al. 1991).
Altogether, this emergence prompted at the regional level a permanent biosurveil-
lance system across the borders, including Senegal, Mali, and Guinea (Thiongane
et al. 1998).
The Filoviruses Serological Signatures of Marburg and Ebola virus in Central
Africa Following the VHF program at the IPB, filoviruses were early identified in
human and vertebrate host (Saluzzo et al. 1980; Gonzalez et al. 1983). From there,
Ebola virus signatures were also identified in Gabon 10 years before the first out-
break in the country (Ivanoff et al. 1982). Ultimately, IPB will be the only institute
after 1976 to maintain more than 30 years of active and passive biosurveillance of
Ebola virus until its reemergence in Gabon. There, such emergence was followed by
two scientists, respectively, former researcher, and director of the IPB (Drs. Marie-
Claude and Alain Georges) in that time in charge at the International Center for
Medical Research of Franceville (CRMF), Gabon (Gonzalez et al. 2000, 2018).
Ultimately, the putative chiropteran reservoir of Ebola virus was identified (Leroy
et al. 2005) and largely consider to date as a main known natural host of Ebola virus
in West and Central Africa (Pourrut et al. 2007; Towner et al. 2007; Gonzalez et al.
2008; Maganga et al. 2011; Sylla et al. 2015).
The Arcane of Ebola virus in the Heart of Darkness In the early 1980s, at the
IPB, in the heart of the African continent, we demonstrated, for the first time, that
the population of central Africa presented natural antibodies against the Ebola virus
strains of Zaire (Zaire Ebola virus, EBOV) and Sudan strain (Sudan ebola virus,
SUDV) (Saluzzo et al. 1980; Gonzalez et al. 1983). Also, we showed for the first
time that several mammal species had Ebola virus-reacting antibodies, including
among others rodents, Cavia (Guinea pigs), and dogs.
However, in that time, these observations did not retain scientific community
attention. Actually, because we used a so-called nonspecific immunofluorescence
antibody test (Ebola virus gamma irradiated infected VeroE6 cell line), and despite
the high cutoff we applied to our test (meaning that we increase the chance to elimi-
nate any nonspecific reaction), the scientific community was not in favor of our
findings (i.e., “too high” Ebola antibody prevalence!) and our hypothesis that either
nonpathogenic Ebola virus strains were circulating in the right bank of the Ubangi
river basin, and/or that people, mostly leaving close to the rain forest (7% positiv-
ity), were exposed to the Ebola virus antigen that didn’t last.
However, we later expanded our survey among several countries of Central
Africa, including Cameroon, Chad, Congo Brazzaville, and Gabon and confirmed
our preliminary observations done in the Central African Republic, showing that
these populations of Central Africa had Ebola virus-reacting antibodies without
recording any past severe clinical Ebola-like syndrome and, moreover, presenting
an Ebola virus seroprevalence-positive gradient from the dry savanna to the rain
forest (Gonzalez et al. 1989).
Then, 10 years after, following this path, we went to explore more specifically
the people leaving on the edge and into the Congolese rain forest in Central Africa.
Again, we consistently observed that 19–20% of these populations shared Ebola
virus-reacting antibodies predominantly, but not only with the Ebola strain isolated
from Zaire, rather than to the Ebola strain isolated from Sudan. We also consistently
demonstrated that hunters were two times more exposed to the Ebola virus antigen
(Johnson et al. 1993a, b).
Ultimately, entering the second millennium, we demonstrated and confirmed our
previous finding, using an ELISA test with purified Ebola virus antigen – a test sup-
posed to be in that time, the gold standard for filovirus serology. Finally, the same
targeted populations and same antibody prevalence were found and confirmed else-
where without epidemic manifestation (Gonzalez et al. 2000). Ultimately, more
than 30 years after our primary observations, using a highly sensitive and specific
ELISA test, it was conclusively and definitively demonstrated that a fifth or the
quarter of the native population of the rain forest had antibody to Ebola virus with-
out, individually, recording any severe disease by the past (Becquart et al. 2010;
Gonzalez et al. 2008).
Conclusively, from our early observation, peoples as well as animals from the
Central African Basin of the Congolese rain forest as well as from the Upper
Guinean Forest of West Africa, the later considered as a relic of the primary
Congolese rainforest, are exposed during their lifetime to the Ebola virus antigen
(infected by the virus or in contact with the viral protein) and produce antibodies
against the virus (Schoepp et al. 2014; Olivero et al. 2016; Gonzalez et al. 2019).
Nowadays, Ebola antibody prevalence appears largely spatially distributed
across the African continent in the absence of severe clinical presentation and/or
outbreak manifestation (Gonzalez 2000, 2008; Bower & Glynn 2017). Although
with these, one time neglected, but fundamental observations as well as the identifi-
cation of potential natural reservoir and hosts form the rain forest, we begin to bring
some light into the darkness of Ebola virus circulation and able to theorize on poten-
tial natural cycle(s) of Ebola virus transmission and the risk of emergence (Allela
et al. 2005; Pourrut et al. 2007; Gonzalez et al. 2008, 2019; Sylla et al. 2015).
Hunting for Lassa fever virus in Central Africa, a new virus from the arenavirus
family (alias Mammarenavirus) was isolated, the Mobala virus, which joined a pre-
vious unidentified arenavirus Ippy virus also from the Central African Republic.
Both were isolated from two rodent hosts in different ecosystems (Gonzalez et al.
1984). Such original findings corroborate by some other from South Africa and led
to two fundamental conceptual considerations including when Lassa fever virus
does not occur in suspected endemic area and micromammals (murine) are present
in these space, other arenavirus became vicariant and infect same or close related
rodent host (Mastomys spp.) (Gonzalez et al. 1986); most of the arenaviruses from
the Old and New World have been isolated and find specifically associated with a
rodent species clades, altogether in favor of a long-term coevolution of both rodents
and Mammarenavirus (Gonzalez and Duplantier, 1999).
Conclusively, the RIIP and IRD teams, with their colleagues and partner institu-
tions devoted to the research on arboviruses, vectors, and viral hemorrhagic fever
viruses, provided, and continue to provide, a tremendous source of knowledge of
such one-time neglected viruses. Convincingly, arbovirologists used such transdis-
ciplinary methodologies – virus, hosts, vectors, and environments – along with
enduring capacity building among research institutes and partners, pioneering the
now widely recognized One Health approach has a compulsory strategy for a com-
prehensive understanding of such complex vector-transmitted diseases and a sus-
tainable biosurveillance which naturally applies.
References
Akakpo AJ, Some MJ, Bornarel P, Jouan A, Gonzalez JP (1989) Epidémiologie de la fièvre de la
vallée du Rift en Afrique de l’Ouest. 1. Enquêtes sérologique chez les ruminants domestiques
au Burkina Faso. Bulletin de la Société de Pathologie Exotique 82:321–331
Allela L, Bourry O, Pouillot R, Délicat A, Yaba P, Kumulungui B, Rouquet P, Gonzalez JP, Leroy
EM (2005) Ebola virus antibody in dogs and human risk. Emerging Infect Dis 11(3):385–390
Becquart P, Wauquier N, Mahlakõiv T, Nkoghe D, Padilla C, Souris M, Ollomo B, Gonzalez JP,
De Lamballerie X, Kazanji M, Leroy E (2010) High prevalence of both humoral and cellular
immunity to Zaire Ebolavirus among rural populations in Gabon. PLoS One 5(2):e9126
Bower H, Glynn JR (2017) A systematic review and meta-analysis of seroprevalence surveys of
ebolavirus infection. Scientific Data 4:160133. https://doi.org/10.1038/sdata.2016.133
Camicas J-L, Cornet J-P, Gonzalez JP, Wilson ML, Adam F, Zeller HG (1994) La fièvre hémor-
ragique de Crimée-Congo au Sénégal: Dernières données sur l’écologie changes in CCHF
virus. Res Virol 146:131–140
Cannon DA, Dewhurst F (1953 Dec) Vaccination by scarification with 17D yellow fever vaccine
prepared at Yaba, Lagos, Nigeria. Ann Trop Med Parasitol 47(4):381–393
Cordellier R, Bouchite B, Roche JC, Monteny N, Diaco B, Akoliba P (1983) Circulation selva-
tique du virus Dengue 2, en 1980, dans les savanes subsoudaniennes de Côte d’Ivoire. Cahier
ORSTOM. Sér Ent Méd et Parasitol 21:165–179
Cornet M, Chateau R, Valade M, Dieng PL, Raymond H, Lorand A (1978) Données bio-écologiques
sur les vecteurs potentiels de virus amaril. Cahier ORSTOM, Sér Ent Méd Parasitol 16:315–341
Cornet M, Robin Y, Adam C, Valade M, Calvo M (1979) Transmission expérimentale comparée
du virus amaril et du virus Zika chez Aedes aegypti L. Cah ORSTOM, Entomol Med Parasitol
17:47–53
Coz J, Valade M, Cornet M, Lemoine M, Lorand L (1977) Utilisation du moustique pour la multi-
plication des arbovirus. Cah. O.R.S.T.O.M., sér. Ent. méd. et Parasitol. XV(3):209–212
Digoutte JP, Saluzzo JF, Adam F (1985) Recent data on hemorrhagic fevers in West Africa. Bull
Soc Pathol Exot Filiales 78(5 Pt 2):874–878
Georges AJ, Saluzzo JF, Gonzalez JP, Dussarat GV (1980) Arboviroses en centrafrique: incidence
et aspects diagnostiques chez l’homme. Med Trop 40:561–568
Georges AJ, Wahid SA, Meunier DY, Georges MC, Saluzzo JF, Peters CJ, McCormick JB,
Gonzalez JP (1983) Serological evidence of endemic Zinga virus and Rift Valley Fever virus in
Central African Republic. Lancet 1(8337):1338
Germain M, Francy DB, Monath TP, Ferrara L, Bryan J, Salaun JJ, Heme G, Renaudet J, Adam C,
Digoutte JP. Yellow fever in the Gambia, 1978–1979: entomological aspects and epidemiologi-
cal correlations. Am J Trop Med Hyg. 29(5):929-40:1980. doi: 10.4269/ajtmh.1980.29.929.
Germain M (1986) La fièvre jaune en Afrique de l’Ouest: une dynamique spatiale. ORSTOM
Actu 14:9–12
Germain M, Francy DB, Monath TP, Ferrara L, Bryan J, Salaun JJ, Heme G, Renaudet J, Adam C,
Digoutte JP (1980) Yellow fever in the Gambia, 1978–1979: entomological aspects and epide-
miological correlations. Am J Trop Med Hyg 29(5):929–940
Germain M, Cornet M, Mouchet JP, Monath TP, Hervé JP, Salaun JJ, Saluzzo JF, Camicas JL,
Hervy JP, Robert V, Deubel V, Gonzalez JP, Digoutte JP, Darwish DO (1982) Recent advances
in research regarding sylvatic yellow fever in West and Central Africa. Bull Inst Pasteur
80:315–330
Gonzalez JP (2005) Ebola virus circulation in Africa: a balance between clinical expression and
epidemiological silence. Epidemiologie 98:210–217
Gonzalez JP, Duplantier JM (1999) The arenavirus and rodent coevolution process: a global view
of a theory. In: Saluzzo JF, Dodet B (eds) Factors in the emergence and control of rodent-borne
diseases. Elsevier, Paris, pp 39–42
Gonzalez JP, Saluzzo JP, Herve JP (1981) Intérêt de la technique d’inoculation intrathoracique à
Aedes aegypti dans l’isolement et le réisolement des arbovirus. Annales de L’institut Pasteur
Virologie 132:519–527
Gonzalez JP, McCormick JB, Saluzzo JF, Georges AJ (1983) Les fièvres hémorragiques afric-
aines d’origine virale en République Centrafricaine. Cah. ORSTOM, Ser. Ent. Méd. et Parasit
XXI(2):119–130
Gonzalez JP, McCormick JB, Georges AJ, Kiley MP (1984) Mobala virus: biological and physico-
chemicals properties of a new arenavirus isolated in the Central African Republic. Annales de
Virologie de l’Institut Pasteur 135E:145–158
Gonzalez JP, Georges AJ, Fiset P, Saluzzo JF, Wisseman JC (1985) Approche sérologique sur
l’incidence des Rickettsioses en République Centrafricaine. Bulletin de la Société de Pathologie
Exotique 78:153–156
Gonzalez JP, Georges AJ, Kiley MP, Meunier DMY, Peters CJ, McCormick JB (1986) Evolutionary
biology of a Lassa virus complex. Med Microbiol Immunol 175:157–159
Gonzalez JP, Bouquety JC, Lesbordes JL, Madelon MC, Mathiot CC, Meunier DMY, Georges AJ
(1987) Rift Valley fever virus and Haemorrhagic fever in the central African Republic. Annales
de Virologie de l’Institut Pasteur 138(385):390
Gonzalez JP, Josse R, Johnson ED, Merlin M, Georges AJ, Abandja J, Danyod M, Delaporte
E, Dupont A, Ghogomu A, Kouka-Bemba D, Madelon MC, Sima A, Meunier DMY (1989)
Antibody prevalence against haemorrhagic fever viruses in randomized representative central

African populations. Res Virol (Annales de l’Institut Pasteur) 140:319–331
Gonzalez JP, Leguenno B, Guillaud M, Wilson ML (1990) A fatal case of Crimean-Congo
Haemorrhagic fever in Mauritania: virological and serological observations suggest epidemic
transmission. Trans R Soc Trop Med Hyg 84:573–576
Gonzalez JP, Nakoune E, Slenczka W, Vidal P, Morvan J (2000) Ebola and Marburg virus antibody
prevalence in selected populations of the central African republic. Microbes Infect 1(1):6
Gonzalez JP, Gouilh MA, Reynes JM, Leroy E (2008) Bat borne viral zoonoses emergence PART
I synthetic analyses chapter 6. In: Pierce Colfer CJ, Kleinau E (eds) People, health and for-
ests. Press
Gonzalez JP, Wauquier N, Vincent T (2018) Revisiting Ebola, a quiet river in the heart of Africa.
Med Sante Trop 28(1):12–17. https://doi.org/10.1684/mst.2018.0751
Gonzalez JP, Souris M, Sylla M, Veas F, Vincent T (2019) Essay on the elusive natural history of
Ebola viruses. Intech Edit. https://doi.org/10.5772/intechopen.88879
Gora D, Thiongane Y, Fontenille D, Maoulouth D, Amadou S, Ruel T, Gonzalez JP (2000) The
potential role of rodents in the enzootic cycle of Rift Valley fever virus in Senegal. Microbes
Infect 4:1–4
Guillaud M, LeGuenno B, Wilson M, Desoutter D, Gonzalez JP, Digoutte JP (1988) Prévalence en
anticorps contre le virus de la Fièvre de la vallée du Rift chez les petits ruminants du Sénégal.
Annales de Virologie de l’Institut Pasteur 139:455–459
Ivanoff B, Duquesnoy P, Languillat G, Saluzzo JF, Georges AJ, Gonzalez JP, McCormick JB
(1982) Haemorrhagic fever in Gabon. I. Incidence of Lassa, Ebola and Marburg viruses in
Haut-Ogoué. Trans R Soc Trop Med Hyg 76(6):719–720
Johnson ED, Gonzalez JP, Georges AJ (1993a) Filovirus activity among selected ethnic groups
inhabiting the tropical forest of equatorial Africa. Trans R Soc Trop Med Hyg 87:536–536
Johnson ED, Gonzalez JP, Georges AJ (1993b) Hemorrhagic fever virus activity in equatorial
Africa: distribution of filovirus reactive antibody in the Central African Republic. Trans R Soc
Karabatsos N (1985) International catalogue of arboviruses, including certain other viruses of
vertebrates. American Society of Tropical Medicine and Hygiene for the Subcommittee on
Information Exchange of the American Committee on Arthropod-borne Viruses, San Antonio
Leroy E, Kumulungui B, Pourrut X, Rouquet P, Yaba P, Délicat A, Paweska J, Zaki SR,
Rollin P, Gonzalez JP, Swanepoel R (2005) Fruit bats as reservoirs of Ebola virus. Nature
438(7068):575–576
Maganga GD, Bourgarel M, Ebang Ella G, Drexler JF, Gonzalez JP, Drosten C, Leroy EM (2011)
Is marburg virus enzootic in Gabon? JID S3:204
Marrama L, Spiegel A, Ndiaye K, Sall AA, Gomes E, Diallo M, Thiongane Y, Mathiot C, Gonzalez
JP (2005) Domestic transmission of Rift Valley fever virus in Diawara (Senegal) in 1998. South
East Asian J. Trop. Med. Public Health 36(6):1487–1495
Molez JF (2006) Question sur l’épidémiologie du virus West-Nile. A propos de la découverte du
virus WN en zone sèche et en dehors des périodes de densité culicidienne et de migration avi-
aire. (PT-EDEN Afro), 10/06, Dakar, Sénégal
Monath TP, Kinney RM, Schlesinger JJ, Brandriss MW, Brès PJ (1983) Ontogeny of yellow fever
17D vaccine: RNA oligonucleotide fingerprint and monoclonal antibody analyses of vaccines
produced world-wide. Gen Virol 64(Pt 3):627–637
Morse SS, Schluederberg A (1990 Jul) From the National Institute of Allergy and Infectious
Diseases, the Fogarty International Center of the National Institutes of Health, and the
Rockefeller University. Emerging viruses: the evolution of viruses and viral diseases. J Infect
Dis 162(1):1–7
Morvan J, Saluzzo JF, Fontenille D, Rollin PE, Coulanges P (1991) Rift Valley fever on the east
coast of Madagascar. Res Virol 142(6):475–482
Morvan JM, Deubel V, Gounon P, Nakouné E, Barrière P, Murri S, Perpète O, Selekon B, Coudrier
D, Gautier-Hion A, Colyn M, Volehkov V (1999) Identification of Ebola virus sequences pres-
ent as RNA or DNA in organs of terrestrial small mammals of the Central African Republic.
Microbes Infect 14:1193–1201
Olivero J, Fa JE, Real R, Farfan MA, Marquez AL, Vargas JM, Gonzalez JP, Nasi R (2016)
Mammalian biogeography and the Ebola virus in Africa. Mammal Rev 47. MAMMAL-15-57. R2
Pourrut X, Gonzalez JP, Leroy E (2007) Spatial and temporal patterns of Ebola virus antibody
prevalence in the putative bat species reservoir. J Infect Dis 196(Suppl 2):S176–S183
Rhodain F, Gonzalez JP, Mercier E, Helynck B, Larouze B, Hannoun C (1989) Arbovirus infec-
tions and viral haemorrhagic fevers in Uganda-results of a serological survey in the Karamoja
district, 1984. Trans R Soc Trop Med Hyg 83:851–854
Robertson SE, Hull BP, Tomori O, Bele O, LeDuc JW, Esteves K (1996) Yellow fever: a decade of
reemergence. JAMA 276(14):1157–1162
Roche JC, Cordellier R, Hervy JP, Digoutte JP, Monteny N (1983) Isolement de 96 souches de
virus Dengue 2 à partir de moustiques capturés en Cote d’Ivoire et en Haute Volta. Ann Virol
(Institut Pasteur) 134E:233–244
Salaun JJ, Germain M, Robert V, Robin Y, Monath TP, Camicas JL, Digoutte JP (1981) Yellow
fever in Senegal from 1976 to 1980. [Article in French] Med Trop (Mars) 41(1):45–51
Saluzzo JF, Gonzalez JP, Georges AJ, Johnson KM (1980) Note préliminaire sur la présence
d’anticorps vis-à-vis du virus Ebola parmi les populations du sud-est de la République
Centrafricaine. Bulletin de la Société de Pathologie Exotique 73(3):238–241
Saluzzo JF, Gonzalez JP, Georges AJ, Johnson KM (1981) Mise en évidence d’anticorps vis-à-vis
du virus Marburg parmi les populations humaines du sud-est de la République Centrafricaine.
Comptes Rendus de l’Académie des Sciences Paris, 292, (IIIn), pp 29–31
Saluzzo JF, Gonzalez JP, Georges AJ (1982) Mise en évidence d'anticorps anti-virus Marburg dans
les populations humaines du sud-est de la République Centrafricaine. Annales de Virologie de
l’Institut Pasteur 133E:129–131
Saluzzo JF, Cornet M, Digoutte JP (1983) [Outbreak of a Chikungunya virus epidemic in western
Senegal in 1982] in French. Dakar Med 28(3):497–500
Saluzzo JF, Aubry P, McCormick J, Digoutte JP (1985a) Haemorrhagic fever caused by Crimean
Congo haemorrhagic fever virus in Mauritania. Trans R Soc Trop Med Hyg 79(2):268
Saluzzo JF, Digoutte JP, Camicas JL, Chauvancy G (1985b) Crimean-Congo haemorrhagic fever
and Rift Valley fever in south-eastern Mauritania. Lancet 12(8420):116
Saluzzo JF, Cornet M, Adam C, Eyraud M, Digoutte JP (1986) Dengue 2 au Sénégal orien-
tal: enquête sérologique dans les populations simiennes et humaines. Bull Soc Pathol Exot
79:313–322
Saluzzo J-F, Tom V, Jay M, Veas F, Gonzalez J-P (2017) Arbovirus discovery in Central
African Republic (19731993): Zika, Bozo, Bouboui, and more. Ann Infect Dis Epidemiol
2(3):Article 10221
Schoepp RJ, Rossi CA, Khan SH, Goba A, Fair JN (2014) Undiagnosed acute viral febrile illnesses,
Sierra Leone. Emerg Infect Dis 20(7):1176–1182. https://doi.org/10.3201/eid2007.131265
Sylla M, Pourrut X, Diatta M, Diop BM, Ndiaye M, Gonzalez JP (2015) Chiropteran and filovi-
ruses in Africa: unveiling an ancient history. Afr J Microbiol Res 22:1446–1472. https://doi.
org/10.5897/AJMR2015.7455. Article Number: 1739F0D53418 ISSN 1996-0808
Theiler M, Sellards AW (1928) The immunological relationship of yellow fever as it occurs in
West Africa and in South America. Ann Trop Med Parasitol 22:449–460
Thiongane Y, Thonnon J, Zeller H, Lo MM, Faty A, Diagne F, Gonzalez JP, Akakpo JA, Fontenille
D, Digoutte JP (1998) Données récentes de l’épidémiologie de la Fièvre de la Vallée du Rift
(F.V.R.) au Sénégal. Dakar Med 1998(96):1–6
Towner JS, Pourrut X, Albarino CG, Nkogue CN, Bird BH, Grard G, Ksiazek TG, Gonzalez JP,
Nichol ST, Leroy EM (2007) Marburg virus infection detected in a common African Bat. PLoS
One 2(1):e764
Traore-Lamizana M, Fontenille D, Zeller HG, Mondo M, Diallo M et al (1996) Surveillance for
yellow fever virus in eastern Senegal during 1993. J Med Entomol 33:760–765
Trape JF, Godeluck B, Diatta G, Rogier C, Legros F, Albergel J, Pepin Y, Duplantier JM (1996) The
spread of tick-borne borreliosis in West Africa and its relationship to sub-Saharan drought. Am
J Trop Med Hyg. 54(3):289–293. https://doi.org/10.4269/ajtmh.1996.54.289. PMID: 8600768
Vasilakis N, Tesh RB, Popov V, Widen SG, Wood TG, Forrester NL, Gonzalez J-P, Saluzzo J-F,
Alkhovsky S, Lam SK, McKenzie JS, Walker PJ (2019) Exploiting the 120 year legacy of the
arbovirus hunters. Virology 11:471
WHO. Ebola Haemorrhagic Fever in Sudan, 1976 (1978) Report of a WHO/International Study
Team. Bull World Health Organ 56(2):247–270. PMID: 307455
Wilson ML, Gonzalez JP, Cornet JP, Camicas JL (1991) Transmission of Crimean-Congo hemor-
rhagic fever virus from experimentally infected sheep to Hyalomma truncatum ticks. Res Virol
142:395–404
From Bwamba to the Present:
The Changing Forest of Arbovirology
Andrew D. Haddow
Abstract The Yellow Fever Research Institute (YFRI), later renamed the East
African Virus Research Institute (EAVRI), in Entebbe, Uganda, served as the key
center of arbovirus research in East Africa in the mid-twentieth century. My grand-
father, Professor Alexander John Haddow, initially a researcher at YFRI, later the
Director of EAVRI, led a small group of researchers who often persevered through
inhospitable conditions and at great risk to themselves, to help lay a foundation of
knowledge for the field of arbovirology. During these research activities, he had
many adventures and sometimes misadventures. These experiences shaped him and
his career and were later recounted to me as bedtime stories by my father when I
was a child. This personal narrative explores how my grandfather’s experiences in
East Africa linked three generations of my family whose fields of study have inevi-
tably focused on infectious diseases. It also provides a testament to some of the
changes the field of arbovirology has undergone in the last 60 years.
When I was about 6 years old, one of my teachers, Mrs. X (name withheld), asked
each of the students in my class what they wanted to be when they grew up. As she
went around the classroom, standard answers were given (doctor, firefighter, astro-
naut, nurse, policeman, artist), but when she got to me, I said, “A researcher.” She
looked at me inquisitively and said, “What is a researcher?” I said, “You know a
researcher.” Mrs. X gave me a stern look and said, “There is no such thing.” I looked
at Mrs. X and told her researchers do exist, and my grandfather had, in fact, been a
researcher. After all, he had studied mosquitoes and viruses in Africa, caught mon-
key’s, saw elephants explode, avoided poisonous snakes, been cursed by a witch
doctor, and climbed mountains. She and I went back and forth for a few minutes, as
I tried to explain to her researchers did exist. She finally cut me off and told me to
come back to class the next day with an actual job.
When I was approached to contribute to this work, the editors asked me to pro-
vide a narrative of some of my grandfather’s experiences in Africa, and how they
influenced me and my career. This stemmed from a talk I gave at the annual meeting
A. D. Haddow (*)
Department of Molecular and Cellular Biology, Kennesaw State University,
Kennesaw, GA, USA
e-mail: ahaddow@kennesaw.edu
206 A. D. Haddow
of the American Committee on Arbovirology (ACAV) some years ago where I

recounted some of the bedtime stories my father told me relating to my grandfather,
Professor Alexander John Haddow, and how they had inspired me to pursue a career
in arbovirology. I only met my grandfather as an infant, and thus everything I know
of him comes from those stories recounted by my father, or my grandfather’s close
friends. These stories captured my imagination and helped inspire me to choose my
present career path. Upon his death, my grandfather’s long-time friend, Professor
P.C.C. Garnham, wrote an excellent biographical memoir of his life which covered
many aspects of his career, including a glimpse into his personality. The reader is
referred to that text for more in-depth information. This chapter will focus on some
of the stories or lessons handed down from my grandfather to my father, and ulti-
mately to me. However, this is an impossible task without also touching on a few
other family members’ “stories,” for these link three successive generations of
Haddow’s whose careers have focused on those pathogens that cause human disease.
My grandfather spent his youth in Glasgow, Scotland, whereas my father
(Alastair Haddow) spent his formative years split between Entebbe, Uganda, and
a boarding school located at the base of Mount Elgon in Kenya. Both knew what
their careers would be before they were 7 years old; my grandfather wanted to study
mosquitoes and the diseases they caused (Fig. 1), while my father wanted to prac-
tice medicine. As a child, my grandfather took my father with him to trap mosqui-
toes at the Zika Tower (known colloquially as the “Haddow Tower”), showed him
how to handle laboratory and trapped animals, survive in the bush, and occasionally
took him into the field during scientific expeditions. Thus, it is of no surprise a sig-
nificant amount of information was imparted to my father, who in turn imparted
what he had learned to me. Consequently, I knew at a young age to sleep with my
feet toward the opening of the tent in case a lion was to drag me out in the middle of
the night (I might have a chance to call for help, or grab the sling of my rifle, as I
was being pulled out of the tent). It would also prevent my skull from being crushed
in the event a hyena wanted the snack sleeping in the tent. Such skills might have
seemed out of place in rural Missouri, the location of my childhood, but they would
be put to use as an adult.
1 The Compound
My grandfather originally started his research career in Kenya focusing on malaria

but, in 1942, transferred to the Rockefeller Foundation’s Yellow Fever Research
Institute (YFRI) in Entebbe, Uganda. After the Rockefeller Foundation divested
itself from most of its overseas laboratories in 1950, this laboratory became known
as the East African Virus Research Institute (EAVRI), and shortly thereafter he
became its Director (Fig. 2).
During the early days of YFRI/EAVRI, Entebbe was a fraction of its current size
and was really a small town rather than the small city it is today. At the time, EAVRI
was located a few miles outside of town, and the Institute was known locally as
From Bwamba to the Present: The Changing Forest of Arbovirology 207
Fig. 1 Drawing of “Stegomyia auther [sic] of yellow fever greatly magnified” by Alexander John
Haddow, age 7. (Reproduced from the Haddow Collection)
“The Compound.” Today, the compound is no longer in the countryside, and in

many ways, it is hard to image it ever was. One entered the compound through a
gate house, which also housed the dark room for developing film. At the time, there
was no “fence” along the compound’s perimeter, although there was a nightwatch-
man. My grandfather’s house (which is still there today) was the first house on
one’s right.
The original buildings are still largely present, though today they are intermin-
gled with the numerous buildings that have cropped up since the late 1980s. EAVRI
was largely self-sufficient, and the compound had a metal shop, a woodworking
shop, and a fabrication shop. The main building housed offices and laboratories.
Originally, the director’s office was just inside the main door, but the room tended
to heat up during the afternoon sun and was moved by my grandfather to a cooler
location in the building. Another benefit was the “new” office also had a direct view
of Lake Victoria. Next to the main building was a garage that housed EAVRI’s
vehicles, ranging from short- and long-wheel base Land Rovers to Volkswagen
Type II Buses or Kombi’s. Behind the garage was a medical clinic which treated
208 A. D. Haddow
Fig. 2 Alexander John Haddow then Director of the East African Virus Research Institute in
Entebbe, Uganda working in his office (circa 1960s). (Reproduced from the Haddow Collection)
persons from the surrounding area. Blood samples were often collected from these
patients, with serology being run to determine previous and recent exposures to
circulating arboviruses.
There were also several nonhuman primate buildings, known as monkey houses.
The Institute maintained a colony of rhesus macaques for disease modeling, and to
use as sentinel animals. One of these rhesus macaques was the animal from which
Zika virus was originally isolated – monkey rhesus 766 (MR766). The laboratory
also kept several other species onsite, including African green monkeys, colobus
monkeys, pottos, and baboons. My grandfather warned my father to be careful
working with the later, as he had been bitten through the thick leather glove he was
wearing while handling one in the laboratory (the canine teeth of an adult baboon
being about 2 inches in length). Colonies of mice were maintained in two separate
mouse buildings, each containing approximately 26,000 mice. There was also a
light-controlled laboratory for animal or mosquito experiments. The atmosphere
within the compound was relaxed and easygoing. Staff that lived onsite would go
home for lunch, while the commuters would often eat sitting under the mango trees
which dotted the compound. There one could watch the family of mongooses that
lived on the compound’s grounds hunting and playing.
The focus of EAVRI was research, and this is evident from the Institute’s publi-
cation output. This was likely helped by my grandfather shielding the Institute and
staff from the bureaucratic red tape created by the British Government. When the
bureaucrats from London became too tiring, EAVRI would attempt to “ignore”
them, the letter in question would have been lost in the mail, or communication
would happen to be down at the time. With this shielding came expectations that the
staff would focus on research and that they did. For example, when talking to
Professor Philip Corbet years ago, he told me my grandfather absolutely despised

meetings as the ultimate time wasters and instead wanted people in the lab working.
Thus, meetings at EAVRI were rare (sometimes once a month), extremely short
(never more than an hour), and to the point. How times have changed.
It is also important to remember that the modern safety devices we take for
granted today (i.e., biosafety cabinets) didn’t exist at the time. Professor Jack
Woodall once joked with me (after showing a picture of himself grinding up infected
mouse brains on a laboratory bench) that they would sit next to open windows when
working with viruses or infected tissues, and only those with the best skills “sur-
vived.” Unfortunately, laboratory-acquired infections were commonplace. Unlike
today, these reports were written up and published. It is unfortunate that in the pres-
ent climate of hyper-regulation, knee-jerk reactions, and policies driven by percep-
tions or feelings rather than empirical data, data on laboratory-acquired infections
and serosurveillance of laboratory staff are not published. The irony is that such data
are critical to determine the effectiveness of implemented biosafety practices.
The research output created by such a small number of staff, using rudimentary
practices by today’s standards, and the general atmosphere of the compound, which
in essence embodied many of the principles of the Enlightenment, is unlikely to be
repeated in the present day. It is therefore important to remember, when researchers
are actually able to focus on research (even with minimal funding), rather than
bureaucratic red-tape, great things can be achieved.
2 The Mountains of the Moon
My grandfather enjoyed many pursuits including mountaineering (Fig. 3). He made

four first accents of peaks within the Ruwenzori Mountains, known as The
Mountains of the Moon. These mountains became a focus of his free time due to
their proximity to his field sites in the Bundibugyo District, where he not only
enjoyed climbing but photographing this beautiful region. He took full advantage of
his time in the mountains and continued his studies on those primate species found
within, in addition to documenting anthropological aspects of those tribes inhabit-
ing this unique ecological landscape. It’s clear he never really stopped “working.” It
was during one of these expeditions he nearly lost his life.
He had previously had a near miss during a thunderstorm. He and the porters
were making their way up one climb, when the sky rapidly darkened and it appeared
that a thunderstorm was imminent. Before he could say a word, all the porters had
thrown down their gear and were frantically setting up the tent. Apparently, this was
the fastest the tent had ever been set up (occurring within a minute). As soon as the
tent was up, the porters and my grandfather ducked inside just as fist-sized hail
began to fall. Evidently, the porters knew how to read the weather, and their quick
thinking likely not only saved their lives, but my grandfathers.
It was during one of these climbs when one thinks everything is going right,
everything inevitably goes wrong. My grandfather and the porters were making
210 A. D. Haddow
Fig. 3 Alexander John Haddow returning from a climb in the Ruwenzori Mountains, Uganda
(circa 1940s). (Reproduced from the Haddow Collection)
their way up a steep slope when my grandfather slipped on a piece of fallen bamboo.
When he finally stopped tumbling and sliding, he discovered he had a spiral com-
pound fracture of his tibia. Mud and filth covered him and the wound. Somehow, he
didn’t go into shock and was able to bind his leg to cover the exposed bone and keep
it from shifting. The porters climbed down to him and assessed the situation. They
immediately cut poles from the patch of nearby bamboo, wrapped him in the tent,
and proceeded to carry him down the mountain (Fig. 4). This took hours, but would
pale to the multi-hour drive on a mud road from Fort Portal to Kampala. The ride
was extremely jarring, and with each bump, the exposed bone would issue forth
unbelievable pain. At the hospital in Kampala, the surgeon debated amputating the
leg with my grandfather. However, the surgeon decided (or more likely was told) to
set the bone as best as possible. My grandfather then spent months in a full leg cast,
Fig. 4 Alexander John Haddow being carried down one of the mountains in the Ruwenzori Range,
Uganda, wrapped in a tent following a fall and spiral compound fracture of his tibia. Notice the
boots sticking out (circa 1940s). (Reproduced from the Haddow Collection)
and it is by a miracle he didn’t get an infection (remember they wouldn’t have had
access to antibiotics).
Unfortunately, between this injury and a previous ankle break that was bad
enough to keep him out of World War 2, he experienced chronic pain for the rest of
his life – often walking with a cane. Of course, when he returned to Glasgow years
later, he didn’t just use a standard cane. After all, Glasgow was quite a “rough” city
at the time; hence, he walked with a sword cane.
3 The Ladders in the Trees
Maybe it was his love of climbing that led him to first scale the tall tropical trees in
search of mosquitoes, or maybe it was his love for exploring the unknown. Either
way, my grandfather was one of the pioneers of vertical trapping to study mosqui-
toes, nonhuman primates, and arboviruses in Africa. His lifelong friend and col-
league Professor William H.R. Lumsden wrote of him:
Working for long stretches as the only white man in Bwamba County, Uganda, beyond the
north-west spur of the Ruwenzori, Haddow improved and extended methods for the study
of the ecology of the mosquitoes which were concerned with the transmission of yellow-
fever virus among the monkeys of the forest canopy and of the forest/cultivation interface.
Forest mosquitoes were to be henceforward his consuming interest. Studies of mosquitoes
of the forest canopy were not made then, as they are nowadays, from safe, steel towers
212 A. D. Haddow
erected by competent engineers, but from flimsy, precarious platforms of poles lashed into
tree forks, often by ladders and footholds nailed to the trunks of trees. Not only did that
work establish clearly the patterns of transmission and survival of yellow-fever virus in East
Africa but also it produced a wealth of knowledge and understanding of the ways of life of
scores of species of forest mosquitoes, and of the monkeys and other vertebrates which
provided them with their blood meals. It set a pattern for field entomological studies of
arbovirus transmission.
Those vertical catches he was involved in were carried out in Uganda from the early
1940s into the mid-1960s. The earliest of these took place in a Banana Plantation in
Bwamba County in the early 1940s, but soon after, vertical collections were being
made in the nearby forests. Initially, these catches were made by lashing wooden
ladders to tree trunks, from which teams of catchers would ascend to small plat-
forms overlooking the abyss, sometimes over 80 feet (~24 m) below (Fig. 5). These
sites then became the location of multiyear studies, which still form the foundation
of several fields of study today. Over time, these techniques and methods were
refined, culminating in the construction of a 120-foot (36.6 m) steel tower (The
Tower), which was built using funds secured by my grandfather from the World
Health Organization.
What is commonly not known is that the Zika Forest was not the original loca-
tion of The Tower. In fact, some of the old black and white pictures of The Tower
often attributed to the Zika Forest were taken from its original location, Mpanga
Forest. The Tower had initially been erected in Mpanga Forest as my grandfather
was hoping to find a location closer to EAVRI for logistical reasons. Ultimately, it
was determined the catches at Mpanga Forest were not optimal, and The Tower was
moved to its current location in the Zika Forest in 1961 (Fig. 6), which was extremely
close to EAVRI. This location also had the benefit of being almost on the equator
which was optimal for studies investigating mosquito circadian rhythms. Zika
Forest was thus the location that my father was able to often visit and even spend
time with my grandfather trapping mosquitoes on the various platforms. One should
not be surprised that many of my bedtime stories revolved around this special forest.
You may be asking yourself if The Tower was only moved to the Zika Forest in
1961, how was the original isolation of the Zika virus made in 1947? Zika Forest
originally had wooden platforms like those found in Bwamba County and else-
where. The sentinel rhesus from which the Zika virus was originally isolated was on
a wooden platform in the canopy of the Forest at the time it was infected.
While both mosquito collections and sentinel nonhuman primates were used to
detect circulating arboviruses using vertical platforms (as well as to study mosquito
circadian rhythms), researchers at YFRI/EAVRI also needed to conduct serosurveil-
lance on those nonhuman primates suspected of serving as amplification or reser-
voir hosts of yellow fever virus. At the time, their only option was to shoot these
animals in the trees (Fig. 7). My father told me he watched my grandfather perform
this task on several occasions. He said “I would see movement in the canopy, and a
tiny dark shape would jump from one tree to another, and then there would be a loud
bang and the monkey would fall to the ground. Everyone would immediately run to
the monkey, and my father would do a quick cardic puncture with a large needle and
Fig. 5 Mosquito-catcher climbing to the 82-foot platform at Mamirimiri, Bwamba County,

Uganda (circa 1940s). (Reproduced from the Alexander John Haddow Collection housed at the
University of Glasgow, Scotland)
214 A. D. Haddow
Fig. 6 Alexander John Haddow (in shorts) standing at the base of 120-foot Tower in the Zika
Forest, Uganda (circa 1960s). (Reproduced from the Alexander John Haddow Collection housed at
the University of Glasgow, Scotland)
syringe and then draw out an immense amount of blood. The blood would then be
stored on ice in a small chest.” The reader should note my father had 20/20 vision,
and he found it difficult to pick out the monkey in the canopy. My grandfather, on
the other hand, had 20/15 vision. In addition to blood, organs would sometimes be
collected and assayed. My grandfather would also collect the animal’s carcass to
make detailed drawings of the species in question (Fig. 8) as well as to make mea-
surements of their skulls, all of which he published. It is important to remember at
that time, there was very little information on many of these nonhuman primate spe-
cies, and his work describing them, and their behaviors are still used today.
4 The Witch Doctor
In the early 1940s, my grandfather led a large mosquito trapping expedition into one
of the forests in Bundibugyo District, Bwamba County, Uganda (likely Semliki or
in proximity too). These were primeval forests, largely untouched by humans. Upon
entry, one might very well have felt they were walking into an impenetrable forest,
never to return.
Somehow, the old trucks managed to hold together on the long journey from
Entebbe, and the team reached their destination, parking along a mud “road.”
Supplies and equipment were offloaded, and last-minute checks were made. Time
Fig. 7 Alexander John Haddow (far left, in the headscarf) and his mentor Alexander Mahaffy (in
the pith helmet) shooting nonhuman primates to collect blood for the purposes of virus isolation
and to carry out serological assays, Uganda (circa 1940s). The box contains collected blood on ice.
(Reproduced from the Alexander John Haddow Collection housed at the University of Glasgow,
Scotland)
was of the essence as it was late in the day, and they needed to set up camp before
nightfall. Once the last-minute checks of supplies and gear were completed, the
team lined up and begin trekking into the forest. As each person entered the forest,
they disappeared into the darkness of the undergrowth. They followed a narrow
ancient path deeper and deeper into the forest, and just when they thought they
would never see the sunlight again, they came upon a small clearing.
To their astonishment, in the middle of this clearing sat a Witch Doctor (tradi-
tional healer) on a fallen tree. The tree had fallen largely intact, with the root ball
attached (hanging over the large hole from which it had once rested). The Witch
Doctor climbed down from his perch and approached my grandfather. A hush
descended upon the team as all wondered what was to be said. The Witch Doctor
told my grandfather this area had been cursed, and whoever was standing on the tree
when it stood up would die. At the time, my grandfather shrugged this off. After all,
why would a fallen tree stand up? With that, the Witch Doctor disappeared into the
forest, and my grandfather began directing the setup of their camp.
To gain a better vantage, my grandfather climbed onto the fallen tree’s trunk. Not
long after, the porters began to chop the branches off the tree for firewood, and the
216 A. D. Haddow
Fig. 8 Drawing of the head of an adult male Cercopithecus ascanius schmidti by Alexander John
Haddow. (Reproduced from the Alexander John Haddow Collection housed at the University of
Glasgow, Scotland)
camp began to take shape. At some point during this process, the weight of the root
ball was more than the trunk, as the tree was now largely devoid of branches, and
the tree began to shudder and creak loudly. The entire camp’s attention was now
directed at the fallen tree and my grandfather who remained standing on it. With one
quick motion, the tree began to stand up, and as the tree neared the vertical, my
grandfather jumped clear.
With that, a certain unease descended upon the camp. Many discussed the events
in hushed whispers for the next few days, all agreeing that death would soon befall
the camp. Soon, my grandfather developed encephalitis. He was carried out of the
forest and taken to the hospital in Fort Portal, Uganda, where he fell into a coma. He
soon heard the words to the effect “Per istam sanctam unctionem et suam piissimam
misericordiam adiuvet te Dominus gratia Spiritus Sancti, ut a peccatis liberatum te
salvet atque propitius alleviet…” At first, he didn’t understand what was going on
but suddenly realized he was being given the last rights. With that, he willed himself
to wake (later recounted to my father), frightening the priest. After a few days, he
regained his strength and was taken to the local cemetery by the District
Commissioner and shown a newly dug grave. This was his grave, and that is how
close he came to death.
Although not as exciting, decades later I would come down with encephalitis
while carrying out mosquito surveillance in Thailand, so it appears getting encepha-
litis in the field is somewhat of a Haddow tradition.
5 Rabies
Rabies has always singularly terrified me, which likely had its start due to some of
my bedtime stories. The story starts off innocently enough. My father was outside
playing with Professor William Lumsden’s children. At some point, a staff mem-
ber’s dog approached the children and bit my father on the hand. The dog in ques-
tion was quickly put down, and its brain tested for rabies. Before waiting for the
results, my father was treated for a rabies exposure by my grandfather. On the first
day, he received two shots of immunoglobulin into his buttocks and abdomen and
subsequently received two shots in the abdomen every day for 2 weeks. The dog
ended up having rabies, and that was my father’s first close call with this deadly
disease.
His second close call came during a rabies outbreak in the region. During this
time, any stray or wild dog was shot on sight. My father and grandmother were
working behind the house in the garden when a rabid dog appeared and started cir-
cling them. They ran to the house, the dog literally just behind them. My father
indicated they barely made it inside before the dog got to them. He ran to my grand-
father’s study and told him what happened. At this point, my grandfather grabbed a
rifle and went out the front door to flank the dog who was now erratically circling
behind the house in the garden. With a single shot, the dog was dead. A few days
later, my father was in a hot water for “borrowing” my grandfather’s shotgun and
proceeding to hunt down other feral animals on the compound. But can anyone
really blame him?
As an infectious disease physician, my father would often relay stories to me of
having to treat people from rabies exposures. Thus, by the time I was an adult, I had
a pretty good understanding of the ecology and transmission modes of rabies virus.
This paid off when I was working part-time at the USDA Parasite Biology,
Epidemiology and Systematics Laboratory in Beltsville, MD, while in graduate
school. Some of the staff members had chosen to homogenize raccoon brains from
a region of high rabies transmission on the benchtop (to assay for Neospora cani-
num). I remember asking the individual in question what they were homogenizing
and upon receiving the response felt sick to my stomach. I immediately told the
laboratory director whose facial expression told me all I needed to know. With that,
one of the other technicians who had been in the room during the homogenization
and I were sent post-haste to the onsite health clinic and vaccinated.
6 Exploding Elephants
My grandfather was an honorary Park Warden (eventually made a “Life Member”)

with the Uganda Park Service. There had been a report of a poached elephant deep
in Queen Elizabeth National Park, and my grandfather and one of his colleagues set
out to investigate. They drove into the Park for several miles (kilometers), but the
218 A. D. Haddow
area soon became impassable, and they had to set off on foot through the bush.
Unfortunately for them, the temperature was about 85 °F (~29 °C), which would
have made carrying their rifles, water, and camping gear quite strenuous. After
walking for about 10 miles (~16 km), they came upon the dead, and now quite
bloated, elephant.
They dropped their gear onto the parched earth and began to investigate the cause
of death. As they stood in front of the head, their timing could not have been worse,
for at that moment, there was a loud roar, and the gastric contents which had been
fermenting for days in the heat exploded out of the mouth and onto both men. They
both stood speechless, dripping from head to toe in the most wretched foul-smelling
bile. To make matters worse, they only had the water which they carried, and they
couldn’t waste it washing up. The stench grew and grew in the afternoon heat, and
eventually, the putrid discharge was baked onto them, forming a thick crust.
Consequently, they spent the remainder of that day and night, as well as the follow-
ing day when they walked the 10 miles back to their truck, covered in this filth. After
hours on the road, they finally arrived back at their origin, where their waiting col-
leagues were covering their noses and telling them to restrict their movements to an
outdoor area until they were able to bathe. My grandfather often recounted this story
to my father whenever they were camping during hot weather, reminding him of the
time he “slept” in a hot tent covered in rotting gastric juices.
7 Elephant Chase
My grandfather was never a sport hunter and only shot game to eat while in the bush
or to study an animal’s internal and external parasites. However, he had to occasion-
ally track rogue elephants or those wounded by poachers to put them down (Fig. 9).
On one of these occasions, he was tracking a wounded bull elephant in the scrub,
when the animal suddenly appeared and initiated a charge. He quickly fired the first
barrel of his double barrel 0.475 No 2 Nitro Express, striking the bull in the center
of the head. This brought the animal to a complete stop, whereupon it began to
sway, and he rapidly dispatched it with the second barrel. He later told my father if
he had been carrying one of the “new” high-velocity rifles of the day, he would have
been killed, as his “elephant rifle” had far great knockdown power.
In the 1960s, my grandfather had another close call with an elephant. This time
he was with my father and uncle, who were making their way through Queen
Elizabeth National Park in one of the Volkswagen Kombis. They stopped to watch
the animals and had put up the top. My father and uncle were both standing on the
table watching a herd of elephants when a bull elephant appeared and began to cir-
cle the herd. After a few minutes, it spotted the Kombi sitting about 70 yards (~64
m) away and began charging. My father, still standing on the table, yelled to my
grandfather who was sitting in the passenger seat, “An elephant’s charging!” My
grandfather turned to the driver and told him to get moving. The driver, Stanley, had
been having a melancholy day and was taking his time starting the vehicle. At that
Fig. 9 Alexander John Haddow in the Bundibugyo District, Uganda (circa 1940s). Note the size
of his Double Barrel 0.475 No. 2 Nitro Express “Elephant” Rifle being carried by one of his assis-
tants. (Reproduced from the Haddow Collection)
point, my grandfather told him to MOVE (and not in the Queen’s English). The
driver turned the key, looked in the mirror, and screamed when he saw the elephant
rapidly approaching (The reader should note, the engine of the early, and for that
220 A. D. Haddow
matter later, Volkswagen Type II Bus was extremely underpowered). The Kombi
began to “speed” off, but the elephant was still gaining. It was only after what
seemed to be an eternity that it began to pull away from the elephant and disaster
was averted.
Not to let the family tradition down, several decades later, I had the unpleasant
experience of being charged by an elephant while on foot in one of the mountain
forests of Khao Chamao-Khao Wong National Park, Thailand.
8 Pygmy Feast
My grandfather had befriended a tribe of pygmies in Bwamba County and happened

to be visiting them during one of their hunts (Fig. 10). As adult pygmies are under
155 cm (5 feet), it was more than amazing they were hunting elephants in the forest!
During this hunt, one of the men silently crept up on a sleeping elephant and stood
underneath it. With a quick motion, he stabbed his spear at an angle into the abdo-
men, under the ribs, and into the elephant’s heart. He immediately jumped from
under the elephant so as not to be trampled, and it trumpeted and started to run, but
shortly thereafter collapsed and died. The tribe then set about butchering the animal.
At one point, one of the men was actually inside the elephant’s chest/abdominal
cavity and was throwing organs out over his shoulder to his wife who was catching
them in baskets. The tribe stayed in this location for a few days eating the elephant
Fig. 10 Visiting a tribe of pygmies in the Bundibugyo District, Uganda (1960). Alexander John
Haddow’s son, then a boy, Alastair Haddow (in a striped shirt, front row), along with their driver
Stanley (in a white shirt, back row). (Reproduced from the Haddow Collection)
and smoking what they were unable to eat before proceeding into the forest for their
next feast.
9 The Deadliest Snake in Africa
I was very lucky as a child to have my father take me to our local zoo probably once
a month when the weather was nice. These adventures were likely different for me
when compared to other children who were visiting the zoo with their parents. We
would make our way around the zoo, and at each animal enclosure, we would stop,
whereupon my father would give a lecture about the animal in question, reciting a
detailed history of their biology, ecology, and behavior. Animals found in Africa
generally had more detailed lectures, and any animal that was considered dangerous
was given special attention. Much of what my father taught me on these topics had
been taught to him by my grandfather. This was how I came to visualize the stories
or anecdotes he told me about lions, leopards, elephants, crocodiles, and other ani-
mals – besides watching nature documentaries with him on the weekends. Many of
my father’s stories or words of wisdom literally revolved around life and death, and
those involving snakes were no different. The zoo in question had a reptile house
where we tended to spend a large amount of time. As we walked around the reptile
house, he would tell stories of the various dangerous snakes found in Africa.
One time, we stopped in front of a cage containing a cobra, and he stared at the
snake and said:
“I was in the garden behind the house when a cobra came out of the wood pile
and went straight for me. I cut its head off with the garden hoe.” He was that direct
and blunt. He told me never to hesitate when I was in danger, as hesitation will get
you killed. I asked him what Grandpa Haddow said after he told him about the
cobra. He answered, “He smiled” (I imagine my grandmother was not too thrilled at
the situation). My father then proceeded to give me an overview of various species
of cobras, followed by a question-and-answer session.
He also relayed his own experiences with green and black mambas, which he
regarded as especially aggressive and dangerous. He had heard of mambas “chas-
ing” people when he was a child, and once had a green mamba come into his class-
room in Kenya, whereupon all the students jumped onto their desks. The teacher at
the time had recently arrived from Britain and was in the process of yelling at the
class for their antics, when the snake started toward him. Lucky for him, he realized
the gravity of the situation in the nick of time, and he too jumped onto his desk.
The last snake we always visited before leaving the reptile house was the puff
adder, which he considered the most dangerous snake in Africa. He would always
say: “Puff adders have killed more people in Africa than any other snake. They like
to lay on paths to warm themselves during cool evenings, and people step on them
and are bitten. Due to the distances involved to reach the hospital most people die.”
I honestly can’t begin to describe how much time was spent on the behavior of this
species, but it saved my life as an adult.
222 A. D. Haddow
In 2013, I was working with a group carrying out human landing catches (I will
just be honest with you, they were biting catches), on Anopheles mosquitoes in
Uganda. I was participating because I was allowed to keep the “by-catch,” or those
non-Anopheles mosquitoes that I was interested in due to their proclivity to transmit
arboviruses. We would arrive at our destination in the early evening when the sun
was still up and then trap throughout the night. I would always receive a gentle rib-
bing due to my “fear of snakes” (I would call it respect). I was generally looking for
puff adders, paying particular attention to where I placed my feet, but truthfully, I
was looking for anything remotely resembling a snake. As puff adders blend in
perfectly with the soil, this made slow going. This was no different than how I
walked when exploring the hills and hollers of the Ozarks as a child, only then I was
looking for copperheads and rattlesnakes (which were more prevalent than one
might imagine).
One night, we arrived late to a new location, as such it was already dark. The
village Chief was walking us around the perimeter of the village showing us possi-
ble trapping locations, when one of my colleagues yelled for me to hurry up (I was
probably 5–10 m behind them walking slowly). That colleague mentioned to the
Chief I was scared of snakes and that’s why I was walking so slowly. The Chief
responded that this was a good thing, as his son had been killed by a snake the previ-
ous year while walking on one of these paths at night. After this event, the gentle
ribbing decreased substantially. I digress with this story, as I wish to convey to the
reader that snakes do kill people in Africa (and throughout the world). Unfortunately,
antivenom research is a neglected area of funding and study, resulting in tens of
thousands of innocent persons paying the ultimate price each year.
A few days later, we were trapping at one of our previous locations, and as usual
arrived at dusk. The local residents had recently been making and drying bricks in
the vicinity of our trap site. Consequently, a large pile of palm leaves remained on
the bare soil, which they had been using to cover the bricks to prevent them from
drying out too quickly and cracking. Unfortunately, we pulled up directly next to
this knee-high pile of leaves. I suggested we should move the truck as there were
likely snakes in the leaves looking for rodents, to which a laugh was received. An
hour or so later, we began trapping. We were denoting the location of biting, and to
this end, I was wearing a thin T-shirt, short shorts, and flip flops. It was unseason-
ably cool, and we would all rush to the truck after each time period, set an alarm to
go off in 5 min, turn the truck on and the heat to full power, and instantly fall asleep
until the alarm woke us up. Then we would then basically run to our locations and
start trapping for the next time period.
It was probably our second or third trapping period of the night, and I was the
first back to the truck. I was walking slowly and paying attention to the ground as I
had a particularly bad feeling that night and my senses were up. I tried to avoid the
leaf pile which was just about a foot and a half away (on my side of the truck), when
I heard a sound that literally made my blood run cold. It was the extremely loud hiss
of a snake – what sounded like a puff adder. I did not move an inch. Everything in
my mind became crystal clear; all my senses were sharpened. I knew if I was bitten,
I would die there. In such situations, people often make the mistake of backtracking
and stepping on the snake. As it was pitch black out, I did not know if I had stepped
over the snake, if it was between my legs, or if it was in front of me. The snake
hissed again; it was blood curdling, but this time I could gauge it was close to my
feet. To this day, I do not know how I achieved what appears to be impossible, but
in a slow swift motion, I twisted my torso, not moving my legs or feet, placed my
hands on the hood of the truck, and lifted my body straight up without so much as
moving my lower extremities. Once I was more than a foot off the ground, I lifted
my legs and torso onto the hood of the truck. I carefully checked the ground on the
far side of the truck, jumped off, and rushed to stop my companions from running
up on the snake. They could see the terror in my eyes, and there were no more snake
jokes after that night.
10 Virus Discoveries
While my grandfather and his colleagues discovered numerous viruses during their
careers in Uganda, it is important to remember researchers during this time didn’t
publish everything. After all, it was the Cold War. During this time, NATO as well
as the Warsaw Pact (i.e., the Soviet Union) had both offensive and defensive bio-
logical weapons programs. Thus, researchers working in this era (prior to the
Biological Weapons Convention treaty) were cognizant of the potential implications
of their discoveries, which would have then been routed through their respective
sides. In my grandfather’s case, NATO. Consequently, one of my bedtime stories
told the tale of the discovery of an unpublished novel virus, which was later “redis-
covered.” Maybe one day I will be willing to shed more light on this, but until then
the reader is provided a partial (redacted) account of the discovery.
----------------------------------------- outbreak involving ---------------------------------------------------
------------------------------------------------------------------------- wild -------------------------------------
--------------- monkeys ------------------------------- which -------------------------------------------------
------------------------------ were ------------------------------------- discovered ----------------------------
------- to be ------------------------------------- infected ------------------------------------------------------
------------------------ with ---------------------------------------------------------------- a -------------------
------------------------------------ novel ------------------------------------------------------------------------

224 A. D. Haddow
11 Then and Now
Early in its history, EAVRI was known as a center of learning and research, and its
activities were widely known outside of East Africa. The laboratory carried enough
prominence that numerous visitors to East Africa would undertake the journey to
Entebbe. Notable scientists such as Professor Max Theiler, Sir Julian Huxley, Sir
Peter Medawar, and Professor Denis Burkitt visited the facilities, as did numerous
famous artists and writers. Princess Elizabeth, later to become Queen Elizabeth II,
planned to tour the laboratory and see my grandmother’s extensive gardens, but her
trip was canceled following the death of her father, King George VI. Today, the now
Uganda Virus Research Institute (UVRI) continues as a center of learning and
research and is host to a wide variety of notable visitors, something that I know
would bring my grandfather great joy.
However, not everything has changed for the better in the field of arbovirology in
the nearly six decades since my grandfather left East Africa. Today, there is clearly
less science and more paperwork (which was already a problem then). Although
there were always bureaucrats (administrators) in the Sciences, it seems in the last
few decades they followed Parkinson’s law and have proliferated at an almost expo-
nential rate. In many ways, we now follow an inverted workplace pyramid (be that
Government, Higher Education, or Industry), where the bureaucrats, supervisors,
managers, and administrators vastly outnumber the persons tasked with carrying out
scientific research. Furthermore, many of these individuals are largely disconnected
from actual research or only maintain a peripheral understanding of its purpose.
This trend has been detrimental to the Sciences (I am certain my grandfather
would agree). This system has reduced innovation, increased the amount of bureau-
cratic red tape, wasted limited funding, and created a culture focused on the inabil-
ity to take risk. No longer can one carry out an experiment or embark on a study that
has a high probability of failure, instead one must carry out “safe-science.” Thus,
true discoveries or breakthroughs are likely stifled when compared to the atmo-
sphere arbovirologists enjoyed when working during the field’s golden years.
On a similar note, today we also contend with a constant stream of new rules and
regulations which did not exist during my grandfather’s time. Although many of
these regulations have been truly necessary, many serve no purpose other than to
slow down research. These offscourings are usually created by persons who inher-
ently believe they possess the knowledge and wherewithal to understand what they
are doing but do not (the most dangerous type of person) or are done to cover one’s
ass. I honestly don’t see how it would be possible to achieve the level of productivity
EAVRI achieved under today’s regulatory burden, and I think that should give us
all pause.
Another massive change is that in my grandfather’s time, one could simply
choose to ignore correspondence, whereas today one is always “connected” through
their mobile phone or by email. While email would have been a benefit during
World War 2, when my grandfather and his colleagues would sometimes not find
out for years if their manuscript had been received, reviewed, or accepted by a
journal; one can only surmise that the inability to disconnect would have had had a
hugely negative impact on their creativity and ability to focus on research activities.
How one ever truly balances our present 7-day work week is beyond me.
This all begs the question, while my grandfather and his colleagues would recog-
nize some of the same techniques we use today, would they recognize anything
else? It is likely they would. Though they would undoubtedly be stunned by the
level of bureaucracy and regulations that exist today, I doubt they would be sur-
prised. Even in their time, the freedom to carry out truly novel work without con-
stant oversight and red tape was rapidly disappearing. Hence, the reason many of
them flocked to a far-off land, which in some ways was one of the last bastions of
The Enlightenment. Lucky for us they did. The methods and techniques they pio-
neered are still used today, while the studies and discoveries they made still serve as
the foundation of our field. In the end, working in sometimes the most inhospitable
conditions, at great risk to themselves, they accomplished something all of us can
be proud of and aspire to achieve.
12 Epilogue
While today we face our own set of unique challenges, just as my grandfather and
his colleagues did over 60 years ago, I would still choose the same career path. It has
allowed me the chance to actually live some of my childhood stories, and along the
way I’ve had the chance to meet, collaborate, and work for some of the most caring
and compassionate scientists, who also embodied the best principles of applied and
pure science. I hope these stories serve as a reminder of a time that is largely a dis-
tant memory while inspiring those still seeking adventure and discovery.
References
Garnham PCC (1980) Alexander John Haddow, 27 December 1912 – 26 December 1978. Biogr
Mems Fell R Soc 26:225–254
Lumsden WHRL (1979) Obituary: Alexander John Haddow. Lancet 133(8108):169–170
Fighting Dengue, Chikungunya,
and Japanese Encephalitis
Scott B. Halstead
Abstract My arbovirus career occurred in four stages, roughly 15 years each: US

Army Medical Corps, University of Hawaii School of Medicine, Rockefeller
Foundation and Pediatric Dengue Vaccine Initiative. I entered the Army through the
Physicians Draft and was assigned to the Department of Viral and Rickettsial
Diseases, 406th Medical General Laboratory, in Tokyo, Japan. This was a world-
leading center of field and laboratory research on Japanese encephalitis and hanta-
virus diseases. After two years stationed at the Department of Viral and Rickettsial
Diseases at the Walter Reed Army Institute of Research, I was assigned to and
founded the Virus Department, SEATO Medical Research Laboratory, Bangkok.
There, I carried out pioneering work to solve the mystery of why Thai hemorrhagic
fever caused by dengue and chikungunya viruses affected native Thais but not for-
eigners, pioneered in tissue culture methods, and discovered antibody-dependent
enhancement. After 3 years at the Yale Arbovirus Research Unit, I left the Army to
find the Department of Tropical Medicine and Medical Microbiology, University of
Hawaii, School of Medicine, where cellular mechanisms of ADE were discovered.
Then, for 12 years, I served as officer and director, Health Sciences Division,
Rockefeller Foundation founding a field program in Honduras and Mexico designed
to reduce dengue virus transmission, and then tested concepts and practices of
community-based control of Aedes aegypti. With Dr. Yu Yong Xin, National Institute
for the Control of Pharmaceutical and Biological Products, Beijing, a 20-year pro-
gram was initiated that demonstrated one-dose lifetime efficacy of SA 14-14-2
Japanese encephalitis vaccine in a Nepal field trial, achieving WHO pre-accreditation
for manufacture and export of this vaccine throughout Asia. In 2003, I founded the
S. B. Halstead (*)
Pediatric Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea
Department of Preventive Medicine and Biostatistics, Uniformed University of the Health
Sciences, Bethesda, MD, USA
Health Sciences Division, Rockefeller Foundation, New York, NY, USA
Department of Tropical Medicine and Medical Microbiology, School of Medicine, University
of Hawaii at Manoa, Honolulu, HI, USA
e-mail: halsteads@erols.com
228 S. B. Halstead
Pediatric Dengue Vaccine Initiative, funded by multimillion dollar grants from the
Gates and Rockefeller Foundations. PDVI supported an international dengue
research program and provided seed grants that built the Thai field site where Sanofi
tested its multivalent dengue vaccine and the Nicaragua field research site still used
by the University of California dengue research program. I have been amazed to
witness the dramatic growth of medical research during my lifetime and feel very
lucky to have had the chance to participate.
This historical reminiscence is dedicated to virus hunters and laboratory manag-

ers—US Army Sergeants Jack McCown, Adam Druzd, Myrlin Funkenbusch, Frank
Munden, and to the many other professional colleagues described in the text.
War introduced me to arbovirology, the study of viruses transmitted by blood-
feeding arthropods. On graduation from Columbia University’s College of
Physicians and Surgeons (P&S) in 1955, I was enrolled in the Berry Plan. This was
a national manpower recruitment system created by the United States at the end of
the Korean War that permitted physicians to defer obligatory military service to dif-
ferent periods in their post-graduate training. After getting a medical degree, I was
selected to take a medical internship at New York’s Bellevue Hospital on Columbia
University’s First Medical Division. Two years later, I was drafted into the US Army
and assigned to the Department of Viral and Rickettsial Diseases (DVRD) at the
406th Medical General Laboratory in Sagami-ono, a suburban Tokyo, Japan (406th
MGL) (Fig. 1).
These are recollections of people, places, events, and accomplishments in a
career that occurred during a unique period in human history. This account is heav-
ily weighted toward the earliest segment of my career, the 11 years in Japan,
Washington D.C., Thailand, and Yale. The Central National Library of Medicine
Biotechnology Information System (PubMed) did not publish abstracts of my
indexed articles until the mid-1970s. Without abstracts in the data base, my early
publications are rarely cited and, more to the point, rarely read. Many have not
entered the mainstream literature. These founding observations and concepts on the
pathogenicity of arboviruses were based on very large data bases. These many
important well-documented observations should not be lost to succeeding genera-
tions of scientists, clinicians, public health administrators, and politicians. Here, I
will review and discuss data from some of the “forgotten” 83 papers I published
from 1957 to 1975.
In Japan and Thailand, in the 1950s and 60s, there were few restrictions imposed
on virus research inside or outside the laboratory. It was easy to move biological
specimens and reagents from lab to lab. Access to individuals, healthy or diseased,
was straightforward and simple. Further, the Army provided ample financial and
personnel resources that made it possible to carry out ambitious research programs.
In Thailand, I was studying a brand-new disease phenomenon. There were no prec-
edents for study of dengue hemorrhagic fever. The Army allowed me to design and
carry out protocols that resulted in new and comprehensive insights into the nature
of dengue (DENV) and chikungunya viral (CHIKV) infections and disease.
Fighting Dengue, Chikungunya, and Japanese Encephalitis 229
Fig. 1 Scott B. Halstead, CAPT MC, 1957
This presentation is organized around those persons whose positions or respon-

sibilities opened access to portions of my career. I thank them for their generous
friendship and support.
1 US Army
Edward L. Buescher, M.D. (1925–1980), M.D., University of Cincinatti.1948.

Ed entered the US Army Medical Corps in 1951, was assigned to the 406th
MGL, 1953–1955, and in 1956 became Chief, Department of Virus and
Rickettsial Diseases, Walter Reed Army Institute of Research (WRAIR)
There are two founding virology research lineages in the United States: The
Rockefeller Institute’s virology laboratory in New York and Princeton directed by
Thomas Rivers from the late 1930s and the laboratory of Albert Sabin who began
his work with Rivers. I entered arbovirology through the lineage of Albert Sabin.
During World War II, Sabin, on active duty with the US Army, was assigned to work
at a Rockefeller Institute laboratory in Princeton, New Jersey. There, he pioneered
fundamental laboratory and clinical research on sandfly fever and dengue. Late in
230 S. B. Halstead
WW II, Sabin was assigned to Okinawa where he studied Japanese encephalitis (JE)
cases and created and tested a formalin-inactivated JE mouse brain vaccine. After
the War, Sabin was sent on temporary duty (TDY) to the 406th MGL in Tokyo,
where a second-generation formalin-inactivated JE vaccine grown in chick embryo
cells was tested in American troops and Japanese children. This vaccine was consid-
ered insufficiently immunogenic, and further development was halted. Prior to WW
II, in 1939, Sabin was a member of the faculty of the School of Medicine, University
of Cincinnati. Ed Buescher, a medical graduate of the University of Cincinnati,
published a paper characterizing JE hemagglutinins with Sabin in 1950. Sabin
described hemagglutinins for several other arboviruses; some of these publications
were co-authored with Bob Chanock. In the early 1950s, Buescher joined the Army
and was assigned to 406th MGL in Tokyo where he, Bill Scherer, and Bob Chanock
undertook comprehensive studies on the ecology of JE. In 1955, Buescher rotated to
Walter Reed Army Institute of Research (WRAIR) where he became chief of
DVRD. Many of the viral strains and laboratory procedures in use at the DVRD at
that time were derived from the Sabin laboratory (Fig. 2).
Fig. 2 Ed Buescher when

Chief, Department of Viral
and Rickettsial Diseases,
WRAIR
1.1 Japan
Ion Gresser, M.D. (1928–2019), M.D., Yale University, 1955. After service in
the US Army, 1956–1958, Ion trained with John Enders then established a
career studying interferon at Institut de Recherches Scientifiques sur le Cancer
in Villejuif, outside Paris.
My internship in 1955 on Columbia University’s First Medical Division at Bellevue

Hospital included a 3-month pathology rotation. I performed autopsies in the 30-table
pathology suite of the New York Medical Examiner. My pathology training partner,
an intern on the Cornell Division at Bellevue Hospital, was Ion Gresser, a graduate of
Yale University School of Medicine. Later in life, he discovered the toxicity of inter-
ferons and contributed importantly to understanding interferon control mechanisms.
In the Berry Plan, Ion was scheduled to enter the Army at the end of his internship. He
approached Dr. Lewis Thomas, then a faculty member of the NYU School of
Medicine, a facility that was adjacent to Bellevue Hospital. Lew introduced Ion to Ed
Buescher who arranged for him to be assigned to the 406th MGL. Sometime in the
autumn of 1956, I received a letter from Ion asking if I would like to join him in Japan.
To be very honest, because we had been engaged in a brutal war with Japan and then
a war in Korea, there was nothing in my head that compelled me to think of Japan as
an attractive assignment. But a dearly loved aunt had been a missionary in China and
I grew up loving China. Japan was near China, so, I thought, why not? (Fig. 3).
Fig. 3 Ion Gresser in France

232 S. B. Halstead
Accordingly, in July 1957, I headed to San Antonio to complete US Army

Medical Corps basic training for physicians at Fort Sam Houston. Shortly thereafter
I arrived in Japan. The 406th MGL was a large facility staffed by more than 250
scientists and technicians. The laboratory, predominantly designed to conduct
research on infectious diseases, had been established as an Army unit toward the
end of the Pacific War. In 1946, it moved from Okinawa to a building in downtown
Tokyo. When I arrived, a new laboratory facility had been constructed outside cen-
tral Tokyo, adjacent to a major US Army hospital in Sagami-ono. The 8th US Army
Headquarters was in nearby Zama. I had no idea how extensively the military infec-
tious disease research program had grown during WW II. Before I arrived, during
the acute phase of the Korean War (1950–1953), allied troops had suffered cases of
severe and fatal Japanese encephalitis and Korean hemorrhagic fever. Investigative
programs for each of these diseases had been established at the DVRD. Indeed, the
JE research program during WW II led to the development of an inactivated JE vac-
cine grown in embryonated eggs, the same substrate used to produce yellow fever
17D vaccine. Unfortunately, this vaccine was poorly protective and was not used
during the Korean War.
I arrived in Japan just after the 1957 Asian influenza outbreak had been recog-
nized in Hong Kong and Singapore. The DVRD ran a general diagnostic virology
laboratory for troops in the Pacific Theater including Korea. There were many
cases of influenza. My very first publication described the detection of antibodies
from an acute influenza infection using a complement fixation (CF) test. In addi-
tion, I worked with Ion on studies attempting to discover where JEV spent the
winter months. I learned many useful skills at the DVRD: how to inoculate baby
mice, harvest, and make virus suspensions from mouse brain tissue; how to prepare
and grow monkey kidney tissue cultures in slanted test tubes (Bill Scherer, also
assigned to the DRVD, was a pioneer in designing and testing tissue culture meth-
ods); and how to collect mosquitoes using light or host-baited Magoon traps, cap-
ture, and bleed wildlife that walked, flew, or slithered—including herons from the
Emperor’s protected colony, as well as capturing, bleeding and banding live birds
recovered in mist nets. Ion returned to the United States during the summer of
1958, and I became chief of a department comprised of administrators, virologists,
ornithologists, entomologists, and a special hepatitis research branch staffed by 8
professionals, 23 enlisted or civilian technical assistants, and 14 Japanese
technicians.
1.2 Korea
Through Army authority, 406 MGL staff had access to military installations through-
out Asia. In 1958, the Republic of Korea experienced the largest JE outbreak in
history, 6767 cases with 1893 deaths. The outbreak occurred over a period of
approximately 2 months. Exceptionally, the disease started in the south and moved
northward. The first case of encephalitis was reported from Pusan in late July. I
spent 4 weeks in August and September in Pusan collecting specimens from Korean
patients and wild-caught mosquitoes.
In early August, JE was reported from Kunsan, a city that was 140 miles north-
west of Pusan. At the US Air Base in Kunsan, from August 20–24, there were three
JE cases, one fatal, all diagnosed serologically and/or virologically. From serologi-
cal surveys of populations in Japan and other JE enzootic countries, it was evident
that there were many more JEV infections than encephalitis cases in humans.
Serological surveys of US troops during the Korean War suggested that nonindige-
nous persons also experienced inapparent JEV infections. The occurrence of cases
in a US military base presented an opportunity to measure the ratio of infections to
overt encephalitis.
In 1958, Kunsan Air Base had an average base population of 806. On visits dur-
ing the Fall of 1958, and early the next year, I bled and interviewed 300 men who
were on base during the epidemic. JE hemagglutination-inhibition (HI) antibody
seroconversions were detected in 28 of these men, allowing the calculation of infec-
tion rates and a JEV case: infection ratio on base. It was estimated that there had
been 25 JEV silent or mild infections for each case of encephalitis (Halstead and
Grosz 1962). This study is the best-designed measure of the ratio of subclinical
infection to clinical encephalitis cases in nonindigenous humans infected with a
north Asian strain of JEV. I bled some of the same men 18 months later and found
that complement-fixing (CF) antibody titers declined rapidly but neutralizing anti-
bodies, measured by the log neutralization index (LNI), actually increased signifi-
cantly! (Halstead and Russ 1962) In my opinion, this reflects an improved strength
(geometric affinity) of interactions between antibodies and neutralizing viral epit-
opes. The prevailing dogma is that titers of viral neutralizing antibodies fall after
infection. Serum dilution neutralization tests measure concentrations of antibodies.
However, the LNI provides a window into the biological reality that as time goes on,
a smaller number of antibody molecules are able to protect against virus reinfection
than previously, and this process of improving protection continues with time.
Among 25 JE-infected personnel, four men attended the dispensary with a febrile
disease during the outbreak; two were hospitalized with mild neurological symp-
toms. Respiratory complaints, described on questionnaires, but not reflected in dis-
pensary visits, occurred at a significantly higher rate among men who seroconverted
to JEV than in uninfected controls. Men with outdoor duties during evening hours
had significantly higher JEV infection rates than those working indoors. Laboratory-
documented JE infections also occurred in Americans assigned to two other
Airbases, Osan and Pusan, both with larger populations than Kunsan. Serological
samples of base personnel allowed measurement of JEV infection rates yielding
estimates of subclinical to clinical ratios of 48:1 and >70:1. Ratios in American
adults can be compared with the >300:1 subclinical to clinical JEV ratio estimated
in Asian children (Halstead and Grosz 1962). JEV causes fatal encephalitis in chil-
dren as well as adults. Our observations prompted an explanatory hypothesis that
the development of encephalitis during JEV infection was under control of host
genetics. Over many generations of exposure to JEV in Asian populations, the high
mortality rate in children would be expected to select for the surviving population
234 S. B. Halstead
that expressed a higher rate of genetic resistance. The Kunsan experience implanted
in me a lifetime appreciation of the power of epidemiology and an interest in viral
pathogenesis.
In a memorable event, I witnessed an abrupt termination of the JE epidemic
immediately after Typhoon Ida hit Pusan on September 26. Ida was one of the larg-
est Pacific typhoons of the twentieth century, inflicting significant damage to Japan,
less to Korea. I attributed the end of the epidemic to mosquitoes “being blown out
to sea.” In truth, the epidemic was waning. The last JE cases occurred during the
first week of October in the northern part of the country.
1.3 Washington, DC
During the summer of 1959, I was transferred to the Walter Reed Army Institute of
Research (WRAIR) in Washington, D.C. The outcome of this assignment was
another episode of “do-it-yourself” learning of basic laboratory virology. I was
assigned to the laboratory just vacated by Maurice Hilleman, who had gone to work
at Merck. Up until that time, my experience was an unusual mix in the laboratory
and field. In just a couple of years, I worked in two of the few virus laboratories in
the world with arbovirus field sites. At WRAIR, the site was in Chincoteague,
Maryland, where the ecology of alphavirus encephalitis was under study by a team
that included Tom Yuill and Phil Russell.
A notable event during my WRAIR tour was a short assignment to Ft. Dix at
Christmastime, 1960. I was part of a team studying the efficacy in recruits of a
DVRD-produced adenovirus vaccine. At the base hospital, I noted that year after
year during the winter months, the wards were filled with rubella cases. We had
available full virus isolation and serum collection kits and access to frozen storage.
I initiated the collection of specimens that were sent to Malcolm Artenstein, M.D.,
a member of DVRD, who pioneered the use of a tissue culture enterovirus challenge
resistance method to achieve the successful first isolation of rubella virus. In his
system, the rubella virus did not produce cytopathic effects (CPE) in infected tissue
culture cells. To detect rubella-infected cells, a cytopathic live enterovirus was
added to specimen-inoculated tubes (Buescher et al. 1962; Parkman et al. 1962,
1964). The presence of the virus was suspected when no cytopathic effect was
observed after inoculating a fully cytotoxic enterovirus when companion tubes
inoculated with control fluid showed cytopathic effect. Suspect tubes were har-
vested, passaged, and tittered, and the agent was identified. In those days, viruses
were identified using type-specific immune serum. For a new virus, convalescent
serum from classical rubella cases was used. A new virus must fail to be neutralized
by a panel of antisera to known viruses. Later in Bangkok, I used a version of the
viral challenge-resistance technique to recover dengue viruses for the first time in
tissue culture (Halstead et al. 1964a).
1.4 Bangkok
Eugene J. Gangarosa, M.D., (1927–). University of Rochester, M.D., 1954.

Completed internship and residency in Internal Medicine at the Walter Reed
Army General Hospital then served in the Department of Enteric Diseases
WRAIR. Subsequently, he was Dean of the School of Public Health, American
University of Beirut; saw lengthy service with CDC; and was on the faculty
of Emory University where he was founding Dean of the School of
Public Health.
A fork in the road of life in 1961 led me to Bangkok. At WRAIR, I developed a

friendship with Dr. Eugene (Gene) Gangarosa in the Department of Bacteriology.
Gene had been assigned TDY (temporary duty) to Bangkok in 1959 to study patients
hospitalized with cholera. At the time, very little was known about the clinical
pathophysiology that led to the massive loss of gastrointestinal fluid. A hypothesis
generated late in the nineteenth century attributed fluid loss in cholera to bacterial
destruction of the intestinal epithelium. Indeed, the “rice water” stools of cholera
were thought to be sloughed mucosa. Gene used an intestinal biopsy capsule
invented by William H. Crosby, Jr., Chief of Hematology at WRAIR, to study duo-
denal tissues from several cholera patients. To everyone’s surprise, intestines in
cholera patients were intact. However, all exhibited intestinal scarring typical of
advanced sprue, a chronic inflammatory disorder thought to be caused by a defi-
ciency of folic acid. There was great excitement over the possibility that overt chol-
era disease, known to occur in some but not all vibrio cholera infections, might
happen in persons with an underlying intestinal pathology (Gangarosa et al. 1960).
This raised hope there might be a way to prevent severe cholera. Sprue could be
treated, and its pathology reversed with a course of folic acid and tetracycline. But
Gene had not biopsied controls. As the cholera outbreak in Bangkok continued into
the summer of 1960, Gene planned to continue his research efforts. He needed
someone to collect intestinal biopsies from normal controls. That would be my job.
I was deployed on TDY to Bangkok in 1960 (Fig. 4).
Bangkok and Thonburi, then exotic twin cities with a total population of 2 mil-
lion, were not much changed from before WWII. Canals ran along tree-shaded
streets filled with bicycle rickshaws and small taxis. The Thais are open and friendly
people with some of the world’s prettiest girls and one of the best cuisines. Western
Medicine had been introduced to Thailand around the turn of the century by profes-
sors from America who staffed Siriraj Hospital Medical College. They had taught
the generation of physicians who were senior staff of most of the city’s hospitals at
the time of my visit. These graduates spoke excellent English and kept their patient
charts in English. I checked into a hotel along with members of the US Navy
Medical Research Unit 2 cholera team led by Captain Robert (Bob) Phillips.
Phillips, at the time, was conducting studies that led to the development of the oral
236 S. B. Halstead
Fig. 4 Gene Gangarosa in 2015
glucose and electrolyte solution now used to treat cholera. This was one of the most
impactful medical discoveries of all time. Dr. Phillips is a rare military medical
research scientist whose accomplishments were recognized by a Lasker Award.
During our TDY, Gene Gangarosa mainly devoted his efforts to the study of cholera
pathophysiology, while I obtained nearly 100 intestinal biopsies from healthy Royal
Thai Army enlisted men. Virtually all controls had “sprue-like” intestinal pathology,
a response subsequently attributed to the consumption of heavily spiced foods
(Sprinz et al. 1962).
During my TDY in Thailand, I was instructed to look for cholera cases in Ubol,
capital of the most easterly province of Thailand. During this trip, I discovered that
at Ubol Hospital, each weekday there were five surgical operations for the removal
of bladder stones from young boys. Back in Washington, I read the literature and
wrote a historical review of this largely forgotten human (noninfectious) surgical
condition (Halstead 1961). I also went on rounds at Bangkok Children’s Hospital
and collected diagnostic specimens from Thai Hemorrhagic Fever (THF) patients.
These yielded strains of chikungunya virus (CHIKV) at WRAIR that ominously
produced gastrointestinal hemorrhages in several species of laboratory rodents. This
virus jumped off the lab bench and hospitalized me, Phil Russell, and Walt Brandt
(Halstead and Buescher 1961; Weiss et al. 1965). My uneventful short rash illness
was “dengue like” with no arthritis. It remained for Don Carey in Vellore, India, in
1963 to be the first recognized that CHIK infections produced profound acute and
post-illness polyarthritis.
In Bangkok, I was attached to the SEATO Cholera Research Laboratory whose
director was Lt Col Oscar Felsenfeld, US Army MC. Later that same year, the
Cholera Laboratory was administratively moved to Dacca, Bangladesh, where it
operates today as the International Centre for Diarrheal Diseases Research. To
replace the SEATO Cholera Laboratory, the SEATO Medical Research Laboratory
(SMRL) was created, retaining Col Felsenfeld in command. Through my involve-
ment with THF cases, I met Dr. Charas Yamarat, Chair of the Department of
Microbiology of the Faculty of Public Health, Mahidol University, Bangkok. He
and Col Felsenfeld urged me to return to the SMRL for a full assignment. By now,
arboviruses were literally and figuratively “in my blood,” and I secured what turned
out to be a 4-year assignment to the SEATO Medical Research Laboratory,
1961–1965.
2 Virus Department, SMRL
Charas Yamarat, M.D., (1908–1987) Chulalongkorn University, M.D. 1929,

Johns Hopkins SPH, MPH,1939, Harvard Univ. DrPH, 1972. Dr Charas was
Chair, Department of Microbiology (1959–1972) and Dean, Faculty of Public
Health (1972–1987), Mahidol University, Bangkok.
Dengue hemorrhagic fever (DHF), an integral component of the twentieth and

twenty-first century dengue pandemic, was recognized as a clinical entity in
September 1954, when Quintos et al. reported 21 cases of a severe febrile disease in
children living in or near Manila. The disease was characterized by fever, flushed
face, abdominal pain, positive tourniquet test, thrombocytopenia, narrow pulse
pressure, shock, gastrointestinal hemorrhages, depression of bone marrow cellular-
ity, depressed maturation of megakaryocytes, and a high case fatality rate (Quintos
et al. 1954; Quintos and Lim 1956). Similarities in presentations between Philippine
cases and those of “epidemic hemorrhagic fever” of Korea (now called hemorrhagic
fever with renal syndrome), a well-recognized acute disease of unknown etiology in
Korean War combatants, led its authors to name this new entity “hemorrhagic fever”
(Quintos et al. 1954). Soon, recognizing that a renal component did not occur, the
authors changed the name to “Philippine hemorrhagic fever (PHF)” (Quintos and
Lim 1956). During the rainy season of 1956, there were an additional 1200 cases of
PHF. By chance, Dr. William McD. Hammon, Director, Commission of Virus and
Rickettsial Diseases, US Armed Forces Epidemiology Board, was in the Philippines
to study vector-borne viral infections. He quickly identified dengue virus (DENV)
as the etiology of PHF. Many cases were attributed to two new viruses, DENV 3 and
4 (Hammon et al. 1960a, b) (Fig. 5).
238 S. B. Halstead
Fig. 5 Charas Yamarat at

my farewell party, 1965
In 1958, an outbreak of 3500 cases of what was called Thai hemorrhagic fever
(THF) resulted in Dr. Hammon’s team being invited to Bangkok by the King of
Thailand. During this visit, CHIKV and multiple DENVs were recovered from clin-
ical cases as well as from Aedes aegypti mosquitoes. Another two “new” viruses
were isolated, designated DENV 5 and 6 (Hammon et al. 1961; Hammon and Sather
1961). It was puzzling that the clinical features of PHF and THF bore little resem-
blance to those of classical dengue fever (DF), a debilitating but nonfatal febrile
exanthem.
In September 1961, seven years after the Quintos publication, I arrived in
Bangkok to establish a US Army—Thai Faculty of Public Health (FPH) research
program on dengue. Remarkably enough, FPH faculty had been trained in arbovi-
rology through Rockefeller Foundation (RF) funding, and the RF had provided
funds to the Microbiology Department to build a small animal house. I review in
some detail my studies on DENV and CHIKV epidemiology and disease in humans
and describe how the modern treatment of DHF/DSS was introduced. Concepts of
virus pathogenesis were changed by this work in Thailand, at Yale and then at the
University of Hawaii into the 1970s. I recognize how fortunate I was to have scien-
tific support and financial resources provided to me that are beyond reach today.
Further, they were under the control of a single individual. Some of this work has
been reviewed (Halstead 2002; Halstead and Cohen 2015).
What did we know about dengue in 1960? Early in the twentieth century, the
dengue fever (DF) syndrome was shown to be caused by a virus transmitted between
humans by bites of Aedes aegypti (Bancroft 1906; Ashburn and Craig 1907; Siler
et al. 1926; Simmons et al. 1931). Careful studies in human volunteers in Australia,
the United States, the Philippines, and the Netherlands (infected mosquitoes had
been shipped from Indonesia) described clinical and laboratory responses to DENV
infection in adults (Siler et al. 1926; Simmons et al. 1931; Cleland et al. 1916;
Chandler and Rice 1923; Snijders et al. 1931). The well-described clinical and epi-
demiological features from these human volunteer studies made it possible to rec-
ognize outbreaks of DF around the globe beginning in the eighteenth century.
During World War II, pan-Pacific outbreaks of DF, particularly among combatants,
led to the recovery in suckling mice of DENV 1 strains from Japan, Hawaii, and
India and of a strain of DENV 2 from New Guinea (Sabin and Schlesinger 1945;
Kimura and Hotta 1944). A decade later, another DENV 2 strain was recovered
from DF cases on Trinidad Island in the Caribbean (Anderson and Downs 1956).
Against this large historical experience, it came as a surprise to find that in SE Asia
DENV infections were causing a fatal disease in children who had few features of
classical DF.
To unravel these complexities, I established research collaborations with Dr.
Charas Yamarat, Chair, Department of Microbiology, Faculty of Public Health
(FPH) of the University of Medical Sciences in Bangkok; Major John Scanlon,
Head, Entomology Department, SEATO Medical Research Laboratory (SMRL);
Dr. Suparb Prasartvinichai, Director, Bangkok Children’s Hospital, plus a virtual
army of scientific colleagues; and, most importantly, Dr Suchitra Nimmannitya,
Chief of the THF Ward. The SMRL headquarters was located on the grounds of the
Thai Army Phramongutklao Hospital directly across Rajavithi Road from the
FPH. The SMRL Virology Department in the FPH Microbiology Department was
next door to Bangkok Children’s Hospital (BCH). The lab itself was in a new build-
ing constructed with Rockefeller Foundation funds.
I arrived in Bangkok equipped with important new research tools. The microtiter
serological system, invented in 1951, was commercially available. During my stay
in Thailand, I was able to take advantage of this microtest by collecting human fin-
gertip blood in capillary pipettes. During the previous decade, the global malaria
eradication program had familiarized entire populations with the routine collection
of fingertip blood for malaria blood smears. Because these were routinely collected
all over the country, authority figures felt comfortable giving permission to bleed
large groups of individuals. Based on this precedent, we obtained permission from
authority figures to collect fingertip blood from persons of all ages. In the 1960s,
tissue culture methods for virology were in their infancy. Antibiotics and antifungal
agents had made it possible to grow cell monolayers in vitro. Asepsis was achieved
by flame sterilization of pipets and glassware. We worked on open benches, laminar
flow hoods having not been invented. With practice and discipline, in the hands of
dedicated and toughened technical staff, these technologies yielded a steady stream
of new results that described the clinical and epidemiological features,
240 S. B. Halstead
pathophysiology, and modern management of human dengue and chikungunya dis-

ease (Halstead and Cohen 2015).
We found CHIKV and all four DENV viruses were endemic throughout Thailand.
CHIKV, but not DENV endemicity, receded near the border with Malaysia. Study
designs made it possible to calculate DENV and CHIKV clinical disease incidence
rates plus subclinical/inapparent infection rates for the entire population of Bangkok
(Halstead 1980).
I made many trips outside Thailand: to NAMRU-2 in Taipei, Taiwan; to labs and
hospitals in the Philippines; to our “sister” US Army lab at the Institute of Medical
Research, Kuala Lumpur, Malaysia; to dengue labs in Singapore, Burma, and India
(Calcutta School of Tropical Medicine, the RF laboratory in Poona); and the RF lab
in Entebbe, Uganda. In March 1963, I attended the 4th Congresses of Tropical
Medicine and Malaria in Rio de Janeiro. My most eventful trip was to Saigon. In
late October 1963, Dr. Emmanuel (Manny) Voulgaropoulous, a health officer for
USAID, invited me to investigate the first recognized outbreak of DHF in South
Vietnam. On November 1, I was having lunch with Manny and Rose, his wife, when
planes flew overhead and bombed the Presidential Palace. The military coup against
Ngo Dhin Diem had begun!
3 Dengue Research
Suchitra Nimmannitya, M.D. (1929–), Faculty of Medicine, Siriraj Hospital,

1954, M.D., Post-Doctoral Fellow, Yale University Department of Pediatrics,
and Department of Epidemiology and Public Health, 1966–1970. M.S., Yale
University School of Medicine, 1966, Chief, Infectious Disease Unit, Bangkok
Children’s Hospital, 1971–1989, Director Bangkok Children’s Hospital,
1989–1991. Founder WHO Centre for Treatment of DHF.
From 1962 to 1964, five different but linked multipart interdisciplinary dengue stud-
ies were designed, initiated, and completed: (1) fever studies of Bangkok children,
hospitalized and nonhospitalized that resulted in the identification of the dengue
shock syndrome (DSS); (2) fever studies on nonindigenous residents of Bangkok,
hospitalized and nonhospitalized; (3) a prospective seroepidemiological cohort
study of DENV and CHIKV infections in Bangkok residents; (4) mosquito vectors
of DENV and CHIKV; and (5) nationwide hospital fever studies on residents of
Thailand outside Bangkok. In these comprehensive clinical studies, staff nurses
were able to collect acute febrile and 2 weeks post-febrile paired sera (syringe and
needle venesection) from nearly 100% of study patients. A newly described method
that differentiated IgM and IgG antibodies by molecular weight on ultracentrifuga-
tion was used to distinguish primary from secondary dengue infections.
Convalescent-phase sera from children with primary DENV infections had
Fig. 6 Professor Suchitra Nimmannitya, clinical teacher of dengue to the world
antibodies in the 19S fraction (IgM) (Russell et al. 1967a). Among children experi-
encing a secondary DENV infection using HI criteria, nearly all early convalescent
phase antibodies studied were in the 7S fraction (IgG) (Russell et al. 1967a). An
important discovery was that anti-DENV IgM antibodies did not fix complement in
the presence of DENV antigens (Russell et al. 1967a). From 1962 to 1964, the
complement-fixation (CF) test was used to all test paired sera thus providing an
unexpectedly sophisticated method of distinguishing primary and secondary DENV
infections: primary, HI but no CF DENV antibodies in early convalescent sera; sec-
ondary, CF DENV antibodies in acute or early convalescent sera (Nimmannitya
et al. 1969; Halstead et al. 1970a) (Fig. 6).
1. Fever Studies on Indigenous Bangkok Children and Dengue Shock
Syndrome
From 1962 to 1964, four complementary studies, all based on random enrollment of
subjects were conducted at BCH: (a) 491 patients with a discharge diagnosis of
dengue hemorrhagic fever (DHF) admitted to hospital for THF, up to two each on
Monday, Wednesday and Friday; (b) 212 children hospitalized with fever (not THF)
from 1962 to 1963; (c) 119 non-febrile surgical patients hospitalized in 1962;
(Nimmannitya et al. 1969) and (d) 323 outpatients with fever enrolled three days a
week in 1962–1964, of whom 26 had CHIKV and 94 had DENV infections
(Halstead et al. 1969a). Remarkably, 7.6% of THF patients had serological evidence
of simultaneous DENV and CHIKV infections. This suggested that vector
242 S. B. Halstead
mosquitoes that had taken two or more viremic blood meals contributed importantly
to infecting humans. Subsequently, wild-caught Aedes aegypti in India were found
to be infected with both viruses (Myers and Carey 1967). We were the first to docu-
ment that DENVs were transmitted in hospitals. From 1962 to 64, 4% of surgical
patients seroconverted to dengue during hospitalization at BCH (Halstead et al.
1969b). Data from BCH were complemented by hospital discharge data from other
Bangkok hospitals. We collected monthly THF admission data from 21 of 42
Bangkok/Thonburi hospitals from 1962 to 1964. The 21 hospitals not visited were
either small (seven beds or less) or did not admit infectious diseases.
4 Discovery of Dengue Shock Syndrome
Sanford “Sandy” N. Cohen, M.D. (1935–), Johns Hopkins University

School of Medicine, M.D., 1960, Pediatric Residency, Johns Hopkins, 1960–
1963. US Army Medical Corps, 1963–1965. Professor and Chair, Department
of Pediatrics, Wayne State University, 1974–1998. Detroit, Michigan.
In 1963, an unexpected visitor appeared in my office at the FPH. “Sandy” Cohen

had been drafted into the US Army, assigned to the Department of Biochemistry,
WRAIR, where he learned about the fatal hemorrhagic fever in Thai children. As
was common in the Army at that time, he talked WRAIR into writing orders for him
to come to Bangkok. He wanted to explore ways to study and improve the clinical
treatment to reduce the mortality of “Thai hemorrhagic fever.” I was thrilled with
his interest, and we immediately launched a plan to get Sandy back during the 1964
THF season. To improve treatment of THF, he wanted direct access to patients with
the ability to control their management. This created a legal problem. He was not
licensed to practice medicine in Thailand. Fortunately, I had a prestigious ally, Dr.
Wilbur Downs (see below). Wil, as Director of the Rockefeller Foundation (RF)
arbovirus research program, made a yearly trip around the world visiting the far-
flung RF network of arbovirus laboratories. He stopped off to visit other arbovirus
laboratories, including mine. Shortly after November 22, 1963, Wil made a third
visit to Bangkok. I immediately recruited him as a supporter of the THF treatment
project. Together, we went to see the Undersecretary of the Thai Ministry of Public
Health, who authorized the creation of a Thai Hemorrhagic Fever Study Center
(THFSC) granting permission for Dr. Cohen to serve as Clinical Director. He
recruited several skilled Thai clinicians and nurses to provide diagnostic and clini-
cal support. The Thai Army Pramongutklao Hospital furnished clinical and labora-
tory facilities, and WRAIR supplied pharmaceuticals, intravenous fluids,
commercially available human plasma proteins, a variety of surgical instruments,
and resuscitation equipment, while SMRL provided the logistical and financial sup-
port for round-the-clock clinical and laboratory services (Fig. 7).
Fig. 7 In Bangkok, 1964, Dr. Sanford Cohen with SBH, Edna Halstead, and Col. Jim Hanson,
Director of the SMRL
Between July 4 and mid-October 1964, the THFSC admitted 149 patients, ages
5 months to 17 years, 123 with DENV and 5 with CHIK infections. Among these
were 23 cases that Dr. Cohen labeled “dengue shock syndrome (DSS)”: narrow
pulse pressure (<20 mm Hg), hypovolemia evidenced by hemoconcentration (>/=
20% increase in hematocrit over recovery value) and idiosyncratically, hypopro-
teinemia (Balankura et al. 1966a; Cohen and Halstead 1966). Shock resulted from
the loss of fluid and small molecular proteins into serosal spaces. This was a viral
pathophysiology never previously described (Cohen and Halstead 1966).Autopsies
performed on the three fatalities revealed pleural, pericardial, and peritoneal effu-
sions; necrosis of parenchymal cells of the liver; focal and petechial hemorrhages;
and in one patient, a sub-arachnoid hemorrhage.
244 S. B. Halstead
Recognition that dengue patients suffered increased vascular permeability led to

a rational life-saving treatment. Patients with DSS were given a rapid infusion of
Ringer’s lactate solution, 10–15 ml/kg body weight per hour, followed by a solution
containing one part Darrow’s potassium lactate solution plus either two or three
parts of 5% glucose in water given at 45 ml/kg/h. When normal blood pressure was
restored, patients were given maintenance fluid therapy followed by oral intake of
fluids. Oxygen was administered to all patients in shock. Eighteen of 23 shock
patients who remained hypovolemic despite fluid therapy were given human plasma
protein at a rate of 10 ml/kg/h until hematocrit value began to decline. One child on
fluid resuscitation after experiencing a gastrointestinal hemorrhage with recurrence
of shock was given a blood transfusion with uneventful recovery. In the decades that
followed, when human plasma protein was no longer available, dextran solutions
came into use. Without significant change, the life-saving procedures described in
1966 continue as the “Gold Standard” treatment of DSS (Cohen and Halstead 1966;
Balankura et al. 1966b; WHO 2009). Three patients admitted late in illness in pro-
found shock died. All had terminal hyperkalemia. Dr. Cohen, who received his
medical training at Johns Hopkins, was particularly well prepared to manage DSS
as it was at Johns Hopkins where fluid resuscitation of shock was pioneered by Dr.
Alfred Blalock beginning in the 1920s.
In 1962–1964, a large percent of THF admissions to the BCH developed shock.
When Dr. Cohen’s case definitions were applied to the entire BCH study, there were
196 virus laboratory-confirmed DSS cases. All had DENV, and none had CHIKV
infections. Crucially, 186 had experienced a secondary dengue infection
(Nimmannitya et al. 1969; Halstead et al. 1967). At BCH, normotensive children
with clinically significant hypovolemia (hemoconcentration of 20% or higher) plus
thrombocytopenia were diagnosed as dengue hemorrhagic fever and those with
shock as dengue shock syndrome (DHF/DSS) (Johnson et al. 1967; Halstead 1966a).
The hemorrhagic diathesis in dengue was studied by a world authority on hemo-
stasis, Dr. Harvey Weiss. He was at that time assigned to the Department of
Hematology, WRAIR (Weiss and Halstead 1965). Laboratory innovations had con-
tributed to successful outcomes. The Virology Department, SMRL was the first to
recover and assay DENVs and to measure DENV-neutralizing antibodies in tissue
cultures (Halstead et al. 1964a, b; Nisalak et al. 1966; Udomsakdi et al. 1966;
Singharaj et al. 1966). SMRL developed the modern DENV plaque assay, which
was quickly adapted to a plaque reduction neutralization test (Sukhavachana et al.
1966; Russell et al. 1967b). These tests continued in use at SMRL with virtually no
changes for decades. An important question remained to be answered. Were there
six or four DENV? This question was resolved by studies at SMRL and YARU
(Udomsakdi et al. 1966). The arbovirus community agreed there were only four
DENV (Fischer and Halstead 1970; Halstead and Simasthien 1970).
Using the HI test, it was often impossible to find a fourfold or greater titer rise
between acute and convalescent because antibody titers were already high early in
illness without further increase in convalescent sera. Clearly, these individuals
were experiencing secondary DENV infections. But what evidence could be used
to classify this as a recent secondary DENV infection? This problem was solved
using epidemiological data. When healthy Bangkok residents of all ages and sexes
were randomly bled late during the rainy season of 1962, fewer than 6% of 1878
sampled had a HI titer > 1:640 (Halstead et al. 1969b). Because the chance of
encountering this titer in normal individuals was low, p <0.05, we could accept an
HI titer of 1:640 or higher as evidence of a recent infection (Udomsakdi and
Halstead 1966). For many years, fixed high HI antibody titers in paired sera were
accepted as evidence of secondary DENV infections until replaced by the capture
IgM/IgG ELISA.
In collaboration with SE Asian and Western Pacific WHO Regional Offices,
WHO Headquarters in Geneva and the Thai Ministry of Public Health the Virology
Department organized a WHO Regional Seminar on Mosquito-Borne Haemorrhagic
Fevers, held in Bangkok, on October 19–26, 1964. More than 150 participants from
40 countries attended the meeting at the Bangkok SEATO Headquarters. Summary
abstracts of 65 papers were published in the Bulletin of the World Health
Organization, Vol. 35, 1: 1 – 104, 1966. Also included was my historical review of
mosquito-borne HF and a meeting summary by the organizers (Halstead 1966a;
Chow et al. 1966).
2. Fever Studies on Nonindigenous Residents in Bangkok
From 1962 to1964, studies on nonindigenous Bangkok residents at the Medical
Unit of the US Embassy-Joint US Military Advisory Group and the Bangkok
Sanitarium identified 81 virologically confirmed DENV and 11 CHIKV infections
among 236 fever cases, and none were DHF/DSS. In young children, CHIKV infec-
tions were relatively more severe than DENV infections. While in adults, the clini-
cal features of DENV and CHIKV infections were quite similar, but the duration of
fever was shorter in CHIK cases than in DENV. Only a few CHIKV patients in our
study group experienced severe arthritic symptoms and signs. Donald Carey, a
Rockefeller Foundation virologist working in Vellore, India, during the 1963–1964
CHIKV epidemic was the first to describe what now are recognized as classical
CHIK clinical symptoms (de Ranitz et al. 1965; Carey et al. 1966, 1969). In our
cases, a maculopapular rash, leukopenia, taste aberration, bradycardia, and palmar
edema characterized DENV and distinguished it from CHIKV disease. During the
non-dengue transmission part of the year, physicians often labeled fever cases “den-
gue.” But only during the rainy season were such diagnoses correct.
From 1962 to 1963, according to the US Embassy data, there were 6000
Americans living in Bangkok. In this population, DENV and CHIKV infection rates
were measured in 288 American school children, 118 parents and pre-school chil-
dren who were bled before and after the rainy season of 1962. In 1963, DENV and
CHIKV seroconversions were studied again in 232 adults and children. In 1962,
DENV infection rates in Americans were 1.3% and 0.7% in 1963. These rates were
starkly different from the 1962 DENV infection rate of 41% among indigenous resi-
dents of Bangkok (Halstead et al. 1969b). Entomologic studies showed that
Americans did not support A. aegypti infestations in their households because water
was seldom stored on compounds. Instead, piped water was used. As documented
below, in Bangkok, high- and low-income groups often lived in close proximity
246 S. B. Halstead
(Halstead et al. 1969b). The marked difference in arbovirus infection rates between
indigenous and nonindigenous residents living side-by-side is evidence that DENV-
infected A. aegypti infrequently moved between compounds. Limited flight range of
A. aegypti protected nonindigenous residents from their arbovirus-infected
neighbors.
From May to July 1964, Ubol, a provincial capital in northeast Thailand, border-
ing on Laos and Cambodia, population 30,000, suffered an outbreak of DENV 1.
Ubol was the site of a joint U.S. – Australian Air Force Base where 46 DENV 1
infections were detected (Halstead et al. 1969c). All of the American military but
none of the Australians routinely received the yellow fever vaccine (YF). All
Australian DENV cases had primary HI and CF antibody responses. Classical sec-
ondary HI responses were observed in virtually all dengue-infected Americans. We
made an immunologically important discovery, CF antibody responses in Americans
were “primary”- DENV 1 CF antibodies were delayed into convalescence. Review
of clinical records and interviews found that nearly 100% of men who circulated
DENV 1 antibodies had experienced overt classical dengue disease. Young adult
Caucasian males experienced primary DENV 1 infections as overt disease at an
infection to disease ratio of 1:1. DENV attack rates were significantly higher in men
working night compared with day shifts. Those men working nights who visited
Ubol during the day were at risk to daytime biting A. aegypti. I used to joke that
dengue was a venereal disease!
3. Prospective Seroepidemiological Cohort Study of DENV and CHIKV
Nineteen areas within Changwat Phranakorn (includes the city of Bangkok)
were selected randomly from 1960 census lists. Each area was standardized to
approximately 400 dwelling units with a total of 48,401 residents. All residents in
19 study areas were separately identified by households. All family members were
given study numbers and identified by age, sex, ethnic group, and economic status
(Halstead et al. 1969b; Halstead 1966b). Ethnic designations were established by
direct observation of distinctive household and displayed religious features.
Special surveys were made to determine household economic status. During May
and December 1962–1964, each house was visited at six- to eight-week intervals
to inquire for occurrence of THF cases. Cases were bled, and the date of onset,
symptoms, and the facility where clinical diagnoses were made were recorded.
Hospitals were visited to verify admissions. Between January and June 1962, a
10% random sample of household members were bled monthly. The plan for con-
tinuous serological surveillance of DENV and CHIKV infections was abandoned
in August 1962 when an outbreak of varicella led residents to actively resist bleed-
ing their children. In Chinese tradition, removing any blood results in serious
weakening. Despite resistance, fingertip blood was collected from all THF cases
among study residents identified during 1962–64. Further, it was possible to obtain
blood from 1888 individuals before and after the rainy season of 1962. These were
tested for HI antibodies to DENV and CHIKV providing a very reliable measure
of arboviral antibody prevalence in the Bangkok metropolitan area for that year.
The extensive management of such a large prospective cohort study was made
possible by the extraordinary organizational abilities and personal skills of Ms

Prabhasi Umpaivit, Chief Nurse. She was assisted by a staff of 14. Intensive sur-
veillance of the 19 study areas over 3 years documented a 20% growth in popula-
tion but also accompanied by an annual 40% turnover. Individuals of all ages
constantly moved between the city and rural Thailand. During the study, in two
areas, all housing was largely replaced by urban renewal. As a result, most analy-
ses and descriptions of the urban epidemiology of DENV and CHIKV infections
and disease were limited to 1962.
The huge prospective cohort study in Bangkok provided many new observations:
(1) Pre-and post-1962 serological studies on a random sample of the total popula-
tion made possible an accurate measure of DENV and CHIKV infection rates for
the whole city. Throughout 1962 many study areas, children with THF were admit-
ted to city hospitals. Identified cases were all confirmed serologically. Because
Virus Department nurses visited virtually all Bangkok/Thonburi hospitals monthly,
we had citywide hospital numerator data. (2) An important unexplained feature of
the biological response to DENV infection was the higher hospitalization and death
rates in females than in males. Serological data showed this not to result from dif-
ferences in infection rates, DENV infection rates in males and females were identi-
cal. A change to female dominance began at age 4 years (Nimmannitya et al. 1969;
Halstead 1970). Later, other careful studies have confirmed female dominance in
DSS cases (Anders et al. 2011). By contrast, in adults experiencing either primary
or secondary DENV disease, males have higher rates of severe dengue and case
fatality than females (Halstead 1997). (3) In 1962, the peak age of admission of
DHF was 3 years, 8.5 per 1000 (secondary DENV infections). (4) DHF/DSS case
fatality rates varied inversely with clinical attack rates. The CFR was significantly
higher in 1963 compared with 1962 or 1964, both years had higher hospitalization
rates than 1963. (5) The higher the case fatality rate, the lower was the rate of recov-
ering DENV viruses from acute phase blood. In 1963, a year with a high case fatal-
ity rate, dengue virus isolation rates from acute phase blood were lower than in
1962 or 1964, years with lower case fatality rates, (6) the modal age of hospitaliza-
tion of infants with primary DENV infections in 1962–1964 was 8 months. Infants
with primary DENV infections were hospitalized at a rate of 16.2 per 1000, twice
the rate of 3 years old with secondary DENV infections. (7) DENV cases, once
recognized, spread from this focus to other nearby houses in brushfire fashion. (8)
Study areas experiencing high endemicity during one year had low numbers of
cases the next year. Many of these observations are unique and have not been fur-
ther investigated.
4. Vectors of Dengue and Chikungunya
Mosquitoes were collected using light and human-baited traps in five study areas
in Bangkok and then processed for viruses (Halstead et al. 1969b; Nisalak et al.
1966; Scanlon 1966a, b). Aedes (Stegomyia) aegypti (L.) was the only member of
the genus Stegomyia found in any numbers of the urban area. Despite special efforts,
fewer than 100 female Aedes albopictus were captured in rural portions of Bangkok
or Thonburi during this study. Based on the percentage of 100 houses with one or
248 S. B. Halstead
more adult A. aegypti, the adult A. aegypti index in Bangkok in 1962–1964 was very
nearly 100%. Mosquitoes bred in a variety of man-made containers but also in tree
holes. No efforts to control A. aegypti were undertaken in any study area during the
report period.
C. quinquefasciatus, C. tritaeniorhynchus, and C gelidus were collected in large
numbers in light traps but few from human baited traps or direct human biting col-
lections in Bangkok, suggesting low anthropophilia. No JEV was recovered from
mosquitoes collected in Bangkok, but JEV was recovered from C. tritaeniorhyn-
chus and C. gelidus in a nearby rural area. In Bangkok, based upon abundant recov-
eries from mosquito pools, CHIKV was transmitted by A. aegypti not C.
quinquefasciatus.
5. Nationwide Studies of Fever in Thailand
Hospital admission data for the years 1962–1964, (name, age, sex, date of admis-
sion, address, and final diagnosis) were collected from 21 Bangkok and Thonburi
hospitals with 10 beds or more and from 98 of 101 provincial and private hospitals
outside Bangkok (Halstead et al. 1969d). In Thai provinces, hundreds to thousands
of children were hospitalized with DHF/DSS with an overall case fatality rate of
6.8%. As early as 1962, THF cases were hospitalized in Central, North Central,
Southeast Coast, and Southwest Coast regions but not in region IX, the seven prov-
inces adjacent to Malaysia. Hospitalization rates varied markedly between prov-
inces, and from year to year, some had rates eight to ten times higher than Bangkok.
In two provinces, peak hospitalizations and the highest case fatality rates occurred
in 1963 compared with nationwide peaks in 1962 and 1964. Initial epidemics of
DHF were recorded in some provinces in North and Northeast Thailand in 1964.
Monthly hospitalization rates usually mirrored those in Bangkok; overall, 1962 and
1964 recorded the largest numbers. Hospitalization and death rates were consis-
tently higher in girls than boys. Serological data were consistent with the conclusion
that DENV and CHIKV had been endemic throughout Thailand for a long period. It
was not possible to guess when CHIKV might have been introduced.
5 Dengue Mysteries
5.1 “Dengue”: What’s in a Name?
The term “dengue” derives from the Swahili, “kidinga pepo” a name that identified
outbreaks of a short-duration febrile disease accompanied by a disabling acute
arthritis that occurred in East Africa in 1823 and again in 1870. (Christie 1872)
James Christie, a physician to the Sultan of Zanzibar in the 1860s and 1870s,
explicitly linked an 1823 epidemic on Zanzibar to the American epidemic of kid-
inga pepo in 1827–1828 (Christie 1881). He reported that the 1823 epidemic
spread from Zanzibar to Gujarat, India, then to Calcutta, and by 1824 on to
Rangoon. He also noted that a similar disease reached St. Thomas in the West
Indies in 1827. Christie states “I am of the opinion that both the disease and its
designation were imported in the West Indian Islands direct from the East Coast of
Africa” (Christie 1881). Christie observed that the medical term “dengue,” was
introduced in the West Indies. An observer of the “dengue” epidemic in New
Orleans in 1828 commented, “The disease alluded to is supposed to have been
brought from Africa, with some slaves imported into the Havana. In that place, it
obtained the name of Dingee, Dengue, Danga, etc. It was there very prevalent and
also in Barbadoes, where it received the name of Dandy fever, from the stiffened
form and dread of motion in patients” (Dumaresq 1828). Accordingly, “dengue” is
a Spanish homonym of “dinga,” a disease that was familiar to East Africans in
Cuba. In New Orleans, disease “spread so rapidly among the inhabitants that in
eight or ten days, at least one-third of the population were laboring under its influ-
ence, including persons of all ages and different sexes” (Dumaresq 1828).
Dumaresq goes on to say “A person on the disappearance of this fever would
attempt to rise from bed, feeling not much loss of strength, and a consciousness of
being able to move about and attend to a little to business; but how egregiously
would he be mistaken when he assumed the upright posture. The joints felt as fet-
tered or anchylosed and the advance of one foot or leg beyond the other, would cost
more pain and effort than the purpose for which may have been advanced was
worth –aye-a thousand times told!” (Dumaresq 1828) The arthritic component of
this febrile exanthema is unique to pandemic human chikungunya infections and
has been variously called “scarlatina rheumatica,” “exanthesis arthrosia,” or “an
eruptive articular or rheumatic fever” (Bernal 1828).
This was not the first epidemic febrile exanthema described in the New World.
Benjamin Rush described a disease he called “bilious remitting fever” that occurred
in Philadelphia mid-August through September 1780, principally among residents
living along the Delaware River waterfront (Rush 1789). Rush said “The fever gen-
erally came on with rigor…In some persons, it was introduced by a slight sore
throat….The pains which accompanied this fever were exquisitely severe in the
head, back and limbs. The pains in the head were sometimes in the back parts of it
and sometime occupied only the eyeballs…. A few complained of their flesh being
sore to touch…the disease was sometimes believed to be a rheumatism. But, its
more general name among all classes of people was breakbone fever…. A nausea
universally, and in some instances, vomiting, accompanied by a disagreeable taste
in the mouth, accompanied this fever…. A rash often appeared on the third and
fourth days.”
Rush’s description of bilious remitting fever was well known to physicians who
attended patients during the 1828 outbreak in the Caribbean. Indeed, George
Stedman, M.D., formerly President of the Royal Medical Society of Scotland, who
practiced medicine on St. Croix, felt that the 1828 “dengue” was quite different
from bilious remitting fever. He observed, “I think that it will be evident to everyone
who pays the least attention to the symptoms, that the diseases, though somewhat
alike in a few symptoms, are essentially different.” The principal distinctions
Stedman made were in the suddenness of the onset and the nature and duration of
250 S. B. Halstead
the after-pains of “dengue” (chikungunya) (Stedman 1828). Christie himself recog-

nized there were two distinct febrile exanthems, with and without post-illness
arthritis.
Throughout the nineteenth century, astute observers in the Americas and India
recognized the differences between “dengue” and “breakbone fever” principally
related to the duration of fever and the occurrence of post-illness arthritis (Wragg
1851; Goodeve 1853; MacKinnon 1854). The term “dengue” was used to describe
the chikungunya epidemic that reached India in 1871–1872 from Zanzibar and East
Africa (Shircore 1872; Verchere and Raye 1872; Sheriff 1873). However, outbreaks
that were more common in India were febrile exanthems “with an almost entire
absence of the articular pains” (Christie 1872; Dengue 1868).
Once Aedes aegypti had been identified as the vector of yellow fever in 1901, the
epidemiological similarities between dengue and yellow fever stimulated workers
in Lebanon, Australia, and the Philippines to investigate the etiology and mode of
transmission of “dengue” (Bancroft 1906; Ashburn and Craig 1907; Reed et al.
1901; Graham 1902). To do this, they studied naturally occurring infections and
used susceptible humans as laboratory hosts. At the time these studies were con-
ducted, it was dengue viruses that were endemic, not chikungunya. All groups iden-
tified the disease they were studying as “dengue fever” or “breakbone fever.” The
existence of two different syndromes had been forgotten.
Initial attention centered on testing a suspicion that Culex fatigans (quinquefas-
ciatus) was a vector. Two groups, in Australia and the Philippines, apparently suc-
cessfully transmitted virus from sick to well humans by bite of an “infected” Culex
fatigans (Bancroft 1906; Ashburn and Craig 1907). Ashburn and Craig successfully
infected human volunteers with an inoculum passed through a diatomaceous earth
filter (Ashburn and Craig 1907). It remained for Siler and Simmons in the Philippines
in 1923 and 1929 to definitively demonstrate that Aedes aegypti was and Culex
quinquefasciatus was not a biological vector of dengue (Siler et al. 1925, 1926;
Simmons et al. 1931). During the first half of the twentieth century, it was dengue
flaviviruses that were studied in many human volunteers whose clinical features
were recorded in detail. All were identified in the literature and in textbooks as
“dengue.”
In 1952, a virus was recovered from an outbreak of an exanthematous febrile
disease in the Southern Province of Tanganyika. It was called “chikungunya,” the
name in the Makonde language that means “that which bends up” (Robinson 1955).
No one suspected its original name had been “dengue” (Halstead 2015a).
5.2 Was THF a Case of Chinese Medicine Poisoning?
At Bangkok Christian Hospital in 1958, “not a single case of THF was diagnosed
among either Americans or individuals of Indian ancestry,” 23% of cases were Thai
and 77% were Chinese children (Nelson 1960). At the time, the ethnic distribution
at this hospital for all conditions was Chinese, 57%, and Thai, 38 %. A physician
had written, “In our hospital we had seen this bizarre clinical pattern, with almost
all of the patients dying rapidly in a shock-like state and responding poorly to sup-
portive therapy. … When we delved into the history of these children, we find all
had been given Chinese medicine, usually for fever … We had previously incrimi-
nated the Chinese medicine as the cause of death” (Nelson 1960). Drug toxicity
seemed plausible in a child with the onset of high fever followed in several days by
restlessness, nausea, vomiting, abdominal pain, and cool extremities delays who
sought medical assistance for irreversible shock. An ethnic imbalance was also
noted in patients seen at Bangkok Children’s Hospital. Of 471 THF cases with con-
firmed DENV infections during 1962–1962, 133 or 28% were Chinese, while only
9.6% of CHIKV patients were Chinese (Nimmannitya et al. 1969). OPD visits for
all conditions were 85.1%, Thai, versus 14.9%, Chinese (Halstead et al. 1969a).
Why were Chinese children admitted at high rates to hospitals for severe DHF/DSS,
while for other febrile illnesses rates of admission of Chinese children were low?
An answer emerged from the Bangkok Area Study. When interviewed, we found
that Chinese parents recognized that the life-threatening signs and symptoms in
their children required treatment by Western medicine. Mild illnesses could safely
be brought to traditional Chinese healers (Nelson 1960). In the 1960s, it was discov-
ered that the time after onset of fever a child was brought for hospital admission was
a variable with major prognostic importance. Delay in medical intervention often
resulted in irreversible shock. Chinese parents came to recognize that a delay in
seeking hospitalization caused by taking ill children to visit a Chinese practitioner
had dire consequences. During the 1960s, there was a steep learning curve about the
course of dengue disease for all parents and physicians in Thailand. Fortunately, this
resulted in parents bringing children to physicians earlier and in turn to earlier fluid
resuscitation, falling case fatality rates, and, ultimately, the disappearance of
“Chinese medicine poisoning.”
5.3 Primary Infection DHF in Infants. Was It Real?
During 1966–1968 the two-infection “hypothesis” had been confirmed two well-
designed prospective cohort studies of children over the age of one year carried out
by my successor at the SMRL (Russell et al. 1968; Winter et al. 1968, 1969).
Hospital data had showed that infants less than 1 year comprised a significant por-
tion of DHF/DSS cases. But their antibody responses were primary. How was it
possible to have secondary immune responses in some and primary dengue anti-
body responses in others? In 1966, the author returned to Bangkok with the explicit
purpose of answering this question. Before he left, he was convinced that infant
cases must have been clinically misclassified. However, in Bangkok, he discovered
Dr. Kamolwat Vinichaikul, a pathologist at Women’s and Children’s Hospital, who
had extensive autopsy and clinical data on more than 200 infants and children who
died of DHF/DSS during 1958–1966. Records included 35 complete autopsies on
infants less than 1 year old. Review of clinical courses, acute phase clinical
252 S. B. Halstead
laboratory results, and gross and microscopic pathologic findings in infants and in
children older than 1 year convinced me that both groups were virtually identical
(Halstead et al. 1970a; Cohen and Halstead 1966; Halstead 1970). This was also the
conclusion of a later Thai pathology study in which gross autopsy findings, organ
involvement, the incidence and distribution, and severity of hemorrhages and tissue
histologic findings in six infants were basically identical to 94 older children
(Bhamarapravati et al. 1967).
In the early 1970s, path-breaking and classical studies on serum complement
activation in DHF/DSS patients were conducted by Drs. Frank Dixon and Hans
Muller-Eberhard, world-class leaders of studies on complement at the Scripps
Institute in San Diego. Their work led to a hypothesis that DHF/DSS was an IgG
antibody-DENV immune complex disease. Complement activated by DENV-
antibody complexes created breakdown products that directly damaged tissues
(Russell et al. 1969; Russell 1970; Bokisch et al. 1973a, b; Memoranda 1973). But
how did infants with primary DENV infections fit into a complement pathogenesis
model? In a 1971 study, a 6-month-old female admitted with DSS caused by DENV
2 circulated C3 and C4 activated by the alternative pathway in acute phase sera. This
infant’s mother circulated neutralizing antibodies to all four DENV (Memoranda
1973). Unfortunately, when this account was written, the pathogenic role of com-
plement in infants with primary DENV infections still had not been defined. A new
mechanism, discovered in 2015, explains the fundamental vascular permeability
pathogenesis, viral toxicosis. This is the cause of DSS during primary and second-
ary dengue infections (Glasner et al. 2018). Today, it is still not clear how much
complement activation contributes to severe dengue disease. Further, there has been
virtually no effort to study complement levels in infants with DHF/DSS.
5.4 When Did Epidemic DHF/DSS Begin?
When did DHF/DSS cases begin in Thailand? Prior to WWII, there was one medi-
cal school in Thailand, Siriraj Hospital in Thonburi. During the 1960s, I inquired to
pediatricians at Siriraj about the whereabouts of hospital charts from before WW
II. Thailand (then known as Siam) was an ally of Japan, and Bangkok had been a
supply point for Japanese troops fighting in Indochina and in Burma. Because it was
a staging area, Bangkok was bombed repeatedly. I was told there had been fires dur-
ing the War that destroyed Siriraj charts. One day, I posed the question to Tranachit
Harinasuta, Professor of Medicine at Siriraj. She was the wife of Chamlong
Harinasuta, Dean of the School of Tropical Medicine, next door to the FPH. Dr.
Tranachit said that “patient records from before the War are in the attic of the main
hospital building.” Sure enough, in the attic scattered over the floor were patient
charts from the 1930s. During that decade, Siriraj medical school was staffed by
American clinical professors paid for by the Rockefeller Foundation. The patient
charts contained descriptions of illness episodes written by medical students using
the same format I learned at P&S. There were supplementary histories and clinical
notes written by residents and attending physicians. More usefully for me, all
records were in English.
There were thousands of charts. I arranged them by month and year, then ran-
domly selected, and read 572 charts from the months of July and August, 1932–1941,
looking for patients with fever, thrombocytopenia, or symptoms or signs of shock or
gastrointestinal hemorrhage (Halstead and Yamarat 1965). No clusters of “DHF-like”
patients were found. Hospital charts from 1942 to 1948 were missing. However, in
charts available from Siriraj records during the hot, rainy seasons of 1950 and after-
ward, there were numerous children admitted with diagnoses of “febrile thrombocy-
topenia” or “febrile thrombocytopenic purpura and cardiovascular collapse” (Halstead
2008a). Siriraj physicians now agree that beginning in 1951, dozens to hundreds of
cases were admitted each year of “influenza with secondary thrombocytopenic pur-
pura” or “influenza with circulatory failure” (Tuchinda 1966). Prior to WW II, there
were at least five instances of outbreaks of classical DHF/DSS throughout the world,
none in Thailand. These were in Australia, Europe, Africa, and Asia, the earliest being
in 1897 in Queensland, Australia (Halstead and Yamarat 1965; Halstead 2008a).
5.5 Yale Arbovirus Research Unit
Wilbur G. Downs, M.D., (1913–1977), Cornell University Medical College

MD, 1938. On duty with the US Army from 1943 to 1946, he was a Malaria
Control Officer in several South Pacific settings and on Okinawa. After the
war, he studied malaria in Mexico with the Rockefeller Foundation (RF). In
1952, he established the Trinidad Regional Virus Laboratory and, in 1961,
was Associate Director of Medical and Natural Sciences Division, and
1963–1971, Director of the Yale Arbovirus Research Unit and Professor of
Epidemiology at Yale.
When the time came to return home to “CONUS” (continental United States) in
1965, I remembered what Elvio Sadun, Chief of Parasitology WRAIR, advised.
Elvio, a good friend during my 1959–1961 tour in Japan, had worked for several
years on lungworm in Korea. When he returned to the United States, he discovered
he was loaded down with administrative duties. Fortunately, he arranged to be
assigned to an academic post that permitted time to write up his field work for pub-
lication. Elvio said, “Don’t go back to WRAIR” (Fig. 8).
With an invitation from Wil Downs, I arranged for the Army to assign me to the
Rockefeller Foundation (RF) Arbovirus Laboratory. The Laboratory was in the pro-
cess of moving lock, stock, and barrel from Rockefeller University in New York
City to New Haven where it became the Yale Arbovirus Research Unit (YARU). In
April 1965, I keynoted a ceremony that inaugurated the new RF-funded building
that housed the Department of Epidemiology and Public Health. I presented an
254 S. B. Halstead
Fig. 8 Dr. Wilbur Downs

as Director of the Yale
Arbovirus Research Unit
early perspective on the epidemiology and pathophysiology of DHF/DSS. My

reward for the talk was dinner with Max Theiler and his wife. Max developed the
17D yellow fever vaccine and received the 1950 Nobel Prize in Medicine. Actually,
his Nobel Prize was for discovering the infant mouse as a laboratory animal for the
study of yellow fever. He had been designated a member of the RF faculty at Yale.
My talk, published promptly by the Yale Journal of Biology and Medicine, con-
tained the first recognition that fatal cases in children during a dengue outbreak in
coastal northeastern Australia in 1897 were an instance of DSS (Halstead 1965).
The address also contains my first published speculation on the cause of dengue
vascular permeability, “A hyperimmune reaction in persons previously sensitized to
dengue antigens may therefore be postulated as an underlying mechanism in the
pathogenesis of hemorrhagic fever” (Halstead 1965).
I spent three happy, useful, and busy years at YARU after my arrival August
1965. The lab was then at full strength with an amazing roster of eminent virolo-
gists: Max Theiler, Wil Downs, Del Clarke, Jordi Casals, Sonya Buckley, Tommy
Aitkin, Loring Whitman, Charles Anderson, and Bob Shope. In addition,
Epidemiology Department faculty member, Dorothy Horstmann, became a lifelong
friend. Many other formidable people were there, Bob McCollum, a hepatitis pio-
neer and later a dean of Dartmouth Medical School; Al Evans, infectious disease
epidemiologist and textbook author; George LeBouvier, immunoprecipitation
expert; and Arwin Diwan, trained in arbovirology at the Poona RF laboratory in
India, who later accompanied me to Hawaii. I should mention that I engineered
bringing Suchitra Nimmannitya to the Department at Yale where she obtained an

MS degree. In New Haven, we were able to discuss and analyze data from the Thai
studies and to prepare manuscripts. From Thailand, I brought as laboratory assis-
tants, Dr. Phinit Simasthien and Lersuang Chavanich who had been in the Virus
Department at SMRL. Both later went on to careers in research and medical
education.
The most important event of my assignment at Yale was a gift of over 125 rhesus
monkeys housed in a brand-new holding facility on the top floor of the Epidemiology
building. Dorothy Horstmann gifted me monkeys used in polio research that she had
done with John Paul and Joseph Melnick. Successful polio vaccines had virtually
eliminated polio research. Also, Dr. Paul retired, and Melnick recently decamped to
the Baylor College of Medicine, Texas. DENV infections in these monkeys led to
the single most important laboratory-based research discovery of my career.
Dorothy’s monkeys were systematically infected in all 12 possible combinations of
two different DENVs at intervals of 2 weeks to 6 months (Halstead et al. 1973a).
Humans with DHF/DSS had thrombocytopenia, altered hemostasis, elevated liver
enzymes, activated complement, hypoproteinemia, and vascular permeability. I
looked for these same responses in monkeys. For 3 years, I spent week after week
pre-bleeding, infecting, then bleeding monkeys on 10 consecutive days; performing
white blood cell counts and differentials, platelet counts, hematocrits, and pro-
thrombin times; and measuring protein concentrations and complement titers.
Monkeys revealed some signs similar to those of human DENV infection. Those
with primary DENV infections had generalized palpable lymphadenopathy, and
some had modest leukopenias. Compared with humans, virus appeared in the blood
in monkeys 2+ days earlier, peak titers were lower, while the duration of viremias
and the kinetics of HI antibody responses were similar. Peak HI antibody titers in
monkeys were twofold lower than those in humans (Halstead et al. 1973b).
Cross-protection against first heterotypic DENV viremia was common. When
two different DENVs were inoculated at an interval of 2 weeks, no heterotypic vire-
mia was detected. At challenge intervals up to 6 months, secondary DENV 1 and 4
viremias were depressed, and no secondary infection DENV 3 viremias was
observed. An analysis of these viremias led to the unexpected discovery of a 13-fold,
statistically significant increase in secondary DENV 2 peak viremias in 44 animals
compared with peak primary DENV 2 viremias in 24 animals (Halstead et al.
1973a). This comprises the first experimental evidence of the phenomenon of
antibody-dependent enhancement of a virus infection (ADE). This is direct evi-
dence of ADE and was discovered in subhuman primates in vivo! Enhancement of
infection, not of disease. The circumstances that led up to the analysis of monkey
viremia data in 1972 have been previously described (Halstead 2002). In addition to
enhanced secondary DENV 2 viremias, monkeys with secondary DENV 1, 2, and 4
infections had mild thrombocytopenia. During the viremic phase of secondary
DENV 2 infections, significant complement consumption was observed in eigth
monkeys. A single animal had a DHF-like pathophysiological response. This ani-
mal had been infected with DENV 4 at a low dose (3000 pfu), then challenged three
months later with DENV 2. At the outset of the secondary DENV 2 infection, this
256 S. B. Halstead
animal circulated monospecific DENV 4 HI antibody. On day 3, this antibody was

replaced by a broadly cross-reactive HI antibody. This broadly reactive secondary-
type HI antibody completely disappeared from circulation on days 4 and 5, replaced
by high-tittered DENV 2 viremia. On day 3, the animal sustained a short-duration
increase in hematocrit, and on day 6, for several days, the animal was thrombocyto-
penic and had prolonged prothrombin times. This exciting outcome of sequential
infection has not been confirmed. Candidly, no one has tried. It is widely stated “that
secondary DENV infection disease does not occur” in monkeys! This simply is not
true. Careful studies in rhesus monkeys given formalin-inactivated dengue vaccines
and challenged with wild-type DENVs have documented several enhanced infec-
tion and disease parameters, enhanced cytokine production, and enhanced viremias
(Borges et al. 2019). Just one of many careless fallacies promulgated by the dengue
research community.
Beginning in August 1967, I began a long period as a consultant on Dengue
Epidemiologic Surveillance for the Division of Communicable Diseases, WHO
Headquarters, and to the two Asian WHO Regional Offices. Recall that in the 1960s,
DHF/DSS was recognized in the Philippines, Thailand, Vietnam, and Singapore—
but we did not know its true geographic distribution in Asia. The actual distribution
of cases was needed to strengthen epidemiological reporting and laboratory services
to improve treatment and strengthen control of disease. At Headquarters, an initia-
tive to strengthen dengue epidemiology had been conceived by an Israeli epidemi-
ologist, who, unfortunately, left WHO before my assignment began. In 1967, I
visited India, Ceylon, East Parkistan, Burma, Thailand, Malaysia, Singapore,
Indonesia, South Vietnam, and the Philippines. Then in 1968, I visited some of these
same countries with a somewhat skeptical Eric Roelsgaard, Chief Medical Officer,
Disease Surveillance at WHO Headquarters. In May 1969, a technical Guide on
Surveillance of Dengue Hemorrhagic Fever based upon these trips was published
by WHO and distributed to the two regions. A major accomplishment of this first
Guide was the introduction of a standardized case report form. The report requested
sharing of DHF data with Regional Offices and suggested improved entomological
surveillance. This Guide was strengthened at 1975–1976 meetings in SE Asia and
reissued as the first WHO Technical Guide for the Diagnosis, Treatment and Control
of Dengue Haemorrhagic Fever.
Thus, began a series of yearly WHO consultant visits beginning and ending in
1983 when I joined the Rockefeller Foundation. Consultant assignments were made
in response to country requests, while travel details were managed by the Southeast
Asian and Western Pacific Regional Offices. Individual country reports were sent to
regional offices, approved, and then forwarded to country representatives and
through them to health ministries. These consultant visits were supplemented by a
series of regional WHO meetings. One in 1973, in Jakarta, prepared for the first
Technical Guide for the Diagnosis, Treatment and Control of Dengue. My visit to
Indonesia occurred before DHF/DSS cases had been recognized in the country.
Indonesia was extremely poor. On rounds in Jakarta hospitals, I saw many children
with severe malnutrition including cases of “noma,” a flesh-eating malnutrition syn-
drome. Many visits over more than a decade resulted in my becoming a colleague
and mentor to a generation of public health leaders, including the celebrated Julie
Sulianti, Director, Communicable Diseases Control and, in 1973, President of the
World Health Assembly in 1975. The WHO consultant component of my activities
in arbovirology is too long and complex to be easily summarized.
6 Hawaii
Emanuel Voulgaropoulos, M.D., MPH. (1932–2006). University of Louvain,

Belgium, MD, 1956. From 1959 to 1964, “Dr. V.” worked in Cambodia for
Tom Dooley’s MEDICO and then for USAID in Vietnam. Recruited to the
University of Hawaii International Health Department, “Manny” was
Associate Dean, School of Public Health, (1965–1984). From 1984 to 1995,
he was a USAID public health officer in Indonesia.
In 1966, I was commissioned into the regular US Army. Sometime in early 1968,
the Army informed me I was to be assigned Director of a small medical research
laboratory at the hospital in the Presidio, San Francisco. In a regular Army career,
such an assignment was an important step toward further command assignments.
From my perspective, my research career in arbovirology had just begun. An admin-
istrative assignment would have ended it. Early in 1968, I received a letter from my
friend Emanuel (Manny) Voulgaropoulos telling me that a new medical school in
Hawaii was recruiting faculty. I had crumpled and discarded his letter, when my
wife said, “Hawaii, you should think about this seriously.” She had enjoyed her
month-long stay in Honolulu in 1957, when we were arranging to get her to Japan.
I answered the letter and met in New York with Dr. Windsor Cutting, the new Dean
in Hawaii and previous Dean of the Stanford University School of Medicine. I
agreed to come to Hawaii as full Professor and Chairman of a Section of Tropical
Medicine. I petitioned the US Army to resign my commission. My petition went to
a Board and was approved, luckily, as the Vietnam War was heating up and the
Army needed Medical Corps officers (Fig. 9).
Dean Cutting had the unenviable task of creating a new medical school with very
little support. Furthermore, there was no office or lab space on the University of
Hawaii campus for the School or its Departments, and there were no hospital affili-
ations. The medical school had a small budget and had encountered a lot of resis-
tance from the University of Hawaii faculty. Dean Cutting had arranged to get an
office and potential laboratory space at Leahi Hospital, a facility originally built by
the Territory of Hawaii to house leprosy patients. When leprosy patients were trans-
ferred to Molokai for long-term custodial care, Leahi was converted to a tuberculo-
sis and chronic disease hospital. When I arrived in July 1968, we were allocated two
unused patient wards on the third floor of the Atherton Building. Our floor had
access to the roof of the Administration Building.
258 S. B. Halstead
Fig. 9 Emanuel Voulgaropoulos in 1959, in Tokyo, on his way to Cambodia to work for Dr.
Tom Dooley
The story of developing a laboratory, furnishing, purchasing equipment, gaining

research funding, recruiting faculty, developing graduate programs, and recruiting
students is a long one and will not be told here. Suffice it to say that much of our
financial support came about because of an epidemiological artifact. There were
almost no rubella infections in Hawaii. As a result, young adults in Hawaii, male
and female, were at risk to rubella infections. We obtained contracts to study rubella
vaccines in this largely nonimmune young adult population. In the laboratory, the
reagents and test methods for measuring rubella and dengue antibodies are nearly
identical. Our ability to perform rubella serology tests gained us a public health
foothold in Hawaii and generated financial support to furnish and construct the
Department of Tropical Medicine and Medical Microbiology (“T3M”).
Subsequently, with funds from the University and the State of Hawaii, we built new
laboratories and animal facilities on the roof of the Administrative Building. These
provided an insectary and a specially designed primate holding facility with a pro-
cedure/operating room, a facility crucial to the success of our extensive dengue
studies in subhuman primates.
In addition to the challenge of establishing a new department and laboratories in
Hawaii, another priority was to analyze and publish research done in Thailand and
Yale. An initial series of five articles was published in 1969, in the American Journal
of Tropical Medicine and Hygiene. A second series of six papers were submitted to
the Journal of Immunology: (1) attempts to recover DENV from human autopsy
tissues; (Nisalak et al. 1970) (2) classifying 295 recently recovered DENV using
three different test systems; (Halstead et al. 1970b) (3) correlating DENV antigenic
and biological attributes with human disease syndromes; (Halstead and Simasthien
1970) (4) a mathematical model of DHF; (Fischer and Halstead 1970) (5) the role
of immune status, virus recovered and other host features controlled the severity of
DENV infections (Halstead et al. 1970a); and (6) explanatory hypotheses. In 1969,
Harry Rose, the editor of JI, my professor of Microbiology from P&S, wrote to say
that as a favor to me, the manuscripts had been sent for review to Ed Buescher.
There, they remained for over a year, and despite repeated attempts, there was no
response and no review. What to do? I decided to take advantage of an offer by the
editor of the Yale Journal of Biology and Medicine to publish the series.
Unfortunately, the Yale Journal had limited global circulation, and these papers
never reached a full potential audience.
7 EUREKA: Antibody-Dependent Enhancement of Dengue

Infection (ADE)
One day in April 1972, describing virus isolation and fluorescent antibody results on
a monkey with a secondary DENV 2 infection, Nyven Marchette said “I have never
seen so much virus in the tissues” (Marchette et al. 1973). Nyven had joined the
Department in 1970, coming from an assignment at the Institute of Medical Research
in Kuala Lumpur. He is now famous for his recovery of a strain of Zika virus from
A. aegypti in Malaysia in 1966 (Marchette et al. 1969). In Hawaii, we had access to
a large monkey colony used for Navy microwave research. In addition, we obtained
large numbers of rhesus monkeys inexpensively through a primate trading network.
Most of our experiments were not lethal, making it possible to trade healthy dengue-
immune monkeys for nonimmunes. In monkeys, we studied the temporal distribu-
tion of DENVs in tissues during first and second DENV infections (Marchette et al.
1972; Marchette and Halstead 1973, 1974). Later the same day of Nyven’s com-
ment, Joyce Chow, a PhD candidate, reported on her attempt to use DENV antigens
to stimulate lymphoblast transformation in peripheral blood leukocytes (PBL) from
DENV-immune monkeys. She reported that she observed incorporation of tritiated
thymidine at a 1:100 and 1:1000 dilutions using live DENV 2 but not at 1:10. I said,
“may be the DENV is killing the PBL.” We looked, and sure enough, DENV 2 grew
to high titer in cultures of PBLs from DENV-immune monkeys, but not in PBL from
nonimmune monkeys (Marchette et al. 1972; Halstead et al. 1973c). Earlier, using
inactivated DENV antigens, Dr. Chow had failed to transform DENV-immune T
cells. Observing growth of DENV in cultures of immune lymphocytes (I thought at
the time) stimulated me to analyze the viremia data from the 118 Yale rhesus mon-
keys that had been infected in all sequences of two sequential DENV infections.
Data for each monkey had been entered on large accounting sheets by my laboratory
technician at Yale, Henry Shotwell. This big roll of data sheets had been sitting on
260 S. B. Halstead
my desk for years. I found that viremia titers of monkeys experiencing a secondary
DENV 2 infection were significantly higher than DENV 2 viremias in susceptible
monkeys (primary infections) (Halstead et al. 1973a, b). All animals had been
infected with exactly the same dose of the same DENV 2 given by the same route.
Thus, the discovery of antibody-dependent enhancement (ADE) of DENV infec-
tion derived from two different research events reported on a single day. The data
were there—it was their analysis that had been stimulated.
For years, I had been looking for evidence of DHF/DSS in monkeys by infecting
them with each of the four DENV in all combinations of sequential infection. Data
from Joyce’s PBL experiments were published in Nature, and soon we showed that
all four DENV readily grew in human and rhesus PBL cultures when obtained from
dengue-immune but not susceptible donors (Marchette et al. 1975, 1976; Halstead
et al. 1976). But exactly what were the cells from DENV-immunes that supported
enhanced DENV infections? To solve this, we needed access to technologies not
available in Hawaii.
8 England: Sabbatical
In 1975–1976, I arranged a 1-year sabbatical assignment to the laboratory of Dr.

Tony Allison in the United Kingdom. When I was at YARU, Tony had been an occa-
sional visitor who expressed interest in my dengue research results and enthusiasti-
cally supported my pathogenesis ideas. At the time of my sabbatical, he was
Director, Division of Cell Pathology at the Clinical Research Centre, Northwick
Park Hospital, London. His lab, focused on parasitic diseases and human macro-
phage biology, was supported by the British Medical Research Council.
Anthony C. Allison, M.D., PhD. (1925–2014), Univ. Witwatersrand, MSc in

Medicine, 1947, Oxford University, PhD, MD, 1950. Medical Research
Council, 1954–1974, Director, International Laboratory for Research on
Animal Diseases, Nairobi, Kenya 1978–1981, V.P. Syntex Corp.1981–1994.
Ed O’Rourke, a third year University of Hawaii medical student, traveled with

me to London. Ed was the son of Dr. Edward O’Rourke, Senior, a close friend who
had been Commissioner of Health in New York City and then Dean, School of
Public Health, University of Hawaii. For our research, we needed a supply of human
PBL. Ed came along as the source of DENV nonimmune PBL, and I supplied
dengue-immune PBL (Halstead and O’Rourke 1977a, b; Halstead et al. 1977, 1980;
O’Rourke et al. 1978). I circulated neutralizing antibodies at titers of around 1:640
against each of the four DENVs. I have no idea where these antibodies came from
as I never experienced an overt clinical dengue illness. I had been exposed to other
flaviviruses, yellow fever vaccine upon induction into the Army, and at WRAIR,
Fig. 10 Dr. Anthony Allison at Syntex
several doses of homemade inactivated JEV and Russian Spring and Summer
Encephalitis (RSSE) virus vaccines. Also, I was born in India. Perhaps I had dengue
infections as a child? Who knew? (Fig. 10).
Dr. Allison had discovered that protein-coated silica particles phagocytosed by
mononuclear phagocytes released silicic acid resulting in cell death. This made pos-
sible an in vitro method to identify cells in the monocytic series. In the mid-1970s,
T and B lymphocytes and monocytes could only be crudely differentiated by fixing
cells and then staining them with identifying antisera. There was no practical way to
identify living T or B lymphocytes or monocytes in cultures. We were able to per-
form in vitro ADE tests on cells cultured with or without silica. Ablation of DENV
infection by silica identified ADE-supporting cells as monocytes (O’Rourke
et al. 1978).
At Northwick Park, our weekly research schedule was as follows: two experi-
ments on PBL from a dengue naïve (Ed) and two on PBL from a dengue-immune
donor (me). In addition, we studied ADE in myeloid cells from susceptible and
DENV-immune rhesus monkeys. This resulted in classic studies published in Nature
and the Journal of Experimental Medicine reporting rigorous scientific evidence of
the phenomenon of antibody-dependent enhancement in human and monkey periph-
eral blood myeloid cells (Halstead and O’Rourke 1977a, b; Halstead et al. 1977).
James S. Porterfield, MD, (1924–2010) University of Liverpool, MD, 1948.

Joined the UK MRC’s Common Cold unit (1949–1952), then assigned to
arbovirus labs in Nigeria and Uganda (1952–1956). At MRC, NIMR, Mill
Hill (1957–1977) and Oxford (1978–2000).
262 S. B. Halstead
An unexpected outcome of my sabbatical in the United Kingdom came from

interactions with Dr. James Porterfield, at the MRC National Institute for Medical
Research at Mill Hill. James and I had become friends at earlier international meet-
ings. Indeed, during our sabbatical stay in 1975–1975, James arranged for my fam-
ily to occupy an MRC apartment in Hampstead (marvelously, it had central heat!).
During the year, as James learned about our work, he wondered if the ADE phenom-
enon was specific to DENV-dengue antibody interactions. Under code, he sent us a
panel of 27 rabbit arbovirus antisera that we tested for neutralization and enhance-
ment of DENV 2. The panel contained an antiserum to one togavirus (Semliki
Forest virus), one bunyavirus (Tahyna), and to normal mouse brain. Those three
failed to enhance DENV 2. However, 23/24 flavivirus antisera did enhance. The
highest titers of DENV 2 ADE were achieved with Modoc, Japanese encephalitis,
and West Nile antisera. An antiserum to Spondweni virus did not enhance DENV 2.
In contrast, only DENV 2 immune serum significantly neutralized DENV 2
(Halstead et al. 1980). In 1968, James moved to Oxford, where all his PhD students,
Jane Cardosa, Malik Peiris, and S.W. Gollins, received PhDs on flavivirus enhance-
ment (Peiris and Porterfield 1979, 1982; Cardosa et al. 1983; Gollins and Porterfield
1985). A review by Porterfield coined the term “antibody-dependent enhancement”
(Porterfield 1986) (Fig. 11).
Fig. 11 James Porterfield

after retirement from
Oxford University
9 Return to Hawaii
There were further notable research achievements in Hawaii: (1) first detection of
antibodies to the DENV-soluble complement-fixing factor (now known as NS1) in
convalescent sera from secondary infection cases of DHF (Falkler Jr. et al. 1973);
(2) finding a lipid inhibitor of DENV infection in milk from human DENV-immunes
(Falkler Jr. et al. 1975); (3) finding that PBL from the acute phase of secondary
human dengue infections did not support ADE DENV infection, in vitro; (Marchette
et al. 1979) (4) finding that rhesus monkeys immunosuppressed with cyclophospha-
mide developed severe and fatal DENV 2 disease (Marchette et al. 1980); (5)
describing the original antigenic sin phenomenon for DENV infections (Halstead
1983); (6) defining the mouse P388D1 FcR-bearing cell line as a ADE correlate of
primary human monocytes (Halstead et al. 1983a); (7) showing that different strains
of DENV 2 produced ADE in PBL cultures; (Halstead et al. 1984a) and (8) demon-
strating Zika virus ADE by flavivirus antibodies in FcR-bearing cells (Fagbami
et al. 1987).
In addition, we conducted a truly “nifty” in vivo ADE experiment. We had shown
that in vitro ADE occurred in cultures of mononuclear phagocytes infected by infec-
tious immune complexes. What happened in vivo? Ralph Hale, Chair, Department
of Ob/GYN at UH (member of the US Olympic Committee) made it possible for us
to collect cord blood sera at Kapiolani Hospital from infants born to dengue-immune
and nonimmune Asian mothers. Five of six cord bloods from mothers who had
recently emigrated to the United States had neutralizing antibodies to all four
DENV, one had relatively monospecific DENV 2 antibodies. Pooled polytypic
immune sera had a DENV 2 HI titer, 1:80, PRNT, 1:14, endpoint ADE titer 1:
2,000,000 and peak ADE titer of 1:300. DENV-immune pooled cord blood sera
were inoculated intravenously in five juvenile rhesus to achieve a dilution of 1:300,
while a flavivirus negative cord blood serum was inoculated into five other mon-
keys. Blood taken immediately after intravenous inoculation and tested undiluted
contained no detectable DENV 2 HI or PRNT antibodies but a DENV 2 ADE titer
of 1: 1,000,000. Fifteen minutes after injecting cord blood serum monkeys were
inoculated subcutaneously with approximately 1000 pfu of DENV 2 16681,
Thailand 1964. Animals given enhancing antibodies had peak DENV 2 viremia
titers 2.7- to 51.4-fold higher than controls (Halstead and O’Rourke 1977a;
Halstead 1979).
A major achievement in our laboratory was to explain and identify the mecha-
nism of an early report of an ADE-like phenomenon. Australian virologists, Hawkes
and Lafferty, observed an increase in plaque numbers when diluted Murray Valley
encephalitis virus (MVEV) had been incubated with varying concentrations of
MVE antibodies then used to infect chick embryo fibroblast (CEF) monolayers
(Hawkes 1964; Hawkes and Lafferty 1967). The authors interpreted the increased
plaque numbers as reflecting the ability of antibodies to stabilize MVEV viability.
In her PhD thesis research, Srisakul (Susie) Kliks provided a different explanation.
She found that chicken “fibroblasts” contained a small percentage of functional
264 S. B. Halstead
chicken macrophages. In the presence of sub-neutralizing IgG chicken anti-MVE-

MVEv immune complexes, an enhanced number of MVEV plaques appeared. This
was because MVEV immune complexes infected macrophages and produced
increased plaque numbers (Kliks and Halstead 1980). Only MVE antibodies raised
in avian species enhanced virus infection. This was explained by the role the Fc
receptor plays in the ADE phenomenon. Immune complexes with mammalian
MVEV IgG antibodies readily enhanced MVEV plaques in mammalian but not
avian FcR-bearing cells. This is because chicken macrophage Fc receptors cannot
accept mammalian IgG antibody Fc termini as the evolutionary distance between
birds and mammals is too great (Halstead et al. 1983a; Kliks and Halstead 1980).
Finally, in Hawaii, we were the first to show that serial passage of wild-type
DENV 1–4 in primary dog kidney cells (PDK) selected for viruses with attenuation
characteristics, both in vitro and when tested in rhesus monkeys. These attenuated
viruses retained protective immunogenicity (Halstead 1978, 2003a). The discovery
of cellular attenuation of DENVs was the result of a systematic evaluation of all the
cell culture systems that had been used to produce viral vaccines that had been
licensed in the United States. In Hawaii, under contracts from the US Army, pro-
longed passaging of two strains of DENV 4 in PDK produced candidate vaccines,
one of which was tested in phase 1 (Halstead et al. 1984b, c, d, e; Eckels et al. 1984;
Marchette et al. 1990).
PDK-passaged of DENVs have contributed attenuated DENVs to four different
tetravalent DENV vaccines that reached phases 1–3 testing in humans:
1) Mahidol Vaccine. This effort started at the University of Hawaii in 1967–1968,
where were serial passage in PDK of DENV 1, 3, and 4 and DENV 3 in African
green monkey kidney (GMK) were initiated. At passage 15, strains were trans-
ferred to Mahidol University, Department of Pathology at Ramathibodi Hospital
for further passage, then for in vitro characterization, monkey, and human phase
1 and 2 efficacy and safety testing. These studies were carried out by Sutee
Yoksan in the laboratory of Natth Bhamaravpravati. The live-attenuated tetrava-
lent vaccine was licensed to Aventis Pasteur (Halstead and Marchette 2003;
Bhamarapravati 1993; Yoksan 2017). After extensive phase 1 and 2 testing in
children and adults, it was recognized that the GMK-passaged DENV 3 compo-
nent was under-attenuated. This halted further development of this first-
generation vaccine (Sanchez et al. 2006).
2) Takeda vaccine. The safe and highly immunogenic DENV 2 16681 PDK 53
component of the Mahidol vaccine is a vaccine component and the backbone
chimeric carrier for DENV 1, 3, and 4 structural proteins in the Takeda tetrava-
lent DENV vaccine that has completed phase 3 testing (Vaughn et al. 1996;
Huang et al. 2003; Osorio et al. 2016; Sirivichayakul et al. 2016; Saez-Llorens
et al. 2017; Tricou et al. 2020).
3) WRAIR vaccine 1. A separate PDK-based attenuation program at WRAIR
achieved attenuation of all four DENVs that were combined into several candi-
date tetravalent vaccines that achieved phase 2 testing (Simasathien et al. 2008;
Sun et al. 2009; Watanaveeradej et al. 2011, 2014; Thomas et al. 2013).
4) WRAIR vaccine 2. A second complete set of University of Hawaii DENV 1, 2,

and 4 viruses were passaged 50 times in PDK by Ravithat Putvatana in
1982–1983. One of these viruses DENV 2 16803 PDK 50 was sent to WRAIR
by Nyven Marchette where it was incorporated as a component of a live-
attenuated tetravalent WRAIR dengue vaccine 2, tested to phase 2 (Sun
et al. 2009).
10 Global Consultations
Every year in Hawaii, I served for weeks to months as a WHO consultant, mostly
for SEARO and WPRO. As part of these consultant assignments, yearly visits were
made to ten dengue-endemic countries (India, Sri Lanka, Burma, Thailand,
Malaysia, Singapore, Indonesia, Vietnam, Cambodia, Philippines). In addition,
T3M had a contract (1968–1973) with the American Medical Association to serve
as an academic partner with the Department of Microbiology, Saigon School of
Medicine. This was an exciting experience. I was in Vietnam shortly after the Tet
offensive in 1968 and made a visit to Saigon in March 1975 just before Vietnam
collapsed. Sadly, members of the Vietnam Medical School Microbiology Department
who were studying in our department were stranded from their families by the fall
of Saigon. It was years before families separated in Vietnam and America were
reunited.
During a WHO consultant assignment in New Delhi, I had an unexpected oppor-
tunity to learn more about the history of dengue. One Sunday in 1970, looking for
something to do, I took a taxi to the headquarters of the National Malaria Control
Programme in Old Delhi. This was a historical institution founded by the British in
the 1900s. I wandered into the library and discovered thick volumes labeled “den-
gue.” These were the 300- and 250-page monographs published by the Philippine
Bureau of Science in 1926 and 1931 that described the human volunteer studies on
dengue conducted by Lt. Col. J. F. Siler and Major James Simmons, respectively.
What attracted my attention was that the clinical record for each experimental den-
gue case provided the full name, rank, and serial number of each volunteer.
When I returned to Hawaii, having found these volumes in the University library,
I selected 10+ names from each group and contacted the National Personnel Records
Center (NPRC) in St. Louis asking for their current addresses. The Records Center
replied, but the only addresses available from the Center were “homes of record”
(address on entering the military). I sent letters to homes of record and received
replies from nine men whose mail reached them via their 1920–1930 residential
addresses. They agreed to take the vacuum tube mailed to them to their physician,
obtain a blood sample, and mail it back to us. Sera were tested for HI, CF, and neu-
tralizing antibodies to DENV 1–4 and to St. Louis encephalitis a flavivirus circulat-
ing in the United States. One or more sera from each group had monospecific DENV
neutralizing antibodies, identifying the 1924–1925 experimental infections as
DENV 4 and 1928–1929 infections as DENV 1. Most men, from their
266 S. B. Halstead
correspondence, said they left the Army 4 to 5 decades earlier. They had lived
mostly in the northwestern United States and had not traveled to dengue endemic
countries. Remarkably, they circulated low to moderate levels of HI and CF and
neutralizing antibodies at titers not much different from those in persons infected
one or two years after a DENV infection. In 1973, a fire destroyed one-third of the
records at the NPRC, including those from the 1920s and 1930s.
During my1975–1976 sabbatical in the United Kingdom, I met George
Papaevangelou, an epidemiologist at the Department of Hygiene, School of
Medicine in Athens. Encouraged by the Siler/Simmons data, we agreed to study the
famous 1928 Athens epidemic of severe and fatal dengue. According to contempo-
rary accounts, there had been 650,000 dengue cases and 1061 deaths among the
704,247 residents of Athens and Pireus. It was reported in 1927 that there had been
an epidemic of mild dengue causing little alarm and no statistics. In 1975, George
bled 62 individuals who were living in Athens in 1928. At the time of the outbreak,
these individuals were 7–50 years old, mostly young adults. Remarkably, 74% were
dengue-immune! Three had monospecific CF and neutralizing antibodies to DENV
2, and 15 had monotypic HI, CF, and PRNT antibodies to DENV 1. Sixty percent of
dengue-immunes had serological evidence of two DENV infections. Ten of these
had CF antibodies suggesting a secondary DENV infection but relatively monospe-
cific DENV 1 neutralizing antibodies, while 17 had classical HI, CF, and PRNT
antibodies against two or more DENV infections (Papaevangelou and Halstead
1977). In a follow-up study, sera were collected from 141 Athenians born in 1927,
1928, or in 1931–1935. DENV 1 or 2 monospecific antibodies were found in
30–40% of individuals born in 1928, further evidence of the intensive epidemic
transmission of dengue viruses in that year. Of the 13 children born after June 1928,
five were immune to DENV 2, none to DENV 1 (Halstead and Papaevangelou
1980). Our original interpretation was that the severe 1928 Greek outbreak was the
result of sequential transmission of DENV 1, then DENV 2. Indeed, we learned
later that this same sequence caused large 1981 and 1997 epidemics of DHF/DSS in
Cuba. In Cuba, the mild virgin soil DENV 1 epidemic occurred 4 or 20 years before
the introduction of DENV 2 (Kouri et al. 1986). Since the publication of the Athens
study, it has been learned that DENV 2 infections can sensitize to secondary DENV
1 infections. Although this sequence is a rare cause of DSS for reasons unknown,
this is what happened in Tahiti in 2001, when DENV 1 produced severe DSS four
years after an island-wide DENV 2 outbreak (Hubert and Halstead 2009).
Of importance in understanding the biology and epidemiology of dengue disease
is to learn what happened in Cuba. In 1981, secondary DENV 2 hospitalizations
occurred across the age range, 3–60+ years. Disease severity peaking at ages
3–4 years, with a second peak of high fatality in the elderly (Guzman et al. 2002a).
In 1928 in Athens, severe disease and deaths were reported predominantly in older
adults, not in children. Our extensive serological data identified that two viruses,
DENV 1 and DENV 2, circulated in 1928. It is possible that DENV 1 followed by
DENV 2 or DENV 2 was followed by DENV 1 infections in 1927 and 1928, respec-
tively. Either scenario explains the 1928 epidemic as an ADE event. There is no
documented instance of sequential transmission of two different DENV at an
interval of one year producing an epidemic of DHS/DSS. The very high rates of

dengue fever reported in 1928 suggest the majority of cases were DENV 1, known
to produce an overt to infection ratio of nearly 1. If there had been a virgin soil
DENV 1 or 2 epidemic 1 year earlier than the introduction of DENV 1 or 2 in 1928,
there would have been severe disease in adults and, notably, in children. When
DENV outbreaks occur at long intervals as in Cuba, where an island-wide DENV 1
epidemic in 1977 was followed by DENV 2 in 1997, there was an especially severe
outbreak of DHF/DSS in persons 20 years and older (Guzman et al. 2000a). The
absence of high mortality of children in Athens in 1928 is consistent with the intro-
duction of different DENVs decades apart (Hare 1898).
WHO consultant visits to dengue-endemic Asian countries provided an opportu-
nitiy to design a Gold Standard prospective seroepidemiological study of human
DENV infections in Rayong, Thailand, in 1980. This was a technically more sophis-
ticated study than was possible in Bangkok in 1962–1964 or the prospective studies
conducted on Ko Samui Island by Phil Russell and colleagues (Russell et al. 1968;
Winter et al. 1968, 1969). DENV infections in Rayong were identified entirely by
neutralizing antibodies. This was made possible by the invention of a single dilution
neutralization test that took advantage of the relative specificity of neutralizing anti-
bodies that circulated for years after primary infections. Monospecific 70% plaque
reduction at a 1:60 final dilution of serum identified a past primary infection.
Significant antibodies at this dilution to two or more DENV signified two or more
past DENV infections. This test was performed at a huge scale because of the
remarkable competence by Miss Suntharee Rojanasuphot, a technician in the Virus
Research Institute of the Thai MPH (Sangkawibha et al. 1984). She, alone in 1980,
completed DENV 1–4 neutralization tests on more than 5000 sera. Although the
study continued over a period of 5 years, only data for one year were compiled into
a manuscript (Sangkawibha et al. 1984). I left Hawaii in 1982 and was unable to
process any of the laboratory data from later years. The 1980 study received only
$15,000 from the Southeast Asia Regional Office of WHO in New Delhi. WHO
funds were supplemented by my NIH funds to the University of Hawaii and contri-
butions of staff of the Virus Research Institute and other units in the Department of
Medical Sciences and hospital staff of the Ministry of Public Health of Thailand.
The key discoveries from the 1980 study were: (1) there is an original antigenic sin
phenomenon in the human DENV neutralizing antibody response; (Halstead et al.
1983b) (2) there were secondary DENV infections in all 12 possible sequences in
1980, but DSS only occurred during secondary DENV 2 infections; (3) these DENV
1–2 infections were very pathogenic; 20% of those were clinically ill were in shock;
(4) secondary DENV infections ending in DENV 1, 3, or 4 did not cause severe
dengue; (5) DSS was observed only during a second not a third or fourth DENV
infection; and (6) low levels of monospecific neutralizing antibodies to DENV 1, 3,
and 4 (and absence of heterotypic DENV 2 neutralizing antibodies) were risk fac-
tors for the occurrence of DSS during secondary DENV 2 infections.
I was encouraged by the collaboration between Ministry of Health scientists,
local health officials, and hospital physicians that produced the Rayong prospective
study. This led to an effort over the next six years promoting the design of
268 S. B. Halstead
prospective epidemiological studies in countries in the two WHO Asian regions.

Several interregional meetings were held, protocols written and funded, but the
needed combination of high-quality clinical, epidemiological, and laboratory com-
petence did not emerge to produce studies of value. A parallel series of WHO meet-
ings were organized to introduce the concepts and practices of community-based
Aedes aegypti control into the two Asian regions. Little of lasting value emerged
from this initiative.
I enjoyed 13 busy and highly productive years in Hawaii, plus two years of sabbati-
cal leave. I spent one year at Northwick Park Hospital in London and another at the US
Army Medical Research Institute for Infectious Diseases (USAMRIID) in Frederick,
Maryland. During my tenure in Hawaii, I recruited to our faculty, Dr. David Morens, a
physician and EIS Officer from CDC, Atlanta. After my departure, he remained at the
Department for 12 years. He and I published a “mind-bending” experiment that has
attracted absolutely no attention. In 1980, at our request, Dr. Donald Burke, then Chief,
Virology Department, AFRIMS, sent us DENV 2 strains that had been recovered from
patients with dengue illnesses of varying severity. Each of these DENVs was studied
under code for ADE versus the human polyclonal dengue cord blood antibodies used
to enhance viremia in rhesus monkeys (Halstead 1979). We observed that the ten
DENV 2s recovered from grade III DHF cases were enhanced to higher titers than
were the three DENV 2s recovered from less severe cases (Morens et al. 1991). This
imbalance resulted from a revision of disease severity in Bangkok after the DENV 2s
had been sent to Hawaii. Despite its profound implications, no attempt has been made
to confirm this result. After I joined the RF, David completed this and other projects,
writing, editing, and publishing a number of manuscripts for which I am grateful
(Morens et al. 1985a, b, 1987a, b, c, d; Morens and Halstead 1987, 1990; Morens 1994).
11 Cuba: Havana
Gustavo Kouri, M.D. (1936–2011). University of Havana, MD, 1962,

Director, Institute Tropical Medicine Pedro Kouri, (1979–2011).
María Guadalupe Guzmán Tirado, MD, PhD, Dr Sci. (1950–). University

of Havana, M.D., 1975, PhD 1985, DrSci 2008. Head, Virology Department,
Director, Reference Center for Research & Diagnosis, Pedro Kourí Tropical
Medicine Institute, Havana (1981–).
While in Hawaii preparing for my 1982–1983 sabbatical leave at the United States
Army Medical Research Institute of Infectious Diseases (USAMRIID), I received a
letter from Gustavo Kouri, Director of the Institute of Tropical Medicine of Havana,
Cuba. He described the severity of the 1981 outbreak, noting that it had been pre-
ceded by an epidemic of DENV 1 in 1977. He agreed that DHF in 1981 was consis-
tent with the phenomenon of immune enhancement. Could I come to Cuba to meet
with him and his staff to discuss research opportunities? I decided to visit Cuba
before starting work at USAMRIID, traveled to Mexico City where I reported to the
Cuban Embassy and awaited a visa and transportation to Havana. The process took
a week, time spent exploring the Anthropology Museum, Chapultepec Castle;
drinking tequila sours; and getting to know Mexican and Cuban history. I had not
known about the Ninos Heroes of Chapultepec. These were military academy
students who fought against the US Marines in 1846 and were national heroes.
Afterward, Chapultepec became a palace of the ill-fated Emperor Maximillian. At
the time of the Mexican War, it was a military academy. Also, I learned that Fidel
Castro met his conspirators at the memorial to the Ninos Heroes in Chapultepec
Park. I read about Fidel’s dash across the Caribbean from Mexico on “Grandma,” a
60-foot motor launch with a small group of comrades. He landed near Santiago,
launching an improbable invasion of Cuba. When I arrived in Havana, I took the
opportunity of visiting the Museum of the Revolution in central Havana where the
Grandma is displayed in all its glory (Fig. 12).
Fig. 12 Gustavo Kouri and “Lupe” Guzman at Varadero Beach in 1982
270 S. B. Halstead
In addition to ubiquitous signs, posters, billboards, and monuments scattered all

over Havana celebrating Che Guevara, the Cuban revolution, and warning the popu-
lation to be vigilant, there were just a few signs along highways outside the city that
accused the Yankees of germ warfare. A year earlier, Fidel made the charge of germ
warfare before the Cuban National Assembly. This had prompted the NY Times and
Wall Street Journal to telephone me in Hawaii asking me to comment on Fidel’s
accusation that the epidemic was the work of the CIA. I told them that I knew more
about the pathogenesis of dengue hemorrhagic fever than anyone at the CIA, and I
had no idea which strain of DENV to introduce into Cuba that would reliably cause
an outbreak of severe secondary DENV infection disease. Ultimately, the Cubans
claimed New Guinea C was the virus, which was introduced by the United States.
But the NGC DENV 2s generally accessible to science were high-passaged mouse
strains, demonstrably attenuated for humans. NGC would not be useful as a germ
warfare agent! It turned out (after years of Cuban deception) that the DENV 2 caus-
ing the 1981 epidemic was an Asian virus probably imported from Vietnam. I was
in Hanoi some years later, booked into a Cuban hotel where I met many Cuban
healthcare workers who had been in Vietnam since the 1970s. What we dengue
scientists didn’t know at the time was that severe dengue disease required an inter-
val of several years between the first and second DENV infections. The four-year
interval between 1977 and 1981 was perfect.
The Institute of Tropical Medicine of Havana, at the time, was located in a pri-
vate house that had belonged to a member of the Bacardi family. In the backyard
was a large swimming pool with the letter B emblazoned on its bottom. Gustavo,
director of the Institute, was the son of its founder and was politically well con-
nected. This made it possible for me to visit many hospitals and talk with physicians
about their clinical experiences with dengue in adults and children. Cuban physi-
cians recognized the danger that patients might die rapidly from vascular permea-
bility. Intensive care with appropriate fluid resuscitation was the key to patient
survival. I visited one of several newly constructed intensive care units, designed,
built, equipped, and staffed during the short period of the epidemic, from April to
September of 1981. Throughout the entire country, among the estimated 20,000
cases of severe dengue, there were only 158 deaths. The 1981 epidemic curve in
Cuba clearly revealed that this outbreak was terminated by an organized interven-
tion. This is a testament to the power of the Cuban government to mobilize and
deploy insecticide spray teams effectively. This is the only successful intervention
that stopped an arbovirus disease that I witnessed in my life.
Between 1982 and 2017, I traveled to Cuba more than 30 times, reviewing clini-
cal and epidemiological data, participating in the design of collaborative research
projects, analyzing data, preparing manuscripts for publication, and designing and
writing research grants. All visits were to the virology group at the Institute of
Tropical Medicine led by Dr. Maria Guadalupe Guzman Tirado, known to everyone
as “Lupe.” Lupe enjoyed the support of her husband Gustavo, also a virologist. We
became close friends. During my first visit in 1982, I helped design a retrospective
seroepidemiological study of the 1981 epidemic published in the AJTMH. From the
standpoint of learning about clinical responses to dengue infections, my amazing
good fortune was that the 1981 outbreak was in full force in July when new resident
physicians arrived and were looking for clinical projects needed to satisfy thesis
requirements for specialty boards. Many of them chose to write about “dengue.”
Because the 1981 outbreak affected persons of all sexes and ages between ages 3
and 60+ years, patients with acute DENV infections were seen by virtually every
medical and surgical specialty. Clinical data had been recorded and reported in a
well-organized fashion by the resident physicians.
The first seroepidemiological study, sited in El Cerro, a district in Havana, was
able to quantify clinical outcomes during secondary DENV infections by age, sex,
and race. This provided objective evidence that blacks experienced less severe dis-
ease during a second heterotypic DENV infection than did Caucasians or Asians
(Bravo et al. 1987; Morier et al. 1987; Guzman et al. 1990). Subsequent studies vali-
dated and sought to explain these differences by identifying dengue resistance
gene(s) in sub-Saharan blacks (de la Sierra et al. 2006; Breugelmans et al. 2013;
Sierra et al. 2007, 2017). The science behind these remarkable studies was made
possible by the accurate measurement of specific DENV-neutralizing antibodies
(Guzman et al. 1990). To do this, the Cubans needed to learn how to perform dengue
plaque assays. I introduced them to the formal modification of Pat Repik’s “shake
and bake” BHK-21 cell suspension dengue plaque assay that we had adopted in
Hawaii (Morens et al. 1985b). This assay was applied to the single dilution neutral-
ization test (1:30) that I devised to study sera from the 1980 dengue outbreak in
Rayong, Thailand (Sangkawibha et al. 1984). Training was provided by Linda Kay
Larsen, a senior medical research technician sent from T3M to Cuba for three
months. This same test has been performed with little change for over 30 years in
Havana. It is the foundation of many important discoveries.
I co-authored 15 papers with the Cuban group (Guzman et al. 2000a, b, 2002a,
b, 2007, 2010, 2012, 2013, 2016; Rodriguez-Roche et al. 2005a, b, c, 2011; Alvarez
et al. 2006; Peeling et al. 2011). In addition, I identified and then commented on a
biologic phenomenon observed in 1981 and again in 1997 and 2001—a month-to-
month increase in the fraction of severe and hospitalized cases and in the case fatal-
ity rates accompanying secondary DENV 2 infections (Halstead 2014a). The
Cubans and I speculated in The Lancet that this might be an example of escape
mutations (Guzman et al. 2000b). This hypothesis proved prophetic. Research
funded by the Wellcome Trust found a single point mutation in the DENV 2 NS1 to
be associated with the rapid increase in disease severity in 1997 (Rodriguez-Roche
et al. 2005b, c, 2011). Later, it was shown that the NS1 mutant enhanced DENV
disease severity in a mouse model by increasing intracellular production of DENV
(Chan et al. 2019).
Important discoveries in Cuba included the first description of classical DHF/
DSS in adults during secondary DENV 2 infections, occurring 18–20 years after a
first infection with DENV 1. Not only did severe disease occur at that long interval,
but disease response was significantly more severe than clinical responses to the
same infection sequence at an interval of four years (Guzman et al. 2000a, 2002b).
We also observed that secondary DENV 3 illness 20 years after a primary DENV 1
infection was very severe. By contrast, secondary DENV 1 or 3 infections 20 years
272 S. B. Halstead
after primary DENV 2 infections were mild (Rodriguez-Roche et al. 2005c, 2016;
Alvarez et al. 2006; Guzman et al. 2012). As was true in Thailand, young children
were at higher intrinsic risk to dengue vascular permeability than older children or
adults during secondary DENV infections (Halstead et al. 1969a; Guzman et al.
2002a; Trung and Wills 2010). This is the only published data showing an age gra-
dation of risk to severe vascular permeability during secondary DENV infections.
Children age 5 or younger were at five times higher risk of developing severe vas-
cular permeability during secondary DENV 2 infections than adults or older chil-
dren. This same gradation was observed in seronegative children sensitized by
Dengvaxia but badly misinterpreted by WHO and Sanofipasteur “experts,” who,
instead, attributed severe breakthrough DENV disease in vaccinated children to
immunological immaturity (Hadinegoro et al. 2016; Wilder-Smith and Byass 2016;
Wilder-Smith and Yoon 2016).
12 Rockefeller Foundation
Ken Warren gets full credit for persuading me to leave academic life in October
1983 to work in international health development at the Rockefeller Foundation
(RF). Dr. Kenneth S. Warren, a renowned parasitologist, had been appointed
Director of Health Services at the Rockefeller Foundation in New York City and
had a strong interest in using science to strengthen and promote health in the
tropics. In 1978, he launched the RF “Great Neglected Diseased of Mankind
Programme.” In 1983, I was working in the lab at USAMRIID during my sabbati-
cal when Ken called one day, asking “How would you like to change your venue?”
I knew a lot about the RF. It had been part of my professional life. When I decided
to go to Bangkok in 1962, I visited RF offices and lab in New York for advice and
reagents. The RF helped build my lab in Bangkok, had trained Thai scientists in
the Microbiology Department, and provided help during my stay. I spent 3 years
at Yale after I returned from Thailand where most of my professional friends were
RF staff at the Yale Arbovirus Research Unit/YARU. Ken wanted me to manage
the RF’s International Clinical Epidemiology Network (INCLEN) project that
had as its mission strengthening research capacity of medical schools in the
developing world by advancing research competence in clinical epidemiology,
biostatistics, health economics, and health social science. This was the dream of
INCLEN’s founder, Kerr White, who wanted to improve patient care by helping
physicians to link health outcomes to their causal roots, teaching, and promoting
disease prevention. I immediately understood that clinical epidemiology would
also help build functional bridges from academic medicine to public health prac-
tice, increasing the visibility and standing of the public health profession. I spent
the next 12 years at the RF expanding and integrating clinical epidemiology into
the structure and curriculum of 24 medical schools in 14 countries on three
continents.
13 Community-Based Dengue Control
Frustrated with the continuing failure of government-based vector control pro-

grams (Halstead 1988, 1993a), in 1988, I initiated one of the first programs
structured specifically to develop and test methods designed to control of DENV
infections using community interventions (Halstead 1993b). We sponsored a
one-year training program for nine Mexican and Honduran candidates sited at
the Johns Hopkins School of Public Health. This training program was devel-
oped by Carl Kendall, Ellie Leontsini, and Peter Winch. Initially, the program
had envisioned recruiting vector control teams that were to be composed of a
team leader, entomologist, epidemiologist, and social scientist. Regrettably, only
physicians were nominated by participating countries. Thus, multidisciplinary
teams were not assembled as planned for the year-long field training devised in
Puerto Rico at the San Juan CDC Dengue Laboratory. All trainees devised field
sites, which were implemented in El Pueblo, Honduras, and Merida, Mexico.
Only in Honduras were graduates of the program integrated into a national Aedes
aegypti control programs. A useful metric was invented to classify A. aegypti
breeding sites in the context of control, the Maya Index: (1) disposable contain-
ers, (2) breeding sites that could not be destroyed but could be controlled, and
(3) breeding sites that could not be controlled by householders. Index 1 contain-
ers could be discarded, while Index 2 controllable containers could be managed
by householders with education and motivation. The last group, Index 3, required
professional attention. In the Americas, the first two container categories
accounted for 90+% breeding sites (Halstead 1993b). A byproduct of faculty
expertise at Johns Hopkins, Linda Lloyd obtained her Dr.PH, becoming a leader
in the field.
The RF program provided important observations on the nature and management
of vector control that were presented and discussed at a meeting in Taipei, May
23–25, 1994, “Dengue Vector Control in the Urban Environment: Toward a New
Alliance in Asia.” The meeting, co-sponsored by the Environmental Protection
Administration and the Department of Health, Executive Yuan, Taiwan, and the RF,
was attended by 50–60 epidemiologists, virologists, vector control specialists, and
public health officials from SE Asian dengue-endemic countries.
The history and perilous nature of national Aedes aegypti control programs was
reviewed by Brian Kay (Kay 1994). Methods of control of A. aegypti had been
invented by Major William Gorgas, Chief Sanitary Officer, US Army in Cuba,
immediately after Walter Reed demonstrated that yellow fever was caused by a
“virus” and transmitted between infected and susceptible humans by bites of a mos-
quito. It was learned that A. aegypti predominantly bred in stored water. Water bar-
rels and cisterns extensively used to store water for drinking and washing purposes
were destroyed or covered. These measures, applied and enforced by US Army
personnel, halted the transmission of yellow fever virus in Havana in three months.
Gorgas, himself, continued to learn and to apply control measures successfully in
the Panama Canal Zone.
274 S. B. Halstead
Gorgas’ methods were meticulously applied in Brazil by the renowned epidemi-

ologist, Fred Soper of the Rockefeller Foundation. Soper discovered that sustained
destruction of breeding sites resulted in eradication of mosquito populations. With
the support of Getulio Vargas, President and Dictator of Brazil, 1930–1945, Soper
had the resources and teams that succeeded in eradicating two different mosquito
species from Brazil, Anopheles gambiae and Aedes aegypti. The former is consid-
ered one of the greatest public health achievements of all time, only surpassed by
the eradication of smallpox. Indeed, Soper’s success in eradication was the inspira-
tion for the global smallpox eradication campaign. From 1947 to 1959, Soper was
Director of the Pan American Sanitary Bureau, predecessor to PAHO, who directed
A. aegypti eradication campaigns throughout the southern hemisphere. These cul-
minated in 1965, in the eradication of this species in 18 South and Central American
countries. Exceptions were Colombia, Venezuela, and Puerto Rico (Halstead
1993b). Aedes aegypti eradication was not implemented in US possessions. As a
result, in 1963, there was a dengue 3 epidemic in Puerto Rico. Without Soper’s
leadership in sustaining vector control, in 1977, DENV 1 was imported into the
Caribbean from SE Asia. Within a short time, it had been transmitted throughout the
American tropics. Eradication of A. aegypti had failed. Why? As Kay noted, the
political conditions of the 1930s and the inspired leadership of Soper were unusual.
Without strong government programs to suspend property rights, the intensive and
continuous inspection of premises required for eradication became impossible.
Only Fidel Castro was able to impose Soper’s methods successfully and then only
after the 1981 DHF epidemic (Kay 1994).
Norman Gratz, former Director of the Vector Biology and Control Division at
WHO, directed attention to the problem of staffing of national vector control pro-
grams. He noted that viable government vector control programs depend upon
access to academic entomology. With support from the RF, he had visited South and
SE Asian universities to inventory departments of medical entomology and/or grad-
uate programs in medical entomology. There were almost no university graduate
programs in medical entomology. As a result, instead of relying on appropriately
trained entomologists, vector control programs in dengue-endemic countries are
staffed by persons with medical training (Gratz 1994). This mismatch led to incom-
petence and unhappiness. The close connections, found in the United States between
public sector vector control programs and relevant departments in state universities,
are missing in dengue-endemic countries (Gratz 1994; Challet 1994).
Successful control of vector mosquitoes can be managed by a public-private sec-
tor partnership. This had been demonstrated by historical events in California. An
entomologist from Orange County, California, described how mosquito abatement
districts were established in the Bay area in California in 1915 to cope with malaria
transmitted by saltwater mosquitoes (Challet 1994). Mosquito abatement districts,
independent agencies created by enabling legislation and overseen by a governing
board and located in cities or counties, were established. Beginning in the 1940s,
abatement was extended to mosquito vectors of California and Western equine
encephalitis viruses. From 1946 onward, the local abatement districts were assisted
by the Bureau of Vector Control in the state Health Department. For each mosquito
abatement district, a governing board sets the terms for staffing and vector control
activities and continuing programmatic oversight. Under supervision of the govern-
ing board, mosquito abatement contracts were awarded to private sector companies
by competitive bids. In California, over a period of 75 years, mosquito abatement
districts have successfully aborted zoonotic outbreaks of California and Western
encephalitis by Culex mosquitoes. These diseases were once annual scourges of the
Central Valley.
The conferees agreed that allocating the dull, repetitive but exacting work of
mosquito control to the private sector solves the motivation problem. It is human
frailty that sabotages public sector programs where performance and pay do not
contribute to productivity. In the mosquito abatement district system, the public sec-
tor provides funding and project oversight, but not day-to-day abatement manage-
ment (Challet 1994). Another example of private mosquito abatement exists. Many
municipalities, homeowner associations, or private homeowners in the United
States hire commercial companies for mosquito and pest abatement. One of the
largest such company in the United States described its services (Clarke 1994).
Commercial vector control has been a major component of mosquito abatement in
the United States after the 1999 introduction of West Nile virus.
Aedes aegypti is an urban mosquito. Fueling the twentieth century, dengue pan-
demic has been the growth of the world’s urban human population from 751 million
in 1950 to 4.2 billion in 2018 (Halstead 1992). Asia, despite having the lowest per-
cent of city dwellers, nonetheless has 54% of the world’s urban population and
many of its largest cities. Of 40 cities with a population of 8 million or more, 19 are
in dengue-endemic countries. A. aegypti prospers in densely populated environ-
ments. Based upon specific mosquito densities, cities support high or low dengue
virus infection rates. When DENV infection rates are high, circa 10–15% annually,
several different DENVs circulate producing stable patterns of severe disease (sec-
ond DENV infections) in both children and adults. In cities with the highest mos-
quito densities and DENV infection rates, disease occurs only in children, adults
being immune. Cities with low-density A. aegypti may experience low annual
DENV infection rates (below 5%). In this setting, DENVs may be successively
introduced resulting in intermittent epidemics of dengue fever interspersed with
severe dengue occurring over a wide age range, disease more evident in adults than
in children (Halstead 1994).
Control of A. aegypti mosquitos has been beset by seven failures: (1) failure to
staff vector control programs adequately, (2) failure to recruit and retain appropriate
professionals, (3) failure to train high-quality professionals, (4) failure to apply
sound managerial and scientific practices, (5) failure to provide adequate funding,
(6) failure to gain public support, and (7) failure of political will. These intersecting
failures was addressed in “Dengue in the Health Transition” (Halstead 1994). Health
Transition describes changes in disease and age profiles in a developing country that
accompany a “demographic transition.” The demographic transition, first identified
in 1929, relates population growth accompanying industrialization to declining
infant mortality rates, declining fertility rates, prolonged human survival, and
change in age structure of the population. These events resulted in profound changes
276 S. B. Halstead
in disease profiles and human health. Many pre-health transition diseases such as
diarrhea, measles, pneumonia, and malaria may disappear as the demographic tran-
sition fully evolves. By contrast, dengue will survive. It is a disease of urban areas
readily spread by modern transportation. “Modern housing and modern industrial
development may provide more rather than fewer breeding places for A. aegypti. To
further complicate matters, greater affluence often results in a population which is
less, rather than more, compliant with mosquito control programs” (Halstead 1994).
Conclusions are worth restating: “The efficient control of dengue will require an
entirely new kind of intersectoral and interdisciplinary mosquito abatement pro-
gram. The following features should be considered:
(a) Incorporate aegypti control programs into urban planning. The architecture of
city park drains, construction sites, and other structure should be designed to
minimize A. aegypti breeding. A special effort must be made to design residen-
tial buildings, which do not have hidden or inaccessible aegypti breeding sites.
Revised construction codes will be needed to reduce the breeding of disease-
bearing insects.
(b) Interagency cooperation. Because a major site of aegypti breeding is in urban
areas, particularly when autonomous municipalities are involved, new forms of
interagency cooperation must be forged. At a minimum, aegypti control requires
cooperation between the ministries of health, environment, the municipal health
department, and urban sold waste disposal agency.
(c) Public and private sector collaboration. The goal of sustained and cost-effective
aegypti abatement poses an especially interesting challenge to the private sec-
tor. In the United States, abatement of nuisance and disease-bearing insects is
routinely assigned to private companies or especially constituted community
agencies. These apply market mechanisms to organize, fund, motivate, and
achieve highly proficient programs.
(d) Stepwise eradication strategy. History has shown that it is easier and cheaper to
sustain aegypti eradication than it is to control breeding at low levels. Tradition
has implanted in the minds of public health workers the concept that aegypti
can be eradicated only on a nationwide basis. This is a false premise. Mosquito
control programs should be opportunistic; in neighborhoods, districts, or
regions where conditions permit, source reduction can and should be carried to
the level of eradication. An area in which aegypti are eradicated permits inex-
pensive, passive monitoring for reinfestation using ovitraps.
(e) Dengue control should be rooted in a “gold standard” method for measuring
dengue infection. A variety of simple sampling mechanisms are available,
which use fingertip blood specimens. Serosurveys might be used on a yearly
basis, especially to check for areas of silent transmission or for detecting spread
to previously uninvolved areas. Day-to-day mosquito abatement should be
based on passive as well as active surveillance for the breeding of mosquitoes
and on case detection as a backup system.
(f) Research and training. The control of A. aegypti is a formidable managerial and
scientific problem. It will be impossible to achieve long-term success in mos-
quito control without new knowledge and a national commitment to research

and training. The technical specialists that national programs required cannot
be properly prepared except in an academic and research environment. It can be
predicted that sustained control of aegypti will require constant innovation for
several reasons, including the fact that the species is plastic in its breeding hab-
its and its insecticide susceptibility. Because no perfect method of abatement
has been discovered, a commitment to research is essential” (Halstead 1994).
14 Australia: Charters Towers
At the end of August, 1989, in Brisbane, I attended the Fifth “Arbovirus Research
Symposium in Australia.” After the meeting, I and Jon Aaskov, Professor of
Immunology and Virology, Queensland University of Technology, motored to north
Queensland, specifically to Charters Towers. Why Charters Towers? It was here in
1897 that Australian physician Dr. F.E. Hare endured and treated patients from a
major dengue epidemic. This was not just an epidemic of dengue fever; it was DHF/
DSS. I had learned about Hare’s report from Max Theiler in 1964, when I was pre-
paring a talk for the American Public Health Association in New York, October 5–9,
1964 (Halstead and Yamarat 1965). Hare stated that dengue had been intermittently
epidemic in Queensland between 1894 and 1897, with particularly severe outbreaks
in 1895 and 1897. In the southern hemisphere, dengue transmission peaks in sum-
mer months, January to March. After the 1897 outbreak, Hare had written to 19
medical practitioners in the affected area who contributed clinical details of 60 fatal
cases. In his report, he noted that many persons experienced dengue attacks both in
1895 and 1897 with “some cases more severe than the first.”
It is important to recall that when chikungunya was introduced into the Caribbean
in 1827, it was called “dengue.” During Hare’s time, descriptions of this outbreak,
which had been characterized by a “post-febrile rheumatic stage,” was fully
described in the medical literature and textbooks and given the name “dengue fever.”
By contrast in the 1897 outbreak, the dominant clinical features in adults were those
of classical dengue fever caused by dengue viruses. Hare carefully noted the absence
of a “post-febrile rheumatic stage,” symptoms we now know are associated with
classical chikungunya disease. He specifically describes two clinical features unique
to human dengue, post-fever depression and excess menstrual bleeding. Hare
believed that fatal outcomes in adults were predominantly associated with pre-
existing conditions. In children, he ascribed deaths directly to the intensity of the
disease, with sudden “collapse occurring on the fifth day of the fever and death
ensued from two to 48 hours later.” Brilliantly, Hare likened these clinical events to
cholera, an acute fluid-losing disease that produced fatal hypovolemic shock. He
had no idea children with dengue were suffering fluid losses internally.
Charters Towers is the “Bethlehem” of dengue hemorrhagic fever. I was amazed
to find in 1989 a town little changed over nearly 100 years. It had grown rapidly
after the discovery of gold in 1872. At that time, Charters Towers had the richest
278 S. B. Halstead
Fig. 13 Charters Towers District Hospital
vein of gold in Australia. During the 1880s, the town reached a population of 30,000.
In 1884, the Charters Towers District Hospital opened. Remarkably, in 1989, the
hospital was still at the same location with only modest changes made over 100 years
(Fig. 13). At the original City Hall (Fig. 14), we obtained complete records for
deaths occurring from December 1896 to December 1897. Numbered burial lots
were listed. I have death records for 16 persons who died, March to June 1897, 13
children and 3 adults. The children’s ages were 1, 3 (3), 4 (3), 5(2), 7, 9, 11, and
13 years, the very age distribution of severe fatal dengue in Asia. More compel-
lingly, we found gravestones memorializing these very children (Figs. 15 and 16).
Data supplied courtesy of The Charters Towers and Dalrymple Family History
Association.
Contemporary records suggest that dengue viruses were endemic in Australia
from the 1860s and quite intensively during the 30 years before WWI. There were
scattered reports of fatal dengue all during that period. In 1906, Australians initiated
modern research on dengue. Thomas Bancroft attempted to transmit dengue virus

by bites of A. aegypti but failed because after taking a blood meal, mosquitoes were
not held for an extrinsic incubation period before feeding experiments commenced.
15 Anti-ADE Sentiment
15.1 Tahiti
Early in 2002, I received an email from Dr. Bruno Hubert, an epidemiologist at the
Directorate of Health, Papeete. Tahiti had experienced a dengue hemorrhagic fever
epidemic that started in Bora Bora in January 2001, then spread through the entire
island chain ultimately affecting 33,000 cases or 16% of the population (Hubert and
Halstead 2009). As the national epidemiologist, Dr. Hubert had collected data from
the entire outbreak. He thought it interesting and worthy of a report in the literature.
Claiming his English was weak, would I help prepare a manuscript for publication?
I readily agreed, and he mailed me a comprehensive report in French. This was a
particularly severe outbreak with 1379 children hospitalized of whom 278 were in
shock with eight deaths. The outbreak was entirely due to DENV 1. The extraordi-
nary epidemiological feature was that all DHF cases were in children ages
5–12 years. Because of sustained laboratory studies over a period of years, all
DENVs circulating in French Polynesia were known from 1964. DENV 1 had cir-
culated in 1988–1989, DENV 3 in 1989–1990, and DENV 2 during 1996–1997.
Then, no dengue viruses circulated until DENV 1 in 2001. Children born before
1988 could have been immune to DENV 1, 3, and 2. Those born in 1989 might have
been immune to DENV 3 and DENV 2. But children born after 1990, ages
4–10 years old, might have been immune to just DENV 2 in 2001. It was this group
that was severely affected during DENV 1 infections. Children younger than 4 years
only experienced primary DENV 1 infections in 2001.This epidemic provided the
first evidence that DENV 1 infection of DENV 2-immunes was pathogenic. Dr.
Hubert carefully identified severe dengue in hospitalized infants. Severe dengue in
infants occurs when their mothers have had two or more DENV infections earlier in
life (Halstead et al. 2002). French Polynesian mothers, age 18 and older, could have
been exposed to multiple DENVs, DENV 3 in 1964–1965 and 1969, DENV 2 in
1971, DENV 1 in 1975–1976, and DENV 4 in 1979 (Hubert and Halstead 2009).
Little did I know it would be 7 years before a manuscript was completed. In
2003, Dr. Hubert informed me that the Director of the Institut Louis Malarde had
decreed that if I was a co-author, Dr. Hubert would not be permitted to prepare and
submit a manuscript for publication. Within a year, he left Tahiti and a year later left
France, working in Montreal for two years. Eventually, he returned to a position at
the Cellule Inter-Régionale d’Épidémiologie in Nantes, and we submitted the man-
uscript to Emerging Infectious Diseases where it was published in 2009 (Hubert and
Halstead 2009).
280 S. B. Halstead
Fig. 14 Original Charter Towers City Hall
Fig. 15 Memorial to Betsy McGrath
15.2 Niue
What was going on? Anti-ADE sentiment in Tahiti began long before 2001. It
started after an outbreak attributed to DENV 2 on Niue in 1972, an isolated island
Fig. 16 Heart-shaped memorial gravestone for Ivy Millicent
located 300 miles east of Samoa and Tonga and a population of 4400, resulted in 12
deaths, seven in children. Based on a post-epidemic serological study, 88% of the
population was infected by DENV 2 from March to June. No patients were studied
during the epidemic, although single serum samples from two patients offered sero-
logical evidence of an acute DENV infection. Nearly all cases were consistent clini-
cally with mild dengue fever. DENV 1 had infected Niue during WWII as evidenced
by DENV 1 neutralizing antibodies in nearly all residents over the age of 25 years
identified when investigations began in August. DENV 2 infections occurred in
Niue in 1972, but did those DENV infections result in deaths? Seven children died
during the epidemic, three in a single family and a forth in nearby relatives. All fatal
cases had extensive petechiae and/or ecchymoses. In some surviving children,
ecchymoses developed into chronic leg ulcers. Prior to death, three children were in
coma and two in “shock.” These clinical features differed importantly from DHF/
DSS. The very high case fatality rate in Niue is at variance with what had been
observed in Bangkok Children’s Hospital in the early 1960s. At BCH, for each child
who died, there were 10 in shock who survived. Hemorrhagic phenomena were
unusually prevalent in Niue. However, among Thai children hospitalized with
severe dengue, only 10% had a petechial rash, and 18% had epistaxis. In the 1897
Australian severe dengue outbreak, physicians, who never heard the term “hemor-
rhagic fever,” found that skin hemorrhages were rare. Importantly, deaths in Niue
occurred early in illness, while in DHF/DSS deaths are delayed, occurring five days
or more after onset of fever. Further, Australian children were alert and often speak-
ing up until their collapse and sudden death. No children in the 1897 Australian
282 S. B. Halstead
outbreak were described as being in coma or in shock. “Shock” in dengue is unique

in that systolic blood pressures remain in the normal range. These classical attri-
butes of dengue “shock” were not reported for Niue cases.
The conclusion by authors of the Niue report was that all seven deaths and five
cases of severe disease in surviving children were due to primary DENV 2 infec-
tions. The investigator was Dr. Leon Rosen, our next-door neighbor at Leahi
Hospital in Honolulu. Leon dedicated his 1977 Presidential Address at the 1977
meeting of the American Society of Tropical Medicine and Hygiene to attack the
epidemiological evidence that supported the DHF/DSS “two-infection hypothesis.”
The alternate hypothesis that he proposed and extended into a series of papers pub-
lished over the next 30 years was that severe hemorrhage, shock, or death were
caused by “virulent” viruses that accompanied either during primary or secondary
DENV infections (Rosen 1986a, b, c, 1989, 1996, 1999). Rosen did not agree that
DHF/DSS was an immunopathology. In his Presidential Address, Rosen contended
that since “more” secondary than primary DENV infections occurred every year in
the population (second, third, and fourth heterotypic infections) the association of
secondary infections with severe disease was an “epidemiological accident.” Rosen
seems not to have understood the strong epidemiological evidence from the 1960s
Bangkok hospital-based studies or two studies in which children experiencing sec-
ond heterotypic DENV infections in prospective serological cohorts developed DSS
during a second, not a third, fourth, or first dengue infection (Russell et al. 1968;
Winter et al. 1968, 1969). Further, in the BCH studies, secondary infections were
significantly correlated with severe, not mild disease. Rosen never acknowledged
nor explained the association of severe dengue with first DENV infections in infants
born to dengue immune mothers.
15.3 Bangkok Conference
One of the strangest scientific experiences of my career occurred at the First

International Conference on Dengue and Dengue Hemorrhagic Fever, Chang Mai,
Thailand, November 20–24, 2000. During the presentation of a talk entitled, “An
epidemiological explanation of the sequential infection scheme” Xavier Deparis
and Bernadette Murge of the Centre National de Reference des Arbovirus et des
fieveres hemorrhagiques virales, Pasteur Institute Paris contended that I was respon-
sible for the deaths of 10,000 SE Asian children. This was the result of the impact
of the erroneous hypothesis that secondary dengue infections produced DHF/DSS,
which severely restricted dengue vaccine development. The authors alleged that a
monotypic DENV 2 vaccine could easily have been produced and when deployed
would have prevented thousands of hospitalizations and deaths. A somewhat similar
thesis was contained in a companion paper by Murge et al, prepared at the virus lab
in Tahiti. I sat down with the entire group after the talk, but no data could shake their
belief. Leon Rosen, whose wife was from French Polynesia, had spent years in the
region. After he retired from Hawaii, he worked the Pasteur Institute in Paris. For
more than 30 years, he and many in the French arbovirus community promoted the
view that the association of DHF/DSS with secondary dengue antibody response
was an artifact related to the high prevalence of two or more dengue infections in
populations of dengue-endemic countries. In their view, all severe dengue disease
cases were due to the continuous circulation of a small population of virulent
DENVs (Chungue et al. 1992; Glaziou et al. 1992; Deparis et al. 1998, 2009;
Murgue et al. 2004).
That being said, Dr. Rosen’s concern that “sensitization” would impact dengue
vaccine development proved to be insightful. I quote, “The sequential infection
hypotheses has very important practical implication with respect to the possible use
of vaccine to prevent dengue infection. If sensitization does indeed occur, it obvi-
ously would be unwise to vaccinate unless protection could be conferred simultane-
ously against all four serotypes. The administration of a monovalent dengue vaccine
would be contraindicated and the application of vaccine prophylaxis would have to
await the development of effective vaccines against all four serotypes. There would
also be an ethical problem with respect to vaccine development since human volun-
teer studies would be necessary and some volunteers would have to receive a mon-
ovalent preparation at some stage of the vaccine trials” (Rosen 1977). This is
perceptive and, in my view, an accurate description of the implications of the den-
gue ADE phenomenon for vaccine developers. However, instead of alerting the sci-
entific community to recognize ADE during vaccine development and testing,
anti-ADE resistance and counter-hypotheses evoked denial after vaccine trials and
further created an environment that crippled constructive research on dengue
pathogenesis.
With anti-ADE, the prevailing scientific view in France, it is not difficult to
understand why Bruno Hubert was not permitted to co-author a paper with me. I
have never met Dr. Hubert and do not know how or if he resolved having published
a dengue paper with me. I do know, that by 2004, the “old gang” of pro-Rosen den-
gue virologists in Paris had moved on and were replaced by a new leader, Felix Rey,
an Argentine. Felix was funded by the PDVI.
16 China: Internationalization of SA 14-14-2 JE Vaccine
Yu Yongxin, (1929–) Foochow University (former Fujian Union University)

BS, 1953. Born in Xianyou, Fujian Province. Deputy Chief, Division of Viral
Vaccine, National Institute for the Control of Pharmaceutical and Biological
Products (NICBP),1956–2005.
284 S. B. Halstead
In July 1983, I visited the National Institute for the Control of Pharmaceutical
and Biological Products (NICBP), China, at the invitation of Dr. Yu Yong Xin. He
was interested in developing a dengue vaccine. I was very excited to learn about his
live-attenuated Japanese encephalitis vaccine, SA 14-14-2, produced in primary
hamster kidney cells. The success of vaccinia and yellow fever vaccines was recog-
nized historically. I had lived through and participated in the development and
deployment of successful live-attenuated vaccines against measles, rubella, mumps,
and polio (Sabin vaccines.) All were host range mutants, selected empirically by
passage in tissue cultures. They were highly effective for very long periods and
accompanied by few adverse events. In my view, a single-dose live-attenuated virus
was the ideal product to meet the huge demand for a permanent, effective, and
affordable JE vaccine throughout Asia (Fig. 17).
Shortly after my visit to China, I was appointed Deputy Director of the Health
Sciences Division of the Rockefeller Foundation (RF) in New York. Based on my
China trip, I created a program that ultimately succeeded in internationalizing SA
14-14-2 vaccine. During my first visit, it was clear that a vaccine produced in pri-
mary baby hamster kidney (BHK) cells would create a regulatory problem. BHKs
had not been used in viral vaccines licensed in the United States or Europe. I empan-
eled a group of experts who suggested growing the SA 14-14-2 vaccine virus in a
Fig. 17 Dr. Yu Yong Xin at the time of the award of the Mahidol Medal in 2009
primary cell substrate that was licensed in the United States. The candidate was
primary dog kidney (PDK) cells. PDK had been used to produce licensed measles
and rubella vaccines. To adapt SA 14-14-2 virus to PDK, in 1985, the RF brought
Dr. Yu to work in the laboratory of Dr. Ken Eckels at WRAIR. Because of limited
relations between the United States and China, it required three years of petitions to
US authorities before we could successfully re-export PDK-adapted SA 14-14-2 to
China. A small field trial of this vaccine in susceptible Chinese adults found an
unacceptably low immunogenicity. Further efforts to adapt SA 14-14-2 to PDK
were abandoned.
Meanwhile, in 1988, Dr. Yu was successful in licensing in China the lyophilized
SA 14-14-2 produced by the Chengdu Institute of Biological Products (CDIBP).
CDIBP was the one vaccine production facility in China with large-scale lyophiliza-
tion equipment. As a step toward internationalization, it was decided to upgrade
their production of SA 14-14-2 in BHK cells. To be licensed by the USFDA, a vac-
cine must be free of adventitious agents and produced using Good Manufacturing
Practices (GMP). A new manufacturing facility must meet FDA or similar WHO
GMP requirements to pre-qualify a product that was available for purchase by inter-
national health agencies for use in poor countries. Two internationally recognized
vaccinologists, Drs. Alex Shelokov and C.J. Lee, visited Chengdu to assess the pro-
duction and safety of BHK cells and to develop plans to bring Chinese vaccine
production to GMP standards. The New Jersey engineering and architectural firm of
John Brown designed a US FDA GMP-compliant facility capable of producing
30 million doses per year. Architectural plans were completed and delivered to
China at a cost of over $1 million. The cost to construct and equip a new facility was
estimated at $25 million. Most of these funds were available to the CDIBP, but a
shortfall of the $5 million in hard currency prevented purchase GMP compliant
lyophilization and filling equipment that aborted immediate plans to construct
this plant.
Meanwhile, there was other important work to be done. In 1990, Dr. Liu Zenghle
of the Department of Pediatrics at the INCLEN Unit, West China Medical University
in Chengdu and Sean Hennessey of the Department of Clinical Epidemiology and
Biostatistics, and the University of Pennsylvania designed and completed a case-
control study that measured the effectiveness of SA 14-14-2 vaccine routinely given
to children in rural Sichuan Province (Hennessy et al. 1996). This study was com-
plemented by a large-scale assessment of vaccine safety (Liu et al. 1997). The
excellent results of these studies were presented at meetings sponsored by the
Dengue and JE Vaccine Task Force of the Initiative for Vaccine Research, WHO,
many chaired by Dr. Theodore Tsai, CDC, Ft. Collins, Colorado. Sadly, on my
retirement in 1995, RF support for internationalization of SA 14-14-2 ended.
Hungsoo Kim (1969–) Yonsei Univ, MSc 1993. Yonsei Univ MPH, 2002.
Chairman, Boran Pharmaceuticals, and Glovax Division, Seoul, Korea,
1995–2010.
286 S. B. Halstead
Fortuitously, the vaccine effectiveness study published in The Lancet attracted

the attention of a Korean businessman, Mr. Hyunsoo Kim, chief executive officer of
Boran Pharmaceuticals. He proposed to market SA 14-14-2 vaccine in Korea and
throughout Asia. Mr. Kim and his staff made many visits to Dr. Yu in Beijing and to
the CDIBP to negotiate export plans and privileges. In 1997, he purchased an export
lot of 200,000 doses. Efforts to obtain a license to use the vaccine in Korea stalled
when the eight manufacturers of JE mouse brain vaccine brought political pressure
against introduction of SA 14-14-2 into Korea. The Korean FDA, citing the “dan-
gers” of growing JE in primary BHK, delayed consideration of SA 14-14-2. The US
FDA and WHO were also skeptical of BHK and sided with the Korean FDA. In
1999, with the expiration date rapidly approaching, I suggested that vaccine be
given to a population at risk to JE. But where? (Fig. 18).
With an introduction from Ted Tsai, Hyunsoo Kim, Sunheang Shin, and I visited
Kathmandu, Nepal, in February 1999. There we learned the northwestern provinces
of the Nepalese Terai region had suffered recurrent epidemics of JE during the past
decade. With Dr. M.K. Banerjee, epidemiologist, and M. B. Bista, Director of the
Epidemiology and Disease Control Division of the Nepal Ministry of Health, a plan
was drawn up to deliver 200,000 doses to children in the Terai. SA 14-14-2 was to
be administered as an unlicensed vaccine upon obtaining written consent of parents
or guardians. An application for study of an investigational new product was sub-
mitted to Nepal Research Council, with permission granted on 16 March 1999. A
Fig. 18 Hyunsoo Kim, Sungheang Shin, Yu Yongxin, and members of Boran Pharmaceuticals
team planning meeting Feb 2000, SA 14-14-2 Nepal Clinical Trial
Sunhaeng Shin (1973–). London School of Hygiene, PhD 2004. Product

Manager, Boran Pharmaceuticals and Glovax Division. 1998–2010.
case-control study to measure vaccine efficacy designed by Dr. Shin (as a compo-
nent of her PhD Thesis) was submitted to the Nepal Research Council and approved
on July 19, 1999.
In April, 1999, pediatrician Dr. Young Mo Sohn, epidemiologist Dr. Heechoul
Oh, public health specialist Dr. Myung Ho Kim, epidemiologist J.B. Tandan, proj-
ect manager Dr. Sunheang Shin, and I visited the Bheri Zonal Hospital, Nepalgunj
in Banke District to recruit the Hospital Director and staff to join the study. A visit
was also made to the Vector Borne Research and Training Center, Hetauda, Nepal,
to arrange for JE IgM-capture ELISA tests to be done on sera from cases and con-
trols. In May 1999, study members Young Mo Sohn and J.B.Tandon revisited Bheri
Zonal Hospital to meet with physicians and nurses to review the clinical records to
be completed on all suspect JE cases.
In May, vaccine was shipped under refrigeration to Nepal. During the third and
fourth weeks of July, more than 160,000 subjects 1–15 years of age were given SA
14-14-2 vaccine at 79 health posts in Bardiya, Banke, and Kailali Districts. An out-
break of JE immediately ensued, peaking the third week of August, with 2934 hos-
pitalized cases and 434 deaths (15%). In November 1999, names, age, sex, home
addresses, admission histories, physical examination, and clinical data of all chil-
dren under 16 years admitted to Bheri Zonal Hospital and resident in Bardiya and
Banke districts were compiled. In the hospital, none of 227 clinically diagnosed nor
any of the 20 serologically proven JE cases had a history of JE vaccination
The designed case-control study was carried out in the field by Dr. J.B. Tandan.
To assure as nearidentical exposure to JEV infection by cases and controls, controls
were selected from children the same age and sex as JE cases living in the same
ward. Villages were visited between January and June, 2000. In hospitals or vil-
lages, parents or guardians of JE cases or controls were interviewed for vaccination
history by a hospital nurse or field worker, respectively. A child was listed as vac-
cinated if parents reported vaccination during 1999, and there was additional proof
of this recall, either a record of the child’s name in the vaccine registry, inspection
of an individual vaccination card, exact recall of vaccination post, or name of vac-
cinator. Individual vaccination records are kept by parents and are in wide use in
Nepal. The vaccine registry data abstractors did not know the health status of chil-
dren or whether the child had suffered encephalitis in 1999. To avoid sampling bias,
complete ascertainment of controls was obtained. Of 557 age and sexmatched con-
trols of the 20 serologically confirmed JE cases, 58% had been vaccinated (Bista
et al. 2001). This meant the efficacy of one dose of SA14-14-2 vaccine given a few
days prior to the onset of a JE epidemic was 99.3%. The case-control study was
repeated one year later, yielding vaccine efficacy of greater than 98% (Ohrr et al.
2005). During the next several years, urban areas of the Terai were invaded by
Maoist rebels. This made travel in the area extremely hazardous. Despite these
288 S. B. Halstead
challenges a 5-year post-vaccination case-control study was completed, recording

protective efficacy at more than 95% (Tandan et al. 2007) SA 14-14-2 proved not
only as a one dose vaccine, but it was effective immediately and could be given to
combat epidemics. From the standpoint of safety and efficacy, SA 14-14-2 vaccine
is comparable to yellow fever 17D, long regarded as a model.
In 2000, the Program for Applied Technology in Health (PATH) in Seattle,
Washington, with support from the Bill and Melinda Gates Foundation, established
a program to boost hepatitis B vaccination and improve immunological services in
Hyderabad, Andhra Pradesh, India. A close collaboration with state and national
health officials on the hepatitis B vaccination program permitted Dr. Julie Jacobson
to successfully approach the Government of India following the disastrous 2005 JE
epidemic in Uttar Pradesh with a proposal to make available millions of doses of
live attenuated JE SA 14-14-2 vaccine. This offer was facilitated by PATH’s land-
mark agreement that CDIBP would offer public-sector pricing to lower-income
endemic countries in Asia (GNP < US$1000) for the next 20 years, subject to
changes in currency and consumer price index. PATH provided support to the
CDIBP to select qualified engineering and design firms to plan and oversee the
construction of a new JE vaccine production facility with the goal of meeting WHO
standards. This placed CDIBP on a list of vaccine producers eligible (WHO pre-
qualified) to bid to sell vaccines to agencies supported by international development
funding.
With the significant technical assistance provided by PATH, SA 14-14-2 vaccine
was licensed by the WHO-approved National Regulatory Authority of India. A
license issued in India, modifications to GMP at CDIBP plus the construction of a
new SA 14-14-2 production facility resulted in WHO pre-qualification of CDIBP
manufacture of SA 14-14-2 in 2013. This was followed in 2014, by the approval of
the SA 14-14-2 vaccine by the Global Alliance for Vaccines and Immunization
enabling purchase of vaccine by development assistance agencies for use in poor
countries. In addition to China, the PATH program promoted the licensing of SA
14-14-2 in Sri Lanka, Thailand, Indonesia, Cambodia, Laos, Philippines, South
Korea, and North Korea. It is estimated that between 1988 and 2015, more than 700
million doses of SA 14-14-2 have been distributed for use in these countries
(Ginsburg et al. 2017).
17 US Navy, Bethesda
In January 1995, just after my 65th birthday, in accordance with prevailing practice,
I retired from the Rockefeller Foundation and accepted a position as Director of
Infectious Disease Research, US Naval Medical and Research Command (NMRDC),
Bethesda, Maryland. NMRDC headquarters were located on the 10th and 11th
floors of the “tower” building on the campus of the Naval Hospital Center. This
appointment was arranged by Capt. Steven Hoffman, whom I met in Indonesia on
WHO consultant trips during my tenure in Hawaii. While I was at the RF, Steve
Fig. 19 Dr. Stephen L. Hoffman, malaria vaccine entrepreneur
attended several annual INCLEN meetings. He was someone who always “thought
outside the box” and is in the process of creating an innovative product, a radiation-
attenuated live P. falciparum sporozoite vaccine, manufactured by his Rockville-
based company, Sanaria (Fig. 19).
Stephen L. Hoffman. MD, DTMH, (1950–) Cornell University School of

Medicine, M.D.,1975. US Naval Medical Corps with duty at NAMRU-2,
NMRC, 1981–2003. Chief Executive and Scientific Officer of Sanaria Inc.,
Rockville, MD., 2003
17.1 Navy Overseas Laboratory Studies
At the Naval R&D Command, I was administratively responsible for research activ-
ities in US Navy medical research laboratories in Cairo NAMRU-3), Peru (now
NAMRU-6), and Jakarta (then NAMRU-2). Because R&D funding came from the
US Army Medical Research and Development Command and because overseas
Naval Research Units are not under the command of the Surgeon General, my rela-
tionship with laboratories was advisory only. Dengue was an important military
290 S. B. Halstead
disease and a research problem for laboratories located in Peru and Indonesia. With
the help of colleagues I had known for many years and travel support from the R&D
Command, I was able to help organize and publish four important dengue studies.
1. NAMRIID, Lima, Peru. Discovery of an “Avirulent Dengue 2 Virus.” A research
opportunity emerged as soon as I arrived in Bethesda. In 1995, an outbreak of
DENV 2 had occurred in Iquitos, Peru, five years after an even larger epidemic
of DENV 1. This exact scenario produced DHF/DSS in Cuba in 1981. Prior to
the DENV 2 outbreak in Iquitos, NAMRU had established a dengue serological
cohort study in 1990. Randomly selected school children and young adults had
been bled in 1993, 1994, and 1995. This made it possible to document an epi-
demic of mild American genotype DENV 2 in 1995. Serological studies of the
cohort estimated a DENV 2 infection rate of 80%, 60.5% with secondary infec-
tions in the sequence DENV 1 then DENV 2. In Cuba, this produced thousands
of DHF/DSS hospitalizations. In Iquitos, there were none (Watts et al. 1999).
Some in the dengue research community excitedly attributed this outcome to the
circulation of an “avirulent” dengue virus (White 1999). We believed this
hypothesis is not correct. A better explanation is the American genotype DENV
2 contained a DENV 1-like epitope on its structural proteins (see below).
2. NAMRIID, Lima, Peru. American DENV 2 retains a DENV 1-like structure.
Unexpectedly, we found that DENV 1 human antibodies efficiently neutralized
American genotype DENV 2, but did not neutralize strains of Asian genotype
DENV 2s that had circulated in the Americas causing DHF/DSS (Kochel et al.
2002). In an in vivo experiment, DENV-1-immune Aotus monkeys were pro-
tected against viremias with American genotype DENV 2 but not against Asian
DENV 2 (Kochel et al. 2005). We attribute the absence of DHF/DSS during
secondary American DENV 2 infections to a DENV-1-like antigenic structure
on American genotype DENV-2 virus. This structure resulted in sufficient cross-
neutralization to prevent ADE. The American genotype DENV 2 (Trinidad
1751) likely circulated in the Americas for centuries and as the zoonotic DENV
2 was imported from West Africa. The exact locus of the DENV-1 epitope(s) on
American DENV 2 virus has not been identified.
3. NAMRU-2, Jakarta, Indonesia. Prospective dengue cohort study in Indonesian
children. A prospective serological dengue cohort study of 4- to 7-year-old chil-
dren was organized in Yogyakarta, Indonesia. There were significant differences
observed in the pathogenicity of specific sequences of secondary DENV infec-
tions between Yogyakarta and Rayong. In Rayong, secondary DENV 2 i nfections
were the sole cause of DSS despite the fact that 62.5% of secondary DENV
infections occurred in other sequences. Remarkably, among children 7 years old
and younger, 20% of DENV 1-immune infected by DENV 2 developed DSS. The
exact opposite was true in Yogyakarta. There were no DSS or DHF cases among
31 DENV 1 children who were then infected with DENV 2. Instead, 6 DHF/DSS
cases occurred in 41 DENV 2-immune children who were then infected with
DENV 1 (Graham et al. 1999). A similar pattern was observed in Tahiti in 2001,
where severe and fatal disease secondary DENV 1 infections occurred in chil-
dren infected with DENV 2 four years earlier (Hubert and Halstead 2009).
4. NAMRIID, Lima, Peru. Dengue resistance gene in Haitian blacks. When United
States and United Nations military personnel were dispatched to Haiti in
1994–1996, they suffered an immediate outbreak of dengue fever. The UN
Command had not prepared troops landing at Port-au-Prince to take measures to
prevent dengue. Dengue cases had not reported from Haiti to PAHO. We specu-
lated that the under-reporting of dengue on Haiti might be due to a genetic resis-
tance to severe dengue disease in the black population of Haiti. Existence of such
a gene had been discovered in Cuba. During the Port-au-Prince outbreak, 185
strains of DENV 1, 2, and 4 were recovered from US and UN military personnel.
The DENV 2 genotypes were identical to those recovered from DHF/DSS cases
in Cuba. But was dengue hyperendemic in Haiti? A collaboration with a filariasis
researcher from Notre Dame gave us access to blood samples from 210 school
children (6–13 years old) resident in Carrefour Borough, Port-au-Prince. All sera
were tested for DENV 1–4 plaque reduction neutralizing antibodies. Nearly 85%
had antibodies to two or more DENV serotypes. This translates into an annual
average DENV transmission rate of 30%, a rate observed only in highly endemic
SE Asia. Despite clear evidence of the virological pre-conditions, no cases of
DHF/DSS had been seen in hospitals based on interviews with well-trained
Haitian pediatricians (Halstead et al. 2001). The absence of DHF/DSS in Haiti is
further evidence of DHF/DSS resistance genes in the sub-Saharan African popu-
lation. A resistance gene is thought to have derived in this population from long
exposure to the structurally and biologically similar flavivirus, lethal for chil-
dren, yellow fever (Sierra et al. 2007, 2017; Oliveira et al. 2018). The yellow
fever resistance gene protects sub-Saharan Africans from severe dengue disease.
18 Pediatric Dengue Vaccine Initiative
The birth of the PDVI was an accident. It unexpectedly emerged from what was the
termination of an in-house Rockefeller Foundation program. In January, 2000, five
years after my retirement as a RF officer, I was an invited by the Veterinary Company
of Colombia (VECOL) in Bogota to celebrate the completion of a RF-funded rabies
vaccine technology transfer project. I had started the project soon after joining the
RF because I was alarmed by the inability of developing countries to produce suf-
ficient quantities of high-quality vaccines. A major initial concern was rabies. A
rabies vaccine that required at least three doses for post-bite treatment was available
on the international market. The problem was that it was expensive. Rabies vaccine
production methods had been pioneered by Louis Pasteur who serially passaged
virus in rabbits to achieve an attenuated live virus. In Pasteur’s initial immunization
sequence, 21 daily doses of vaccine were given subcutaneously around the umbili-
cus. Initially, virus “fixed” with formalin was injected. Then, progressively less and
less attenuated live virus was given. Subsequently, rabies vaccines were made from
292 S. B. Halstead
virus grown in mouse, rabbit, or monkey brains then killed by formalin or betapro-
priolactone. Pasteur Institutes, widely distributed in developing countries, produced
rabies vaccines used predominantly as post-bite treatments. In Europe and the
United States, rabies virus grown in tissue cultures and fixed with betapropriolac-
tone was given in a three-dosage regimen, usually with rabies immune serum given
immediately at the site of the bite. In developing countries locally produced rabies
vaccines often of very poor quality and in most countries, there was no effective
national vaccine quality control management. Only the well-to-do could afford
effective treatment, while poor people had to use locally produced rabies vaccines,
dooming many to deaths making rabies a significant burden to most developing
countries.
In November 1984, the Health Sciences Division initiated a program to transfer
tissue culture-based virus vaccine production technology to several large or medium-
sized developing countries. Vaccines on the list were rabies, Japanese encephalitis,
dengue, and yellow fever. It was reasoned that if the ability to produce viral vaccines
in tissue cultures could be mastered in developing countries, there were important
spinoff benefits: (1) mastery of all steps in production, safety, and efficacy testing of
human vaccines provided experience needed to design and implement national bio-
logics quality control safeguarding the quality of many domestic and imported bio-
logic products and (2) laboratory techniques for production of human viral vaccines
are virtually identical to those used to make veterinary vaccines. We agreed that if
technology transfer could be shared between the health and agriculture sectors,
quality veterinary and human rabies vaccines could be produced in a single facility.
A WHO Group of Experts, supported by the RF, chose as technology transfer
laboratory the Netherlands national vaccine production facility Rijksinstituut voor
Volksgezondheid en Milieu (RIVM) and VECOL as the first vaccine technology
transfer recipient. The premature death in 1987 of the RIVM Director, Dr. A.L.Van
Wezel, led to selection of the Institute Armand Frappier in Montreal as the training
center. There, vaccine production was based on a sepharose bead microcarrier tissue
culture system developed by Bill Thilly at MIT. After many technical, logistic, and
terrorism-based delays, human rabies vaccine of suitable quality was produced by
VECOL, licensed and approved for use in Colombia, and thus the celebration.
Dr. Ariel Pablos-Mendez, previously an infectious disease officer at the Ministry
of Health of Mexico and now a RF officer, had been invited to the Bogota meeting.
Due to illness, he was unable to attend. After the meeting, I visited the RF to brief
Ariel on its outcome. I discovered he was not interested in extending the rabies vac-
cine technology transfer project, but did want to do something about dengue. On
March 3, 2000, I wrote to outline a program to accelerate dengue vaccine develop-
ment. It described existing candidate dengue vaccines, noting that most were
designed for adults, with few resources allocated for development and testing of
vaccines for children. The Mahidol University (University of Hawaii) tissue culture
passaged tetravalent dengue vaccine, licensed to Pasteur-Merieux, was in its third
year of phase 2 testing (Halstead and Marchette 2003). It had recently become clear
that this vaccine when given to susceptible adult volunteers was moderately reacto-
genic and only produced antibodies to DENV 3 (Kanesa-thasan et al. 2001). Further
studies showed the DENV 3 was under-attenuated and consistently over-grew the
more attenuated companion viruses (Sanchez et al. 2006; Kitchener et al. 2006).
There seemed no way to solve this problem, accordingly this vaccine was aban-
doned. Fortunately, there were alternative candidates: a lab in the National Institute
for Allergy and Infectious Diseases (NIH) had constructed a genetically altered
strain of DENV 4, the Navy had a monotypic dengue DNA vaccine candidate, while
the Army had adapted all four live DENV to PDK cells. All these vaccines needed
to be tested for safety and immunogenicity in humans. None of the developers had
plans to test dengue vaccines in children, the population most affected. My letter
concluded, “I will end by agreeing strongly with your view that something like IAVI
(International AIDS Vaccine Initiative) must come into existence if a safe and effec-
tive dengue vaccine is to become a reality.”
On August 21, 2000, I sent the following memorandum to the RF: “A Dengue
Vaccine Initiative to accelerate the development, manufacture and deployment of a
safe and effective multivalent dengue vaccine for children.” There was an inaugural
organizational meeting at the RF in NYC on October 6, 2000. For the next three
years with RF support, I met with dengue vaccine developers and other stakeholders
to plan and raise interest and the political needed to generate funds for what became
the Pediatric Dengue Vaccine Initiative (PDVI). The crucial organizational event
was an international meeting held in Ho Chi Minh City, December 5–8, 2001, (3
months after “9/11”). This Conference was co-sponsored by the Rockefeller
Foundation and the International Vaccine Institute (IVI) with support from
Children’s Hospital No. 1 and the Pasteur Institute in Ho Chi Minh City. Other con-
tributors were the World Health Organization’s Initiative for Vaccine Research, the
UNDP/World Bank/WHO Special Programme for Research and Training in
Tropical Diseases, Aventis Pasteur, and GlaxoSmithKline. The meeting convened
more than 150 pediatricians, vector control specialists, immunologists, virologists,
vaccine scientists, economists, international health development specialists, and
representatives of funding agencies from 30 industrialized and developing coun-
tries. A summary describes the burden of dengue, a first pharmacoeconomic analy-
sis of a pediatric dengue vaccine, and provided a scientific blueprint to support the
accelerated development of a pediatric dengue vaccine (Almond et al. 2002).
Conferees recommended that the Pediatric Dengue Vaccine Initiative (PDVI) carry
out country surveys of dengue burden, phase 3 field site development; develop
dengue-scientific infrastructure; support the development of safe and effective den-
gue vaccines; and promote dengue vaccine advocacy and partnerships at a total
10-year budget of $240 million. [Final Report: Accelerating the Development and
Introduction of Dengue Vaccines for Poor Children. The Road from Vietnam: Vision
and Goals. Reports of Working Groups: (1) Need for a Pediatric Dengue Vaccine.
(2) Pharmacoeconomic Analysis of a Pediatric Dengue Vaccine. (3) Scientific
Blueprint for Pediatric Dengue Vaccine Development. International Vaccine
Institute, 2002.]
With momentum and interest generated in Vietnam, there were two follow-up
meetings, Antibodies in dengue: the problem and the challenge to science, attended
by 57 vaccine and antibody experts from fifteen countries held at the Lawton Chiles
294 S. B. Halstead
Fig. 20 Dr. Sally Stansfield after retiring from the Gates Foundation
International House (Stone House), NIH Campus on June 3 and 4, 2002, and A
Burden of Dengue Workshop, sponsored by the Ellison and Rockefeller Foundations
and on November 5–7, 2002, at PAHO in Washington, D.C., convened by PAHO
and the Rockefeller Foundation, a first assessment of the cost effectiveness of den-
gue vaccine attended by 55 participants from 14 Asian and Latin American dengue
endemic countries plus the United States. Information from this Workshop (Shepard
et al. 2004) (Fig. 20).
Sally Stansfield, M.D. (1950–) University of Washington, MD, 1975. Assoc

Dir Global Health Strategies, Bill & Melinda Gates Foundation, 1999–2006.
Exec Sec WHO Health Metrics Network, Managing Dir. Social Impact
Practice, Deloitte Consulting, 2006–2018.
In March 2002, with RF support, a PDVI Secretariat was established at the IVI
and a Board of Councilors (Chairman, Duane Gubler), Scientific Advisory
Committees, and an interim Director (SBH) appointed and a survey of dengue vac-
cine development published (Halstead and Deen 2002). On August 1, 2002, an
application to support the PDVI program was submitted to the Bill and Melinda
Gates Foundation (BMGF). Discussions over the next few months revealed the
Foundation was interested in supporting PDVI. However, a final program descrip-
tion required knowing how much the BMGF was willing to provide. This was
accomplished at the annual meeting of the American Society of Tropical Medicine
and Hygiene at the Adam’s Mark Hotel in Denver, Colorado. At the President’s
Reception on November 12, 2002, Sally Stansfield whispered in my ear “fifty-six
million dollars.” Sally, an infectious diseases physician, had been a CDC EIS officer
assigned to USAMRIID in 1982–1983 the year I was there on sabbatical leave. In
2002, she was associate director, Global Health Strategies at the BMGF.
In late December, a formal letter of inquiry to the Gates Foundation was sent
from the IVI requesting $56 million to support the activities of the PDVI for a six-
year period. On June 4, 2003, a letter from Regina Rabinovich, Director, Infectious
Diseases, BMGF, to John Clemens, Director of IVI, announced a grant of $55 mil-
lion for the period July 1, 2003 to December 31, 2008. I was fortunate to be able to
recruit a former PhD student, Susie Kliks, PhD, an administrator in the office of
President of the University of California, who served capably for ten years as
Deputy Director of the PDVI Targeted Research Program.
2003 was the busiest of my career in arbovirology. Visits were made to Cuba in
January to edit research papers and again in August to participate in a biannual
hemispheric dengue scientific meeting. In January, I attended the Novartis Institute
of Tropical Disease inaugural meeting in Singapore where Duane Gubler and I dis-
cussed the complexities of siting PDVI headquarters. After much deliberation and
discussion with the BMGF officers, PDVI was sited in Seoul, South Korea, at the
International Vaccine Institute. I returned via Bangkok and the BMGF in Seattle
where I discussed grant planning with Dr. Rabinovich. In March, I visited IVI to
interview candidates for PDVI office staff. Returning on March 16, I had a short
bout of angina waling up the off-ramp at Dulles Airport. This led to the discovery
the next day that I had a nearly obstructed left coronary artery. Instead of travelling
to Vienna to plan a June PDVI scientific meeting and then to Geneva for the PDVI
Board of Councilors (BoC) meeting, I participated via conference call on Monday,
March 30. I was in my room at the Washington Hospital Center two days after triple
coronary bypass surgery. Four weeks later, on April 30, I was back in the air, making
the postponed trip to Vienna to plan the inaugural meeting, “Dengue Virus:
Molecular Basis of Cell Entry and Pathogenesis.” This was held in Vienna on June
25–27, 2003, at the Haus der Musik, attended by 85 researchers from 15 countries.
We discussed the unique efficacy and safety issues of human dengue virus infec-
tions in the context of the mechanisms of viral cell entry and immune protection.
The meeting was co-sponsored by the World Health Organization’s Initiative for
Vaccine Research, the Centers for Disease Control at Ft. Collins, Colorado,
Acambis, Inc, Cambridge, Massachusetts, Aventis Pasteur, Lyon, France,
GlaxoSmithKline Biologicals, Rixensart, Belgium, Novartis Institute of Tropical
Medicine, Singapore, Henry M. Jackson Foundation for the Advancement of
Military Medicine, Rockville, Maryland, and the Rockefeller, Ellison Medical and
the Bill and Melinda Gates Foundations. The PDVI Targeted Research Program
emerged from this meeting (Halstead et al. 2005).
An early priority of PDVI was to improve data on the burden of dengue illnesses.
Following formal solicitation, Brandeis University health economics faculty mem-
bers, Donald Shepard and Jose Suaya, were engaged as a project management team
to plan and conduct exploratory visits to Asian and Latin American countries, to
draft and carry out a program. This team published some of the most accurate and
insightful descriptions of disabilities caused by dengue viruses (Lum et al. 2008;
296 S. B. Halstead
Martelli et al. 2011) and emerged as the world’s authority on the economic burden
imposed by dengue viruses and the cost-effectiveness of dengue vaccines (Shepard
et al. 2004, 2011, 2012a, b, 2013, 2014a; b, 2016; Lum et al. 2008; Martelli et al.
2011; Halstead et al. 2007; Suaya et al. 2007, 2009; Armien et al. 2008; Beatty et al.
2011; Carrasco et al. 2011; Lam et al. 2011; Halasa et al. 2012; Lozano et al. 2012;
Murray et al. 2012; Undurraga et al. 2013, 2015, 2017; Hotez et al. 2014; Edillo
et al. 2015; Packierisamy et al. 2015; Rouvinski et al. 2015; Stanaway et al. 2016;
Thalagala et al. 2016; Tiga et al. 2016; Bangert et al. 2018; Zeng et al. 2018a, b;
Zubieta-Zavala et al. 2018; Hariharan et al. 2019; O’Reilly et al. 2019).
A second objective of PDVI was to design and establish “Dual Use Cohort
Studies,” field sites where dengue infections could be observed and fully studied
prospectively in a vaccine-eligible population, the same sites to be available for a
phase 3 vaccine clinical trial. A Scientific Advisory Group II (SAG II) was appointed
to implement this program component. Chair: David Vaughn, members: Robert
Edelman, Mary Ann Lansang, Douglas Watts, Maria Joan Wawer, Peter Winch and
Zhu Yi Xu. SAG II published a request for program proposals in the Lancet, dead-
line for letter of intent (LOI) of November 15. Follow-up site visits were carried out
and sites selected in Managua, Nicaragua, managed by Eva Harris and UC Berkeley
and in Rajburi, Thailand, managed by Arunee Sabchareon, School of Tropical
Medicine, Mahidol University. This latter site was used to study various parameters
of dengue epidemiology and antibody responses to dengue infections (Pengsaa
et al. 2003, 2006, 2008; van Panhuis et al. 2011; Sabchareon et al. 2012a;
Sirivichayakul et al. 2014). As planned, the first phase 3 vaccine clinical trial con-
ducted by Sanofipasteur was in Rajburi, Thailand (Sabchareon et al. 2004, 2012b,
2013; Chokephaibulkit et al. 2010; Plennevaux et al. 2016).
The Nicaraguan pediatric dengue cohort was established as a major component
of the PDVI program. After termination of PDVI funding, the site was supported by
the NIH plus several foundations and is still active (2019). This field site and deriva-
tive studies are the most important outcomes of PDVI funding, using high-quality
research to study sequential human dengue virus infections prospectively in chil-
dren (Gordon et al. 2013). The Nicaragua site had been selected as a potential site
of a phase 3 vaccine trial. This did not happen, although Takeda tested vaccine
efficacy in a nearby location in Nicaragua. The published output from the PDVI
Nicaragua pediatric cohort is too vast to be included in this manuscript.
During 2003, a recruitment effort mounted by the Board of Counsellors resulted
in the selection of a Director, Harold Margolis, a CDC officer, pediatrician, and
hepatitis expert. PDVI grew into a large and complex organization managed by
Director Margolis and staff from offices in the IVI. The name PDVI was changed
after 2010 when feedback from the Gates Foundation raised concerns about the
focus on a vaccine for children, when global epidemiology showed that in many
countries, dengue was predominantly a disease burden for adults. Accordingly, the
name was changed to the Dengue Vaccine Initiative (DVI).
The purpose of this historical account is to describe my activities in arbovirus
research and vaccinology. For this reason, this history will focus on the design and
implementation of the PDVI dengue-scientific research program. A Scientific
Advisory Group III (SAG III)—John Roehrig (Chair), Rafi Ahmed, Alan Barrett,
Burton, Siamon Gordon, James Strauss—in September 2003, announced a PDVI
Supportive R&D Program in Nature, with a Request for Proposals and deadlines for
submission of LOIs (November 15) and final proposals (15 February 2004). The
program was designed to gain a fundamental understanding of immune protection
against dengue illness/infection targeting five areas of research: (1) DENV struc-
ture, envelope and subunits; (2) DENV target cells, receptors; (3) neutralization
mechanisms and assays for protective antibodies; (4) ADE mechanisms and assays
for ADE; and (5) pathology and animal models.
LOIs and final proposals were reviewed by an augmented SAG III who evaluated
then awarded research grants or contracts to 20 laboratories in the United States,
Cuba, and Europe. This group comprised a PDVI Dengue Research Network,
administratively managed by Dr. Kliks. A founding premise of this program was
that protection against dengue virus infection required innate and acquired immu-
nity, the latter being predominantly mediated by antibodies. As reported at annual
Network meetings, 2004–2013, gratifying progress was made in identifying virus
and cell receptors, mechanisms of attachment and viral entry, animal modes of
antibody-dependent enhancement (ADE) and protection and in understanding the
rules governing how dengue antibody-virus complexes permit infection, neutraliza-
tion, and infection enhancement in Fc receptor-bearing cells. The founding of PDVI
was accompanied by an astounding increase in research on dengue. PDVI Network
researchers have contributed several hundred scientific papers to high-impact jour-
nals describing research supported by PDVI. Some PDVI laboratories continue
(2019) to dominate global research on dengue (and Zika)—Eva Harris, University
of California at Berkeley, Aravinda de Silva, University of North Carolina, Michael
Diamond, Washington University, St. Louis, Richard Kuhn and Michael Rossman,
Purdue University, Felix Rey, Pasteur Institute Paris, Theodore Pierson, NIAID,
NIH, Carol Blair, Colorado State University, Ft. Collins, CO, Timothy Endy, Upstate
Medical Center, Syracuse, N.Y., Duane Gubler and Shi Mei Lok, Emerging
Infectious Diseases, Duke-Singapore University Graduate Medical School, Steve
Whitehead at NIAID and Anna Durbin, Johns Hopkins School of Public Health,
plus virologists at WRAIR, AFRIMS and NAMRI. A contract for isolating mono-
clonal dengue IgG antibodies was awarded to Antonio Lanzavecchia, Institute for
Research in Biomedicine in Bellinzona, Switzerland. Recognizing constraints to
progress imposed by limited scientific research capacity and the isolation of small
research groups in dengue-endemic countries, beginning in 2004, PDVI promoted
the growth of relevant dengue research communities in dengue-endemic countries
of Asia and the Americas by organizing a series of semiannual international dengue
research network meetings. The series in the American region has continued, a sev-
enth biannual meeting will be held in Lima, Peru, in April 19–23, 2020. The scien-
tific output of various components of the PDVI Targeted Research Program is too
extensive to include in this report.
A mid-point program evaluation was conducted in late 2006 by a committee
chaired by Dr. Stanley Lemon, former Dean, University of Texas Medical Branch in
Galveston. The committee observed that the PDVI had developed a robust scientific
298 S. B. Halstead
research portfolio that had achieved substantial advances in identifying neutraliza-

tion targets on the viral envelope and cell receptors, mechanisms of attachment,
viral entry, and understanding the rules governing how dengue antibody-virus com-
plexes permit neutralization and infection enhancement. These findings provided
the potential to avert delay and keep the vaccine development and evaluation on
track toward providing a foundation for improving second-generation vaccines
through a “tool box” of well-validated, high-throughput laboratory tests for vaccine
evaluation including an effective small animal model that illustrates in vivo protec-
tion against ADE.
Did the PDVI Targeted Research Program succeed or fail? Despite our efforts to
study and understand mechanisms of ADE and immune protection in dengue, there
was no information developed that led anyone in PDVI to predict that the leading
candidate, the chimeric yellow fever tetravalent dengue vaccine, would fail, not
only fail but raise non-neutralizing antibodies that enhanced the severity of break-
through disease in vaccinated seronegative children. A small, vibrant dengue T cell
immunity research group emerged after PDVI was born. This group successfully
demonstrated the crucial role of nonstructural viral protein epitopes to generate
competent T cell immunity. The Sanofipasteur vaccine construct presented yellow
fever rather than dengue nonstructural proteins to the immune system. It is likely
this omission raised an incompetent T cell response. However, PDVI was designed
to promote research cooperation. Our meetings and research management were
designed to provide intra-investigator sharing of research results and collaborations.
PDVI created enduring research partnerships. Not only did PDVI make a valuable
beginning, but a similar new program is needed to understand immunological
mechanism(s) of (1) long-lasting homotypic protective dengue immunity, (2) short-
term protective heterotypic immunity follow a single DENV infection, or the (3)
long-lasting heterotypic protection that follows infections with two or more dengue
viruses.
After the termination of the PDVI Targeted Research Program, I continued to
engage with vaccine developers as a consultant and as a continuing critic of prog-
ress in developing and testing dengue vaccines. I continue to try to describe the
knowns and unknowns of dengue pathogenesis. ADE has proved to be a very com-
plicated phenomenon (Guzman et al. 2013; Halstead 2003b, 2006, 2007, 2008b,
2009, 2012a, 2013a, b, c, 2014a, b, 2015b, c, 2019a, b, c; Chan et al. 2019; Halstead
et al. 2002, 2005, 2010, 2020a; Hung et al. 2004, 2005; Nguyen et al. 2006; Nishiura
and Halstead 2007; Robinson et al. 2007; Halstead and Lum 2009; Ubol and
Halstead 2010; Althouse et al. 2014; Aye et al. 2014; Halstead and Wilder-Smith
2019). These complications have been discussed from the context of lessons learned
from my experiences in field studies around the world over the past 60 (Halstead
and Deen 2002; Halstead 2012b, 2014c, d, 2016a, b, c, 2017a, b, c, 2018a; b;
Halstead and Thomas 2013, 2017; Mahalingam et al. 2013; Aguiar et al. 2016a, b,
2017; Halstead and Aguiar 2016a, b, c; Halstead and Russell 2016a, b, Russell and
Halstead 2016; Schwarz 2016; Dans et al. 2018; Gubler and Halstead 2019; Halstead
and Dans 2019; Halstead et al. 2020b).
Acknowledgments I am grateful to Eileen Jong, who patiently counseled ways to make this a
better and more interesting work.
References
Aguiar M, Stollenwerk N, Halstead SB (2016a) The risks behind Dengvaxia recommendation.

Lancet Infect Dis 16(8):882–883
Aguiar M, Stollenwerk N, Halstead SB (2016b) The impact of the newly licensed dengue vaccine
in endemic countries. PLoS Negl Trop Dis 10(12):e0005179
Aguiar M, Halstead SB, Stollenwerk N (2017) Consider stopping dengvaxia administration with-
out immunological screening. Expert Rev Vaccines 16(4):301–302
Almond J, Clemens JD, Engers H et al (2002) Accelerating the development and introduction of
a dengue vaccine for poor children, 5-8 December 2001, Ho Chi Minh City, Vietnam. Vaccine
20:3043–3046
Althouse BM, Durbin AP, Hanley KA, Halstead SB, Weaver SC, Cummings DA (2014) Viral
kinetics of primary dengue virus infection in non-human primates: a systematic review and
individual pooled analysis. Virology 452–453:237–246
Alvarez M, Rodriguez-Roche R, Bernardo L et al (2006) Dengue hemorrhagic Fever caused by
sequential dengue 1-3 virus infections over a long time interval: Havana epidemic, 2001–2002.
Am J Trop Med Hyg 75(6):1113–1117
Anders KL, Nguyet NM, Chau NV et al (2011) Epidemiological factors associated with dengue
shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Vietnam.
Am J Trop Med Hyg 84(1):127–134
Anderson CR, Downs WG (1956) Isolation of dengue virus from a human being in Trinidad.
Science 124:224–225
Armien B, Suaya JA, Quiroz E et al (2008) Clinical characteristics and national economic cost of
the 2005 dengue epidemic in Panama. Am J Trop Med Hyg 79(3):364–371
Ashburn PM, Craig CF (1907) Experimental investigations regarding the etiology of dengue fever.
J Infect Dis 4:440–475
Aye KS, Charngkaew K, Win N et al (2014) Pathologic highlights of dengue hemorrhagic fever in
13 autopsy cases from Myanmar. Hum Pathol 45(6):1221–1233
Balankura M, Valyasevi A, Kampanart-Sanyakorn C, Cohen SN (1966a) Dengue infection in Thai
children: a pathopysiological study. Bull World Health Organ 35(1):51–53
Balankura M, Valyasevi A, Kampanart-Sanyakorn C, Cohen SN (1966b) Treatment of dengue
shock syndrome. Bull World Health Organ 35(1):75
Bancroft TL (1906) On the etiology of dengue fever. Australasian Med Gazette 25:17–18
Bangert M, Latheef AT, Dev Pant S et al (2018) Economic analysis of dengue prevention and case
management in the Maldives. PLoS Negl Trop Dis 12(9):e0006796
Beatty ME, Beutels P, Meltzer MI et al (2011) Health economics of dengue: a systematic literature
review and expert panel’s assessment. Am J Trop Med Hyg 84(3):473–488
Bernal MJ (1828) Memoria sobre la epidemic que ha sufrido esta ciudad nombrada vulgalmente el
dengue. In: Oficina del Gobierno y Capitania General H, Cuba, editor. Havana, Cuba
Bhamarapravati N (1993) Dengue vaccine development. In: Thongcharoen P (ed) Monograph
on dengue/dengue haemorrhagic fever. Regional Office for South-East Asia. World Health
Organization, New Delhi, pp 161–163
Bhamarapravati N, Tuchinda P, Boonyapaknavik V (1967) Pathology of Thailand haemorrhagic
fever: a study of 100 autopsy cases. Ann Trop Med Parasitol 61:500–510
Bista MB, Banerjee MK, Shin SH et al (2001) Efficacy of single dose SA 14-14-2 vaccine against
Japanese encephalitis: a case-control study. Lancet 358:791–795
300 S. B. Halstead
Bokisch VA, Muller-Eberhard HJ, Dixon FJ (1973a) The role of complement in hemorrhagic
shock syndrome (dengue). Trans Assoc Amer Phys 86:102–110
Bokisch VA, Top FH Jr, Russell PK, Dixon FJ, Muller-Eberhard HJ (1973b) The potential patho-
genic role of complement in dengue hemorrhagic shock syndrome. N Engl J Med 289:996–1000
Borges MB, Marchevsky RS, Carvalho Pereira R et al (2019) Detection of post-vaccination
enhanced dengue virus infection in macaques: an improved model for early assessment of
dengue vaccines. PLoS Pathog 15(4):e1007721
Bravo JR, Guzman MG, Kouri GP (1987) Why dengue haemorrhagic fever in Cuba? 1. Individual
risk factors for dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Trans R Soc
Breugelmans JG, Lewis RF, Agbenu E et al (2013) Adverse events following yellow fever pre-
ventive vaccination campaigns in eight African countries from 2007 to 2010. Vaccine
31(14):1819–1829
Buescher EL, Parkman PD, Artenstein M, Halstead SB (1962) Isolation of an agent associated
with rubella in military recruits. Fed Proc 21:466
Cardosa MJ, Porterfield JS, Gordon S (1983) Complement receptor mediates enhanced flavivirus
replication in macrophages. J Exp Med 158(1):258–263
Carey DE, Myers RM, Reuben R, Rodrigues FM (1966) Studies on dengue in Vellore, South India.
Bull World Health Organ 35(1):61
Carey DE, Myers RM, de Ranitz CM, Jadhav M (1969) The 1964 Chikungunya Epidemic at
Vellore, South India, including observations on Concurrent Dengue. Trans R Soc Trop Med
Hyg 63(4):434–445
Carrasco LR, Lee LK, Lee VJ et al (2011) Economic impact of dengue illness and the cost-
effectiveness of future vaccination programs in Singapore. PLoS Negl Trop Dis 5(12):e1426
Challet GL (1994) Mosquito abatement district programs in the United States. Kaohsiung J Med
Sci 10:S67–S73
Chan KWK, Watanabe S, Jin JY et al (2019) A T164S mutation in the dengue virus NS1 protein is
associated with greater disease severity in mice. Sci Transl Med 11(498)
Chandler AG, Rice L (1923) Observations on the etiology of dengue fever. Am J Trop Med
3:233–262
Chokephaibulkit K, Sirivichayakul C, Thisyakorn U et al (2010) Safety and Immunogenicity of a
single administration of live-attenuated Japanese encephalitis vaccine in previously primed 2-
to 5-year-olds and naive 12- to 24-month-olds: Multicenter randomized controlled trial. Pediatr
Infect Dis J 29(12):1111–1117
Chow CY, Gratz NG, Halstead SB et al (1966) Mosquito-borne hemorrhagic fevers of South-East
Asia and the Western Pacific. Bull World Health Organ 35(1):17–33
Christie J (1872) Remarks on “kidinga Pepo” a peculiar form of exantematous disease. Epidemic
in Zanzibar, East Coast of Africa, from July 1870 till January 1871. BMJ (1):577–579
Christie J (1881) On epidemics of dengue fever: their diffusion and etiology. Glasgow Med J
3:161–176
Chungue E, Glaziou P, Spiegel A, Martin PM, Roux JF (1992) Estimation of dengue infection
attack rate in a cohort of children during a dengue 3 outbreak in Tahiti (1989–1990). Southeast
Asian J Trop Med Public Health 23:157–158
Clarke JL (1994) Privatization of mosquito control services in urban areas. Kaohsiung J Med Sci
10(Supplement):S74–SS7
Cleland JB, Bradley B, McDonald W (1916) On the transmission of Australian dengue by the
mosquito Fasciata fasciata. Med J Australia 2:179–184
Cohen SN, Halstead SB (1966) Shock associated with dengue infection. I. Clinical and physi-
ologic manifestations of dengue hemorrhagic fever in Thailand, 1964. J Pediatrics 68:448–456
Dans AL, Dans LF, Silvestre MAA, Halstead SB, Guyatt GH (2018) Cause and consequence of
loss in vaccine trust. Hum Vaccin Immunother 15(3):628–629
de la Sierra B, Garcia G, Perez AB et al (2006) Ethnicity and difference in dengue virus-specific
memory T cell responses in Cuban individuals. Viral Immunol 19(4):662–668
de Ranitz CM, Myers RM, Varkey MJ, Isaac ZH, Carey DE (1965) Clinical impression of chikun-
gunya in Vellore gained from study of adult patients. Ind J Med Res 53(8):756–763
Dengue AW (1868) The science and practice of medicine. Lindsay and Blakeston, Philadelphia,
pp 323–325
Deparis X, Roche C, Murgue B, Chungue E (1998) Possible dengue sequential infection: dengue
spread in a neighbourhood during the 1996/97 dengue-2 epidemic in French Polynesia. Trop
Med Int Health 3(11):866–871
Deparis X, Marechal V, Matheus S (2009) Pathophysiological mechanisms of dengue fever: criti-
cal review of current concepts. Med Trop (Mars) 69(4):351–357
Dumaresq PJ (1828) An account of dengue, danga or dandy fever, as it occurred in New Orleans
and in the person of the writer, communicated in a letter to one of the editors. Boston Med Surg
J 1:497–502
Eckels KH, Scott RM, Bancroft WH et al (1984) Selection of attenuated dengue 4 viruses by serial
passage in primary kidney cells. V. Human response to immunization with a candidate vaccine
prepared in fetal rhesus lung cells. Am J Trop Med Hyg 33(4):684–689
Edillo FE, Halasa YA, Largo FM et al (2015) Economic cost and burden of dengue in the
Philippines. Am J Trop Med Hyg 92(2):360–366
Fagbami AH, Halstead SB, Marchette NJ, Larsen K (1987) Cross-protection and enhancement
among African flaviviruses by immune mouse ascitic fluids. Cytobios 49:49–55
Falkler WA Jr, Diwan AR, Halstead SB (1973) Human antibody to dengue soluble complement-
fixing (SCF) antigens. J Immunol 111(6):1804–1809
Falkler WA Jr, Diwan AR, Halstead SB (1975) A lipid inhibitor of dengue virus in human colostrum
and milk; with a note on the absence of anti-dengue secretory antibody. Arch Virol 47(1):3–10
Fischer DB, Halstead SB (1970) Observations related to pathogenesis of dengue hemorrhagic
fever. V. Examination of age specific sequential infection rates using a mathematical model.
Yale J BiolMed 42:329–349
Gangarosa EF, Beisel WR, Benyajati C, Sprinz H, Piyaratn P (1960) The nature of the gastrointes-
tinal lesion in asiatic cholera and its relation to pathogenesis: a biopsy study. Am J Trop Med
Hyg 9:125–135
Ginsburg AS, Meghani A, Halstead SB, Yaich M (2017) Use of the live attenuated Japanese
Encephalitis vaccine SA 14-14-2 in children: a review of safety and tolerability studies. Hum
Vaccin Immunother 13(10):2222–2231
Glasner DR, Puerta-Guardo H, Beatty PR, Harris E (2018) The good, the bad, and the shocking:
the multiple roles of dengue virus nonstructural protein 1 in protection and pathogenesis. Ann
Rev Virol 5(1):227–253
Glaziou P, Chungue E, Gestas P et al (1992) Dengue fever and dengue shock syndrome in French
Polynesia. Southeast Asian J Trop Med Public Health 23:531–532
Gollins SW, Porterfield JS (1985) Flavivirus infection enhancement in macrophages: an electron
microscopic study of viral cellular entry. J Gen Virol 66:1969–1982
Goodeve E (1853) Observations on the epidemic fever with scarlet eruption, prevalent in Calcutta
in the hot and rainy season of 1853. Indian Ann Med Sci 1:248–268
Gordon A, Kuan G, Mercado JC et al (2013) The nicaraguan pediatric dengue cohort study: inci-
dence of inapparent and symptomatic dengue virus infections, 2004-2010. PLoS Negl Trop
Dis 7(9):e2462
Graham H (1902) Dengue: a study of its mode of propagation and pathology. Med Rec NY
61:204–207
Graham RR, Juffrie M, Tan R et al (1999) A prospective seroepidemiologic study on dengue in
children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995–1996. Am J Trop
Med Hyg 61(3):412–419
Gratz NG (1994) Education and employment of medical entomologists in Aedes aegypti control
programmes. Kaohsiung J Med Sci 10:S19–S27
Gubler DJ, Halstead SB (2019) Is Dengvaxia a useful vaccine for dengue endemic areas? BMJ
367:l5710
302 S. B. Halstead
Guzman MG, Kouri GP, Bravo J, Soler M, Vazquez S, Morier L (1990) Dengue hemorrhagic fever
in Cuba, 1981: a retrospective seroepidemiologic study. Am J Trop Med Hyg 42:179–184
Guzman MG, Kouri G, Valdes L et al (2000a) Epidemiologic studies on dengue in Santiago de
Cuba, 1997. Am J Epidemiol 152(9):793–799
Guzman MG, Kouri G, Halstead SB (2000b) Do escape mutants explain rapid increases in dengue
case-fatality rates within epidemics? Lancet 355(9218):1902–1903
Guzman MG, Kouri G, Bravo J, Valdes L, Vazquez S, Halstead SB (2002a) Effect of age on out-
come of secondary dengue 2 infections. Int J Infect Dis 6:118–124
Guzman MG, Kouri G, Valdes L, Bravo J, Vazquez S, Halstead SB (2002b) Enhanced severity of
secondary dengue 2 infections occurring at an interval of 20 compared with 4 years after den-
gue 1 infection. PAHO J Epidemiol 81:223–227
Guzman MG, Alvarez M, Rodriguez-Roche R et al (2007) Neutralizing antibodies after infection
with dengue 1 virus. Emerg Infect Dis 13(2):282–286
Guzman MG, Halstead SB, Artsob H et al (2010) Dengue: a continuing global threat. Nat Rev
Microbiol 8(12 Suppl):S7–S16
Guzman MG, Alvarez A, Vazquez S et al (2012) Epidemiological studies on dengue virus type 3 in
Playa municipality, Havana, Cuba, 2001–2002. Int J Infect Dis 16(3):e198–e203
Guzman MG, Alvarez M, Halstead SB (2013) Secondary infection as a risk factor for dengue
hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-
dependent enhancement of infection. Arch Virol 158(7):1445–1459
Guzman MG, Gubler DJ, Izquierdo A, Martinez E, Halstead SB (2016) Dengue infection. Nat Rev
Dis Primers 2:16055
Hadinegoro SR, Arredondo-Garcia JL, Guy B, Bouckenooghe A, Noriega F, Jackson N (2016)
Answer to the review from Halstead and Russell “Protective and immunological behavior
of chimeric yellow fever dengue vaccine”. Vaccine 34(36):4273-4. https://doi.org/10.1016/j.
vaccine.2016.02.004
Halasa YA, Shepard DS, Zeng W (2012) Economic cost of dengue in Puerto Rico. Am J Trop Med
Hyg 86(5):745–752
Halstead SB (1961) Bladder stone in Thailand. A review of the problem. Am J Trop Med Hyg
10:918–925
Halstead SB (1965) Dengue and hemorrhagic fevers of Southeast Asia. Yale J Biol Med 37:434–454
Halstead SB (1966a) Mosquito-borne haemorrhagic fevers of South and South-East Asia. Bull
World Health Organ 35(1):3–15
Halstead SB (1966b) Epidemiological studies of Thai haemorrhagic fever, 1962–64. Bull World
Halstead SB (1970) Observations related to pathogenesis of dengue hemorrhagic fever.
VI. Hypotheses and discussion. Yale J Biol Med 42:350–362
Halstead SB (1978) Studies on the attenuation of dengue 4. Asian J Infect Dis 2:112–117
Halstead SB (1979) In vivo enhancement of dengue virus infection in Rhesus monkeys by pas-
sively transferred antibody. J Infect Dis 140(4):527–533
Halstead SB (1980) Immunological parameters of Togavirus disease syndromes. In: Schlesinger
RW (ed) The togaviruses, biology, structure, replication. Academic Press, New York,
pp 107–173
Halstead SB (1983) Etiology of infant shock syndrome: enhancement of viral infection by mater-
nal antibody. In: Tildon JT, Roeder LM, Steinschneider A (eds) Sudden infant death syndrome.
Academic Press, New York, pp 567–578
Halstead SB (1988) Aedes aegypti: why can’t we control it? Bull Soc Vector Ecol 13:304–311
Halstead SB (1992) The XXth century dengue pandemic: need for surveillance and research.
World Health StatQ 45:292–298
Halstead SB (1993a) Global epidemiology of dengue: Health systems in disarray. Trop Med
35:137–146
Halstead SB (1993b) Community-based dengue control: a description and critique of the
Rockefeller Foundation program. Trop Med 33(4):285–291
Halstead SB (1994) Dengue in the health transition. Kaohsiung J Med Sci 10(Suppl):2–14
Halstead SB (1997) Epidemiology of dengue and dengue hemorrhagic fever. In: Gubler DJ, Kuno
G (eds) Dengue and dengue hemorrhagic fever. CAB, Wallingford, pp 23–44
Halstead SB (2002) Dengue hemorrhagic fever: two infections and antibody dependent enhance-
ment, a brief history and personal memoir. Rev Cubana Trop Med 54(3):171–179
Halstead SB (2003a) Neutralization and antibody dependent enhancement of dengue viruses. Adv
Virus Res 60:421–467
Halstead SB (2003b) Neutralization and antibody-dependent enhancement of dengue viruses.
In: Chambers TJ, Monath TP (eds) The flaviviruses: pathogenesis and immunity. Elsevier
Academic Press, New York, pp 422–467
Halstead SB (2006) Measuring dengue enhancing antibodies: caveats. J Infect Dis 193(4):601
Halstead SB (2007) The dengue case definition dilemma: a commentary. Pediatr Infect Dis J
26:291–292
Halstead SB (2008a) Dengue: overview and history. In: Halstead SB (ed) Dengue. Imperial
College Press, London, pp 1–28
Halstead SB (2008b) Dengue virus-mosquito interactions. Annu Rev Entomol 53:273–291
Halstead SB (2009) Antibodies determine virulence in dengue. Ann NY Acad Sci 1171(Suppl
1):E48–E56
Halstead SB (2012a) Controversies in dengue pathogenesis. Paediatr Int Child Health
32(Suppl 1):5–9
Halstead SB (2012b) Dengue vaccine development: a 75% solution? Lancet 380(9853):1535–1536
Halstead SB (2013a) Dengue vascular permeability syndrome: what no T cells? Clin Infect Dis
56(6):900–901
Halstead SB (2013b) Dengue: the syndromic basis to pathogenesis research. Inutility of the 2009
WHO case definition. Am J Trop Med Hyg 88(2):212–215
Halstead SB (2013c) Chapter 19: Pathogenic exploitation of Fc activity. In: Ackerman M,
Nimmerjahn F (eds) Antibody Fc: linking adaptive and innate immunity. Academic Press,
London, pp 333–350
Halstead SB (2014a) Chapter 5: Intraepidemic increases in dengue disease severity: applying les-
sons on surveillance and transmission. In: Whitehorn J, Farrar J (eds) Clinical insights: dengue
fever: transmission, diagnosis & surveillance. Future Medicine, London, pp 84–101
Halstead SB (2014b) Dengue antibody-dependent enhancement: knowns and unknowns. Microbiol
Spectr 2(6)
Halstead SB (2014c) Dengue vaccines. In: Gubler DJ, Ooi EE, Vasudevan S, Farrar J (eds) Dengue
and dengue hemorrhagic fever, 2nd edn. CAB International, Wallingford, pp 548–576
Halstead SB (2014d) Stumbles on the path to dengue control. Lancet Infect Dis 14(8):661–662
Halstead SB (2015a) Reappearance of chikungunya, formerly called dengue, in the Americas.
Emerg Infect Dis 21(4):557–561
Halstead SB (2015b) Pathogenesis of dengue: dawn of a new era. F1000 Res:4
Halstead SB (2015c) Comment on “Dengue virus NS1 protein activates cells via Toll-like receptor
4 and disrupts endothelial cell monolayer integrity” and “Dengue virus NS1 triggers endo-
thelial permeability and vascular leak that is prevented by NS1 vaccination”. Sci Transl Med
7(318):318le4
Halstead SB (2016a) Dengue vaccine efficacy: not a zero sum game. J Infect Dis 214(12):2014
Halstead SB (2016b) Critique of World Health Organization recommendation of a dengue vaccine.
J Infect Dis 214(12):1793–1795
Halstead SB (2016c) Licensed dengue vaccine: public health conundrum and scientific challenge.
Halstead SB (2017a) Biologic evidence required for Zika disease enhancement by dengue antibod-
ies. Emerg Infect Dis 23(4):569–573
Halstead SB (2017b) Dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless
of age. Vaccine 35(47):6355–6358
304 S. B. Halstead
Halstead SB (2017c) Achieving safe, effective, and durable Zika virus vaccines: lessons from
dengue. Lancet Infect Dis 17(11):e378–ee82
Halstead SB (2018a) Safety issues from a Phase 3 clinical trial of a live-attenuated chimeric yellow
fever tetravalent dengue vaccine. Hum Vaccin Immunother 14(9):2158–2162
Halstead SB (2018b) Which dengue vaccine approach is the most promising, and should we be
concerned about enhanced disease after vaccination? There is only one true winner. Cold
Spring Harb Perspect Biol 10(6)
Halstead S (2019a) Recent advances in understanding dengue. F1000 Res:8
Halstead SB (2019b) Insights from direct studies on human dengue infections. Proc Natl Acad Sci
U S A 116(1):17–19
Halstead SB (2019c) Travelling arboviruses: a historical perspective. Travel Med Infect Dis 101471
Halstead SB, Aguiar M (2016a) Dengue vaccines: are they safe for travelers? Travel Med Infect
Dis 14(4):379–3783
Halstead SB, Aguiar M (2016b) Dengue vaccine and the 2016 Olympics. Lancet
388(10041):237–238
Halstead SB, Aguiar M (2016c) Is discussion of dengue vaccination for the 2016 Olympics neces-
sary? Authors’ reply. Lancet 388(10054):1881–1882
Halstead SB, Buescher EL (1961) Hemorrhagic disease in rodents infected with virus associated
with Thai hemorrhagic fever. Science 134(3477):475–476
Halstead SB, Cohen SN (2015) Dengue hemorrhagic fever at 60 years: early evolution of concepts
of causation and treatment. Microbiol Mol Biol Rev 79(3):281–291
Halstead SB, Dans LF (2019) Dengue infection and advances in dengue vaccines for children.
Lancet Child Adolesc Health 3(10):734–741
Halstead SB, Deen J (2002) The future of dengue vaccines. Lancet 360:789–800
Halstead SB, Grosz CR (1962) Subclinical Japanese encephalitis I. Infection of Americans with
limited residence in Korea. Am J Hyg 75:190–201
Halstead SB, Lum LC (2009) Assessing the prognosis of dengue-infected patients. F1000
Med Rep:1
Halstead SB, Marchette NJ (2003) Biologic properties of dengue viruses following serial passage
in primary dog kidney cells: studies at the University of Hawaii. Am J Trop Med Hyg 69(6
Suppl):5–11
Halstead SB, O’Rourke EJ (1977a) Antibody-enhanced dengue virus infection in primate leuko-
cytes. Nature 265(5596):739–741
Halstead SB, O’Rourke EJ (1977b) Dengue viruses and mononuclear phagocytes. I. Infection
enhancement by non-neutralizing antibody. J Exp Med 146:201–217
Halstead SB, Papaevangelou G (1980) Transmission of dengue 1 and 2 viruses in Greece in 1928.
Halstead SB, Russ SB (1962) Subclinical Japanese encephalitis. II. Antibody responses of
Americans to single exposure to JE virus. Am J Hyg 75:202–211
Halstead SB, Russell PK (2016a) Protective and immunological behavior of chimeric yellow fever
dengue vaccine. Vaccine 34(14):1643–1647
Halstead SB, Russell PK (2016b) Response to Hadinegoro et al. Vaccine 34(36):4275
Halstead SB, Simasthien P (1970) Observations related to the pathogenesis of dengue hemorrhagic
fever. II. Antigenic and biologic properties of dengue viruses and their association with disease
response in the host. Yale J Biol Med 42(5):276–292
Halstead SB, Thomas SJ (2013) Dengue vaccines. In: Plotkin SA, Orenstein W, Offit PA (eds)
Vaccines, 6th edn. Elsevier Saunders, Philadelphia, pp 1042–1051
Halstead SB, Thomas SJ (2017) Dengue vaccines. In: Plotkin SA, Orenstein W, Offit PA (eds)
Vaccines, 7th edn. Elsevier, Philadelphia
Halstead S, Wilder-Smith A (2019) Severe dengue in travellers: pathogenesis, risk and clinical
management. J Travel Med 26(7)
Halstead SB, Yamarat C (1965) Recent epidemics of hemorrhagic fever in Thailand. Observations
related to pathogenesis of a “new” dengue disease. J Am Pub Health Assoc 55(9):1386–1395
Halstead SB, Sukhavachana P, Nisalak A (1964a) In vitro recovery of dengue viruses from natu-
rally infected human beings and arthropods. Nature 202(4935):931–932
Halstead SB, Sukhavachana P, Nisalak A (1964b) Assay of mouse adapted dengue viruses in mam-
malian cell cultures by an interference method. Proc Soc Exp Biol Med 115:1062–1068
Halstead SB, Nimmannitya S, Yamarat C, Russell PK (1967) Hemorrhagic fever in Thailand;
recent knowledge regarding etiology. Jpn J Med Sci Biol 20s:96–103
Halstead SB, Nimmannitya S, Margiotta MR (1969a) Dengue and Chikungunya virus infection in
man in Thailand, 1962–1964: II. Observations on disease in outpatients. Am J Trop Med Hyg
18(6):972–983
Halstead SB, Scanlon J, Umpaivit P, Udomsakdi S (1969b) Dengue and chikungunya virus infec-
tion in man in Thailand, 1962–1964: IV. Epidemiologic studies in the Bangkok metropolitan
area. Am J Trop Med Hyg 18(6):997–1021
Halstead SB, Udomsakdi S, Singharaj P, Nisalak A (1969c) Dengue and chikungunya virus infec-
tion in man in Thailand, 1962-1964. III. Clinical, epidemiology, and virologic observations on
disease in non-indigeneous white persons. Am J Trop Med Hyg 18(6):984–996
Halstead SB, Udomsakdi S, Scanlon J, Rohitayodhin S (1969d) Dengue and chikungunya virus
infection in man in Thailand, 1962–1964: V. Epidemiologic observations outside Bangkok. Am
J Trop Med Hyg 18(6):1022–1033
Halstead SB, Nimmannitya S, Cohen SN (1970a) Observations related to pathogenesis of dengue
hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered.
Yale J Biol Med 42:311–328
Halstead SB, Udomsakdi S, Simasthien P, Singharaj P, Sukhavachana P, Nisalak A (1970b)
Observations related to pathogenesis of dengue hemorrhagic fever. I. Experience with clas-
sification of dengue viruses. Yale J Biol Med 42(5):261–275
Halstead SB, Shotwell H, Casals J (1973a) Studies on the pathogenesis of dengue infection in
monkeys. II. Clinical laboratory responses to heterologous infection. J Infect Dis 128(1):15–22
Halstead SB, Shotwell H, Casals J (1973b) Studies on the pathogenesis of dengue infection in
monkeys. I. Clinical laboratory responses to primary infection. J Infect Dis 128(1):7–14
Halstead SB, Chow J, Marchette NJ (1973c) Immunologic enhancement of dengue virus replica-
tion. Nat New Biol 243(122):24–26
Halstead SB, Marchette NJ, Sung Chow JS, Lolekha S (1976) Dengue virus replication enhance-
ment in peripheral blood leukocytes from immune human beings. Proc Soc Exp Biol Med
151(1):136–139
Halstead SB, O’Rourke EJ, Allison AC (1977) Dengue viruses and mononuclear phagocytes:
II. identity of blood and tissue leukocytes supporting in vitro infection. J Exp Med 146:218–228
Halstead SB, Porterfield JS, O’Rourke EJ (1980) Enhancement of dengue virus infection in mono-
cytes by flavivirus antisera. Am J Trop Med Hyg 29(4):638–642
Halstead SB, Larsen K, Kliks S, Peiris JS, Cardosa J, Porterfield JS (1983a) Comparison of
P388D1 mouse macrophage cell line and human monocytes for assay of dengue-2 infection-
enhancing antibodies. Am J Trop Med Hyg 32(1):157–163
Halstead SB, Rojanasuphot S, Sangkawibha N (1983b) Original antigenic sin in dengue. Am J
Trop Med Hyg 32(1):154–156
Halstead SB, Venkateshan CN, Gentry MK, Larsen LK (1984a) Heterogeneity of infection
enhancement of dengue 2 strains by monoclonal antibodies. J Immunol 132(3):1529–1532
Halstead SB, Diwan AR, Marchette NJ, Palumbo NE, Srisukonth L (1984b) Selection of attenu-
ated dengue 4 viruses by serial passage in primary kidney cells. I. Attributes of uncloned virus
at different passage levels. Am J Trop Med Hyg 33(4):654–665
Halstead SB, Eckels KH, Putvatana R, Larsen LK, Marchette NJ (1984c) Selection of attenuated
dengue 4 viruses by serial passage in primary kidney cells. IV. Characterization of a vaccine
candidate in fetal rhesus lung cells. Am J Trop Med Hyg 33(4):679–683
Halstead SB, Marchette NJ, Diwan AR, Palumbo NE, Putvatana R (1984d) Selection of attenuated
dengue 4 viruses by serial passage in primary kidney cells. II. Attributes of virus cloned at dif-
ferent dog kidney passage levels. Am J Trop Med Hyg 33(4):666–671
306 S. B. Halstead
Halstead SB, Marchette NJ, Diwan AR, Palumbo NE, Putvatana R, Larsen LK (1984e) Selection
of attenuated dengue 4 viruses by serial passage in primary kidney cells. III. Reversion to viru-
lence by passage of cloned virus in fetal rhesus lung cells. Am J Trop Med Hyg 33(4):672–678
Halstead SB, Streit TG, Lafontant JG et al (2001) Haiti: Absence of dengue hemorrhagic fever
despite hyperendemic dengue virus transmission. Am J Trop Med Hyg 65:180–183
Halstead SB, Lan NT, Myint TT et al (2002) Infant dengue hemorrhagic fever: research opportuni-
ties ignored. Emerg Infect Dis 12:1474–1479
Halstead SB, Heinz FX, Barrett AD, Roehrig JT (2005) Dengue virus: molecular basis of cell entry
and pathogenesis, 25-27 June 2003, Vienna, Austria. Vaccine 23(7):849–856
Halstead SB, Suaya JA, Shepard DS (2007) The burden of dengue infection. Lancet
369(9571):1410–1411
Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM (2010) Intrinsic antibody-
dependent enhancement of microbial infection in macrophages: disease regulation by immune
complexes. Lancet Infect Dis 10(10):712–722
Halstead SB, Russell PK, Brandt WE (2020a) NS1, dengue’s dagger. J Infect Dis 221:857–860
Halstead SB, Russell PK, Katzelnick LC et al (2020b) Ethics of a partially effective dengue vac-
cine: lessons from the Philippines. Vaccine 38(35):5572–5576
Hammon WM, Sather GE (1961) Chikungunya and unidentified viruses from Thai haemor-
rhagic fever cases. In: Felsenfeld O (ed) Symposium on haemorrhagic fever. SEATO Medical
Research Laboratory, Bangkok, pp 39–41
Hammon WM, Rudnick A, Sather GE, Rogers KD, Morse LJ (1960a) New hemorrhagic fevers of
children in the Philippines and Thailand. Trans Assn Amer Phys 73:140–155
Hammon WM, Rudnick A, Sather GE (1960b) Viruses associated with epidemic hemorrhagic
fevers of the Philippines and Thailand. Science 131:1102–1103
Hammon WM, Sather GE, Rudnick A (1961) Identification and classification of the Dengue
Groups of Viruses. In: Felsenfeld O (ed) Symposium on haemorrhagic fever. SEATO Medical
Research Laboratory, Bangkok, pp 30–36
Hare FE (1898) The 1897 epidemic of dengue in North Queensland. Australasian Med Gazette
21(March):98–107
Hariharan D, Das MK, Shepard DS, Arora NK (2019) Economic burden of dengue illness in India
from 2013 to 2016: A systematic analysis. Int J Infect Dis 84:S68–s73
Hawkes RA (1964) Enhancement of the infectivity of arboviruses by specific antisera produced in
domestic fowls. Aust J Exp Biol Med Sci 42:465–482
Hawkes RA, Lafferty KJ (1967) The enhancement of virus infectivity by antibody. Virology
33:250–261
Hennessy S, Liu Z, Tsai TF et al (1996) Effectiveness of live-attenuated Japanese encephalitis vac-
cine (SA14-14-2): a case-control study. Lancet 347(9015):1583–1586
Hotez PJ, Alvarado M, Basanez MG et al (2014) The global burden of disease study 2010: inter-
pretation and implications for the neglected tropical diseases. PLoS Negl Trop Dis 8(7):e2865
Huang CY, Butrapet S, Tsuchiya KR, Bhamarapravati N, Gubler DJ, Kinney RM (2003) Dengue
2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. J Virol
77(21):11436–11447
Hubert B, Halstead SB (2009) Dengue 1 virus and dengue hemorrhagic fever, French Polynesia,
2001. Emerg Infect Dis 15(8):1265–1270
Hung NT, Lei HY, Lan NT et al (2004) Dengue hemorrhagic fever in infants: a study of clinical
and cytokine profiles. J Infect Dis 189(2):221–232
Hung NT, Lan NT, Lei HY et al (2005) Association between sex, nutritional status, severity of
dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever. Am J
Trop Med Hyg 72(4):370–374
Johnson KM, Halstead SB, Cohen SN (1967) Hemorrhagic fevers of Southeast Asia and South
America: a comparative appraisal. Prog Med Virol 9:105–158
Kanesa-thasan N, Sun W, Kim-Ahn G et al (2001) Safety and immunogenicity of attenuated den-
gue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine 19(23-24):3179–3188
Kay BH (1994) Intersectoral approaches to dengue vector control. Kaohsiung J Med Sci
10(Suppl):S56–S61
Kimura R, Hotta S (1944) On the inoculation of dengue virus into mice. Nippon Igaku 3379:629–633
Kitchener S, Nissen M, Nasveld P et al (2006) Immunogenicity and safety of two live-attenuated
tetravalent dengue vaccine formulations in healthy Australian adults. Vaccine 24(9):1238–1241
Kliks S, Halstead SB (1980) An explanation for enhanced virus plaque formation in chick embryo
cells. Nature 285:504–505
Kochel TJ, Watts DM, Halstead SB, Hayes CG, Espinosa A, Felices V, Caceda R, Bautista T,
Montoya Y, Douglas S, Russell KL (2002) Effect of dengue-1 antibodies on American dengue-
2 viral infection and dengue haemorrhagic fever. Lancet 360:310–312
Kochel TJ, Watts DM, Gozalo AS, Ewing DF, Porter KR, Russell KL (2005) Cross-serotype neu-
tralization of dengue virus in Aotus nancymae monkeys. J Infect Dis 191(6):1000–1004
Kouri G, Guzman MG, Bravo J (1986) Hemorrhagic dengue in Cuba: history of an epidemic. Bull
Pan Am Health Organ 20:24–30
Lam SK, Burke D, Capeding MR et al (2011) Preparing for introduction of a dengue vaccine:
recommendations from the 1st Dengue v2V Asia-Pacific Meeting. Vaccine 29(51):9417–9422
Liu ZL, Hennessy S, Strom BL et al (1997) Short-term safety of live attenuated Japanese enceph-
alitis vaccine (SA14-14-2): results of a randomized trial with 26,239 subjects. J Infect Dis
176(5):1366–1369
Lozano R, Naghavi M, Foreman K et al (2012) Global and regional mortality from 235 causes
of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of
Disease Study 2010. Lancet 380(9859):2095–2128
Lum LC, Suaya JA, Tan LH, Sah BK, Shepard DS (2008) Quality of life of dengue patients. Am J
Trop Med Hyg 78(6):862–867
MacKinnon K (1854) On the epidemics of the Bangal and North-West Presidencies. Indian Ann
Med Sci 3:147–181
Mahalingam S, Herring BL, Halstead SB (2013) Call to action for dengue vaccine failure. Emerg
Infect Dis 19(8):1335–1337
Marchette NJ, Halstead SB (1973) Survival of dengue virus in post mortem samples of tissues
from experimentally infected Rhesus monkeys. Am J Trop Med Hyg 22(2):242–243
Marchette NJ, Halstead SB (1974) Immunopathogenesis of dengue infection in the Rhesus mon-
key. Transplant Proc 6(2):197–201
Marchette NJ, Garcia R, Rudnick A (1969) Isolation of Zika virus from Aedes aegypti mosquitoes
in Malaysia. Am J Trop Med Hyg 18(3):411–415
Marchette NJ, Halstead SB, Nash DR, Stenhouse AC (1972) Recovery of dengue viruses from tis-
sues of experimentally infected rhesus monkeys. Appl Microbiol 24(3):328–333
Marchette NJ, Halstead SB, Falkler WA Jr, Stenhouse A, Nash D (1973) Studies on the pathogen-
esis of dengue infection in monkeys. 3. Sequential distribution of virus in primary and heter-
ologous infections. J Infect Dis 128(1):23–30
Marchette NJ, Sung Chow JS, Halstead SB, Lolekha S, Pongpanich B (1975) Dengue virus rep-
lication in cultures of peripheral blood leukocytes during the course of dengue haemorrhagic
fever. Southeast Asian J Trop Med Public Health 6(3):316–321
Marchette NJ, Halstead SB, Chow JS (1976) Replication of dengue viruses in cultures of periph-
eral blood leukocytes from dengue-immune Rhesus monkeys. J Infect Dis 133(3):274–282
Marchette NJ, O’Rourke T, Scott RM, Nimmannitya S, Halstead SB, Bancroft WH (1979) Absence
of leukocytes permissive to dengue 2 virus in the acute phase of dengue hemorrhagic fever. Am
J Trop Med Hyg 28(3):570–576
Marchette NJ, O’Rourke T, Halstead SB (1980) Studies on dengue 2 virus infection in
cyclophosphamide-treated rhesus monkeys. Med Microbiol Immunol (Berl) 168(1):35–47
Marchette NJ, Dubois DR, Larsen LK et al (1990) Preparation of an attenuated dengue 4 (341750
Carib) virus vaccine. I. Pre-clinical studies. Am J Trop Med Hyg 43(2):212–218
Martelli CM, Nascimento NE, Suaya JA et al (2011) Quality of life among adults with confirmed
dengue in Brazil. Am J Trop Med Hyg 85(4):732–738
308 S. B. Halstead
Memoranda (1973) Pathogenic mechanisms in dengue hemorrhagic fever. Report of an interna-

tional collaborative study. Bull World Health Organ 48:117–132
Morens DM (1994) Antibody-dependent enhancement of infection and the pathogenesis of viral
disease. Clin Infect Dis 19:500–512
Morens DM, Halstead SB (1987) Disease severity-related antigenic differences in dengue 2 strains
detected by dengue 4 monoclonal antibodies. J Med Virol 22:169–174
Morens DM, Halstead SB (1990) Measurement of antibody-dependent infection enhance-
ment of four dengue virus serotypes by monoclonal and polyclonal antibodies. J Gen Virol
71:2909–2914
Morens DM, Halstead SB, Larsen LK (1985a) Comparison of dengue virus plaque reduction
neutralization by macro and “semi-micro” methods in LLC-MK2 cells. Microbiol Immunol
29(12):1197–1205
Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N (1985b) Simplified plaque
reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells:
comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin
Microbiol 22(2):250–254
Morens DM, Halstead SB, Marchette NJ (1987a) Profiles of antibody-dependent enhancement of
dengue virus type 2 infection. Microb Pathog 3:231–237
Morens DM, Larsen LK, Halstead SB (1987b) Study of the distribution of antibody-dependent
enhancement determinants on dengue 2 isolates using dengue 2-derived monoclonal antibod-
ies. J Med Virol 22:163–167
Morens DM, Sather GE, Gubler DJ, Rammohan M, Woodall JP (1987c) Dengue shock syndrome
in an American traveler with primary dengue 3 infection. Am J Trop Med Hyg 36(2):424–426
Morens DM, Venkateshan CN, Halstead SB (1987d) Dengue 4 virus monoclonal antibodies iden-
tify epitopes that mediate immune infection enhancement of dengue 2 viruses. J Gen Virol
68:91–98
Morens DM, Marchette NJ, Chu MC, Halstead SB (1991) Growth of dengue type 2 virus isolates
in human peripheral blood leukocytes correlates with severe and mild dengue disease. Am J
Trop Med Hyg 45(5):644–651
Morier L, Kouri G, Guzman G, Soler M (1987) Antibody-dependent enhancement of dengue 2
virus in people of white descent in Cuba. Lancet 1:1028–1029
Murgue B, Cassar O, Roche C, Deparis X (2004) Pathogenesis of dengue: the emperor is still
naked! Med Mal Infect 34(Suppl 1):S31–SS3
Murray CJ, Vos T, Lozano R et al (2012) Disability-adjusted life years (DALYs) for 291 diseases
and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease
Study 2010. Lancet 380(9859):2197–2223
Myers RM, Carey DE (1967) Concurrent isolation from patient of two arboviruses, chikungunya
and dengue type 2. Science 157:1307–1308
Nelson ER (1960) Hemorrhagic fever in children in Thailand: Report of 69 cases. J Pediat
56:101–108
Nguyen TH, Nguyen TL, Lei HY et al (2006) Volume replacement in infants with dengue hemor-
rhagic fever/dengue shock syndrome. Am J Trop Med Hyg 74(4):684–691
Nimmannitya S, Halstead SB, Cohen S, Margiotta MR (1969) Dengue and chikungunya virus
infection in man in Thailand, 1962–1964. I. Observations on hospitalized patients with hemor-
rhagic fever. Am J Trop Med Hyg 18(6):954–971
Nisalak A, Singharaj P, Halstead SB (1966) Evaluation of sucking mice and tissue culture for recov-
ery of viruses from haemorrhagic fever patients in Thailand. Bull World Health Organ 35:67
Nisalak A, Halstead SB, Singharaj P, Udomsakdi S, Nye SW, Vinijchaikul K (1970) Observations
related to pathogenesis of dengue hemorrhagic fever. III. Virologic studies of fatal disease. Yale
J Biol Med 42:293–310
Nishiura H, Halstead SB (2007) Natural history of dengue virus (DEN)-1 and DEN-4 infections.
Reanalysis of classical studies. J Infect Dis 195:1007–1013
O’Reilly KM, Hendrickx E, Kharisma DD et al (2019) Estimating the burden of dengue and the
impact of release of wMel Wolbachia-infected mosquitoes in Indonesia: a modelling study.
BMC Med 17(1):172
O’Rourke EJ, Halstead SB, Allison AC, Platts-Mills TA (1978) Specific lethality of silica for human
peripheral blood mononuclear phagocytes, in vitro. J Immunol Methods 19(2–3):137–151
Ohrr HC, Tandan JB, Sohn YM, Shin SH, Pradhan DP, Halstead SB (2005) Effect of a single dose
of SA 14-14-2 vaccine 1 year after immunization in Nepalese children with Japanese encepha-
litis: a cose-control study. The Lancet 366:1375–1378
Oliveira M, Saraiva DP, Cavadas B et al (2018) Population genetics-informed meta-analysis in
seven genes associated with risk to dengue fever disease. Infect Genet Evol 62:60–72
Osorio JE, Wallace D, Stinchcomb DT (2016) A recombinant, chimeric tetravalent dengue vaccine
candidate based on a dengue virus serotype 2 backbone. Expert Rev Vaccines 15(4):497–508
Packierisamy PR, Ng CW, Dahlui M, Venugopalan B, Halasa YA, Shepard DS (2015) The cost of
dengue vector control activities in Malaysia by different service providers. Asia Pac J Public
Health Asia Pac Acad Consort Public Health 27(8 Suppl):73s–78s
Papaevangelou G, Halstead SB (1977) Infections with two dengue viruses in Greece in the 20th
century. Did dengue hemorrhagic fever occur in the 1928 epidemic? J Trop Med Hyg 80:46–51
Parkman PD, Buescher EL, Artenstein MS (1962) Recovery of rubella virus from army recruits.
Proc Soc Exp Biol Med 111:225–230
Parkman PD, Buescher EL, Artenstein MS, McCown JM, Mundon FK, Druzd AD (1964) Studies
of Rubella. I. Properties of the virus. J Immunol 93:595–607
Peeling RW, Artsob H, Pelegrino JL et al (2011) Evaluation of diagnostic tests: dengue. Nat Rev
Microbiol 8(12 Suppl):S30–S38
Peiris JS, Porterfield JS (1979) Antibody-mediated enhancement of flavivirus replication in
macrophage-like cell lines. Nature 282:509–511
Peiris JS, Porterfield JS (1982) Antibody-dependent plaque enhancement: its antigenic specificity
in relation to Togaviridae. J Gen Virol 58:291–296
Pengsaa K, Yoksan S, Limkittikul K et al (2003) Maternally transferred neutralising dengue anti-
bodies in Thai infants: a pilot study. Ann Trop Paediatr 23(3):159–165
Pengsaa K, Luxemburger C, Sabchareon A et al (2006) Dengue virus infections in the first 2 years
of life and the kinetics of transplacentally transferred dengue neutralizing antibodies in thai
children. J Infect Dis 194(11):1570–1576
Pengsaa K, Limkittikul K, Luxemburger C et al (2008) Age-specific prevalence of dengue antibod-
ies in Bangkok infants and children. Pediatr Infect Dis J 27(5):461–463
Plennevaux E, Sabchareon A, Limkittikul K et al (2016) Detection of dengue cases by serological
testing in a dengue vaccine efficacy trial: utility for efficacy evaluation and impact of future
vaccine introduction. Vaccine 34(24):2707–2712
Porterfield JS (1986) Antibody-dependent enhancement of viral infectivity. AdvVirus Research
31:335–355
Quintos FN, Lim LE (1956) Philippine hemorrhagic fever. Santo Tomas J Med 11(5):319–328
Quintos FN, Lim LE, Juliano L, Reyes A, Lacson P (1954) Hemorrhagic fever observed among
children in the Philippines. Philipp J Pediatrics 3(3):1–19
Reed W, Carroll J, Agramonte A (1901) The etiology of yellow fever: an additional note. JAMA
36:431–440
Robinson MC (1955) An epidemic of virus disease in Southern Province, Tanganyika Territory in
1952–1953. I. Clinical features. Trans R Soc Trop Med Hyg 49:28–32
Robinson JL, Wills B, Halstead SB, Matthew JL (2007) Three commentaries on ‘corticosteroids
for treating dengue shock syndrome,’ with introduction by ECBH editor. Eviden Based Child
Health Cochrane Rev J 2:1080–1086
Rodriguez-Roche R, Alvarez M, Gritsun T et al (2005a) Virus evolution during a severe dengue
epidemic in Cuba, 1997. Virology 334:154–159
310 S. B. Halstead
Rodriguez-Roche R, Alvarez M, Gritsun T et al (2005b) Dengue virus type 2 in Cuba, 1997: con-
servation of E gene sequence in isolates obtained at different times during the epidemic. Arch
Virol 150:415–425
Rodriguez-Roche R, Alvarez M, Holmes EC et al (2005c) Dengue virus type 3, Cuba, 2000–2002.
Rodriguez-Roche R, Sanchez L, Burgher Y et al (2011) Virus role during intraepidemic increase in
dengue disease severity. Vector Borne Zoonotic Dis 11(6):675–681
Rodriguez-Roche R, Blanc H, Borderia AV et al (2016) Increasing clinical severity during a
dengue virus type 3 cuban epidemic: deep sequencing of evolving viral populations. J Virol
90(9):4320–4333
Rosen L (1977) The emperor’s new clothes revisited, or reflections on the pathogenesis of dengue
hemorrhagic fever. Am J Trop Med Hyg 26:337–343
Rosen L (1986a) Dengue in Greece in 1927 and 1928 and the pathogenesis of dengue hemorrhagic
fever: new data and a different conclusion. Am J Trop Med Hyg 35(3):642–653
Rosen L (1986b) The pathogenesis of dengue haemorrhagic fever. A critical appraisal of current
hypotheses. S Afr Med J (Suppl):40–42
Rosen L (1986c) [Pathogenesis of hemorrhagic dengue: critical discussion of current hypotheses]
La pathogenese de la dengue hemorragique: discussion critique des hypotheses actuelles. Bull
Soc Pathol Exot Filiales 79:342–349
Rosen L (1989) Disease exacerbation caused by sequential dengue infections: myth or reality? Rev
Infect Dis 11(Suppl 4):S840–S8S2
Rosen L (1996) Dengue hemorrhagic fever. Bull Soc Path Ex 89:91–94
Rosen L (1999) Comments on the epidemiology, pathogenesis, and control of dengue. Med Trop
59(4):495–498
Rouvinski A, Guardado-Calvo P, Barba-Spaeth G et al (2015) Recognition determinants of broadly
neutralizing human antibodies against dengue viruses. Nature 520(7545):109–113
Rush B (1789) An account of the bilious remitting fever, as it appeared in Philadelphia, in the sum-
mer and autumn of the year 1780. In: Rush B (ed) Medical inquiries and observations, 1st edn.
Prichard and Hall, Philadelphia, pp 89–100
Russell PK (1970) Pathogenesis of the dengue shock syndrome: evidence for an immunologic
mechanism. Immunopathology:426–435
Russell PK, Halstead SB (2016) Challenges to the design of clinical trials for live-attenuated tetra-
valent dengue vaccines. PLoS Negl Trop Dis 10(8):e0004854
Russell PK, Udomsakdi S, Halstead SB (1967a) Antibody response in dengue and dengue hemor-
rhagic fever. Jpn J Med Sci Biol 20:103–108
Russell PK, Nisalak A, Sukhavachana P, Vivona S (1967b) A plaque reduction test for dengue virus
neutralization antibodies. J Immunol 99(2):285–290
Russell PK, Yuill TM, Nisalak A, Udomsakdi S, Gould D, Winter PE (1968) An insular out-
break of dengue hemorrhagic fever. II. Virologic and serologic studies. Am J Trop Med Hyg
17(4):600–608
Russell PK, Intavivat A, Kanchanapilant S (1969) Anti-dengue immunoglobulins and serum beta 1
c/a globulin levels in dengue shock syndrome. J Immunol 102(2):412–420
Sabchareon A, Lang J, Chanthavanich P et al (2004) Safety and immunogenicity of a three dose
regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai
children. Pediatr Infect Dis J 23(2):99–109
Sabchareon A, Sirivichayakul C, Limkittikul K et al (2012a) Dengue infection in children in
Ratchaburi, Thailand: a cohort study. I. Epidemiology of symptomatic acute dengue infection
in children, 2006-2009. PLoS Negl Trop Dis 6(7):e1732
Sabchareon A, Wallace D, Sirivichayakul C et al (2012b) Protective efficacy of the recombinant,
live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, con-
trolled phase 2b trial. Lancet 380(9853):1559–1567
Sabchareon A, Wallace D, Lang J, Bouckenooghe A, Moureau A (2013) Efficacy of tetravalent
dengue vaccine in Thai schoolchildren - Authors’ reply. Lancet 381(9872):1094–1095
Sabin AB, Schlesinger RW (1945) Production of immunity to dengue with virus modified by
propagation in mice. Science 101:640–642
Saez-Llorens X, Tricou V, Yu D et al (2017) Safety and immunogenicity of one versus two doses of
Takeda’s tetravalent dengue vaccine in children in Asia and Latin America: interim results from
a phase 2, randomised, placebo-controlled study. Lancet Infect Dis 17(6):615–625
Sanchez V, Gimenez S, Tomlinson B et al (2006) Innate and adaptive cellular immunity in
flavivirus-naive human recipients of a live-attenuated dengue serotype 3 vaccine produced in
Vero cells (VDV3). Vaccine 24(23):4914–4926
Sangkawibha N, Rojanasuphot S, Ahandrik S et al (1984) Risk factors in dengue shock syndrome:
a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol
120:653–669
Scanlon JE (1966a) Bangkok haemorrhagic fever investigations: the 1962–63 mosquito collec-
tions. Bull World Health Organ 35(1):82–83
Scanlon JE (1966b) The distribution of Aedes aegypti in Thailand. Bull World Health Organ
35(1):81–82
Schwarz TF (2016) Answer to “Dengue vaccine and the 2016 Olympics”, Halstead and Aguiar.
Lancet 388:237–238
Shepard DS, Suaya JA, Halstead SB et al (2004) Cost-effectiveness of a pediatric dengue vaccine.
Vaccine 22(9-10):1275–1280
Shepard DS, Coudeville L, Halasa YA, Zambrano B, Dayan GH (2011) Economic impact of den-
gue illness in the Americas. Am J Trop Med Hyg 84(2):200–207
Shepard DS, Undurraga EA, Lees RS, Halasa Y, Lum LC, Ng CW (2012a) Use of multiple data
sources to estimate the economic cost of dengue illness in Malaysia. Am J Trop Med Hyg
87(5):796–805
Shepard DS, Undurraga EA, Lees RS, Halasa Y, Lum LCS, Ng CW (2012b) Use of multiple data
sources to estimate the economic cost of dengue illness in Malaysia. Am J Trop Med Hyg
87(5):796–805
Shepard DS, Undurraga EA, Halasa YA (2013) Economic and disease burden of dengue in
Southeast Asia. PLoS Negl Trop Dis 7(2):e2055
Shepard DS, Halasa YA, Tyagi BK et al (2014a) Economic and disease burden of dengue illness in
India. Am J Trop Med Hyg 91(6):1235–1242
Shepard DS, Undurraga EA, Betancourt-Cravioto M et al (2014b) Approaches to refining esti-
mates of global burden and economics of dengue. PLoS Negl Trop Dis 8(11):e3306
Shepard DS, Undurraga EA, Halasa YA, Stanaway JD (2016) The global economic burden of
dengue: a systematic analysis. Lancet Infect Dis 16(8):935–941
Sheriff M (1873) History of the epidemic of dengue in Madras in 1872. Med Times Gaz 2:543
Shircore SM (1872) Notes on the eruptive fever prevailing in and around Calcutta. Indian Med
Gaz 7:33–34
Sierra BL, Kouri G, Guzman MG (2007) Race: a risk factor for dengue hemorrhagic fever. Arch
Virol 152(3):533–542
Sierra B, Triska P, Soares P et al (2017) OSBPL10, RXRA and lipid metabolism confer African-
ancestry protection against dengue haemorrhagic fever in admixed Cubans. PLoS Pathog
13(2):e1006220
Siler JF, Hall MW, Hitchen AP (1925) Transmission of dengue fever by mosquitoes. Proc Soc Exp
Biol and Med 23:197–201
Siler JF, Hall MW, Hitchens AP (1926) Dengue: its history, epidemiology, mechanism of transmis-
sion, etiology, clinical manifestations, immunity, and prevention. Philipp J Sci 29:1–304
Simasathien S, Thomas SJ, Watanaveeradej V et al (2008) Safety and immunogenicity of a tet-
ravalent live-attenuated dengue vaccine in flavivirus naive children. Am J Trop Med Hyg
78(3):426–433
Simmons JS, St John JH, Reynolds FHK (1931) Experimental studies of dengue. Philipp J Sci
44:1–252
312 S. B. Halstead
Singharaj P, Simasathien P, Sukhavachana P, Halstead SB, Scanlon JE (1966) Recovery of dengue

and other viruses in mice and tissue culture from Thai mosquitos. Bull World Health Organ
35(1):67–68
Sirivichayakul C, Sabchareon A, Limkittikul K, Yoksan S (2014) Plaque reduction neutralization
antibody test does not accurately predict protection against dengue infection in Ratchaburi
cohort, Thailand. Virol J 11:48
Sirivichayakul C, Barranco-Santana EA, Esquilin-Rivera I et al (2016) Safety and immunogenic-
ity of a tetravalent dengue vaccine candidate in healthy children and adults in dengue-endemic
regions: a randomized, placebo-controlled phase 2 study. J Infect Dis 213(10):1562–1572
Snijders EP, Dinger EJ, Schuffner WAP (1931) On the transmission of dengue in Sumatra. Am J
Trop Med 11(2):171–197
Sprinz H, Sribhibhadh R, Gangarosa EJ, Benyajati C, Kundel D, Halstead S (1962) Biopsy of
small bowel of Thai people. With special reference to recovery from Asiatic cholera and to an
intestinal malabsorption syndrome. Am J Clin Pathol 38:43–51
Stanaway JD, Shepard DS, Undurraga EA et al (2016) The global burden of dengue: an analysis
from the Global Burden of Disease Study 2013. Lancet Infect Dis 16(6):712–723
Stedman GW (1828) Some account of an anomalous disease that raged in the islands of St.
Thomas and Sta. Cruz in the West Indies during the months of September, October, November,
December and January, 1827-8. Edinb Med Surg J 30:227–248
Suaya JA, Shepard DS, Chang MS et al (2007) Cost-effectiveness of annual targeted larvicid-
ing campaigns in Cambodia against the dengue vector Aedes aegypti. Trop Med Int Health
12(9):1026–1036
Suaya JA, Shepard DS, Siqueira JB et al (2009) Cost of Dengue cases in eight countries in the
Americas and Asia: a prospective study. Am J Trop Med Hyg 80(5):846–855
Sukhavachana P, Nisalak A, Halstead SB (1966) Tissue culture techniques for the study of dengue
viruses. Bull World Health Organ 35:65–66
Sun W, Cunningham D, Wasserman SS et al (2009) Phase 2 clinical trial of three formulations of
tetravalent live-attenuated dengue vaccine in flavivirus-naive adults. Hum Vaccin 5(1):33–40
Tandan JB, Ohrr H, Sohn YM et al (2007) Single dose of SA 14-14-2 vaccine provides long-term
protection against Japanese encephalitis: a case-control study in Nepalese children 5 years after
immunization. Vaccine 25(27):5041–5045
Thalagala N, Tissera H, Palihawadana P et al (2016) Costs of dengue control activities and hospi-
talizations in the public health sector during an epidemic year in Urban Sri Lanka. PLoS Negl
Trop Dis 10(2):e0004466
Thomas SJ, Eckels KH, Carletti I et al (2013) A phase II, randomized, safety and immunogenicity
study of a re-derived, live-attenuated dengue virus vaccine in healthy adults. Am J Trop Med
Hyg 88(1):73–88
Tiga DC, Undurraga EA, Ramos-Castaneda J, Martinez-Vega RA, Tschampl CA, Shepard DS
(2016) Persistent symptoms of dengue: estimates of the incremental disease and economic
burden in Mexico. Am J Trop Med Hyg 94(5):1085–1089
Tricou V, Low JG, Oh HM et al (2020) Safety and immunogenicity of a single dose of a tetrava-
lent dengue vaccine with two different serotype-2 potencies in adults in Singapore: A phase 2,
double-blind, randomised, controlled trial. Vaccine 38(6):1513–1519
Trung DT, Wills B (2010) Systemic vascular leakage associated with dengue infections - the clini-
cal perspective. Curr Top Microbiol Immunol 338:57–66
Tuchinda P (1966) Therapy and therapeutic studies of Thai haemorrhagic fever. Bull World Health
Organ 33:74
Ubol S, Halstead SB (2010) How innate immune mechanisms contribute to antibody-enhanced
viral infections. Clin Vaccine Immunol 17(12):1829–1835
Udomsakdi S, Halstead SB (1966) Serological studies of dengue haemorrhagic fever. Bull World
Udomsakdi S, Nisalak A, Halstead SB (1966) Identification of dengue and chikungunya viruses.
Bull World Health Organ 35:68–69
Undurraga EA, Halasa YA, Shepard DS (2013) Use of expansion factors to estimate the burden of
dengue in Southeast Asia: a systematic analysis. PLoS Negl Trop Dis 7(2):e2056
Undurraga EA, Betancourt-Cravioto M, Ramos-Castaneda J et al (2015) Economic and disease
burden of dengue in Mexico. PLoS Negl Trop Dis 9(3):e0003547
Undurraga EA, Edillo FE, Erasmo JNV et al (2017) Disease burden of dengue in the Philippines:
adjusting for underreporting by comparing active and passive dengue surveillance in Punta
Princesa, Cebu City. Am J Trop Med Hyg 96(4):887–898
van Panhuis WG, Luxemburger C, Pengsaa K et al (2011) Decay and persistence of maternal den-
gue antibodies among infants in Bangkok. Am J Trop Med Hyg 85(2):355–362
Vaughn DW, Hoke CH Jr, Yoksan S et al (1996) Testing of a dengue 2 live-attenuated vaccine
(strain 16681 PDK 53) in ten American volunteers. Vaccine 14(4):329–336
Verchere AM, Raye DO (1872) Remarks on some of the symptoms of dengue. Indan Med Gaz
7:132–133
Watanaveeradej V, Simasathien S, Nisalak A et al (2011) Safety and immunogenicity of a tet-
ravalent live-attenuated dengue vaccine in flavivirus-naive infants. Am J Trop Med Hyg
85(2):341–351
Watanaveeradej V, Gibbons RV, Simasathien S et al (2014) Safety and immunogenicity of a
rederived, live-attenuated dengue virus vaccine in healthy adults living in Thailand: a random-
ized trial. Am J Trop Med Hyg 91(1):119–128
Watts DM, Porter KR, Putvatana P et al (1999) Failure of secondary infection with American
genotype dengue 2 to cause dengue haemorrhagic fever [see comments]. Lancet
354(9188):1431–1434
Weiss HJ, Halstead SB (1965) Studies of hemostasis in Thai hemorrhagic fever. J Pediatrics
66(5):918–926
Weiss HJ, Halstead SB, Russ SB (1965) Hemorrhagic disease in rodents caused by chikungunya
virus. 1. Studies of hemostasis. Proc Soc Exp Biol Med 119:427–432
White NJ (1999) Variation in virulence of dengue virus [comment]. Lancet 354(9188):1401–1402
WHO (2009) Dengue: guidelines for diagnosis, treatment prevention and control. WHO, Geneva
Wilder-Smith A, Byass P (2016) The elusive global burden of dengue. Lancet Infect Dis
16(6):629–631
Wilder-Smith A, Yoon IK (2016) Edging closer towards the goal of a dengue vaccine. Expert Rev
Vaccines 15(4):433–435
Winter PE, Yuill TM, Udomsakdi S, Gould D, Nantapanich S, Russell PK (1968) An insular
outbreak of dengue hemorrhagic fever: I. epidemiologic observations. Am J Trop Med Hyg
17(4):590–599
Winter PE, Nantapanich S, Nisalak A, Udomsakdi S, Dewey RW, Russell PK (1969) Recurrence of
epidemic dengue hemorrhagic fever in an insular setting. Am J Trop Med Hyg 18(4):573–579
Wragg WT (1851) History of the break-bone fever; an epidemic that prevailed in Charleston in the
summer of 1851. Charleston Med J Rev 6:153–182
Yoksan S (2017) Short history of dengue and Mahidol dengue vaccine. South East Asian J Trop
Med Pub Health 48(Supplement 1):20–32
Zeng W, Halasa-Rappel YA, Baurin N, Coudeville L, Shepard DS (2018a) Cost-effectiveness of
dengue vaccination in ten endemic countries. Vaccine 36(3):413–420
Zeng W, Halasa-Rappel YA, Durand L, Coudeville L, Shepard DS (2018b) Impact of a nonfatal
dengue episode on disability-adjusted life years: a systematic analysis. Am J Trop Med Hyg
99(6):1458–1465
Zubieta-Zavala A, Lopez-Cervantes M, Salinas-Escudero G et al (2018) Economic impact
of dengue in Mexico considering reported cases for 2012 to 2016. PLoS Negl Trop Dis
12(12):e0006938
Chronicles of Hantaviruses: Foundations
of Epidemiology and Ecology
James W. Le Duc and James E. Childs
Abstract The story of the hantaviruses is among the most exciting in all of virol-
ogy. The disease now known as hemorrhagic fever with renal syndrome was likely
present for decades in Asia. It gained attention of western medicine when United
Nations soldiers suffered this new illness during the Korean Conflict. Through US
Army sponsored research, Hantaan virus was identified as the cause and a link was
born with the US military that continues to this day. Many discoveries were made
by scientists at the US Army Medical Research Institute of Infectious Diseases
while collaborating with experts worldwide. What was originally thought to be a
single disease of Asia is today recognized as a complex of related infections found
nearly worldwide. Here, we share our personal experiences conducting field studies
on the hantaviruses, with special attention to Seoul virus that is found virtually
worldwide where it exists in close association with its rodent host, Rattus norvegi-
cus. It is not our goal to provide a comprehensive summary of the many break-
throughs made; rather, we hope to share the excitement and challenges we witnessed
firsthand during this period of discovery as the scientific community came to better
understand the complexity of the hantaviruses.
1 Introduction
The story of the hantaviruses and the diseases they cause is among the most exciting
in all of virology. The disease now known as hemorrhagic fever with renal syn-
drome (HFRS) was likely present for decades and perhaps even centuries in Asia
where it was referred to by various names such as epidemic hemorrhagic fever
J. W. Le Duc (*)
Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX, USA
e-mail: jwleduc@utmb.edu
J. E. Childs
Epidemiology of Microbial Diseases Department, Yale University School of Public Health,
Yale University, New Haven, CT, USA
e-mail: james.childs@yale.edu
316 J. W. Le Duc and J. E. Childs
(EHF), hemorrhagic nephroso-nephritis, Korean hemorrhagic fever (KHF), and oth-

ers (Gajdusek 1962; Johnson 2001). The disease gained the attention of western
medicine when United Nations soldiers suffered this mysterious new illness while
in combat during the Korean Conflict. In response to UN soldiers, sick and dying
from what they called epidemic hemorrhagic fever (EHF) and later became known
as Korean hemorrhagic fever (KHF), the US military created the Hemorrhagic
Fever Commission that brought together the greatest medical minds of the era to try
to find the cause of this life-threatening disease (Earle 1954). The Commission did
a remarkable job of characterizing the clinical disease, acquiring specimens from
acutely ill and convalescent patients, and defining the epidemiology of the disease,
but they were unsuccessful in their attempts to isolate the causative agent (Sheedy
et al. 1954). Through these historic studies, a link was born between HFRS, and
specifically KHF and the US military, and that bond continues to this day. Many of
the major discoveries about this group of viruses were facilitated by grants from the
US Army or were the result of research conducted by scientists affiliated with the
US Army while collaborating with experts from around the world. What was origi-
nally thought to be a single disease of Asia caused by an unknown pathogen is today
recognized as a complex of related viruses found nearly worldwide and caused by
an ever-growing number of related viruses belonging to a new family of viruses, the
Hantaviridae (Maes et al. 2009; Laenen et al. 2019).
Included in this fascinating history is the discovery of a completely novel, often
fatal clinical disease found initially in the southwestern United States. Described in
1993, the clinical course of this disease diverged from classic HFRS as the lungs,
rather than the kidneys, were predominantly affected. The disease was named hanta-
virus pulmonary syndrome (HPS or hantavirus cardiopulmonary syndrome, HCPS
in 2010—(Nichol et al. 1993)) and consistent with later findings, a species-specific
reservoir host was rapidly identified, the deer mouse, Peromyscus maniculatus
(Childs et al. 1994). The identification of a novel New World hantavirus causing
mortality and morbidity as high or much higher than the better-known Old World
HFRS-causing viruses was exceptional. The initial identification of the disease and
rapid recognition of the infecting virus was heralded as a phenomenal achievement
by virologists and epidemiologists (Nichol et al. 1993). As described below, many
of the key players involved in the discovery and the essential reagents and tests
needed to establish the diagnosis had only recently moved from the United State
Army Medical Research Institute of Infectious Diseases (USAMRIID) to the
Centers for Disease Control and Prevention (CDC).
The only other indigenous hantavirus previously described in the United States
was Prospect Hill virus (PHV) isolated from the meadow vole, Microtus pennsylva-
nicus, a virus related to the Old World Puumala virus (PUUV) (Pyung-Woo et al.
1985). PUUV also utilizes a reservoir arvicolid rodent, Clethrionomys (Myodes)
glareolus. Seoul virus (SEOV) maintained by the Norway rat (Rattus norvegicus) is
the only other Old World hantavirus found in the Americas (LeDuc et al. 1986b).
Chronicles of Hantaviruses: Foundations of Epidemiology and Ecology 317
1.1 The Isolation of Hantaan Virus-Etiologic Cause of Korean

Hemorrhagic Fever
This remarkable story starts with the isolation of Hantaan virus (HTNV), the cause
of HFRS, by Ho Wang Lee and his colleagues and published in 1978 (Lee et al.
1978). Lee had been supported for many years by a grant from the US Army to
identify the cause of HFRS, but he was unsuccessful in isolating a causative virus,
and his funding was scheduled to end in 1976. Just prior to his grant being termi-
nated, he was able to demonstrate that convalescent sera from recovered HFRS
patients reacted specifically with an antigen found in the lung tissues of the striped
field mouse, Apodemus agrarius. Upon sharing his findings with his DOD program
managers, he was given a short 6-month extension to replicate his findings and
independently verify them in a second laboratory. He was successful in doing this,
and as a result, his funding continued, and the modern era of hantavirology began.
Lee’s 1978 publication describing the first isolation of HTNV provided an antigen
that could be used for the diagnosis of suspect cases and an opportunity for charac-
terization of the virus (see Schmaljohn chapter in this book). Thus began a scientific
partnership that continued for more than a decade between Ho Wang Lee and his
colleagues in Korea working closely with Joel Dalrymple and a talented team based
at USAMRIID in Frederick, Maryland (Fig. 1).
Many others from around the world also made important contributions to the
unfolding story of the hantaviruses, and these advances were reported in hundreds
of scientific publications and numerous excellent reviews. Ho-Wang Lee’s personal
discovery of HTNV was meticulously documented in a two-volume book that cap-
tures the personal correspondence between Ho Wang Lee and his many collabora-
tors between 1970 and 1999 and itself represent a fascinating perspective of this
discovery as it unfolded (Lee 2000). His letters ranged from routine
Fig. 1 Both panels. Joel Dalrymple and Ho-Wang Lee enjoying a meal and discussions in Korea
communications with his Army program officers to quite personal exchanges with
Nobel Prize winner Carlton Gajdusek, strategy discussions with hemorrhagic fever
expert Karl Johnson, and technical exchanges with USAMRIID investigators as
they worked together to tease out HTNV from infected Apodemus rodent tissues.
It is not our goal in this chapter to provide a comprehensive summary of these
many breakthroughs. Rather, we hope to share our personal experiences and the
excitement and challenges that we witnessed firsthand as part of the USAMRIID
team during this period of innovation and discovery in the 1980s and early 1990s as
the scientific community came to better understand the complexity of the
hantaviruses.
1.2 The Search for the Rodent Reservoirs of Hantaan Virus:

A Connection Established by Early
Epidemiological Studies
KHF is a member of a class of syndromic diseases regionally known as EHF, Songo

fever, hemorrhagic nephroso-nephritis, and nephropathia epidemica (NE), among
other names. All were of unidentified etiology and contained under the umbrella
term HFRS. Although the viruses causing these geographically and often clinically
distinct diseases had not yet been isolated, numerous studies had determined by
epidemiological methods that risk of human infection was intimately linked to
proximity to field mice and voles (for early reviews dating from the 1950s to1960s
see: (Gajdusek 1962; Smadel 1953; Smorodintsev et al. 1963)). The striped field
mouse, A. agrarius, was commonly encountered by military troops bivouacking
within its preferred environment in Korea. This rodent is a generalist but preferen-
tially inhabits grass, scrub land, and agricultural areas (Youngman 1956). It is ubiq-
uitous on the Korean Peninsula and is considered the most common of rodents (Won
and Smith 1999). Of note, early studies of HFRS occurring among Soviet and
Japanese troops bivouacking in the field had produced strong epidemiological links
to outbreaks often focused in clusters where this mouse was most common. In 1956,
a study of the epidemiology of KHF, including studies of the possible role of an
arthropod vector and sylvatic reservoir host in maintaining the agent and complicit
in the transmission of the zoonotic agent to humans, was supported by the US mili-
tary as a result of KHF among troops engaged in the conflict. After years of effort,
the identification of antigens within the lungs of A. agrarius that responded to sera
obtained from KHF patients provided a means for detailed epidemiological investi-
gations of human and rodent infections and disease and led to the subsequent isola-
tion of HTNV (Lee et al. 1978). The isolation of other viruses from various species
of murid (mice and rats) and arvicolid (voles) rodents across the Eurasian Continent,
causing a range of clinically diverse forms of HFRS, quickly followed, and new Old
World viruses continue to be identified.
2 Clinical Disease
The clinical course of HFRS was perhaps best first described in detail in western
medicine during a Symposium on Epidemic Hemorrhagic Fever published in May
1954 in the American Journal of Medicine (Earle 1954). More than 1000 cases of
EHF (=KHF) occurred among United Nations forces during the fall of 1951, lead-
ing to the establishment of a dedicated treatment center in Korea where patients
could be cared for and the new disease better understood (Sheedy et al. 1954).
Hospitalized patients formed the basis for the clinical description of EHF and led to
the observation that the disease typically followed five phases, each of variable
duration and severity (Fig. 2a).
The disease begins typically with rapid onset of chills and fever, lethargy, and
generalized weakness, heralding the start of the febrile phase of the disease, which
lasted for about 5–7 days. During this phase, patients might suffer lumbar aching
and diffuse abdominal pain, sometimes severe, accompanied by facial flushing to
include the shoulders, neck, and head and conjunctival injection. Toward the last
day or two of the febrile phase, some patients may develop hypotension or shock,
indicating the onset of the hypotensive phase. This varies in duration and may last
only a few hours in patients suffering a milder course of illness to 1–3 days in those
severely ill. Marked proteinuria occurs at about day 5 of illness in most patients,
independent of severity of illness. The oliguric phase begins as patient’s blood pres-
sure returns to normal or increases to hypertensive levels and may last 2–3 days.
The diuretic phase begins with diuresis and general improvement, although fluid
and electrolyte imbalances may be present. The convalescent phase may last for
several months in the most severely ill patients. Mortality among UN forces was
about 10% initially; however, this proportion dropped to about 5% as clinicians
gained familiarity with the disease. Importantly, a wide range of severity of illness
was noted, with some patients skipping phases completely in milder cases. This
variation caused some confusion later as additional hantaviruses were discovered
that caused different clinical presentations. Decades later, the clinical course of dis-
ease caused by prototype HTNV was again reviewed and summarized (McKee et al.
1985). Their findings differed little from the original descriptions reported in 1954,
a testimony to the clinical skills and attention to detail shown by these pioneer phy-
sicians and support staff.
The availability of isolation methods and antigen substrates for the detection of
antibodies to HTNV not only permitted specific epidemiological studies of HFRS
but also provided the means to study the course of infection within the reservoir
host. Following challenge with HTNV, Apodemus mice develop IFA and neutraliz-
ing antibodies at about 12 days (Fig. 2b). The clinical disease was minimal. However,
viral antigens, while disappearing from the blood concurrent with first antibody
production, could be detected for >200 days within lung tissue and were present in
urine over the same extended interval. These observations indicated that immunity
in this species was not sterilizing and viral persistence in solid tissues was accom-
panied by chronic shedding of virus in urine and in the feces (for a shorter duration).
Fig. 2 Schematics of the time-course of disease in humans and development of tissue infection
and immune responses developing in a reservoir rodent host when inoculated with Hantaan virus.
(a) The five-phase clinical course of severe HFRS in humans as ascertained by studies of KHF
(reprinted from JAMA with permission). (b) The course of infection and infectivity of Hantaan
virus in Apodemus agrarius. Shaded area of bars for lung, parotid glands, and kidneys indicates
presence of antigen, but not virus. Similar chronic infection and persistent shedding of virus is
thought to occur in most primary hosts of hantaviruses. (Reproduced with permission from
Lee et al. 1981)
This was the initial observation of what was to become a hallmark of hantaviral
infection among their specific rodent reservoirs, minimal or no clinical disease,
establishment of a chronic viral infection among certain tissues concomitant with
the presence of neutralizing antibodies and persistent or intermittent shedding of
virus over the life span of the infected individuals.
3 Diagnostics
In addition to the clinical documentation that the hemorrhagic fever commission

provided in describing EHF to western medicine, a dedicated team systematically
collected acute and convalescent sera from patients seen at the hemorrhagic fever
hospital. These were painstakingly stored in glass vials, lyophilized, carefully
labeled, and ultimately stored in three US Army issue olive drab metal footlockers.
Each vial was labeled with the patient’s name and number, date of collection, vol-
ume, and a “DD” number. The information was typed on white adhesive tape, and
one can imagine the team members stretching the adhesive tape the length of the
typewriter carriage, carefully typing each record, and then pealing the tape off,
spreading it across a glass or plastic sheet, using a scalpel to cut each individual
label, and then finally attaching each label to the appropriate vial. In all, over 600
sera from 245 patients were preserved in this manner, and these were stored for
nearly three decades awaiting the discovery of the pathogen causing EHF.
The isolation of prototype HTNV provided the opportunity for these sera to be
opened, rehydrated, and tested against HTNV at USAMRIID using a newly devel-
oped IgM and IgG enzyme immunoassay (LeDuc et al. 1990). The “DD” on each
label was assumed to be “disease day” and of the specimens from the 245 patients
tested, all but 15 were shown to have antibodies specific to HTNV. This serves as a
testament to the diagnostic and clinical skills of the commission physicians. The
IgM capture assay was positive very early in the course of infections, and all KHF
patients were positive in this test by day 7 post-onset of disease (see table below).
IgG titers rose more slowly as expected and typically reached the highest titers in
the last sample tested (see Fig. 3). Two key points emerged from this study. First,
Fig. 3 Contemporary serological results obtained from lyophilized acute and convalescent serum
samples collected from patients seen at the hemorrhagic fever hospital in Korea three decades before
the discovery of Hantaan virus. (a) All patients were IgM positive by day 7 post-onset of disease. (b)
IgG titers rose more slowly and typically reached the highest titers in the last convalescent samples
tested. (Reproduced with permission from Le Duc et al. 1990)
these results provided strong evidence that HTNV was very likely the cause of EHF
(=KHF) seen during the Korean Conflict. Second, it showed that the IgM capture
immunoassay was a reliable diagnostic tool for hantavirus-induced diseases. This
was especially important, since prior to these studies, most diagnostics relied on
immunofluorescent antibody assays that suffered from limited availability of slides
to allow testing and nonspecific reactions.
4 Treatment
Treatment for HFRS patients in the mid-1980s was limited to supportive care and
careful fluid management, with no virus-specific interventions available to alter the
course of the disease. As a result, HFRS mortality rates in China at that time ranged
from about 5–10% in major hospitals and was higher in less developed parts of the
country. USAMRIID investigators led by John Huggins were interested in the anti-
viral drug ribavirin as a possible treatment for HFRS based on earlier success in the
treatment of Lassa fever and promising in vitro results that showed HTNV sensitiv-
ity to the drug in laboratory testing. Huggins was able to convince leaders in both
the US Department of Defense and China to conduct a prospective, double-blind,
placebo-controlled clinical trial of intravenous ribavirin therapy of HFRS in Chinese
patients. The study was conducted during the 1985–1986 and 1986–1987 fall and
winter transmission seasons in and around Wuhan, Hubei Province, an area endemic
for HFRS in central China. A total of 242 serologically confirmed HFRS patients
were enrolled, and results found a sevenfold decrease in the risk of death among
treated patients as compared to those receiving a placebo. A significant beneficial
impact on the clinical course of disease was also seen (Huggins et al. 1991). The
study was considered a major breakthrough at that time and subsequently led to the
use of ribavirin in the treatment of HFRS in China (Fig. 4a).
The story of how this study came to be is noteworthy. China had only recently
emerged from the Cultural Revolution and the China of the mid-1980s stood in stark
contrast to the modern country we know today. There were very few private cars,
and most people got around by bicycle. Hospital and laboratory facilities were out-
dated, and access to modern laboratory instrumentation was limited. The idea of
conducting a formal clinical trial in partnership between US Army investigators
from USAMRIID and Chinese collaborators was thought by some to be fanciful,
yet Huggins was able to convince leaders on both sides to try. What followed was an
amazing clinical trial that met rigorous international standards, yet it was conducted
under less-than-ideal conditions and involved Chinese partners who had little to no
experience in the conduct of a modern human clinical trial.
The collaborations began with a series of long flights between the US and China,
arrivals and departures in yet-to-be-modernized Chinese airports, and a lot of tea
drinking as details were arranged and agreements reached. The first formal visit was
in 1985, and the USAMRIID team members were met with great fanfare. The
American guests were housed at the most luxurious lakeside lodge previously used
Fig. 4 Glimpses of the extraordinary teams of international virologists and biologists who eluci-
dated many of the key elements in the diversity of hantaviruses causing a range of human diseases,
their global incidence and prevalence, and the diverse rodent hosts that serve as reservoir hosts. (a)
The ribavirin study team at Hubei Medical College, Wuhan, China, 1986? L➔R, “Amy,” Translator,
Joe Smith, MD, John Huggins, PhD, “Keith” lab tech, MAJ Reed, clinical lab officer, Shu-Yuan
Xiao, MD, Tom Cosgriff, MD. (b) First International Symposium on Hantaviruses and Crimean-
Congo hemorrhagic fever virus, Halkidiki, Greece, 1988. (L➔R standing, Bob Swanepoel, Tatjana
Avsic-Zupanc, Antonios “Tony” Antoniadis; seated, Loredona Nicoletti; Yong Kang, Ana Gligic;
foreground, Paula Veroni). (c) Adnan Sarcevic, Tom Ksiazek and Alemka Markotic (L➔R) in
Sarajevo establishing laboratory diagnostics for HFRS, June 1989. (d) Shu-Yuan Xiao, Jim Le
Duc, Tatjana Avsic-Zupanc, Gordana Diglisic, Alan Schmaljohn, (L➔R), USAMRIID, ca 1992
by Chairman Mao. Subsequent visitors were offered rooms at other progressively

lesser quality accommodations, and those of us participating in the final lab wrap-up
stayed in the medical school’s very humble foreign student dormitory.
In order to generate the well-documented laboratory results that would be
required for international acceptance of the study, modern laboratory instrumenta-
tion needed to be purchased, imported, and installed in the Wuhan collaborating
hospitals. Export regulations, transportation challenges, maintenance, and instilla-
tion in the absence of manufacturer’s technical representatives were all significant
challenges that had to be overcome. It soon became apparent that reliable electrical
power was a major limitation, and this necessitated the instillation of uninterrupted
power supplies for most critical equipment. “Room temperature” varied consider-
ably according to the seasons, from blistering hot in the summer to freezing cold in
the winter. Wuhan was known as one of the ovens of China, with very high sum-
mertime temperatures and oppressive humidity. The lab where the IgM capture
immunoassay was housed to confirm HFRS infections became one of the most
popular places on the medical campus when dedicated heating and air conditioning
was installed.
Meetings took place with clinical staff at collaborating hospitals as the study
began and these often concluded with a banquet involving exotic foods (for the
Americans) and locally produced high-potency drinks. GI repercussions were com-
mon, but life-long friends were made, and a true spirit of friendship and collabora-
tion developed. Indeed, these relationships continue to this day (Fig. 4b).
5 Hemorrhagic Fever with Renal Syndrome in Europe

and the Americas
5.1 Nephropathia Epidemica in Sweden
A less severe form of HFRS called nephropathia epidemica (NE) was known to
occur in Scandinavia, European Russia, and much of Europe. The disease was first
described in the 1930s, and the similarities in clinical presentation and course of
illness to HFRS of Asia were also well documented (Lähdevirta 1971). In the early
1980s, the virus causing NE had not yet been isolated, although the association
between the human illness and a rodent reservoir host, the bank vole, Myodes glare-
olus (then called Clethrionomys glareolus) was suspected. Lung tissues of infected
bank voles collected in the Puumala region of Finland reacted with convalescent
sera from NE patients from the same region by IFA testing (Brummer-Korvenkontio
et al. 1980).
Collaborations were begun between USAMRIID investigators and a young
Swedish physician-scientist, Bo Niklasson, with the goal of isolating the virus caus-
ing NE, and to determine the geographic distribution of the disease and virus
throughout Sweden. Over the course of the next several years, field teams trapped
rodents across Sweden, from the southernmost tip of the country to well above the
Arctic Circle. Human NE cases were documented and blood and tissues (lung,
spleen) from rodents were collected and processed for virus isolation attempts and
antibody testing. Serological surveys documented the high prevalence of infections
among bank voles captured in the northern two-thirds of Sweden, and this distribu-
tion was reflected in the human sera tested, with the highest antibody prevalence
found in residents of northern Sweden as well.
The high antibody prevalence among voles was restricted to northern Sweden,
and the zone discriminating between regions of high and low prevalence corre-
sponded to a previously well-defined ecological boundary. This biogeographical
boundary formally called the Limes Norrlandicus runs from ~59°N on the western
coast of Sweden to 61°N on the eastern shore and separates the northern boreal zone
from the southern boreal-nemoral zone. Populations of voles north of this divide are
strongly cyclic, while populations of south are noncyclic or cycle at unpredictable
periods. These observations suggested that environmental and climatic features
influenced not only the physiological, behavioral, and reproductive biology of
Myodes but also parameters related to viral prevalence and maintenance within this
vole and hence the epidemiology of NE (Niklasson and Le Duc 1987).
Attempts to isolate PUUV were successful after repeated co-cultivation with
Vero E-6 cells and several blind passages. The isolate was designated the 83–227
strain (Niklasson and Le Duc 1984). Concurrently, across the Fort Detrick campus
where USAMRIID was located, a second group led by Richard Yanagihara was
working in Carlton Gajdusek’s NIH lab, and they too succeeded in isolating PUUV,
which they designated the Hallnas strain (Yanagihara et al. 1984).
The field work involved in defining the distribution of PUUV in Sweden and the
collection of rodent tissue for virus isolation attempts was an adventure in itself.
Small mammals were live-trapped using neat little traps designed by a Swedish
zoologist specific for small rodents and shrews. It used a counterbalanced platform
that opened under the weight of the voles, taking them into the caged area where
they had access to the apple and peanut butter bait. Once inside, the weight raised
the entrance platform, capturing the voles. Collections were done in the summer
months of 1982–1984, in and around areas where human cases had occurred.
Captured animals were processed in temporary field sites under less-than-ideal bio-
containment conditions. Blood was collected, and lung, spleen, and kidneys asepti-
cally removed and stored in plastic vials on dry ice pending return to the National
Bacteriological Laboratory in Stockholm or USAMRIID for testing. A Swedish
military jeep-like vehicle, which drew stares and comments every time the gas tanks
were filled, was our means of transportation. The team stayed in government quar-
ters, cheap hotels, and occasionally in isolated locations. The most noteworthy was
the site far above the Arctic Circle in Abisko National Park in Swedish Lapland
where the terrain was rugged, the mosquitoes were hungry, and the weather was
unpredictable—it even snowed in August. The work was hard, but the scenery was
beautiful and the occasional sauna and refreshments with the locals made for great
adventures.
These studies contributed to the understanding of HFRS in Europe in two pri-
mary ways. First, the isolation of PUUV from Myodes glareolus confirmed the
long-suspected importance of voles as a natural reservoir of PUUV. This facilitated
a better understanding of the epidemiology of NE and provided a scientific basis for
rational guidelines for disease prevention and risk mitigation. Second, it provided a
specific virus that could be interrogated for susceptibilities to antiviral drugs, used
in diagnostic assays, and development of animal models to facilitate vaccine, drug,
and therapeutic development.
5.2 HFRS in Greece
In the 1980s, isolated cases of HFRS had been reported in southern and central
Europe, outside the traditional endemic area of NE in Scandinavia and western
Russia. The clinical presentation in these areas was unique, with some presenting as
a mild HFRS quite similar to NE caused by PUUV, while a more severe and
life-threatening form more similar to HFRS due to HTNV was also present. Initial
studies have begun between USAMRIID investigators and Dr. “Tony” Antoniades
following the report of two HRFS cases near Thessaloniki in northern Greece
(Antoniadis et al. 1984), and subsequently, collaborative outbreak investigations
were started following HFRS infections of eight shepherds in northwestern Greece
in 1983. All eight patients were hospitalized, three were severely ill, and one died.
Subsequent studies found over 6% of residents of the small village of Tsepelovo
were seropositive by IFA for antibody to HTNV. Tsepelovo was the nearest village
to the mountaintop where the shepherds had tended their flock and were likely
infected. More specific plaque reduction neutralization tests of patient’s convales-
cent sera found the highest titers to HTNV, with low, apparently cross-reactive titers
to SEOV and PUUV (LeDuc et al. 1986a, b). These findings, and others from else-
where in the region, suggested that a new virus similar or identical to HTNV might
be present and responsible for the more severe disease seen.
As part of the outbreak investigations, small rodents were captured in and around
Tsepelovo and in the high meadows of nearby Tempfi Peak where the infected shep-
herds tended their sheep. The entire area is very rocky, and most of the village
houses and roads were made of local stone. We stayed in the village and set up the
field laboratory in one of the rooms of the mayor’s newly built house that he had
kindly allowed us to share. Each morning we checked our field boots for the small
scorpions that were common in the house and seemed to take a special fancy to the
dark interior of our boots, then we would drive a few miles to the base of Tempfi
Peak. There we would lug our live traps up the stone path to the highland meadow
where we collected the mice captured the previous night. In the afternoon, we would
again make the trek up the mountain to bait the traps for the night. We made this trip
back and forth for several days, generally with few animals caught. In spite of our
poor capture rate, we felt ourselves to be quite the outdoorsmen, braving the ele-
ments and strenuously hiking up the mountain, until one morning we met an older
lady, probably in her 70 s, who was setting at the watering trough about half-way up
the peak. She was crocheting a doily and looked up to greet us smiling as we huffed
and puffed up the hill with our load of traps. After catching our breath, we continued
our trek to the top, and she marched up the hill with us, stride for stride talking all
the while as she continued to crochet her doily, never once missing a beat or show-
ing any signs of the exhaustion that we felt. After this experience, we decided that
the local residents were indeed hardy souls (and that maybe we were wimps).
We were able to capture only 85 rodents and one shrew, and only 2 of 33 cap-
tured Apodemus flavicollis were positive for antibody to hantaviruses by IFA. All
other species were negative, and we were unsuccessful in our attempts to isolate a
virus from the seropositive rodents. Nonetheless, the studies contributed to the
growing body of knowledge that there existed in the region a likely new hantavirus
capable of causing severe infections and death in humans and that the suspected
new virus may be maintained in nature by the field mouse, A. flavicollis.
Given the rapid advances being made around the world on the hantaviruses,
Antoniadis and LeDuc organized what turned out to be a very unique meeting in
1988 in Halkidiki, on the coast of northern Greece. Participants came from the
Soviet Union and other communist bloc nations, Albania, North Korea, and other
countries not normally represented at international scientific meetings in the West.
The scientific presentations were fantastic and culturally diverse, reflecting differ-
ences in local conditions and resources as well as varying attitudes as to what con-
stituted a 10-min presentation—hard to cram in 20 pages of written text before the
timer goes off! One of the highlights was the informal discussions between scien-
tists with very different backgrounds but shared curiosity and goals. These led to
productive scientific collaborations and lasting friendships. Indeed, the contacts
made at this meeting helped some Soviet scientists find homes in the West following
the collapse of the Soviet Union.
5.3 HFRS in the Former Yugoslavia
In the early 1980s, there was an opportunity for military-to-military collaborations

in medicine between the United States and Yugoslavian armed forces. USAMRIID
investigators took this opportunity to work with our Yugoslav military and civilian
scientist counterparts to further investigate hantaviruses found in what was then
Yugoslavia. Collaborations started with Dr. Ana Gligic from Belgrade, along with a
young virologist from Ljubljana, Tatjana Avsic-Zupanc, and a recently graduated
physician from the medical school in Sarajevo, Alemka Markotic, among others
(see Fig. 5b, c above).
Ana Gligic was perhaps the premier virologist in Yugoslavia at that time. She
was famous for her leadership in combating the smallpox epidemic of 1972 in
Yugoslavia when 175 patients were infected and 35 ended in death (Ristanović et al.
2017). In addition, she was a pioneer in the study of HFRS in Yugoslavia, and her
young colleagues were eager to work with her. Her laboratory at Torlak in Belgrade
was well known, and she worked closely with the Yugoslavia Military Medical
Academy where one of us (JWL) was even invited to lecture. HFRS was first recog-
nized in Yugoslavia in 1952 (Simic 1952) and outbreaks and individual cases of
both mild and severe HFRS had been seen since then (Gligić et al. 1989). PUUV
was recognized to cause the milder forms of HFRS in Yugoslavia, but when our col-
laborations began, the cause of the more severe HFRS had not yet been discovered.
Jointly, we captured rodents in several parts of Yugoslavia including in what is today
Slovenia, Croatia, Serbia, and Bosnia-Hercegovina, and we investigated severe
HFRS patients and their likely sites of exposure. Over the course of these studies,
Dobrava virus (DOBV) was isolated by Avsic-Zupanc from the lungs of A. flavicol-
lis captured near the Slovenian village of Dobrava, an area where severe HFRS
cases had occurred. The virus was shown to be distinct from other known hantavi-
ruses, and convalescent sera from recovered severe HFRS cases reacted to high titer
to this new virus (Avsic-Zupanc et al. 1992, 1994). At about the same time, a second
new hantavirus was isolated by Ana Gligic, which she named Belgrade virus. This
virus was recovered from severe HFRS patients in Serbia (Gligic et al. 1992).
Dobrava and Belgrade viruses turned out to be the same, and there was considerable
debate if the name Dobrava or Belgrade had priority for the new virus. Over time,
the name Dobrava virus has been generally accepted, although some references to
Dobrava-Belgrade virus exist in the literature.
Our studies in the former Yugoslavia offered a unique glimpse of history in the
making. Our collaborations began prior to the onset of the civil war and continued
through much of the hostilities. Our collaborators came from Serbia, Croatia, and
Slovenia, and these new countries became warring factions leaving our efforts to
collaborate especially difficult. Before the war started, we began to develop the
diagnostic laboratory capacity needed to conduct a ribavirin efficacy trial in
Sarajevo. This would provide us with a second site to replicate the ribavirin treat-
ment study that had been successfully completed in China. However, as overt war
broke out, our field studies were obviously halted, and the idea of doing a second
ribavirin trial was abandoned. We did continue to host our partners from all parts of
the region as visiting scientists to USAMRIID as we worked together to character-
ize the hantaviruses found there and to interrogate the characteristics of the severe
disease caused in humans. The informal social gatherings at home that were
arranged for our visitors were admittedly a bit stressful, however, as the competition
moved from the battle fields in Yugoslavia to the dinner table where the quality of
regional cuisine was at times nearly weaponized.
These were very difficult times for everyone involved. Some of our collaborators
who were working as physicians in Sarajevo risked their lives every day walking
through “sniper alley” just to reach the hospital to care for the sick, wounded, and
injured. Indeed, one of our team was severely wounded but fortunately survived.
With the collapse of Yugoslavia and the birth of several new countries, our original
collaborators have since gone on to establish themselves as leaders in the fields of
virology and infectious diseases, each making substantial contributions to the field
of hantavirology and others. The discovery and isolation of Dobrava virus repre-
sents one of the major accomplishments of the decade, and it helped resolve the
question of what caused the severe HFRS found in the Balkan Region.
5.4 HFRS in Latin America
The US Army Medical Research Unit located in Belem, Brazil (USAMRU-Belem,

or sometimes referred to as USAMRU-TransAmazon), was created in the early
1970s to work with Brazilian scientists to investigate the causes of illness seen in
settlers that moved into new communities in the Amazon basin as the TransAmazon
highway was constructed. Our studies focused primarily on small villages of colo-
nists that were established in the forests in the area between Maraba and Altamira
and the connecting road to Santarem in Para State. Begun in 1972, the TransAmazon
“highway” was in reality a dirt road that was nearly impassable during the rainy
season and not much better during the dry season.
USAMRU-Belem was co-located with the Instituto Evandro Chagas (IEC) in
Belem, a historic city near the mouth of the Amazon. The IEC is a Brazilian medical
research facility that was historically affiliated with the Rockefeller Foundation
overseas laboratory network and has a rich and illustrious history of virus discovery
that continues to this day. The Belem laboratory was actively involved in research
on yellow fever virus, but scientists working at the IEC also discovered many, many
new arboviruses during their investigations. During its relatively short tenure, the
USAMRU-Belem was highly productive, but unfortunately, it was forced to close in
1979 following international disputes at the highest political level between the
United States and Brazil. These issues were not related to the laboratory itself, but
USAMRU-Belem nonetheless became an unintended causality. As LeDuc was the
commander of USAMRU-Belem, he had the dubious honor of accounting for sev-
eral million dollars worth of scientific equipment and instrumentation shipped or
purchased to outfit the lab, ensuring that all local national employees were dis-
charged appropriately and of sending all the US personnel, their families, and their
household belongings back home safely. These were challenging tasks, especially
accounting for microscopes, centrifuges, and all sorts of other laboratory equipment
that had been deployed to field stations in remote corners of the Amazon basin.
Fortunately, the US Embassy official assigned the task of closing the USAMRU-
Belem property book elected to donate all the equipment to the Brazilians, much to
the relief of many. In spite of the sad ending of USAMRU-Belem, the collaborations
that were undertaken and the friendships made were incredibly valuable and contin-
ued long after the lab closed.
With this background, when the question of the global distribution of the newly
discovered hantaviruses was begun, it was logical to work with scientists from the
IEC. We attempted to determine if Seoul virus (SEOV) was present in domestic rats
in Belem and elsewhere in Latin America working with many of our same col-
leagues from the USAMRU-Belem days. We were able to provide diagnostic
reagents from USAMRIID to IEC scientists, and we worked together with them to
examine locally captured rats, ultimately finding that more than half of the rats cap-
tured in and around Belem had antibody to a hantavirus, most likely SEOV. Similar
studies were conducted elsewhere in Brazil and in Argentina, again finding sero-
positive rats in all the areas sampled. Further, we were able to isolate a Seoul-like
virus from rats captured in Belem, thus representing the first isolation of a hantavi-
rus from South America (LeDuc et al. 1985). Later studies found associated human
disease in southern Brazil (Iversson et al. 1994). All of this was done prior to the
discovery of hantavirus pulmonary syndrome and the realization that other rodent
species throughout Latin America were infected with their own distinct hantavi-
ruses, some of which have now been shown to cause serious, life-threatening dis-
ease in humans.
6 HFRS in Cities and Discovery of SEOV
In the early 1980s, when cases of HFRS were identified in Seoul and four nearby
South Korean cities by confirming the presence of IFA antibodies reactive with
HTNV, the likelihood of an alternative reservoir host was immediately suspected.
Ho Wang Lee and his Korean and USAMRIID collaborators turned their attention
to these urban cases of HFRS that occurred in places largely inhospitable to field
mice, which are better suited to rural environments. The required habitat for
A. agrarius, the host of HTNV, was clearly not the urban environment of Seoul
(Youngman 1956). The most ubiquitous urban rodents are the Norway rat (Rattus
norvegicus), the black rat (Rattus rattus), and the house mouse (Mus musculus).
These three murid rodents enjoy a cosmopolitan distribution as globally introduced
by Eurasian countries during the exploration and colonization of every Continent,
with the exception of Antarctica, and the subsequent establishment of trading routes
(Long 2003). Studies in Seoul naturally focused on rats, which were widely distrib-
uted in the city. Serum antibodies were detected in 13% of samples from 477 R. nor-
vegicus and from 11% of 47 R. rattus. Viral antigens fluorescing with anti-HTNV
antibodies were found in pulmonary tissues of 42 animals, and a virus was isolated
from 23 rats, all but two of which were R. norvegicus (Lee et al. 1982). This virus,
antigenically grouped with HTNV but shown to be distinct by serological testing,
was named Seoul virus, in keeping with the tradition of naming arboviruses (the
directly transmitted hantaviruses and arenaviruses are generally included among
arboviruses for convenience) after the geographic location where first obtained. Of
note, this study also reported that sera from the Asian rodents, Bandicota bengalen-
sis and Bandicota indica, were antibody positive by IFA to HTNV, but PRNTs were
inconclusive suggesting that neither HTNV or SEOV were the likely source of
infection (LeDuc et al. 1986b). Bandicoots are known to live in close association
with humans in Asia. These observations and those by others led to further testing
of tissues from wild-caught bandicoot rats captured in Thailand and ultimately led
to the isolation of Thai virus (Thailand 749), yet another novel hantavirus previ-
ously unknown to science (Elwell et al. 1985; LeDuc et al. 1986b).
The implications of this finding had worldwide public health implications as at
this time all hantaviral diseases were only recognized from the Old World. With the
discovery of SEOV within Norway and black rat populations, the specter of a poten-
tially globally distributed virus capable of causing HFRS had to be confronted.
7 Global Distribution of Hantaan-Related Viruses Among

Peridomestic Rodents with Special Reference to SEOV
Infection Among Norway Rats
Within two years, virologic and serologic evidence emerged that a SEOV-like virus
was present among wild Norway rats in several cities of the United States–
Philadelphia, Houston, and New Orleans (LeDuc et al. 1982; Tsai et al. 1982).
Initial screening by IFA tests indicated the greatest reactivity to SEOV, which was
confirmed by PRNT. By1984, isolates of the SEOV-like virus had been obtained
from rats captured from port cities at granary storage facilities in Philadelphia
(Girard Point virus (GPV)) and New Orleans (Tchoupitoulas virus (TCHV)) (LeDuc
et al. 1984, 1985; Tsai et al. 1982).
In addition to the isolation of SEOV-like viruses from the United States, other
parallel studies identified the magnitude of this public health threat through estab-
lished collaborations between USAMRIID investigators, other US DOD overseas
laboratories, national public health organizations, and colleagues at academic cen-
ters. Over 1700 rodent sera were collected from in and around residences in urban
and peri-urban settings from more than 20 sites around the world (LeDuc et al.
1986b). More than 20% of rodent sera were positive by IFA against prototype
HTNV, and a subsample of positive sera was further tested by plaque reduction
neutralization tests against both HTNV and GPV. Rats of the genus Rattus provided
the majority of positive sera and infections caused by SEOV. Positive rats were
found from sites in North and South America, Europe, Africa, Asia, and Australia.
Rats infected with SEOV had a global distribution although not every site yielded
positive animals. Sites with the highest antibody prevalence against hantaviral anti-
gens were Baltimore, MD, USA (64%), and Belem, Brazil (56%) (LeDuc et al.
1986b). The global distribution of a virus capable of causing HFRS maintained by
a ubiquitous and prolific species was of global health concern, and in response,
many international studies were initiated focusing on the epidemiological link
between SEOV-infected rats and the potential of human infection and disease. We
illustrate these types of efforts by concentrating on particularly intensive and exten-
sive studies conducted by USAMRIID and JHU in Baltimore, MD.
7.1 JHU, USAMRIID, and SEOV
James (Jim) LeDuc, then the Chief of Epidemiology at USAMRIID, contacted

Professor Keerti Shah at Johns Hopkins University (JHU) School of Hygiene and
Public Health. Shah, who died at age 90 in 2019, had mentored scientists working
at USAMRIID, and several had received PhDs from JHU through this collaborative
agreement. Of note, and relevant to the history of arbovirology, prior to joining the
faculty at JHU, Dr. Shah had been employed as a virologist at the Poona, India,
laboratory funded by The Rockefeller Foundation in 1952. He had been involved in
studies identifying novel and previously described arboviruses responsible for
human disease in India including Kyasanur Forest, Ganjam, and Chikungunya
viruses. He was deeply interested in vector-borne and zoonotic pathogen mainte-
nance and cross-species transmission, for example, “spillover,” to humans. LeDuc
asked, was he aware of anyone catching wild rats in urban Baltimore? This request
could not have been better directed to any other individual or Institution.
7.2 Sites Are Critical for Arboviral and Zoonotic Viral

Study-JHU and a Long Legacy of Field
and Microbiological Research on Urban Norway Rats
Individual private and public institutions have been or are synonymous with world-
renowned excellence in the study of arboviral and zoonotic diseases (several are
discussed in more detail within this volume). Without reference to the dates of
establishment, the names of the international and national Rockefeller-Institute
sites, the NAMRU locations funded by the US military and federally funded pro-
grams at CDC Fort Collins standout. Of historical note, the Rockefeller Foundation
became the first major source of funds for professional education in public health
and gave $1 million to JHU between 1916 and 1922 to organize the first full-fledged
school of public health in the United States (Evans 2009; Brown 1976).
Other universities and federal laboratories were vitally important sites for the
training of many of the most influential arbovirologists of that era. The most influ-
ential research and training program was established in 1964 at the Yale School of
Public Health—again funded by the Rockefeller Foundation (see contributions by
other authors within this volume). This center, named the Yale Arbovirus Research
Unit (YARU), trained numerous individuals competent in both field and laboratory
techniques and, through collaboration with the web of internationally supported
sites, collected an invaluable and irreplaceable library of arthropod-borne and zoo-
notic viruses, in addition to developing an inventory of immune reagents for their
study (Downs 1982). The list of medical entomologists and other biologists who
were trained at or were once on the faculty at YARU is a Who’s Who list of the most
eminent arbovirologists of that era, and some are authors contributing chapters to
this volume. The demise of YARU could have been a scientific disaster for the inte-
grated study of arbovirology. However, the integration of the principal scientific
leaders into programs at the University of Texas Medical Branch at Galveston
ensured some continuity, and the extensive collections of viruses and reagents found
homes in Texas and at the CT Agricultural Field Station in New Haven.
Rockefeller supported programs, and investigators concentrated on collecting
and classifying arboviruses, establishing vector/vertebrate reservoir associations,
and elucidating the prevalence of infection/disease among humans. Although rats of
the genus Rattus were identified as potential viral hosts within these diverse studies,
most of the research was conducted within relatively pristine environments such as
rainforests. Studies of the microbiological threats posed by urban populations of
Norway and black rats, the most-cosmopolitan and successful invasive mammalian
colonizers in history (with the exception of humans), were not a priority. However,
some institutions, most notably JHU Medical School and School of Hygiene and
Public Health (collectively referred to as JHU), were the home of many researchers
who had initiated intensive studies of the urban rats and their zoonotic micro-and
mega-parasites. As of 2019, this tradition has carried on for a century.
Early studies (1920–1927) conducted by JHU faculty, or individuals who would
later join the faculty, focused not only on the role of urban rats in the maintenance
and transmission of zoonotic pathogens to humans but also on rodent ecology by

conducting field studies in diverse sites across the city where human-rat interactions
varied in intensity. As early as 1920, investigators had studied rat control and popu-
lation rebound in Baltimore (Emlen et al. 1920). Investigations of Leptospira ictero-
haemorrhagiae infecting humans (Weil’s Disease; Walch and Walch-Sorgdrager
1927) quickly followed the extraordinarily comprehensive studies conducted of
human disease and the role of Norway rats as a key reservoir host of leptospires in
Japan (Inada 1915; Ido 1917). Studies on helminths (Luttermoser 1936) and proto-
zoa (Andrews and White 1936) among urban rats were some of the first to attempt
to describe the diversity of parasitic communities among these hosts. Studies of
arthropod-transmitted agents included defining the prevalence of Rickettsia typhi
infection among fleas obtained from rats trapped from an endemic foci (Dyer et al.
1931a, b) and investigating a risk factor for plague emergence by quantifying
Xenopsylla cheopis infestation prevalence on rats (James and Davis 1950). Detailed
studies on rat physiology and, most significantly, their inability to taste active chem-
icals of potent rodenticides led to some of the earliest descriptions evaluating con-
trol measures for reducing rat populations at targeted sites within the urban
environment (Dieke and Richter 1946; Richter and Emlen 1946).
Of note, early studies of the incidence of rat bites and rat-bite fever, caused by a
bacterium (Spiralis) spp. requiring direct interaction between humans and Norway
rats, provided insights into the epidemiology of human-rodent interactions
(RICHTER 1945b). These studies conducted at JHU resulted in detailed maps of rat
infestations and risk of disease stratified by human demographic characteristics.
Observations linking high risk of infections to primarily black residents of low
socioeconomic status neighborhoods of Baltimore was consistent with all future
studies of rat infestation and risk of exposure to SEOV.
7.3 Studies of Urban Rat Ecology from Baltimore
Beginning in the 1940s, and prior investigations by individuals recruited to the cen-
ter, David E Davis and colleagues published dozens of papers from The Rodent
Ecology Project (funded in part by the Rockefeller Foundation) on the characteris-
tics and size estimates of rat populations (Davis 1949, 1951; Emlen et al. 1949;
Davis and Fales 1950), home range (Davis et al. 1948), population control and
rebound (Emlen et al. 1948; Emlen 1947), the caloric quality of human garbage
providing a major food resource (Schein and Orgain 1953), methods of capture and
developing metrics to estimate the population size, and distribution of Norway rats
within urban environments. This research provided valuable methods and informa-
tion informing studies of urban rats, including critical demographic and ecological
data to inform later analyses of Norway rat populations and insights into behavioral
and ecological attributes of urban Norway rats that proved relevant for rapid gains
in our knowledge of the intraspecific dynamics of the acquisition, maintenance, and
transmission of SEOV.
7.4 SEOV and Norway Rats in Baltimore
When Jim LeDuc first contacted Keerti Shah at JHU, the timing was perfect. In the
early 1980s, James (Jamie) Childs, a JHU doctoral student, was involved in an eco-
epidemiological study of urban cats and the prevalence and potential transmission
of zoonotic parasites to humans. Childs mapped the distribution of dead cats picked
up by the Municipal Animal Shelter (MAS), which responded to reports from city
residents identifying the location of carcasses, hoping to identify neighborhoods
predicted to support high densities of cats. Sites of various levels of predicted “cat-
tiness” were visited to validate this mapping method, and it became immediately
apparent that this method reliably identified areas harboring the largest population
of free-ranging cats. However, what was not expected was finding large numbers of
Norway rats at the same locations. Although it was initially surprising to find these
two species often cast as mortal enemies of near mythological proportions living in
what appeared happy coexistence (Fig. 7), it was obvious that these neighborhoods
offered the same suite of critical resources (e.g., food and shelter mostly inadver-
tently supplied by humans) required by both urban denizens (Figs. 5, 7).
This provided a new opportunity to enhance research on cat-borne pathogens to
capitalize on these communal settings by capturing rats and obtaining tissue and
blood samples to investigate their role as intermediate hosts for Toxoplasma gondii.
This collection of rat serum and tissues soon proved to have a totally unintended but
significant use in the study of SEOV—a striking example of serendipity and
collaboration.
The first collaboration with USAMRIID was a serological analysis of the demo-
graphic characteristics of infected rats captured from several locations scattered
through Baltimore (Childs et al. 1985). This cross-sectional study indicated that
SEOV was ubiquitous among rat populations throughout the city, and, in what was
to become a common feature of all the diverse hantaviruses maintained in their
species-specific reservoir rodent hosts, that active infection was low among juvenile
rats prior to a steady increase among the oldest animals as determined by mass/age
approximations. The smallest and youngest rats had declining antibody prevalence
and titers before both markers of infection consistently increased among aging ani-
mals reaching an asymptote of ~80–90%.in the oldest age class (Fig. 8.)
A simple explanation was that acquired maternal immunity, declining steadily
among subadult rats <300 g, influenced the age at which initial acute infection
occurred and therefore the intraspecific dynamics of SEOV acquisition and mainte-
nance. Subsequent studies challenging laboratory-raised rats with SEOV indicated
that this explanation was correct. Neonatal rats inheriting maternally derived anti-
SEOV antibodies were protected from acute infection by viral challenge for up to
8–10 weeks (Zhang et al. 1988).
Additional studies of a variety of hantaviruses infecting different rodent-specific
hosts have found similar patterns where demographic disparities in age-specific and
often sex-specific infection prevalence indicate the complex dynamics of virus
acquisition and maintenance. As an example, Peromyscus maniculatus infected by
Fig. 5 Images of sites within Baltimore city supporting high densities of Norway rats. (a)
Occupied residences with accumulations of refuse-the red oblong circumscribe an extensive
mound of dirt surrounding a Norway rat burrow excavated from under the concrete backyard bor-
dering an alley. (b) Occupied residences in these blighted neighborhoods were interspersed
between abandoned row-houses. (c) One of the authors (Jamie Childs) stands outside an aban-
doned garage filled with refuse; the red circle surrounds a posted notice by the Baltimore Dept. of
Health indicating that this alley was recently treated with a rodenticide—however, rats still
abounded
Fig. 6 (a) Johns Hopkins University “rat catchers” holding rat trap used for live collections (note
the animal in the trap is a long-tailed weasel (Mustela frenata)-not a rat). (b) A large sexually
mature Norway rat with scrotal testes—the red arrows show the locations of several severe wounds
typical of the intraspecific aggression; also note the badly infected right rear paw. L➔R James
Childs, George Korch, Greg Glass (ca. 1986)
Fig. 7 Typical interactions between cats and Norway rats observed in alleys and backyards of
Baltimore study sites. (a) Two rats, one on the ground and the other atop an open garbage can, do
not appear concerned by the presence of a mother cat and her kitten. (b) and (c) Similar reactions
from a subadult and adult cat, respectively. (Figure b and c reproduced with permission from
Childs 1986)
Fig. 8 The prevalence of antibody and SEOV-RNA among Baltimore Norway rats has a U-shaped
distribution. Larger subadult and sexually mature adult rats have the highest prevalence of anti-
body and viral carriage. (Reproduced with permission from Childs et al. 2019)
Sin Nombre virus show the same U-shaped pattern in antibody acquisition as do
Norway rats, but there is also a significant male bias in infection prevalence, indicat-
ing other underlying factors are in play in determining the intraspecific dynamics of
virus transmission and maintenance (Childs et al. 1994).
Like all preliminary studies, major questions emerged from these intriguing
results. Although the IFA and PRNT tests indicated the highest reactivity to SEOV
antigens, was this Baltimore rat virus the same as SEOV? Was SEOV a “recent” Old
World introduction or had the virus been present for centuries within Norway rats,
perhaps dating to the 1770s when Norway rats were first documented within the
United States (Long 2003)? What were the temporal and seasonal dynamics of
intraspecific SEOV maintenance and transmission among rats? And, of overwhelm-
ing importance, was there a significant public health threat to residents in urban
locations where rats abound? Were domestic cases of HFRS going undetected in the
United States? If so, how do rat distribution and the likelihood of close human con-
tact vary across the urban landscape of Baltimore (and other cities), and might these
interactions define a pattern of subsequent risk of infection or disease among resi-
dents? And how do demographic and ecological properties of Norway rat popula-
tions influence the risk of virus spillover to humans?
Solutions to some of these questions appeared straightforward and could be
addressed by cross-sectional studies obtaining blood and tissue samples from rats
and initiating serological studies of humans. But where and how to study rats and
how to obtain sufficient serum samples from humans when antibody prevalence was
assumed to be low was an issue. What human population should be targeted for such
studies to improve the likelihood of detecting antibody and where could reliable
samples be obtained along with the collection of personal data, disease history, and
history of rat exposure? These turned out to be solvable problems; however, ques-
tions concerning the dynamics of acquiring and maintaining SEOV infection within
urban rat populations of Baltimore could only be addressed by far more intrusive
and controversial means—the use of capture-mark-recapture (CMR) techniques
(see section below).
7.5 Closer USAMRIID and JHU Collaborations
To conduct more detailed analyses, long-term studies were required. Jamie Childs
joined USAMRIID in 1983 as a National Research Council candidate working with
and mentored by Jim LeDuc. A draft grant to intensively study SEOV dynamics
among Baltimore’s urban rat population and to investigate the possibility of human
infection/disease was occurring had been written by Shah, Childs, and George
Korch, a PhD student at JHU. Soon this grant was funded through USAMRIID and
Childs was hired as an Assistant Professor at JHU to run the grant along with Korch
who had just received his doctorate. Within a few months, a mammologist, Gregory
Glass, who had just received a PhD from the University of Kansas, joined this team
(Fig. 6). Along with Jim LeDuc at USAMRIID, in addition to his lab’s expert
technical support from Greg Smith and Cindi Rossi, this group of individuals
worked on various aspects of SEOV epidemiology over the next 8 years.
7.6 Isolation of Baltimore Rat Virus (BRV) and Development

of a RT-PCR Identifying Persistent Carriage of SEOV
The first isolates of a SEOV-related virus from a wild Norway rat in the United
States were in Philadelphia and New Orleans. A SEOV-like virus was isolated from
Norway rats captured at a granary in Philadelphia and named Girard Point virus
(GPV) in recognition of the port site yielding the isolate (LeDuc et al. 1984), and
coincidentally, Tchoupitoulas virus (THCV) was obtained from the pancreas of a
naturally infected animal captured in New Orleans (Tsai et al. 1985). Of particular
importance to future observations on the dynamics of acquisition and maintenance
of SEOV-related viruses within Norway rat populations was the finding that this
virus was most readily isolated from tissue-antigen and antibody-positive
Norway rats.
Baltimore rat virus was also isolated from seropositive rat tissues (Childs et al.
1987b). The persistent finding that seropositive rats with high titers of anti-SEOV
antibody proved to be the best source for viral isolation suggested that this virus
might establish a chronic infection, with persistent or sporadic shedding throughout
the life span of a rat. This theory appeared to be the most parsimonious explanation,
as a similar phenomenon occurred under certain circumstances when house mice
were infected with the Arenavirus, lymphocytic choriomeningitis virus (LCMV)
(Traub 1936).
The potential for SEOV to establish a chronic infection among rats, even coinci-
dent with a significant immune response, was explored when the first RT-PCR was
developed by Ray Arthur and colleagues at JHU and USAMRIID (Arthur et al.
1992). The assay targeted a conserved region in the nucleocapsid gene carried by
the S-Segment of the virus. Isolates tested included representatives of the four well
characterized hantaviruses, HTNV, SEOV, PUUV, and PHV, as well as Bandicota
indica (Thailand 749 virus), and from an Old World insectivore Suncus murinus
(Thottopalayam virus), the common house shrew endemic to Southeastern Asia
(Carey et al. 1971). Amplicons were all 281 nucleotide pairs (np) in length, with the
exception of Thottopalayam virus, which had an amplification product of approxi-
mately 320 np. Restriction endonuclease analysis of amplicons revealed that two
distinct groups of hantaviruses were discernible by a consistent pattern, HTV-like
viruses and SEOV-like viruses, while other viruses showed variable and unique pat-
terns precluding grouping.
Of particular epidemiological importance, 12 of 13 RNA extracts from the lung
and kidney of seropositive rats were positive by RT-PCR, while none of the tissues
from eight seronegative rats were positive (Arthur et al. 1992). The largest and old-
est rats were the source of positive hits, while smaller juvenile animals were
negative. The evidence for the persistence of SEOV was clear—the oldest demo-
graphic class of rats were most commonly infected (Fig. 8). As our knowledge of
hantavirus infection and maintenance among reservoir rodent hosts evolved, the
presence of virus or viral RNA, concurrent with the presence of circulating antibod-
ies, became a consistent finding indicating chronic infection associated with shed-
ding of virus in the excreta/secreta of infected reservoir hosts. A significant example
of this commonality was demonstrated in 1993 when SNV sero-positive deer mice
(Peromyscus maniculatus) were also found to be PCR-positive/virus during the out-
break of HPS in the Southwestern United States.
7.7 Prospective Studies of the Dynamics of Intraspecific

Acquisition and Maintenance of SEOV Within Urban Rat
Populations of Baltimore
Longitudinal studies of SEOV dynamics among the urban population of Norway

rats in Baltimore required capturing rats, transporting them to a JHU laboratory,
anaesthetizing them, obtaining morphometric data for demographic studies, acquir-
ing blood, marking them with numbered ear tags for CMR studies, letting them
regain their wits, and then releasing them back into the environment at their exact
place of capture-all during the same night. This was obviously an exacting protocol
that required long nights by researchers as release of rats generally occurred after
1 AM. Local residents were glad to see rats removed, but knowledge that we were
just “borrowing” them for a few hours might have altered this opinion. Obvious
ethical concerns were apparent; we were releasing infected rats back on the streets
of Baltimore; however, the protocol was approved by IRB and Animal Use commit-
tees at JHU, USAMRIID, and from the Baltimore City Department of Health.
Imagine attempting to get such a protocol approved today!
Recapturing Norway rats is difficult as this species are notoriously neophobic
and trap-shy (Barnett 1958). The number of previous prospective studies of Norway
rats within an urban environment were few; most were reported from Baltimore and
were conducted three decades earlier. Although the social, ecological, and environ-
mental changes to the urban landscape of Baltimore were substantial, these earlier
studies conducted at JHU provided important information on the movements and
home-range of rats and data on survivorship, which were readily complemented by
our own findings. Additionally, we included “nonresidential” urban sites designated
parkland. Based on trap success (number of rats caught per night/number of traps
set; corrected for missing or triggered but empty traps), the number of rats within
residential locations far exceeded those in the parkland.
Over the course of our studies, we captured 762 small animals of which 381
(58.3%) were R. norvegicus. A total of 78, 48% of the small mammals recaptured
and rebled at intervals >1 month, were Norway rats (Childs et al. 1987a). Based on
capture-mark-release studies, 17 of the 78 recaptured rats seroconverted from
negative (reciprocal IFA titer <32) to positive, six initially positive (all at low initial
titers; five at antibody titers of 1:32, 1 at 1:64) rats sero-reverted, and the remainder
were consistently negative or positive over their entire capture histories. A high rate
of seroconversion was apparent at both urban and parkland sites, with a mean of
27.4% of the at-risk population (seronegative at first capture) becoming infected
during subsequent recapture. Over the entire study duration, the incidence of sero-
conversions varied from 7% to 46% among rats captured from residential sites,
compared to 9% for those captured in parklands (Childs et al. 1987a).
Not surprisingly, rats that seroconverted had significantly longer inter-capture
intervals (mean = 4 months) than animals, which did not seroconvert. Confirming
patterns of increasing prevalence of antibody with rat mass/age found in cross-
sectional surveys (Childs et al. 1985), rats that seroconverted or were consistently
seropositive were significantly larger (older) than rats that remained negative. Mass/
age estimates indicated that infection rates increased significantly among animals
reaching 300 g, or approximately 4–5 months of age.
Population trends of rats were similar in both residential and parkland habitats.
In general, maximal populations occurred from October to January. Survivorship
curves based on the number of recaptures/month, the rate at which rats disappeared
from the marked trappable population, indicated that 50% of rats were lost by
1 month, 90% by 3 months with a maximal longevity of 13 months (Glass 1989).
Less than 50% of rats survived long enough to become infected. Earlier cross-
sectional studies indicated that ~90% of the largest and oldest rats had antibody, but
with half of the population dying or disappearing prior to becoming infected, the
importance of overwintering infected adult rats in maintaining SEOV was evident.
7.8 Identifying Routes of SEOV Transmission Among Rats
Further analyses of capture rats revealed a significant pattern suggesting a mecha-

nism of intraspecific transmission of SEOV. Animals wounded, that is, animals with
signs of open lesions graded on a four-point scale, were significantly more likely to
seroconvert between captures (see Fig. 6b) (Glass et al. 1988). Overall, 33% of rats
seroconverting were wounded compared to 8% of unwounded animals. Based on
mass/age estimates, infection was highly associated with the onset of sexual matu-
rity and aggression at masses >200 grams.
Although aerosol transmission of SEOV to laboratory rats was demonstrated at
USAMRIID, the median infectious dose required was 2 logs greater than that pro-
duced by i.m. inoculation (Nuzum et al. 1988). Although the relevance of aerosol
transmission within natural populations of Norway rats is impossible to tease out
from other transmission routes, the sensitivity of animals to i.m. exposure, coupled
with the epidemiological data linking the incidence of wounding to seroconversion
to SEOV, suggests that inoculation of infectious saliva is probably the dominant
route. Later studies confirmed the presence of SEOV RNA in the saliva of
antibody-positive wild rats, indicating the potential for bite-transmission, although

urine and feces were also positive at a lower frequency (Hinson et al. 2004).
Of interest, the presence of wounds on Norway rats trapped in urban slums of
Salvador, Brazil, is also a significant and independent risk factor for rats acquiring
leptospiral infection (Minter et al. 2017). In this situation, mechanical transmission
through contamination of saliva through genital grooming may be the means of
transmission, as identifying leptospires in rat saliva has proved inconclusive.
7.9 SEOV Persistence and Impact on Norway Rats
A major question was how persistent SEOV infection might influence the life his-
tory of rats? Was growth rate, mortality, or fertility modified by infection status? In
another rodent-borne virus system, arenaviruses such as Machupo and LCMV can
significantly affect growth, survival, and fertility in their respective rodent reservoir
hosts (Childs and Peters 1993). By means of data collected from both cross-sectional
and prospective studies, SEOV infection had little to no effect of these parameters
within Norway rats (Childs et al. 1989).
No differences in the ultimate body size of SEOV-infected versus uninfected rats
were found. Fertility was not affected by SEOV infection. The number of embryos
carried by seropositive females was 11 (range 1–16), indistinguishable from that
found among seronegative females. Examination of external evidence of sexual
maturity among males found that scrotal testes, a measure of sexual maturity, was
found at similar frequency in uninfected versus infected rats of different size classes
(Childs et al. 1989).
Similar to the growth and fertility findings refuting the postulation that SEOV
infection has a negative effect on fertility, the survival rates of seronegative and
positive individual rats were essentially identical (Childs et al. 1989).
7.10 SEOV Pathology and Mortality Among Neonatal

Laboratory Rats and Relevance to Field Studies
While field studies suggested that SEOV infection had little impact on rat survival,
Wistar rat pups raised from seronegative dams showed varying degrees of mortality
and clinical disease when challenged by intraperitoneal inoculation (i.p.) of 104
FFU SR-11 virus, a strain of SEOV isolated from a laboratory rat. Mortality varied
from 100% with rats challenged at age <1 day, 71% at 3 days, and 18 and 16% at
ages of 5–7 days (Zhang et al. 1989). Clinical signs included ruffled fur, weight loss,
ataxia, limb paralysis, and convulsions. None of the 10- to 15-day-old rats died or
showed any significant clinical signs. Of note, challenge by a different SEOV,
KI-262 virus isolated from a wild rat and a strain nearly identical to prototype
SR-11, originally isolated by H. W. Lee, did not induce clinical disease and caused
no mortality.
The epidemiological significance of these laboratory studies to natural infection
and morbidity/mortality among wild rat populations is unclear. The identification of
extreme variation among strain-specific SEOV isolates to induce disease and death
in laboratory experiments precludes any direct comparisons or generalizations to
data collected by observations obtained from field-collected wild Norway rats. The
ages of the rats challenged in the laboratory studies ranged from <1–15 days, a
period of time in which growing pups have not ventured from their neonatal bur-
rows, which reliably occurs at 21–25 days (Calhoun 1962). Prior to leaving the natal
burrow, virtually all contact is between pups and dams, and if the female is already
seropositive and therefore also persistently infected (see sections above), the off-
spring is protected by maternal antibody, and any morbidity/mortality is likely to be
rare (Schountz and Prescott 2014). Exceptions may occur when a dam is acutely
infected during pregnancy and offspring are born prior to the dam’s seroconversion,
but these events would have a narrow window of opportunity.
7.11 SEOV and Human Infection/Disease in Baltimore
The crucial question remained, how does the great abundance of rats, the high fre-
quency of SEOV infection across all sampled rat populations in Baltimore, and
strong evidence of frequent human-rat interaction among inner-city residents trans-
late into human infection or disease (Childs et al. 1991b)? To address this issue, a
series of targeted studies were implemented by JHU and USAMRIID colleagues.
We first conducted a serosurvey of more than 2000 persons who visited a sexu-
ally transmitted disease clinic (STD) located in inner-city Baltimore. The sample
was predominantly composed of black men in their mid-20 s whom, based on ques-
tionnaire data, were from low SES neighborhoods. All of these individuals were
born in Baltimore, resided in areas known to have infected rats, and none had trav-
eled outside the United States (LeDuc et al. 1992). The results indicated a low prev-
alence of indigenous exposure to SEOV of 2.4 infections per thousand individuals
in this cohort.
We next examined sera from more than 4500 patients seen at the JHU Hospital.
Only patients with elevated proteinuria, a consistent laboratory finding in all forms
of HFRS (McKee et al. 1985), were included in this study as we had no knowledge
of the severe pulmonary disease associated with New World hantaviruses prior to
the discovery of HPS and SNV in 1993 (Khan et al. 1996). Again, most of the indi-
viduals (~75%) were Black and from low SES neighborhoods of inner-city
Baltimore, but in this sample population, Black women in their mid-40 s predomi-
nated. The SEOV antibody prevalence of 12 per thousand was fivefold higher than
that seen at the STD clinic. As a control population, sera collected from individuals
using the JHU Hospital emergency room were tested and the age-corrected
seroprevalence of SEOV antibodies was low and did not differ significantly from
participants drawn from the STD clinic (LeDuc et al. 1992).
Most of the seropositive patients did not have IgM antibodies or a rising IgG titer
based on testing sequential samples from participants indicating past rather than
acute infection. Seropositive individuals consistently reported a history of disease
involving nausea, vomiting, epigastric pain, and low-grade fever. Laboratory find-
ings from seropositive patients indicated the prevalence of renal and liver involve-
ment exceeding that found in seronegative patients, as measured by elevated BUN,
serum creatinine, AST, ALT, and total bilirubin (Glass et al. 1990; LeDuc et al.
1992). Thrombocytopenia occurred in some seropositive individuals, and pleural
effusion was also observed (a finding that took on a greater significance after the
discovery of HPS!). Hemorrhagic manifestations were rare and mild, although
blood in the sputum and melena was reported. This spectrum of illness associated
with changes in hantaviral antibody status was similar to reports of mild HFRS due
to rat-borne hantaviruses from the Far East and Europe (see section above on clini-
cal disease).
On detailed review of medical charts by a nephrologist who lacked prior knowl-
edge of the patients’ serological status, it was observed that many of the JHU
patients with antibodies to a hantavirus suffered some form of chronic renal disease.
By matching each seropositive person (N = 15) by age and sex to five seronegative
controls from the same patient population, we found that the seropositive group was
significantly more likely to suffer from a specific form of chronic renal disease,
hypertension, or a history of stroke (Glass et al. 1990; LeDuc et al. 1992). The asso-
ciation was specific for conditions that could conceivably be linked to past kidney
disease, whereas the prevalence of other chronic illnesses such as diabetes did not
differ significantly between groups. Hypertensive nephrosclerosis was the most
common diagnosis among the seropositive group (70%), significantly greater than
the 9% found among the matched seronegative controls. One patient continued to
show evidence of chronic renal insufficiency 13 months after samples were initially
tested. The possibility that prior SEOV infection/disease could produce sequelae
resulting in chronic renal disease posed a potentially significant public concern.
There was little to nothing known of whether acute hantaviral infection/disease
could be associated with later sequelae manifested by chronic kidney disease.
To further investigate the possible linkage between evidence of past SEOV infec-
tion and chronic renal disease, we then examined more than 400 patients enrolled in
a renal dialysis program in Baltimore. The population age and sex distribution were
similar to that of the JHU Hospital sample. Prevalence of antibody among this group
was 20 per 1000, significantly higher than the 12 per thousand in the JHU cohort
and 2.2 per thousand in the STD cohort (Glass et al. 1993).
These studies were the first to indicate that HFRS does occur within the United
States and produces an illness similar to that seen in other parts of the world where
disease is due to rat-borne hantaviruses. The possibility that prior SEOV infection/
disease could produce sequelae resulting in chronic renal disease posed a poten-
tially significant public concern. There was little to nothing known of whether acute
hantaviral infection/disease could be associated with later sequelae manifested by
chronic kidney disease. One study that examined Korean Conflict veterans with a
history of KHF 3–5 years after acute disease and a matched control group found a
significant increase in the rate of genitourinary hospital admissions among the
HFRS cases, which increased with the severity of their original disease (Rubini
et al. 1960). Additional findings included hyposthenuria, persistent mild albumin-
uria, and hypertension. A study of NE patients (a milder form of HFRS) 1–6.5 years
post recovery found evidence of hypertension in nearly 75% of the individuals, in
addition to depressed renal tubular function (Lahdevirta 1982; Lähdevirta et al.
1978). These indications of chronic renal dysfunction and hypertension are consis-
tent with the findings from Baltimore and a hypothesis that prior hantaviral infec-
tion is a risk factor for future renal complications.
The public health and medical communities in Baltimore and throughout the
United States had no knowledge that a zoonotic virus was being maintained at high
frequency within our own urban rat populations caused a HFRS-like disease within
cities of Asia. A US ability to mount investigative surveillance was precluded at this
time by the total lack of clinical suspicion among the medical community and the
nonexistence of laboratories capable of providing diagnostic confirmation outside
of USAMRIID facilities. CDC had not yet inherited or implemented the knowledge,
reagents and expertise in laboratory testing methodology necessary for investigating
the epidemiology of hantaviruses spurred by the arrival of former USAMRIID per-
sonnel. But the successful and quick integration of USAMRIID and CDC talents
was on national display when a mysterious new, highly fatal disease emerged in the
Southwestern United States and was rapidly identified as a novel hantavirus unique
to the New World (Nichol et al. 1993; Khan et al. 1996).
Of significant importance to our ongoing understanding of if or how hantaviral
infection/disease can result in chronic renal sequelae are results being obtained by
prospectively following a cohort of persons recovering from infection/disease
caused by the New World hantavirus SNV. The results from a study involving 30
individuals recovering from HPS/HCPS (29 caused by SNV and one caused by
Bayou virus) found over half of the subjects met the standard clinical definition for
chronic kidney disease in long-term follow-up (Pergam et al. 2009). Fifteen subjects
had proteinuria 4–96 months (four being the earliest follow-up time) after acute
disease, of which 11 were followed for 2 years or more. All subjects who developed
proteinuria remained proteinuric on follow-up testing, with the exception of one
with transient improvement. The median time between the initial illness and the
detection of proteinuria was 18 months. The authors conclude “We feel that these
data support careful screening of patients with HCPS for long-term renal
involvement.”
However, other studies have not found similar associations between chronic
renal sequelae following hantaviral infection ((e.g., from Egypt (Botros et al. 2004)
and Sweden (Settergren et al. 1998)). More longitudinal studies, such as that per-
formed by Pergam (Pergam et al. 2009), including additional survivors infected by
other hantaviruses (such as Andes virus in South America), are critical for clarifying
these associations.
8 Control of Rats and SEOV Transmission to Humans
The maintenance of SEOV requires the presence of rats and the control of transmis-
sion of this virus to humans can only be achieved by effective wild rat control.
Aspects of potential methods to limit SEOV to persons who choose to keep rats as
pets or for commercial uses are discussed in a separate section below.
Although methods for the attempted control or eradication of urban rat popula-
tions have “advanced” over the centuries from the use of domesticated predatory
animals, such as dogs, cats, and ferrets, to the era of chemical rodenticides, any
gauge of success has only briefly flickered above the overall record of failed out-
comes. Even modest successes have been of limited duration owing to the lack of
continuous support, fueled by competition for funds for local public health con-
cerns, biological factors such as the evolution of global pesticide resistance among
urban rat populations, and, most importantly, the intractability of untangling issues
concerning delivery of health-related services across heterogeneous urban land-
scapes where socioeconomic disparities always exist (Parsons et al. 2017;
UN-Habitat 2004). In impoverished and marginalized populations, often occurring
at the fringes of more developed urban centers, environmental, infrastructural, and
health delivery system deficiencies translate into the highest risk for acquiring many
communicable diseases particular to humans in addition to vector-borne and zoo-
notic pathogens such as SEOV and leptospirosis (Riley et al. 2007; Costa et al.
2015; Childs et al. 1991a, 1998).
Predators Of all the potential predators of rats domestic, cats have been champi-
oned as a natural biological means for controlling rat populations, but evidence of
their effectiveness is limited at best. As a rare example of positive effects, the pres-
ence of cats reduced the risk of re-population following rat depopulation on farms
and adjacent agricultural lands by trapping and poisoning (Elton 1953). In Baltimore,
feral cats and Norway rats share the urban environment, existing off the same
resources, mostly in peaceful coexistence (Fig. 7). However, some predation by cats
on rats occurs. Childs and colleagues noted a significant difference in the mass/age
distribution of predated rats deposited and collected near the dens of feral cats when
compared to the results obtained from trapped rats. Cats predate only on the small-
est and youngest rats, all <200 g, and these predatory events exerted no influence on
the size of urban Norway rat populations (Childs 1986; Glass et al. 2009).
Additionally, size-dependent predation of rats by urban cats had no effect on larger/
older animals (>200 g), which become infected with SEOV (or leptospires) as
young adults and maintain chronic infections throughout their life span.
However, rats can respond to the presence and number of cats within a defined
area by shifting their temporal and spatial behaviors to avoid cats. Interrupting typi-
cal behavior patterns of urban rats could shape an urban resident’s impressions of
decreasing rat abundance, generating the impression of effective control by cats
(Parsons et al. 2018).
Rodenticides In theory, control of rat populations by chemical means would

impact all demographic classes of rats, therefore reducing the prevalence of infec-
tion and the transmission of SEOV from adults to juveniles and eventually to
humans. Following WW II, studies on chemical approaches to rat control were
stimulated by the effectiveness of agents, principally DDT, for the control of vectors
of arthropod-borne diseases (Keiner 2005). Wartime efforts to synthesize new
rodenticides to control rat populations and therefore the risk of transmission of
rodent-borne pathogens choose Baltimore as the testing grounds of the powerful
new compound, alpha naphthyl thiourea (ANTU) developed by Curt Richter at JHU
(Richter 1945a). ANTU affects the lungs of rats resulting in animals drowning in
fluid accumulating in this tissue. From 1942 to 1946, experimental campaigns con-
ducted by JHU and representatives of several Baltimore city agencies found that this
approach, although cheap, invariably failed in long-term efforts to effectively reduce
populations. Additionally, ANTU was toxic to other mammals, including dogs and
cats. These results were typical of the failures encountered when new generations of
anti-coagulant-based rodenticides were used in isolation without regard to other
complementary preventative measures.
The conclusions drawn from these efforts fully endorsed the concept of mixed
pesticide and ecological approaches to control urban rats and canonized under the
term integrated pest management (IPM). The words of the Baltimore experts in the
mid-1940s were as true then as they are today.
“Experience has demonstrated that it is futile to endeavor to eliminate rats by
poisoning alone. An alteration of the rat’s environment so he will have no place to
eat or live is the first essential step in effective control measures. Essential in these
measures are the understanding and willingness of the people of the city in prevent-
ing the feeding and breeding of rats on their premises as far as this is possible”
(Keiner 2005).
Integrated Pest Management (IPM) IPM is an “ecologically” based approach to
vermin control integrating poison control, environmental modification to reduce
harborage and food sources, and necessitating the recruiting and active involvement
of urban residents in the effort to maintain modified conditions and ensure proper
refuge disposal in rat-proof containers. These interventions are costly, logistically
complicated to implement and difficult to sustain, but improve quantitative mea-
sures of population reduction. Recommendations for implementing this approach
are fully described in a 2006 document published by the CDC (CDC 2006): see
appendices B1–B8 for forms.
Improvements in Targeted Rat Control The development of molecular methods

to “fingerprint” alleles associated with geographically linked or distinct rat popula-
tions within urban environments has led to the concept of identifying rat-eradication
units (REU). By identifying gene-flow patterns—a measure of immigration/disper-
sal—to define an interconnected REU could provide a better means of effective
control by directing control efforts to areas requiring simultaneous action (Kajdacsi
et al. 2013; Richardson et al. 2017; Combs et al. 2018). In modeling the impact of
different levels of IPM on Norway rats to reduce the risk of humans contracting
leptospirosis, outcomes indicate that an initial reduction of rat populations by
rodenticides followed by environmental modifications provided the best promise for
sustained benefits (Minter et al. 2018). The implications of these models suggest
why block-by-block programs or targeting limited areas for control have failed. But
again, such approaches are expensive but require further study as they present a
rational approach to urban rodent control and subsequent reduction of the risk of
rat-borne pathogen.
Methods of Surveillance for Determining the Distribution of Urban Rats A

basic prerequisite for urban rat control requires accurate methods for surveilling
large areas to determine high-risk sites for targeting control efforts. Surveillance
methods typically include trapping at different sites, implementing rodent surveys
to detect evidence of rat infestation (e.g., burrows, feces, skid marks) (CDC 2006),
using rodent complaint data to identify areas of high rat density (Murray et al. 2018)
and video/photographic imaging of rat activity (Parsons et al. 2018). One measure
can be substituted for another after validation. As an example, using tracking boards
to monitor rat activity is a cheaper approach to trapping and can be readily scaled up
to cover large areas (Hacker et al. 2016). Integrating GIS approaches to identify
environmental features of urban neighborhoods, coupled with census data, can
leverage findings from field-collected data to develop predictive models of where
urban rat populations are greatest. Such models have been developed and validated
using data on rat bites, a condition legally mandated to be reported to health authori-
ties in NYC, coupled with ground-truthing-based rat survey methods (Childs et al.
1998). An additional study using reports of rat sightings in NYC as the outcome
found that the same suite of environmental and sociological variables identified by
modeling rat bites was significant indicators of problem areas (Walsh 2014). The
identification of similar suites of covariates predicts different quantitative indices of
rat-human interaction and holds hope that different assessment strategies can pro-
duce robust outcomes. However, as these joint efforts were only performed in NYC,
any generalizations are precluded, especially among cities in less developed tropical
locations. Further investigations of variables predicting the geographical distribu-
tion and abundance of urban rats are obviously required, but the limited or nonexis-
tent public health support of field-based studies of urban rats is a major problem to
implementing surveillance (Desvars-Larrive et al. 2018).
9 A New Twist: Pet Rats and SEOV Infection and Disease
In 2013, simultaneous reports from Sweden and the United Kingdom documented
SEOV infection and disease among owners of “fancy rats” (Lundkvist et al. 2013;
Jameson et al. 2013). In 2016–2017, the first cases of SEOV among pet rat owners
were described from the United States (Kerins et al. 2018). During follow-up inves-
tigations, household members other than the principal rat owner, workers at
breeding facilities, and those at pet stores supplying pet rats were found to be
infected by serological means. Commercial breeding of rats as a food source for
reptile owners also carries a risk of SEOV infection/disease (Swanink et al. 2018).
Serosurveys in several countries have elucidated the extent to which pet rat own-
ers were at exceptional risk for SEOV infection and disease. In the United Kingdom,
hantavirus antibody prevalence based on IFA tests was 34.1% (N = 79) among fancy
rat owners compared to 3·3% (N = 300) in a baseline control group of blood donors,
2·4% (N = 170) among persons with occupational exposure to rats destined for the
pet trade, and 1·7% (N = 295) among rodent control personnel with occupational
exposure to wild rats (Duggan et al. 2017). Overall, the prevalence of antibodies
among the general public, individuals occupationally involved in the pet rat trade
and those involved in wild rat control, was indistinguishable and on the order of ten
times lower than that found among pet rat owners. Truly intimate contact between
humans and rats appears to be the prerequisite for effective SEOV transmission and
subsequent infection/disease (Duggan et al. 2017) helping to explain why frequent,
but more casual, human-rat interactions in urban locations do not generate large
numbers of infections (Childs et al. 1991b).
In the United States, studies targeting approximately 100 facilities in 21 states,
which had been linked to persons acquiring pet rats and subsequent SEOV disease/
infection, identified 31 facilities in 11 states with confirmed human or rat infections
(Kerins et al. 2018). Seventeen of 163 (10.4%) sampled persons had evidence of
acute infection (IgM and IgG positive), and four (2.5%) were infected in the past
(IgG only). Eight of the 17 persons with evidence of acute infection reported a
recent febrile illness of which three had required hospitalization. As six of the
infected US facilities reported exchanging rats with Canada, a follow-up study
found three infected Canadians: one individual had both IgM and IgG antibodies,
and two had only IgG antibodies, but no history of recent serious illness was uncov-
ered (Kerins et al. 2018).
A serological study of rats in a breeding facility in the United Kingdom, which
supplied pets to owners who subsequently became ill with SEOV infection, found
>80% of the animals seropositive—a figure far higher than that found among most
wild rat populations (with the exception of Baltimore!) (McElhinney et al. 2017).
The major finding from studies of SEOV infection/disease among pet owners
and other persons with varying degrees of exposure to domesticated or wild rats was
that intimate contact with rats was required for SEOV transmission and infection by
SEOV was not the rare occurrence found among the general public. The number of
pet rat owners with clinical disease eclipsed the few sporadic cases of disease/infec-
tion reported within the general public interacting with wild rat reservoirs of SEOV
(for review see (Clement et al. 2019)). The public health concerns of the risk of
SEOV infection among humans from rat contact were elevated but largely restricted
to pet owners. What are the actual numbers of persons at high risk of SEOV infec-
tion and how do recommendations for limiting exposure to SEOV based on experi-
ence with wild rats translate into prevention measures for pet owners?
In the United States alone, an estimated half million persons own pet rats or mice
(http://www.petrats.org/home_.aspx accessed August 2019). SEOV is present and
variably prevalent among rats in breeding environments, which vary in size from the
cottage industry to the commercial level. Although recognition of SEOV infection
among rat owners is limited to Europe and North America, many countries such as
Japan have a long history of owning and breeding domestic rats (Serikawa 2004). It
is abundantly clear that the domesticated Norway rat has adorers around the world,
and every April 4 since 2002, worldwide rat fanciers have celebrated the virtues and
enjoyment of owning a pet Norway on “World Rat Day” (https://happydays365.org/
world-rat-day/world-rat-day-april-4/ accessed August 2019).
Information on the extent of the international trade in Norway rats is not readily
available, although some countries, such as Australia, ban the importation of rats
(http://www.daff.gov.au/aqis/cat-dogs/other accessed August 2019).
10 Reducing SEOV Infection Among Pet Rat Owners
Individuals who place themselves at risk of SEOV infection by intentionally adopt-

ing rats as pets pose a unique and challenging group for intervention. As noted by
Rubin (2017), this group has a perception that their domesticated rats pose little or
no risk. Veterinary oversight and routinely scheduled diagnostic testing for a variety
of pathogens are required for rats held in commercial breeding facilities, but such
precautions are not readily scalable to the small cottage industries intent on breed-
ing fancy rats. As no apparent disease occurs among rats infected with SEOV, pet
owners and small-scale breeders may be unaware that chronic SEOV infection can
be maintained over generations of closely housed colonies. However, visiting the
websites of fancy rat advocates, it is evident that this community has acted proac-
tively to the SEOV threat, and practical information for reducing risk of exposure to
SEOV is readily available on these sites. In addition, educational seminars are
offered as a regular component of meetings of fancy rat societies.
Basically, recommendations for owners of pet rats to limit the risk of trans-
mission of SEOV are similar to those developed for any other high-risk occupa-
tion. Owners need to be aware that exposure to soiled bedding within cages is
likely to be a significant risk factor for acquiring infection, and materials should
be soaked with a 10% bleach solution or a commercial disinfectant before cleans-
ing. Hand cleansing after handling rats or potentially contaminated material
should be a standard procedure, and persons should be especially aware that
touching their faces can contaminate mucosal membranes. The use of HEPA-
filtered masks and gloves will reduce the risk of exposure to SEOV and should
be considered when cleansing cages and removing used bedding materials
(Kerins et al. 2018). These recommendations are easy to state, but assessing how
successful they are in reaching individuals involved in the diverse activities sur-
rounding the domestic rat trade is challenging.
11 The End of an Era
After more than a decade of exciting discoveries and adventures studying the newly
discovered hantaviruses, the “dream team” that had been the foundation of the
USAMRIID program began to dissipate. In July of 1992, Joel Dalrymple passed
away. Joel’s passing was perhaps the most devastating loss to USAMRIID, an orga-
nization that was already teetering due to reduced funding and changing priorities
within DOD. Joel had been the driving force for collaborations with Ho Wang Lee,
and his work in partnership with Connie Schmaljohn and others had pioneered dis-
coveries on the characteristics of the new group of viruses. As described elsewhere,
Dalrymple and Schmaljohn had defined the hantaviruses as a new genus within the
family Bunyaviridae (recent taxonomic revisions elevated the hantaviruses to fam-
ily status: Family Hantaviridae (see Laenen et al. 2019). They explored develop-
ment of animal models to replicate human disease, and they initiated studies to
develop a protective vaccine. These efforts continue to this day, primarily under the
direction of Connie and her colleagues. Nonetheless, the loss of Joel, who was such
a charismatic and dynamic leader, was a stunning blow felt throughout USAMRIID
and beyond.
At about the same time, the CDC began a new initiative driven by the increasing
recognition of new infectious diseases appearing nearly annually around the world,
not the least of which was the discovery of HIV and AIDS. CDC unveiled a new
program focused on “Emerging Infectious Diseases” that was founded in part on the
1992 National Academies of Science report of the same name (Oaks et al. 1992).
Changes in the leadership of CDC’s Special Pathogens Branch were underway, and
as a result, Tom Ksiazek and C. J. Peters left USAMRIID to join and lead the
Branch. Ksiazek and Peters had been key leaders in the discoveries surrounding the
hantaviruses, as well as being involved in many other exciting events during the
decade of the 1980s, and their loss represented another major blow to USAMRIID’s
human resources.
Similarly, Jamie Childs joined the CDC in 1992 eventually to lead the Branch
working on rabies, rickettsial diseases, and poxviruses. Shortly, thereafter, LeDuc
joined the CDC and was deployed to the World Health Organization to initiate the
emerging infectious diseases strategy globally.
The offer of a permanent positions at CDC for former USAMRIID personnel and
Childs was an exciting and timely opportunity. Peters, Ksiazek, and LeDuc were all
military officers and were each facing mandatory retirement within a few years and
would be forced to leave USAMRIID or become civilian scientists, a very unlikely
option given the budget crisis at that time. CDC had at that time the only other
BSL-4 laboratory facility in the United States, outside USAMRIID, where work on
hantaviruses and arenaviruses, in addition to many other high hazard agents could
be performed. The USAMRIID team of experts brought laboratory expertise in han-
dling BSL-4 agents, and many assay methods and viral reagents developed in house.
The significance of the shift in the center of gravity of hantavirus expertise is hard
to overestimate, and it was soon apparent how important that expertise was.
In the spring of 1993, an outbreak of what came to be known as hantavirus pul-

monary syndrome (HPS) emerged in the southwestern United States. The experi-
ence, expertise, and critical reagents that had been developed by these experts while
at USAMRIID were quickly called to action in support of CDC’s response to this
new disease. CDC’s recently recruited hantavirus experts worked closely with exist-
ing CDC staff to rapidly respond and identify a completely new hantavirus previ-
ously unknown to science and demonstrate that it was responsible for the disease
that was making headlines across the nation. They also initiated field studies to
identify its rodent host and develop effective public health messaging to reduce the
risk of further infections. As more was learned about this new disease, it became
apparent that the HPS-like diseases were actually present in many areas of Central
and South America. Building on the network of scientific collaborators that had
developed while investigating SEOV and other viruses in the region, reagents were
provided for local outbreak investigations and ecological studies. Through these
partnerships, a completely new group of pathogenic viruses was discovered, and the
diseases they cause are identified and characterized. Obviously, many, many players
contributed to these discoveries, but the foundational studies done at USAMRIID
during the 1980s and early 1990s contributed greatly to the growth of the field and
the understanding of the important diseases that hantaviruses cause.
12 Where Are They Now?
George Korch, originally from JHU, joined the Army after receiving his PhD and
advanced to the rank of Colonel leading to his command of USAMRIID for two
terms. There is little doubt that his participation with joint JHU-USAMRIID in the
1980s made George an extremely attractive candidate for the job. George went on
to join JHU and then served as a top advisor to the Department of Homeland
Security and then the Department of Health and Human Services, often dealing with
the emergence of high-consequence pathogens and helping coordinate diverse
approaches to disease amelioration and control. Greg Glass continued to work on
rodent-borne viruses and became a full professor in the Department of Molecular
Immunology and Microbiology at JHU before moving to the University of Florida
with joint appointments in the Geography Department and Emerging Pathogens
Institute. Jamie Childs joined the Epidemiology of Microbial Disease Department
at the Yale School of Public Health, where he still has the title of Senior Research
Scientist/scholar. C.J. Peters, Tom Ksiazek, and Jim Le Duc all left CDC to join the
faculty of the University of Texas Medical Branch in Galveston, in part to be associ-
ated with the newly constructed Galveston National Laboratory, a maximum con-
tainment (BSL4) facility. C.J. Peters has since retired, while Ksiazek and Le Duc
continue as professors at UTMB. Tatjana Avsic-Zupanc is a full professor at the
medical school in Ljubljana, Slovenia, where she continues to lead investigations
into the hantaviruses and is a member of the Slovenian National Academy of
Sciences. Alemka Markotic is a physician and the director of the University Hospital
for Infectious Diseases in Zagreb, Croatia, where she too continues to lead her
research team studying the hantaviruses, while managing her demanding clinical
responsibilities. Tony Antoniadis retired from his university position in Thessaloniki,
Greece; became the President of the Institut Pasteur Hellenique in Athens; and is
now involved in clinical laboratory medicine in Greece. Shu-Yuan Xiao from
Wuhan, China, earned his US medical license and was a faculty member at UTMB,
then moved to the University of Chicago where he is a professor of pathology and
recognized leader in liver disease. Many other colleagues and friends not mentioned
here continue to make significant contributions to the expanding knowledge base of
the hantaviruses.
We were all extremely fortunate to have been part of such an exciting era at
USAMRIID, JHU, and the CDC. The strong leadership, guidance, and financial
support provided by USAMRIID and the US Army Medical Department were criti-
cal to achieving the breakthroughs in the study of hantaviruses. The military and
civilian scientists working at USAMRIID continue to provide advice and essential
product development in combating severe and highly fatal disease outbreaks such as
those caused by the infamous Ebola virus.
References
Andrews J, White HF (1936) An epidemiological study of Protozoa parasitic in wild rats in

Baltimore, with special reference to Endamoeba histolytica. Am J Hyg 24(1):184–206
Antoniadis A, Pyrpasopoulos M, Sion M, Daniel S, Peters CJ (1984) Two cases of hemorrhagic
fever with renal syndrome in northern Greece. J Infect Dis 149(6):1011–1013
Arthur RR, Lofts RS, Gomez J, Glass GE, LeDuc JW, Childs JE (1992) Grouping of hantaviruses
by small (S) genome segment polymerase chain reaction and amplification of viral RNA from
wild-caught rats. Am J Trop Med Hyg 47(2):210–224
Avsic-Zupanc T, Xiao SY, Stojanovic R, Gligic A, Van der Groen G, LeDuc JW (1992)
Characterization of Dobrava virus: a hantavirus from Slovenia, Yugoslavia. J Med Virol
38(2):132–137
Avsic-Zupanc T, Poljak M, Furlan P, Kaps R, Xiao S-Y, LeDuc JW (1994) Isolation of a strain of
a Hantaan virus from a fatal case of hemorrhagic fever with renal syndrome in Slovenia. Am J
Trop Med Hyg 51(4):393–400
Barnett SA (1958) Experiments on ‘neophobia’ in wild and laboratory rats. Br J Psychol
49(3):195–201. https://doi.org/10.1111/j.2044-8295.1958.tb00657.x
Botros BA, Sobh M, Wierzba T, Arthur RR, Mohareb EW, Frenck R, El Refaie A, Mahmoud I,
Chapman GD, Graham RR (2004) Prevalence of hantavirus antibody in patients with chronic
renal disease in Egypt. Trans R Soc Trop Med Hyg 98(6):331–336. https://doi.org/10.1016/
s0035-9203(03)00063-4
Brown ER (1976) Public health in imperialism: early Rockefeller programs at home and abroad.
Am J Public Health 66:897–203
Brummer-Korvenkontio M, Vaheri A, Hovi T, von Bonsdorff C, Vuorimies J, Manni T, Penttinen
K, Oker-Blom N, Lahdevirta J (1980) Nephropathia epidemica: detection of antigen in bank
voles and serologic diagnosis of human infection. J Infect Dis 141:131–134
Calhoun JB (1962) The ecology and sociology of the Norway rat, vol 1008. US Government
Printing Office Washington, DC
Carey DE, Reuben R, Panicker KN, Shope RE, Myers RM (1971) Thottapalayam virus: a pre-
sumptive arbovirus isolated from a shrew in India. Indian J Med Res 59:1758–1760
CDC (2006) Integrated pest management: conducting urban rodent surveys. US Department of
Health and Human Services, Atlanta
Childs JE (1986) Size-dependent predation on rats (Rattus norvegicus) by house cats (Felis catus)
in an urban setting. J Mammal 67(1):196–199. https://doi.org/10.2307/1381025
Childs JE, Peters CJ (1993) Ecology and epidemiology of arenaviruses and their hosts. In: Salvato
MS (ed) The Arenaviridae. Plenum Press, New York, pp 331–384
Childs JE, Korch GW, Smith GA, Terry AD, LeDuc JW (1985) Geographical distribution and age
related prevalence of antibody to Hantaan-like virus in rat populations of Baltimore, Maryland,
USA. Am J Trop Med Hyg 34:385–387
Childs JE, Glass GE, Korch GW, LeDuc JW (1987a) Prospective seroepidemiology of hantavi-
ruses and population dynamics of small mammal communities of Baltimore, Maryland. Am J
Childs JE, Korch GW, Glass GE, LeDuc JW, Shah KV (1987b) Epizootiology of hantavirus infec-
tions in Baltimore: isolation of a virus from Norway rats, and characteristics of infected rat
populations. Am J Epidemiol 126(1):55–68
Childs JE, Glass GE, Korch GW, LeDuc JW (1989) Effects of hantaviral infection on survival,
growth and fertility in wild rat (Rattus norvegicus) populations of Baltimore, Maryland. J Wildl
Dis 25:469–476
Childs JE, Glass GE, Ksiazek TG, Rossi CA, Barrera Oro JG, Leduc JW (1991a) Human-rodent
contact and infection with lymphocytic Choriomeningitis and Seoul viruses in an Inner-City
population. Am J Trop Med Hyg 44(2):117–121. https://doi.org/10.4269/ajtmh.1991.44.117
Childs JE, Glass GE, LeDuc JW (1991b) Rodent sightings and contacts in an inner-city popula-
tion of Baltimore, Maryland, U.S.A. Bulletin of the Society of Vector. Ecology 16(2):245–255
Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin GO, Gage KL, Rollin
PE, Sarisky J, Enscore RE (1994) Serologic and genetic identification of Peromyscus manicu-
latus as the primary rodent reservoir for a new hantavirus in the southwestern United States. J
Infect Dis 169(6):1271–1280
Childs JE, McLafferty SL, Sadek R, Miller GL, Khan AS, DuPree ER, Advani R, Glass GE
(1998) Epidemiology of rodent bites and prediction of rat infestation in New York City. Am J
Epidemiol 148(1):78–87
Childs JE, Klein SL, Glass GE (2019) A case study of two rodent-borne viruses: not always the
same old suspects. Front Ecol Evol 7:35. https://doi.org/10.3389/fevo.2019.00035
Clement J, LeDuc JW, Lloyd G, Reynes J-M, McElhinney L, Van Ranst M, Lee H-W (2019) Wild
rats, laboratory rats, pet rats: global Seoul hantavirus disease revisited. Viruses 11(7):652
Combs M, Puckett EE, Richardson J, Mims D, Munshi-South J (2018) Spatial population genom-
ics of the brown rat (Rattus norvegicus) in New York City. Mol Ecol 27(1):83–98
Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-
Ridder B, Ko AI (2015) Global morbidity and mortality of leptospirosis: a systematic review.
PLoS Negl Trop Dis 9(9):e0003898
Davis DE (1949) The weight of wild brown rats at sexual maturity. J Mammal 30(2):125–130.
https://doi.org/10.2307/1375259
Davis DE (1951) The relation between level of population and pregnancy of Norway rats. Ecology
32(3):459–461. https://doi.org/10.2307/1931721
Davis DE, Fales WT (1950) The rat population of Baltimore, 19491. Am J Epidemiol 52(2):143–146.
https://doi.org/10.1093/oxfordjournals.aje.a119414
Davis DE, Emlen JT, Stokes AW (1948) Studies on home range in the Brown rat. J Mammal
29(3):207–225. https://doi.org/10.2307/1375387
Desvars-Larrive A, Baldi M, Walter T, Zink R, Walzer C (2018) Brown rats (Rattus norvegicus) in
urban ecosystems: are the constraints related to fieldwork a limit to their study? Urban Ecosyst
21(5):951–964
Dieke SH, Richter CP (1946) Comparative assays of rodenticides on wild Norway rats: I. toxicity.
Public Health Rep (1896–1970):672–679
Downs WG (1982) The Rockefeller Foundation virus program: 1951-1971 with update to 1981.
Annu Rev Med 33(1):1–30
Duggan JM, Close R, McCann L, Wright D, Keys M, McCarthy N, Mannes T, Walsh A, Charlett A,
Brooks TJG (2017) A seroprevalence study to determine the frequency of hantavirus infection
in people exposed to wild and pet fancy rats in England. Epidemiol Infect 145(12):2458–2465.
https://doi.org/10.1017/S0950268817001480
Dyer RE, Ceder ET, Lillie RD, Rumreich A, Badger LF (1931a) The experimental transmission
of endemic typhus fever of the United States by the rat flea Xenopsylla cheopis. Public Health
Rep (1896–1970) 46(42):2481–2499. https://doi.org/10.2307/4580215
Dyer RE, Ceder ET, Rumreich A, Badger LF (1931b) Typhus fever : the rat flea, Xenopsylla
cheopis, in experimental transmission. Public Health Rep 46(32):1869–1870. https://doi.
org/10.2307/4580131
Earle DP (1954) Analysis of sequential physiologic derangements in epidemic hemorrhagic fever:
with a commentary on management. Am J Med 16(5):690–709
Elton CS (1953) The use of cats in farm rat control. Br J Anim Behav 1(4):151–155. https://doi.
org/10.1016/S0950-5601(53)80015-8
Elwell MR, Ward GS, Tingpalapong M, LeDuc J (1985) Serologic evidence of Hantaan-like virus
in rodents and man in Thailand. Southeast Asian J Trop Med Public Health 16(3):349–354
Emlen JT (1947) Baltimore’s community rat control program. Am J Public Health Nat Health
37(6):721–727. https://doi.org/10.2105/AJPH.37.6.721
Emlen JT, Stokes AW, Winsos CP (1920) The rate of recovery of decimated populations of brown
rats in nature. Ecology 29:133
Emlen JT, Stokes AW, Winsor CP (1948) The rate of recovery of decimated populations of brown
rats in nature. Ecology 29(2):133–145. https://doi.org/10.2307/1932809
Emlen JT, Stokes AW, Davis DE (1949) Methods for estimating populations of brown rats in urban
habitats. Ecology 30(4):430–442
Evans D (2009) The role of schools of public health: learning from history, looking to the future. J
Public Health 31(3):446–450. https://doi.org/10.1093/pubmed/fdp065
Gajdusek DC (1962) Virus hemorrhagic fevers. Special reference to hemorrhagic fever with renal
syndrome (epidemic hemorrhagic fever). J Pediatr 60:841–857
Glass G (1989) Comparative ecology and social interactions of Norway rat (Rattus norvegicus)
populations in, Baltimore, Maryland
Glass GE, Childs JE, Korch GW, LeDuc JW (1988) Association of intraspecific wounding with
hantaviral infection in wild rats (Rattus norvegicus). Epidemiol Infect 101:459–472
Glass GE, Childs JE, Watson AJ, LeDuc JW (1990) Association of chronic renal disease, hyperten-
sion, and infection with a rat-borne hantavirus. Arch Virol (Suppl 1):69–80
Glass GE, Watson AJ, LeDuc JW, Kelen GD, Quinn TC, Childs JE (1993) Infection with a ratborne
hantavirus in US residents is consistently associated with hypertensive renal disease. J Infect
Dis 167:614–620
Glass GE, Gardner-Santana LC, Holt RD, Chen J, Shields TM, Roy M, Schachterle S, Klein SL
(2009) Trophic garnishes: cat–rat interactions in an urban environment. PLoS One 4(6):e5794.
https://doi.org/10.1371/journal.pone.0005794
Gligić A, Obradović M, Stojanović R, Vujošević N, Ovčcarić A, Frušic M, Gibbs CJ Jr, Calisher
CH, Gajdusek DC (1989) Epidemic hemorrhagic fever with renal syndrome in Yugoslavia,
1986. Am J Trop Med Hyg 41(1):102–108
Gligic A, Dimkovic N, Xiao S-Y, Buckle GJ, Jovanovic D, Velimirovic D, Stojanovic R, Obradovic
M, Diglisic G, Micic J (1992) Belgrade virus: a new hantavirus causing severe hemorrhagic
fever with renal syndrome in Yugoslavia. J Infect Dis 166(1):113–120
Hacker KP, Minter A, Begon M, Diggle PJ, Serrano S, Reis MG, Childs JE, Ko AI, Costa F (2016)
A comparative assessment of track plates to quantify fine scale variations in the relative abun-
dance of Norway rats in urban slums. Urban Ecosyst 19(2):561–575. https://doi.org/10.1007/

s11252-015-0519-8
Hinson ER, Shone SM, Zink MC, Glass GE, Klein SL (2004) Wounding: the primary mode of
seoul virus transmission among male norway rats. Am J Trop Med Hyg 70(3):310–317. https://
doi.org/10.4269/ajtmh.2004.70.310
Huggins JW, Hsiang CM, Cosgriff TM, Guang MY, Smith JI, Wu ZO, LeDuc JW, Zheng ZM,
Meegan JM, Wang QN (1991) Prospective, double-blind, concurrent, placebo-controlled clini-
cal trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome. J Infect
Dis 164(6):1119–1127
Ido Y (1917) A report on the discovery of the causative organism (a new species of spirochet.) of
Nanukayami (7-day fever). Nippon Naika Gakkai Zasshi (J Jpn Soc Inten Med) 5:255–263
Inada R (1915) Areport on the discovery of the causative organism (a new spesies of spirochete) of
Weil’s disease. Tokyo Ijishinshi (Tokyo Med J) 1908:351–360
Iversson L, Rosa M, Lomar A, Sasaki GM, LeDuc J (1994) Human infection by hantavirus in
southern and southeastern Brazil. Rev Assoc Med Bras (1992) 40(2):85–92
James Y, Davis DE (1950) Seasonal changes in abundance of fleas on rats at Baltimore. Md Public
Health Reports (1896-1970) 65(10):337–342. https://doi.org/10.2307/4587270
Jameson LJ, Taori SK, Atkinson B, Levick P, Featherstone CA, van der Burgt G, McCarthy N, Hart
J, Osborne JC, Walsh AL, Brooks TJ, Hewson R (2013) Pet rats as a source of hantavirus in
England and Wales, 2013. Eur Secur 18(9):20415. https://doi.org/10.2807/ese.18.09.20415-en
Johnson K (2001) Hantaviruses: history and overview. In: Hantaviruses. Springer, pp 1–14
Kajdacsi B, Costa F, Hyseni C, Porter F, Brown J, Rodrigues G, Farias H, Reis MG, Childs JE, Ko
AI, Caccone A (2013) Urban population genetics of slum-dwelling rats (Rattus norvegicus) in
Salvador, Brazil. Mol Ecol 22(20):5056–5070. https://doi.org/10.1111/mec.12455
Keiner C (2005) Wartime rat control, rodent ecology, and the rise and fall of chemical rodenticides.
Endeavour 29(3):119–125. https://doi.org/10.1016/j.endeavour.2005.07.004
Kerins JL, Koske SE, Kazmierczak J, Austin C, Gowdy K, Dibernardo A, Seoul Virus Working
G, Canadian Seoul Virus Investigation G, Canadian Seoul Virus Investigation G, Contributors
(2018) Outbreak of Seoul virus among rats and rat owners - United States and Canada, 2017.
MMWR Morb Mortal Wkly Rep 67(4):131–134. https://doi.org/10.15585/mmwr.mm6704a5
Khan AS, Khabbaz RF, Armstrong LR, Holman RC, Bauer SP, Graber J, Strine T, Miller G, Reef
S, Tappero J, Rollin PE, Nichol ST, Zaki SR, Bryan RT, Chapman LE, Peters CJ, Ksiazek TG
(1996) Hantavirus pulmonary syndrome: the first 100 US cases. J Infect Dis 173(6):1297–1303.
https://doi.org/10.1093/infdis/173.6.1297
Laenen L, Vergote V, Calisher CH, Klempa B, Klingström J, Kuhn JH, Maes P (2019) Hantaviridae:
current classification and future perspectives. Viruses 11(9):788
Lähdevirta J (1971) Nephropathia epidemica in Finland. A clinical, histological and epidemiologi-
cal study. Ann Clin Res 3(Suppl. 8)
Lahdevirta J (1982) Clinical features of HFRS in Scandinavia as compared with East Asia. Scand
J Infect Dis Suppl 36:93–95
Lähdevirta J, Collan Y, Jokinen E, Hiltunen R (1978) Renal sequelae to nephropathia epidemica.
Acta Pathol Microbiol Scand A Pathol 86(1–6):265–271
LeDuc JW, Smith GA, Bagley LR, Hasty SE, Johnson KM (1982) Preliminary evidence that
Hantaan or a closely rtelated virus is enzootic in domestic rodents. N Engl J Med 307:624–624
LeDuc JW, Smith GA, Johnson KM (1984) Hantaan-like viruses from domestic rats captured in
the United States. Am J Trop Med Hyg 33(5):992–998
LeDuc JW, Smith GA, Pinheiro FP, Vasconcelos PF, Rosa ES, Maiztegui JI (1985) Isolation of a
Hantaan-related virus from Brazilian rats and serologic evidence of its widespread distribution
in South America. Am J Trop Med Hyg 34:810–815
LeDuc J, Antoniades A, Siamopoulos K (1986a) Epidemiological investigations following an out-
break of hemorrhagic fever with renal syndrome in Greece. Am J Trop Med Hyg 35(3):654–659
LeDuc JW, Smith G, Childs JE, Pinheiro FP, Maiztegui J, Niklasson B, Antoniades A, Robinson
D, Khin M, Shortridge K (1986b) Global survey of antibody to Hantaan-related viruses among
peridomestic rodents. Bull World Health Organ 64(1):139
LeDuc JW, Ksiazek TG, Rossi CA, Dalrymple JM (1990) A retrospective analysis of sera col-
lected by the Hemorrhagic Fever Commission during the Korean Conflict. J Infect Dis
162(5):1182–1184
LeDuc JW, Childs JE, Glass GE (1992) The hantaviruses, etiologic agents of hemorrhagic fever
with renal syndrome: a possible cause of hypertension and chronic renal disease in the United
States. Annu Rev Public Health 13:79–98
Lee HW (2000) Correspondence on the discovery and original investigations on hemorrhagic fever
with renal syndrome and hantaviruses. In Lee HW (ed) The Hantaan Life Science Foundation,
1–136 Tongsoong-dong Chongno-ku, Seoul, Vols 1 and 2, pp 1–2811
Lee HW, Lee PW, Johnson KM (1978) Isolation of the etiologic agent of Korean hemorrhagic
fever. J Infect Dis 137(3):298–308
Lee HW, Lee PW, Baek KJ, Song CK, Seong IW (1981) Intraspecific transmission of Hantaan
virus, etiologic agent of Korean hemorrhagic fever, in the rodent Apodemus agrarius. Am J
Trop Med Hyg 30(5):1106–1112
Lee H, Baek L, Johnson K (1982) Isolation of Hantaan virus, the etiologic agent of Korean hemor-
rhagic fever, from wild urban rats. J Infect Dis 146:638–644
Long JL (2003) Introduced mammals of the world. CSIRO Publishing Melbourne, Australia and
CABI Publishing, Wallingford, 589 pgs
Lundkvist Å, Verner-Carlsson J, Plyusnina A, Forslund L, Feinstein R, Plyusnin A (2013) Pet rat
harbouring Seoul hantavirus in Sweden, June 2013. Eur Secur 18(27):20521
Luttermoser GW (1936) A helminthological survey of Baltimore house rats (Rattus norvegicus).
Am J Hyg 24(2):350–360
Maes P, Klempa B, Clement J, Matthijnssens J, Gajdusek DC, Krüger DH, Van Ranst M (2009)
A proposal for new criteria for the classification of hantaviruses, based on S and M segment
protein sequences. Infect Genet Evol 9(5):813–820
McElhinney LM, Marston DA, Pounder KC, Goharriz H, Wise EL, Verner-Carlsson J, Jennings
D, Johnson N, Civello A, Nunez A, Brooks T, Breed AC, Lawes J, Lundkvist Å, Featherstone
CA, Fooks AR (2017) High prevalence of Seoul hantavirus in a breeding colony of pet rats.
Epidemiol Infect 145(15):3115–3124. https://doi.org/10.1017/S0950268817001819
McKee KT Jr, MacDonald C, LeDuc JW, Peters CJ (1985) Hemorrhagic fever with renal
syndrome—a clinical perspective. Mil Med 150(12):640–647. https://doi.org/10.1093/
milmed/150.12.640
Minter A, Diggle PJ, Costa F, Childs J, Ko AI, Begon M (2017) Evidence of multiple intraspecific
transmission routes for Leptospira acquisition in Norway rats (Rattus norvegicus). Epidemiol
Infect 145(16):3438–3448. https://doi.org/10.1017/S0950268817002539
Minter A, Diggle PJ, Costa F, Childs J, Ko AI, Begon M (2018) A model for leptospire dynamics
and control in the Norway rat (Rattus norvegicus) the reservoir host in urban slum environ-
ments. Epidemics 25:26–34. https://doi.org/10.1016/j.epidem.2018.05.002
Murray MH, Fyffe R, Fidino M, Byers KA, Ríos MJ, Mulligan MP, Magle SB (2018) Public
complaints reflect rat relative abundance across diverse urban neighborhoods. Front Ecol Evol
6(189). https://doi.org/10.3389/fevo.2018.00189
Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann H, Sanchez A, Childs
J, Zaki S, Peters CJ (1993) Genetic identification of a hantavirus associated with an outbreak
of acute respiratory illness. Science 262(5135):914–917
Niklasson B, Le Duc J (1984) Isolation of the Nephropathia Epidemica agent in Sweden. Lancet
323(8384):1012–1013. https://doi.org/10.1016/S0140-6736(84)92344-4
Niklasson B, Le Duc J (1987) Epidemiology of Nephropathia Epidemica in Sweden. J Infect Dis
155(2):269–176
Nuzum EO, Rossi CA, Stephenson EH, LeDuc JW (1988) Aerosol transmission of Hantaan and
related viruses to laboratory rats. Am J Trop Med Hyg 38(3):636–640. https://doi.org/10.4269/
ajtmh.1988.38.636
Oaks SC Jr, Shope RE, Lederberg J (1992) Emerging infections: microbial threats to health in the
United States. National Academies Press
Parsons MH, Banks PB, Deutsch MA, Corrigan RF, Munshi-South J (2017) Trends in urban rat
ecology: a framework to define the prevailing knowledge gaps and incentives for academia,
pest management professionals (PMPs) and public health agencies to participate. j Urban Ecol
3(1):jux005
Parsons MH, Banks PB, Deutsch MA, Munshi-South J (2018) Temporal and space-use changes
by rats in response to predation by feral cats in an urban ecosystem. Front Ecol Evol 6(146).
https://doi.org/10.3389/fevo.2018.00146
Pergam SA, Schmidt DW, Nofchissey RA, Hunt WC, Harford AH, Goade DE (2009) Potential
renal sequelae in survivors of Hantavirus Cardiopulmonary Syndrome. Am J Trop Med Hyg
80(2):279–285. https://doi.org/10.4269/ajtmh.2009.80.279
Pyung-Woo L, Amyx HL, Yanagihara R, Gajdusek DC, Goldgaber D, Gibbs CJ (1985) Partial
characterization of Prospect Hill Virus Isolated from meadow voles in the United States. J
Infect Dis 152(4):826–829
Richardson JL, Burak MK, Hernandez C, Shirvell JM, Mariani C, Carvalho-Pereira TSA, Pertile
AC, Panti-May JA, Pedra GG, Serrano S, Taylor J, Carvalho M, Rodrigues G, Costa F, Childs
JE, Ko AI, Caccone A (2017) Using fine-scale spatial genetics of Norway rats to improve con-
trol efforts and reduce leptospirosis risk in urban slum environments. Evol Appl 10(4):323–337.
https://doi.org/10.1111/eva.12449
Richter CP (1945a) The development and use of Alpha-Naphthyl Thiourea (ANTU) as a rat poi-
son. JAMA 129(14):927–931. https://doi.org/10.1001/jama.1945.02860480007002
Richter CP (1945b) Iincidence of rat bites and rat bite fever in Batltimore. JAMA 128(5):324–326.
https://doi.org/10.1001/jama.1945.02860220006002
Richter CP, Emlen JT (1946) Instructions for using ANTU as a poison for the common Norway rat.
Public Health Rep (1896-1970) 61(17):602–607. https://doi.org/10.2307/4585643
Riley LW, Ko AI, Unger A, Reis MG (2007) Slum health: diseases of neglected populations. BMC
Int Health Hum Rights 7(1):2. https://doi.org/10.1186/1472-698X-7-2
Ristanović E, Gligić A, Atanasievska S, Protić-Djokić V, Jovanović D, Radunović M (2017)
Smallpox as actual biothreat: lessons learned from its outbreak in ex-Yugoslavia in 1972.
Annali dell’Istituto superiore di sanita 52(4):587–597
Rubin R (2017) Pet rats infect daughter, mother with Hantavirus. JAMA. 318:1969. https://doi.
org/10.1001/jama.2017.17645
Rubini ME, Jablons S, McDowell ME (1960) Renal residuals of acute epidemic hemorrhagic fever.
JAMA Intern Med 106(3):378–387. https://doi.org/10.1001/archinte.1960.03820030066011
Schein MW, Orgain H (1953) A preliminary analysis of garbage as food for the Norway rat. Am J
Trop Med Hyg 2(6):1117–1130
Schountz T, Prescott J (2014) Hantavirus immunology of rodent reservoirs: current status and
future directions. Viruses 6(3):1317–1335
Serikawa T (2004) Colourful history of Japan’s rat resources. Nature 429(6987):15–15. https://doi.
org/10.1038/429015b
Settergren B, Stegmayr BG, Elgh F (1998) Lack of serologic evidence for an association between
Hantavirus infection and end-stage renal disease. Nephron 80(1):93
Sheedy JA, Froeb HF, Batson HA, Conley CC, Murphy JP, Hunter RB, Cugell DW, Giles RB,
Bershadsky SC, Vester JW (1954) The clinical course of epidemic hemorrhagic fever. Am J
Med 16(5):619–628
Simic M (1952) Successful application of peritoneal dialysis in a case of renal insufficiency.
Vojnosanit Pregl 9(9–10):285
Smadel JE (1953) Epidemic hemorrhagic fever. Am J Public Health Nations Health
43(10):1327–1330
Smorodintsev AA, Kazbintsev LI, Chudakov VG (1963) Virus Hemorrhagic Fevers (in Russian,
translated by IPST Cat. No. 1210). Gosudarstvennoe Izdatel’stvo Meditsinskoi Literatury,
Leningrad
Swanink C, Reimerink J, Gisolf J, de Vries A, Claassen M, Martens L, Waegemaekers T, Rozendaal
H, Valkenburgh S, Hoornweg T, Maas M (2018) Autochthonous human case of Seoul Virus
infection, the Netherlands. Emerg Infect Dis 24(12):2158–2163. https://doi.org/10.3201/
eid2412.180229
Traub E (1936) The epidemiology of Lymphocytic Choriomeningitis in white mice. JExpMed
64(2):183–200
Tsai TF, Bauer SP, Sasso DR, McCormick JB, Bradford H, Caraway CT, McFarland LM, Medrano
O, Soulie G (1982) Preliminary evidence that Hantaan or a closely related virus is enzootic in
domestic rodents. N Engl J Med 307:623–624
Tsai TF, Bauer SP, Sasso DR, Whitfield SG, McCormick JB, Caraway TC, McFarland L, Bradford
H, Kurata T (1985) Serological and virological evidence of a Hantaan Virus-related enzootic in
the United States. J Infect Dis 152(1):126–136. https://doi.org/10.1093/infdis/152.1.126
UN-Habitat (2004) The challenge of slums: global report on human settlements 2003. Manag
Environ Qual Int J 15(3):337–338. https://doi.org/10.1108/meq.2004.15.3.337.3
Walch E, Walch-Sorgdrager G (1927) Observations on Leptospira icterohaetnorrhagiae in the
wild rats of Baltimore. Am J Hyg 7(4):393–406
Walsh MG (2014) Rat sightings in New York City are associated with neighborhood sociodemo-
graphics, housing characteristics, and proximity to open public space. PeerJ 2:e533
Won C, Smith KG (1999) History and current status of mammals of the Korean Peninsula.
Mammal Rev 29(1):3–36
Yanagihara R, Svedmyr A, Amyx HL, Lee P, Goldgaber D, Gajdusek DC, Gibbs CJ, Ström K
(1984) Isolation and propagation of nephropathia epidemica virus in bank voles. Scand J Infect
Dis 16(3):225–228
Youngman PM (1956) A population of the striped field mouse, Apodemus agrarius coreae. Central
Korea J Mammal 37(1):1–10. https://doi.org/10.2307/1375520
Zhang XK, Takashima I, Hashimoto N (1988) Role of maternal antibody in protection from hem-
orrhagic fever with renal syndrome virus infection in rats. ArchVirol 103:253–265
Zhang XK, Takashima I, Mori F, Hashimoto N (1989) Comparison of virulence between two
strains of Rattus serotype hemorrhagic fever with renal syndrome (HFRS) virus in newborn
rats. Microbiol Immunol 33(3):195–205
Yellow Fever and Other Viruses in West
Africa
Thomas P. Monath
Abstract This chapter chronicles experiences in field investigations of outbreaks

of yellow fever, Lassa fever and other arboviruses over a number of years during my
career at the Centers for Disease Control (CDC) and acknowledges many of the
remarkable people who contributed to these activities. The catalyst and impetus for
my career pathway has always been my curiosity and fascination with the complex
underlying ecology and pathogenesis of yellow fever and other arboviruses.
I sometimes reflect on how fortunate I was in finding a path forward in my profes-

sional life. The dye was cast early on for a career in tropical medicine. Interested in
natural history since early childhood, I had organized annual expeditions to the
tropics to collect zoological specimens for the Museum of Comparative Zoology
between 1961 and 1964, supported in part by the National Geographic Society. The
last of these expeditions in 1964 focused on the Omo River basin of Ethiopia, where
(unbeknownst to me at the time) only 2 years before one of the largest epidemics of
yellow fever had occurred. At Harvard Medical School (HMS), I attached myself to
the Department of Tropical Medicine and soon fell under the wing of Professor and
Nobel Laureate in virology, Thomas Weller, as well as Tom Frothingham (who was
interested in arboviruses) and Frank Neva. Andrew Spielman, later to become a
world-renown medical entomologist, had just joined the Department. In my third
year at HMS, Weller arranged for me, accompanied by my wife, Jennifer, to spend
a semester in the Gambia, at the Medical Research Council laboratories under the
direction of Ian MacGregor, one of the leading figures in malaria research. This
experience was formative, as I fell in love with the country and the culture, and the
work, much of it in the field, was an extension of my adventures on my zoological
trips. All other possible career directions were excluded when I attended a lecture
and film in my last year at HMS by Telford Work, then the director of the Virology
Section at the Communicable Disease Center (CDC), describing the discovery of
Kyasanur Forest Disease in 1957 and its investigation by the Rockefeller Foundation
T. P. Monath (*)
Crozet BioPharma LLC, Lexington, MA, USA
e-mail: Tom.monath@crozetbiopharma.com
360 T. P. Monath
Laboratory in Poona. It was hard to believe that there existed an occupation so well
suited to my interests in natural history, research, medicine, and the tropical world. This
career path remained a clear goal through the next years of clinical training, and when I
was finally drafted in 1968 during the Vietnam war, I was able to secure a commission
in the US Public Health Service (USPHS) and an assignment to the Arbovirology Unit
at the Center for Disease Control (CDC), as it was then named, reporting for duty on
June 1. It was hard to imagine a more perfect alignment of the stars.
Arbovirology at CDC in 1968 was the leading discipline in the Virology Section,
largely due to the cast of characters there and the legacy of Tel Work. Tel’s office
was as he’d left it, the walls papered with a huge small-scale map of the world, with
colored pins showing locations of newly discovered viruses and ongoing field stud-
ies but occupied now by Bob Kissling (Calisher et al. 2014) who had assumed the
role of Director. Roy Chamberlain was his deputy. Those reading this chapter will
undoubtedly know of the pioneering work by Kissling and Chamberlain on eastern
equine encephalitis, St. Louis encephalitis (SLE), and other arboviruses at the Virus
and Rickettsia Section of CDC, previously located in Montgomery AL
(Chamberlain 1980).
The Marburg virus incident had occurred in Germany and Yugoslavia in 1967,
and the first outbreak of Lassa virus was soon reported in Nigeria in 1969. Kissling
played an important role in these investigations, establishing the first BSL-4 labora-
tory at CDC. Other workers in arbovirology at CDC at the time included Fred
Murphy, Dan Sudia, Charlie Calisher, Rex Lord, and Brian Henderson. It was an
exciting time, and I had a steep learning curve in arbovirus taxonomy and epidemi-
ology, vector biology, laboratory and field techniques as well as taking the Epidemic
Intelligence Service course. I was determined to be proficient in all laboratory meth-
ods for virus isolation, characterization, and serological testing, as well as field work.
The 1968 National Convention of the Republican Party was held at the Miami
Beach Convention Center in Miami Beach FL, on August 5–8. CDC was tasked to
determine if there was a risk to the delegates from SLE virus, which had caused
major outbreaks in Florida in 1962. This was my first introduction to mist-netting
birds and trapping mosquitoes, and it was a bonding experience with legendary
CDC field research staff, Vern Newhouse, Rex Lord, and Gib Johnston. The next
2 years were filled with other remarkable opportunities. I spent time at the Yale
Arbovirus Research Unit, where Jordi Casals and Bob Shope helped me work up
some unidentified viruses isolated by Brian Henderson in East Africa (Monath et al.
1972a). Here, I was in the central node of the famed Rockefeller Virus Program.
Max Theiler, who had won the Nobel prize for the discovery of yellow fever 17D
vaccine, was active and in the lab. Wilbur Downs was Director of YARU. I was awed
in the presence of these accomplished arbovirologists but eager to learn.
Back in Atlanta, opportunity after opportunity presented itself. I was sent off to
Harrisburg, Illinois, to investigate an outbreak of SLE and to Winona, Minnesota, to
investigate an unusual cluster of LaCrosse virus infections (Monath et al. 1970). My
interest in SLE was fostered by these experiences, and it became a focus of experi-
mental and epidemiological studies over the next decade and the subject of my first
book (Monath 1980). And in 1969, I spent 2 months in Guayaquil investigating one
of the most devastating epidemics of Venezuelan equine encephalitis, in which the
Yellow Fever and Other Viruses in West Africa 361
infectious disease hospital was filled with severely affected patients, many requiring
iron lung support (Gutierrez et al. 1975). Years later, when I was Director at CDC,
Ft. Collins, we started a multiyear epidemiological program in Ecuador with the
goal of uncovering the maintenance cycle of epizootic VEE subtypes.
Yellow fever was the topic of much discussion at the time. In 1969, a widespread
epidemic occurred in Nigeria, Togo, Ghana, Mali, and Upper Volta (Burkina Faso),
with an estimated 100,000 cases. Don Carey, Director of the Virus Research
Laboratory in Ibadan (the last of the Rockefeller Virus Program laboratories), and
his team investigated the outbreak on the Jos Plateau (Carey et al. 1972). Brian
Henderson, who had previously served as a medical officer under Tel Work, had just
returned from a 3-year posting to the East African Virus Research Institute in
Entebbe, where he had worked on yellow fever, including surveys to determine how
far south into Kenya the 1962 Ethiopian outbreak had extended. He had also under-
taken experimental infection studies in nonhuman primates on the interaction of
yellow fever and other flaviviruses. Henderson was Chief of the Arbovirology Unit
from 1969 to 1970, and his stories whet my appetite for arbovirus investigations in
Africa. I started promoting the idea of following in his footsteps to secure a posting
in West Africa. This seemed sensible to Kissling, Chamberlain, and Henderson and
also to Rockefeller colleagues at YARU who passed on the idea to Don Carey in
Ibadan. I met with the CDC Director, David Sencer, who was highly supportive, and
told me that CDC always needed to have one expert in yellow fever since it was one
of the world’s three quarantinable diseases and since CDC had a mission to prevent
introduction into the United States. He intimated that I could fill that slot. Within a
few months, Don Carey was invited to Atlanta to discuss the assignment, and in
1969, I spent a few weeks in Ibadan preparing plans for a research program. On the
way over, I stopped in Dakar to visit Paul Brès, Director of the Institut Pasteur labo-
ratory, which was then and still is a hub for research on yellow fever and other
arboviruses. Brès, who was a leading figure in yellow fever epidemiology and pre-
vention and subsequently Chief of the Viral Disease Branch at WHO, became a
lifelong friend, and we corresponded frequently. He later tried to recruit me to
WHO, but I wasn’t ready for an administrative job.
In mid-1970, I arrived in Ibadan, together with my wife, daughter Andrea (then
about 10 months old), our Bassett hound, and two cats. Though assigned to the
Rockefeller laboratory, I was supported administratively by the CDC-led smallpox
eradication program, which was in full swing at the time and supplied me with,
among other things critical to my plans, a four-wheel drive extended-cab Power
Wagon. The Rockefeller Virus Research Laboratory was located in several build-
ings on the grounds of the University College Hospital. The laboratories were
orderly and well-lit, with wooden benches and cabinets constructed by local carpen-
ters, and equipped with the basics, water baths, centrifuges, Revco freezers, and the
like. All work was on the open bench. There were no mechanical pipettors, and
serological pipettes were manipulated using rubber pipetting fillers with valves.
Hemagglutination inhibition, complement fixation, neutralization tests, and virus
isolation were the main research tools. Neutralization tests and virus isolation
attempts were performed exclusively in suckling mice. The animal facility consisted
362 T. P. Monath
of two large rooms, one for the mouse colony, and another for housing suckling
mouse litters following inoculation. Window air conditioners provided some tem-
perature control, but airflows were haphazard. There were three Rockefeller staff
scientists at the time: Don Carey (Director), Graham Kemp (veterinarian, virolo-
gist), and Vernon Lee (entomologist) (Fig. 1). Don had replaced Ottis Causey who
(with his wife Calista) had just left Ibadan, their final posting with the Rockefeller
Virus Program. Don was wiry and fit, a gentle, and somewhat ascetic pediatrician-
virologist, whose previous assignment was in the Rockefeller Virus Research Center
in Poona, where his work centered on dengue and chikungunya in south India. In
1969, Carey and colleagues had isolated dengue types 1 and 2 for the first time in
Africa (Moore et al. 1975).
Don was a great mentor; he was a man of ideas but was always ready for action.
A story in circulation at the time was Don’s attempt to get a viscerotomy sample
from a man who had succumbed to yellow fever during the 1969 epidemic. When
other approaches failed, Don followed the funeral procession and had jumped into
the open grave to successfully obtain the sample. Of course, these were the days of
cowboy virology, when there were few of the present-day constraints on research
methods and behaviors.
Fig. 1 Staff of the Rockefeller Foundation Virus Research Laboratory, University of Ibadan, 1971.
Front row, 5th position from left is Graham Kemp. Third row, first position on left partially hidden
is Don Carey. Fifth position, partially hidden is David Wilson, Yale undergraduate who partici-
pated in the Okwoga investigations. Eleventh position is the author, followed by Vern Lee,
Rockefeller entomologist, Edward O’Connor, senior technician, Dr. Tam David-West, unknown,
and Dorothy Moore, a staff scientist at the laboratory
In the laboratory on a typical day, Don retired to his office at noontime, where he
played the flute and then, like Churchill, napped for an hour allowing him to work
late into the evening. In 1969, when I stayed with Don in his lovely house on the
Ibadan University campus, I observed his work habits. We retired for coffee and talk
after dinner, after which he excused himself and went upstairs to read journals until
well after midnight. A source of amusement after dinner was the house-pet vervet
monkey that invariably defecated on the oriental rug and then immediately leaped
out the window. Don explained that he had spent months trying to train it by physi-
cally throwing it out the window when it had made a mess, but instead of stopping
the latter habit, it had learned to mimic its master’s response to it.
My immediate research goal was to determine, through serological surveys, the
geographic extent of the 1969 epidemic, particularly in southeastern Nigeria (for-
mer Biafra) where it was suspected the outbreak had arisen, but where there was
little information because of the war of secession, which had just ended. This objec-
tive was temporarily put aside, when the lab received a visit from a young man who
had recently visited relatives in a remote village in Okwoga, Benue State, about
100 km north of Enugu the former capital of Biafra, well south of the areas of north-
ern Nigeria affected in 1969 and 64 km by poor roads to the nearest physician and
hospital. The relative said there was substantial illness and many deaths and that
those affected had jaundice. Accompanied by David Wilson, a Yale undergraduate
student working at the Ibadan lab, I reached the area by road just before Christmas,
1970. The population of Okwoga District was 19,772 according to the last census,
scattered across 12 villages comprised of thatched huts. The population was engaged
in subsistence farming. As always in these situations, the first priority was a meeting
with the village chiefs. They were friendly, openly grateful that someone had come
to check on them and confirmed that there were many cases that fit a uniform pattern
of symptoms consistent with yellow fever. We explained the general plan, whereby
we would map and perform a census of the affected area, search for ill persons,
attempt to determine the time frame of the outbreak and the incidence of disease,
take blood samples, and advise the Ministry regarding vaccination. The chiefs
promised to spread the word and to ensure the population would cooperate with our
activities. It is important to note that it was only 8 years since the end of British
colonial rule, and there was a general respect for doctors, missionaries, and admin-
istrators. No individual consent process or consent forms would be used; this was
consistent with CDC’s practice at the time for epidemiological investigations of
disease outbreaks where only diagnostic blood sampling was performed.
Our sleeping quarters were provided by the chief of Okwoga village, a simple
hut with a dirt floor and thatched roof. David and I set up folding cots, sleeping
bags, and mosquito netting, which ensured relative comfort, and brought in our sup-
plies of vacutainer tubes, cryovials, 50 L liquid nitrogen tank, and other items
required for the work. We estimated we would be there for 2–3 weeks, at the least.
The Okwoka village chief assigned a schoolboy, aged about 14, as an interpreter
and general majordomo. We had a stove, lanterns (the area was not electrified), rice,
and tinned meat, to be supplemented by locally sourced bananas, oranges, and
onions. Water brought from a village well was boiled. Beer was sporadically
364 T. P. Monath
available. This problem was mitigated by two-fifth of Johnnie Walker Red, which,
other than the nitrogen tank and DEET, was our most precious possession. Many
people don’t know the most important attribute of DEET, which is that it repels
houseflies much more efficiently than mosquitoes. Its application to the face allows
one to go about one’s business fly-free, whereas the local population has swarms
around eyes, nose, and mouth to which they are mostly inured. This was noted by
the people who sometimes commented that, astonishingly, “Oyimbo [white man] no
have flies.”
Our liquid nitrogen tank was sufficient for a month-long stay, including enhanced
losses due to sloshing around in the truck. Our practice was to simply drop cryovials
containing serum into the tank, to be retrieved on return to Ibadan by emptying the
tank contents into a sink with dry ice and organizing the hoard of samples. This
worked well, although some leakage of serum from cryovials with external thread
caps was observed, forming floating blobs of frozen serum. Similarly, the method of
separating serum was adapted to the situation with no available centrifuge. Blood in
vacutainer tubes was allowed to clot and retract, at which point the rubber cap was
popped, and the serum decanted by simply pouring into cryovials with hand-written
on tape produced on a board with slots allowing the tape to be divided into labels
with a scalpel. Pasteur pipettes were rarely employed, as they just created more
biowaste, and could not be used when serum separation was performed in the back-
seat of the Power Wagon bumping along roads mutilated by the rainy season.
Biowaste was accumulated in hard containers for the needles and plastic bags for
the rest, burned in a pit near our hut, then buried.
On Christmas eve, a traveling priest came to Okwoga to perform mass in the
local church. David and I attended the service, conducted in the local language with
an oddment of Latin. All seemed to go well, and we concluded that the locals faith-
fully adhered to a Catholic doctrine. However, there were significant bumps in the
night that followed and a general stir in the village that interrupted our repose. The
next morning, David and I explored the deserted village. The citizenry was sleeping
off an orgy of sacrificial offerings to the pagan gods. Chicken heads and feet, feath-
ers stuck in blood, goat body parts, and betel nut offerings were arrayed in front of
most huts. The Catholic service had been followed by a pantheistic ritual to the
original gods. No chances were being taken.
Fever clinics were held on successive days in four villages, followed by house-
to-house surveys to gather histories of illness and determine the extent of the out-
break. Two simple case definitions were used, febrile illness with jaundice or
without jaundice occurring in the past 2 months. Two yellow fever virus isolations
were subsequently made from individuals with mild undifferentiated febrile illness.
Twenty-two cases were confirmed by the rise or fall of CF antibodies in paired sera;
another 91 individuals were presumptive infections based on CF antibody titers
≥16 in single sera. Among 243 persons, 48 (19.8%) had reported fever with jaun-
dice, 69 (28.4%) undifferentiated febrile illness, and 125 (51.4%) no illness during
the preceding 2 months (Monath et al. 1973a). The serological data supported the
predictive value of history of illness, since 71% and 61% of those with fever and
jaundice or fever without jaundice, respectively, were CF-positive versus 12% of
those with no history of illness, a finding that was highly statistically significant.
These data also provided a clue to the ratio of inapparent:apparent infections, which
was 3:1 if fever with jaundice is considered. As such data were not previously avail-
able for yellow fever, confirmation of the ratio would be a focus of future outbreak
investigations. Based on the survey, the attack rate for fever with jaundice was 14%,
and the overall incidence of yellow fever infection in the population was 37%.
Entomological investigations proved that this was a purely sylvatic outbreak
(Monath et al. 1973a). Interestingly, very similar epidemiological parameters were
found across a series of yellow fever outbreaks that I was to investigate over the
coming years, but one central feature of the Okwoga epidemic had an overarching
impact— it was a “silent” epidemic that came to light only by rumor and chance and
would have otherwise been missed. How often was this kind of event occurring in
Africa? What was the true underlying burden of endemic and epidemic disease?
Was yellow fever enzootic in Nigeria and where did transmission occur in between
outbreaks?
Okwoga sparked a series of activities that were aimed at these questions. I had to
single-handedly work up all specimens on return to the lab. However, when a
focused multidisciplinary team was required, I was fortunate to engage others at the
Ibadan laboratory, in particular Rockefeller scientists Vernon Lee (entomology) and
Graham Kemp (veterinarian-virologist). A noteworthy event in 1971 was the arrival
at the lab of two young veterinary graduates, Oyewale Tomori and Ademola
Fagbami, who quickly became friends and colleagues. Both made a name for them-
selves in arbovirology, and in particular, Tomori became a yellow fever and hemor-
rhagic fever expert and gained international prominence. Tomori and Fagbami
assisted me on several yellow fever field investigations in Nigeria in 1971–1972,
and Wale and I would collaborate on yellow fever outbreak investigations in the
1980s. Today, we are still both engaged in WHO working groups on the subject.
Discovery of the “silent” Okwoga outbreak indicated the importance of defining
the extent of yellow fever activity across a much larger area in southeastern Nigeria,
which had not been affected by the 1969 epidemic. Serological surveys were
extended to multiple locations extending outward from Okwoga in Benue State and
southwards in eastern Nigeria as far as Cross River State (Monath and Wilson 1973).
The methodology for the fast-paced sero-geographic survey was simple conve-
nience sampling, although an effort was made to include children as well as adults
and to obtain detailed identifying data to enable us to find the same persons on a
return visit in a longitudinal study, as well as a history of yellow fever-like illness
and yellow fever vaccination. This was complicated by the fact that adults rarely
knew their birthday (not a celebrated event), and almost never had written records
of immunizations. Moreover, in contrast to a situation with a near-term outbreak,
illness history was unreliable, and jaundice due to chronic forms of hepatitis and
hemolytic anemias was quite prevalent.
On a typical day, we took blood samples and histories from hundreds of people
lined up in a queue in the tropical sun, often surrounded by curious children, flies
everywhere, and, importantly, often faced with requests for medical attention. We
had few means of dealing with the latter but did our best as it was an implicit quid
366 T. P. Monath
pro quo of complying with our survey. The Rockefeller lab approach (probably
stemming from its emphasis on blood sampling in fever clinics) was to dispense
aspirins, which were provided in large tins as pink, green, blue, yellow, and white
tablets so that one would think different medications were being provided on an
individual basis. This generally was a satisfactory solution to the expectation for
medical attention, usually for “pains all over the body,” but we would also dispense
antimalarials and antibiotics if strongly indicated and supplies were adequate. On
one occasion, an elderly village chief requested a private meeting to discuss his
impotence (apparently the result of adding a young wife and enraging an old one).
This was approached with a regimen of blue pill in morning and pink pill at night.
The outcome was not ascertained as we left on that evening, but we hoped for a
strong placebo effect.
Things did not always go smoothly in this sanguinary campaign. The bloodlet-
ting itself was relatively easy, as I had been trained in the old days when house staff,
not phlebotomists, took blood samples and started intravenous drips, and I was
pretty adept at cannulating the tiniest of veins in dehydrated nonagenarians. To
digress a moment, in an early investigation of St. Louis encephalitis in Harrisburg,
Illinois, I had similarly gathered people to get blood samples in the Town Hall. A
leading Baptist preacher who had helped to gather the crowd offered to go first. As
the needle went in, he had a vasovagal episode and fell over backward, blood now
spurting from his tourniqueted arm. This was visible to many people in line and so
disquieted the crowd that most ran away. While no fainting episodes happened dur-
ing the Nigerian surveys, there were other problems stemming in part from supersti-
tions about blood, and, in particular, that white men sell it or use it for nefarious
purposes and witchcraft. On one memorable occasion, we had arranged to bleed the
children in an elementary school, with the approval of the chief the night before and
with the support of the teacher. However, quite soon after starting work, a roar was
heard from down the hill in the village, as a battle charge of matronly mothers with
sticks and machetes ascended toward the school. My driver said to drop everything
NOW, and we leaped into the Power Wagon and raced away to contemplate our nar-
row escape. At the next village, we took pains to ensure ladies were present at the
“town hall” meetings.
Only months before, in January 1970, the Biafran war had ended. This civil war,
which began in 1967, was a nationalist movement by the Igbo population in eastern
Nigeria for independence from the northern-dominated federal government. In the
larger towns, there were rubble, burned-out vehicles, bullet-ridden compounds and
storefronts, and other remnants of the lost battle for secession. It was still a danger-
ous place, and we had been warned to exercise caution because of bands of former
Biafran soldiers who now survived by criminal activity, armed robbery, and hostage-
taking. The most danger came when driving at night. On two occasions, we spotted
a roadblock ahead, did a quick U-turn, and then took a long detour.
Our survey of children and adults at locations over a wide area in former Biafra
uncovered recent and remote yellow fever infections at prevalence rates (for neutral-
izing antibodies) ranging from 0% to 38%. Flavivirus cross-reactions, even by CF
and neutralization, complicated interpretation (Monath and Wilson 1973; Monath
et al. 1973b). This was addressed by including multiple flaviviruses in the
serological testing plan. One location, Mwansi, in southern East-Central State, an

area of lowland forest modified by human activity and far distant from Okwoga,
appeared to be a focus of recent yellow fever activity, once again suggesting occur-
rence of “silent” outbreaks escaping notice.
In an attempt to uncover endemic yellow fever transmission, I established a lon-
gitudinal laboratory-based surveillance in selected hospitals across eastern Nigeria,
in which samples from admissions for acute jaundice were collected. The logistics
of properly storing, shipping, or physically retrieving samples and case histories
were extremely challenging in those days. Nonetheless, we found that 1% of jaun-
dice cases admitted to hospital had laboratory evidence for yellow fever (Monath
et al. 1972b). Interestingly, between 2005 and 2011, the African Regional Office of
WHO established a yellow fever surveillance database of reports of suspected yel-
low fever cases (based primarily on a case definition of fever with jaundice) across
21 countries in West and central Africa using PCR and IgM ELISA diagnostic
methods. Thousands of suspected yellow fever cases were investigated, with 1%
confirmatory hits, an identical rate to that found in the Nigerian sample in the 1970s.
In addition to the surveys, it appeared critical to investigate isolated and sporadic
reports of individual cases, since these might augur more widespread transmission
that was being missed and provide clues to where and how yellow fever was main-
tained. The enzootic cycle had been characterized in East and Central Africa, but not
in West Africa. Following up two confirmed sporadic cases in the surrounding
savanna, we became interested in an area of high forest along the Niger river, the
so-called Nupeko Forest. From an ecological perspective, the area appeared promis-
ing as a site for continuous yellow fever transmission. Investigation of the human
population in Nupeko forest revealed cumulative yellow fever immunity with age,
indicating an annual rate of infection of about 1%. Yellow fever seroprevalence in
nonhuman primates was 57% (Monath et al. 1974a; Lee et al. 1974). Sylvatic vec-
tors were found but no virus isolations made. We concluded that the virus likely
circulated year-round but at a low level.
In 1972, I was winding up the 2-year assignment in Nigeria. It had been an enor-
mously rewarding experience to work at the last of the great Rockefeller virus labs
with colleagues who would become lifelong friends and to meet many arbovirus
experts across West Africa, especially in the Instituts Pasteur and ORSTOM in
Cameroun, Senegal, Cote d’Ivoire, and Burkina Faso. I was able to publish the work
on Okwoga, Nupeko, and surveillance of jaundiced cases. Perhaps more important
in terms of the future, I sent monthly reports back to Roy Chamberlain at CDC in
Atlanta. That effort was richly rewarded, as later I was invited to stay on in the
USPHS and to take over as Chief of the Arbovirology Unit on my return from Africa
in 1972 and soon thereafter to become Director of the Division of Vector-Borne
Viral Diseases in Fort Collins.
Just before returning from Nigeria in March 1972, however, I received a call
from Atlanta that a suspected Lassa fever outbreak was occurring at Curran Lutheran
Hospital in Zorzor, Liberia, population 2500. The diagnosis had been made clini-
cally by Dr. Paul Mertens, the hospital physician. Within a month, we had packed
up our house in Ibadan, my wife and daughter moved in with friends, and I assem-
bled gear for an investigation in Zorzor, including traps, mist nets, bleeding and
368 T. P. Monath
dissecting equipment, and liquid nitrogen, with the goal of trying to uncover the
natural transmission of Lassa virus. Most important, I was able to obtain the invalu-
able assistance of nurse Penny Pinneo, the first Lassa fever survivor (Monath et al.
1973c) who, being immune, was of great assistance in undertaking high-risk proce-
dures. This was a nosocomial epidemic with 11 cases and 4 deaths (36%) (Monath
et al. 1973c; Mertens et al. 1973). The index case was a patient from Zigida, 24 miles
north of Zorzor on a barely passable track. She had survived, and I was able to focus
collection of rodents and bats in and around her house, these animals being sus-
pected as virus reservoirs. I had high expectation that it would be possible to find the
source of infection, although without a team to do intensified trapping, my single-
handed efforts were certainly limited. No one in the village had seen mist nets
before and watched with interest every morning as I extracted many large Eidolon
helvum fruit bats, which were somewhat aggressive, and smaller insectivorous spe-
cies, which became hopelessly entangled in the nets. Dealing with samples from
these animals was another matter, as the work had to be done at night by lantern
light after daytime activities were completed. Personal protective equipment was
primitive; I wore a multipurpose respirator mask and gloves and was observant and
careful with sharp instruments. Rodents had been captured in snap traps (Fig. 2), but
Fig. 2 The author

collecting rodents in
Zigida, Liberia, at the
home of the index case of
Lassa Fever in the Zorzor
outbreak, 1972
the live bats had to be euthanized. Blood was obtained from bats and spleen, liver,
and kidney from all the animals, frozen in liquid nitrogen, and the carcasses pre-
served in formalin for later identification. On return to Atlanta, I attempted virus
isolation in Vero cells, but unfortunately, none of the samples was positive. In con-
trast, four Lassa virus isolates were made from patients at Curran Hospital (Monath
et al. 1973c).
This investigation over, I flew in a single-engine plane from Zorzor to Monrovia,
to find my family having just arrived from Nigeria. We hopped a flight to Rome and
spent some R&R in Sardinia before returning to CDC. Within a few months, how-
ever, a new disease emergence occurred that brought me back to an area in Sierra
Leone not far from Zorzor.
In August 1972, Paul Goff, a Peace Corps physician in Sierra Leone, alerted
CDC to an outbreak in Panguma, Eastern Province, suspected of being Lassa fever.
I was asked to stand up a team and was once again keen to discover the natural
transmission cycle of the virus but this time with adequate resources to do so. The
team included CDC Epidemic Intelligence Officers David Fraser and Kent
Campbell; Rockefeller veterinary-virologist Graham Kemp; CDC vertebrate ecolo-
gist Vern Newhouse; Penny Pinneo; and Jordi Casals, the famed arbovirologist from
Yale (who also had recovered from Lassa and was immune). After formalities at the
Ministry of Health in Freetown and changing dollars into huge stacks of hyperin-
flated high-denomination Leones that literally filled a duffel bag, we traveled by
road to the Eastern Province.
Because of fear of the fatal disease, Panguma town (population ~3000) was
locked down and fearful. Schools were closed and vehicles hurried through town
with windows closed. The shop owners and traders were of Lebanese origin, most
of whom were engaged in the diamond trade and anxious to curry favor with the
arriving medical team. We were heartily welcomed by the sisters at Panguma
Catholic Hospital (Fig. 3), given housing and space for Jordi to set up a laboratory
for CF antibody testing on site (Fig. 4). A priority was to establish an isolation ward
in the hospital, segregate suspected Lassa patients, and advise on supportive care.
We had with us a few units of convalescent plasma, and of course, we had a pair of
willing donors. In fact, Jordi had received Pinneo plasma when he was ill, and at the
time, this treatment was thought to be effective (Frame et al. 1984). Two of the nurs-
ing sisters with early symptoms and signs were subsequently treated.
The investigation over the next month led to the following conclusions: first,
most of the 63 Lassa fever cases identified were community-acquired and had
occurred over at least 2 years, a dramatically different picture than in previous noso-
comial outbreaks (Fraser et al. 1974); second, multiple potential reservoir hosts
were trapped and sampled, and the culprit was ultimately identified when samples
were tested at CDC—the multimammate rat Mastomys natalensis (Fig. 5) (Monath
et al. 1974b); third, the incidence of infection in the community could be estimated
(2.2 per 1000); fourth, persons in case households were more often seropositive
than in noncase households, indicating transmission hot spots; fifth, inapparent
infections were common, with an estimated ratio of asymptomatic to symptomatic
370 T. P. Monath
Fig. 3 Arrival at the Panguma Hospital, epicenter of the 1972 Lassa fever epidemic in Sierra
Leone. Third from left, Vern Newhouse CDC vertebrate ecologist; to his right Kent Campbell,
CDC EIS Officer; next Jordi Casals, Yale arbovirologist and Lassa survivor; far right, David Fraser,
CDC EIS Officer
cases of 30:1; and finally, simple patient isolation methods abrogated nosoco-
mial spread.
One evening, we were invited to the house of a Lebanese trader for dinner; our
host certainly motivated by a wish to ingratiate himself in case of medical need. The
discussion naturally centered on the intricacies of diamond trading. Our host offered
to show us some rough stones, one of which was a rare black diamond. When the
latter was passed around the dark room where we were sitting cross-legged on car-
pets, Jordi managed to drop it. Despite a massive effort, it could not be located. The
trader became quite vexed, and we later concluded that he had thought Jordi had
swallowed the stone. We left assuring our host that it would show up in the light of
day. No more of the incident was heard, but we apparently had become persona non
grata in this stratum of the community and received no additional dinner invitations.
By the time we left Panguma, the residual Leones in our duffel bag had depreci-
ated further. We still managed to make a handsome donation to the hospital. Once
again, these were the “good old days,” and we did not need itemized receipts for
most distributions. In any case, I am sure we saved the taxpayer some accounting
costs by not turning in receipts for charitable gifts to the hospital or bounties on
mice paid to our village assistants.
A memorable experience occurred after the team dispersed to go home. Kent
Campbell, one of the two EIS Officers, had decided to go to the United Kingdom to
track down nuns who had previously served in Panguma, with the objective of
determining whether Lassa was new to the area or an old disease that had been
Fig. 4 Jordi Casals,

internationally acclaimed
Rockefeller arbovirologist
in the laboratory
established at Panguma
Hospital during the Lassa
Fever epidemic in 1972.
Complement fixation tests
were performed real time,
allowing on site
identification of confirmed
human cases
Fig. 5 Processing specimens of rodents and other suspected reservoir hosts of Lassa virus,
Panguma, Sierra Leone, 1972. Graham Kemp (left) and Vern Newhouse (right) taking organ tis-
sues for preservation in liquid Nitrogen. On the right, a typical sampling of rodents captured during
the investigation. Ultimately multiple isolations of Lassa virus were made from a single species
(Mastomys natalensis)
372 T. P. Monath
missed. He was joined in London by his wife, Liz. While there, Kent became ill with
headache, fever, and sore throat, early symptoms of Lassa to which he obviously
had been recently exposed. Kent went to the Hospital for Tropical Diseases, where
he was admitted and placed in isolation under the care of Professor Anthony
Woodruff (a renown tropical medicine clinician) and Chief Resident Adel Mahmoud
(later Professor of Geographic Medicine at Case Western Reserve and President of
the American Society of Tropical Medicine & Hygiene). Over the next couple of
days, Kent felt increasingly ill, and his anxiety was heightened by the orientation of
the hospital to tropical conditions other than acute viral infections requiring inten-
sive care. He called his father, a prominent Memphis physician, saying he was con-
fined in a “cross between a museum and a chronic care facility.”
Within a day of returning to Atlanta from Sierra Leone, I received an urgent call
from the CDC Director, Dave Sencer, and his deputy, John Bagby, requesting I pro-
ceed to London to diplomatically extract Kent from the hospital and accompany
him in the capsule used to quarantine the astronauts on return from the moon (to
prevent escape of lunar pathogens), which was being flown over on a C-137 to a
military base near London. When I got to the Hospital for Tropical Diseases,
Woodruff and I went to see Kent. Isolation consisted of gowning outside Kent’s
room. Professor Woodruff rolled up his paper facemask so it resembled a mustache
with nose and mouth exposed. After examining Kent, I was pretty sure he did not
have Lassa. Notwithstanding, the evacuation proceeded in the middle of the night
without a hitch, thanks largely to Adel Mahmoud’s efforts. We had an uneventful
flight to Kennedy airport and proceeded by ambulance to Columbia Presbyterian
where Pinneo and Casals had previously been treated for Lassa fever. I took Kent’s
samples back to CDC and worked them up, no Lassa, some unidentified respiratory
virus. Someone told me the medevac alone cost $40,000 ($200,000 in today’s
money). Professor Woodruff had been exceptionally gracious about me filching his
patient, and we became good friends. In 1976, at the ASTMH meeting in
Philadelphia, he presented me with a woodcut he had made depicting Lassa fever
(Fig. 6).
Except for some short trips, I had no opportunity to do any substantive work in
Africa until 6 years later. In October 1978, CDC smallpox eradication personnel
suspected the occurrence of yellow fever in the Gambia. Preliminary investigations
and serological testing confirmed the etiology by early December, when a country-
wide mass vaccination campaign was initiated. Because of the reported high disease
incidence, an apparent westward movement of the outbreak (toward the capital,
Banjul), and uncertainty of the vector species involved in virus transmission, a mul-
tinational team was organized under the auspices of WHO. I led the team, beginning
investigations on January 2, 1979. I was joined by Bob Craven (medical officer) and
Bruce Francy (entomologist/virologist) from CDC Ft. Collins; Louis Ferrara and
Max Germain (an internationally known yellow fever expert) from L’Office de Ia
Récherche Scientifique et Technique Outre-Mer (ORSTOM), Institut Pasteur,
Dakar, Senegal; and, later on, the well-known hemorrhagic fever scientists Ernie
Bowen and David Simpson from Porton Down in the United Kingdom and Akinyele
Fabiyi, who had taken over direction of the former Rockefeller lab in Ibadan. We
Fig. 6 Lithograph prepared by Prof. Anthony W. Woodruff, Hospital for Tropical Diseases,
London, depicting the impact of Lassa virus in Africa, presented to the author in 1976
were graciously hosted by the Medical Research Council laboratory and its director,
Ian MacGregor, with whom I had done my medical school semester in the Gambia
back in 1966, sponsored by Tom Weller.
Over the next month, we surveyed nine villages from west to east, determining
an attack rate of 2.6–4.4% and case fatality rate of 19%. The overall incidence of
infection defined by CF antibodies was 32.6%, and we were able to estimate an
inapparent:apparent infection ratio (for illness with jaundice) of 12:1 (Monath et al.
1980). The entomological investigations, including human bait collections, revealed
that sylvatic vectors (Aedes luteocephalus and A. furcifer-taylori) were primarily
responsible but that as the dry season proceeded, A. aegypti-borne transmission had
occurred (Germain et al. 1980a, b).
The epidemiological investigation required an extended camping trip for the core
team, and we made heavy use of the MRC’s camp gear, which wasn’t much appreci-
ated on its return—something I still regret today. Dinners were invariably corned
beef cooked on a Coleman stove with tomato paste and served over rice. The Johnnie
Walker Red that had been obtained from the US Embassy commissary ran out half-
way through the mission, but we were grateful that it had enlivened our discussions
374 T. P. Monath
with the erudite Max Germain on yellow fever ecology and the copulatory behavior
of Aedes furcifer males. Many nights, camped by the Gambia River, I heard hippos
grunting and wheezing near the tent only to wake up and find it was Bob Craven
snoring loudly.
At one point, we learned that Bowen, Simpson, and Fabiyi had finally arrived to
join the team. I found Fabiyi in a schoolhouse with two men holding down a villager
from whom Akinyele (a PhD virologist) was trying to extract blood from the left
jugular vein. I was able to rescue the poor fellow without offending Akinyele. He
and I were great friends, and I had pierced his daughters’ ears when in Ibadan. We
often laughed about this episode subsequently, and the story was retold many times
over campfires and, of course, embellished.
The Gambia is the one country in West Africa that has taken ironclad steps to
protect its population against yellow fever through strong immunization policies
and practices.
My last major adventure in Africa happened about 10 years later in 1986, when
yellow fever was again suspected in Nigeria, centered in Oju District, Benue State,
a short distance to the east from the Okwoga area affected in 1970. I led a team from
CDC Atlanta (Kevin DeCock, later a well-known HIV investigator), Peter Tukei
from the East Africa Virus Laboratory, Entebbe, Wale Tomori, Abdulsalami Nasidi
who ran the Vaccine Production Laboratories in Yaba (Lagos) and was subsequently
the Senior Epidemiologist for Nigeria, and Roger Cordellier from the Institut
Pasteur, Cote d’Ivoire. The area was very remote, characterized by moist savanna
and gallery forest, and scattered small villages.
This outbreak was in full swing when we arrived in August. Schools had been
closed and turned into makeshift hospitals (Fig. 7). Patients at one school were lined
up to see a nurse who was overwhelmed with the acutely ill who were laid on mats
as there were no beds (Fig. 8). Many of those waiting outside were overtly ill or
prostate. Fresh graves were found outside affected villages (Fig. 9), and anecdotally,
it was said that one in every 10 residents had perished. We were led by townsfolk to
see sick and dying patients in their compounds. One young man about 20 had just
died as we arrived and had been propped up in a chair his nostrils plugged with cot-
ton, sclera deeply jaundiced (Fig. 10). A liver biopsy was allowed by the family, and
we later isolated yellow fever virus. There were no hospitals in the area, and in any
case, most people preferred native medicines and avoided hospital.
We found a small empty government building for shelter. There was no running
water, and a small supply was brought daily for bathing. Work in the tropics is best
accomplished with at least one shower a day and a start in fresh clothes. In Oju, I
learned to bathe with one teacup of water.
Our work was directed at determining the extent of the outbreak, an epidemic
curve, and the likely vectors involved. It appeared that the outbreak involved nearly
10,000 persons and extended into Cross River State (DeCock and Monath 1988;
DeCock et al. 1988). It was a sylvatic outbreak, with A. africanus breeding in man-
made containers as well as tree holes as the vector and humans the predominant
host. Also, as a yellow fever vaccination campaign had been initiated by mobile
teams from the Expanded Program of Immunization, we tried to estimate vaccine
Fig. 7 Make-shift hospital in a school in Oju, Nigeria, epicenter of a sylvatic yellow fever epi-
demic in 1986 that spawned one of the largest recorded urban outbreaks in the country from 1987
to 1992 (acknowledgements American Journal of Tropical Medicine & Hygiene)
effectiveness. A separate effort was made to determine whether there were any
adverse outcomes of vaccination during pregnancy and whether vaccine efficacy
was reduced in pregnancy (Nasidi et al. 1993). These were all important questions
with no or few existing data.
Food was always the topic of conversation especially at the end of the long work-
days heading home on rutted and muddy laterite roads. After a couple of hours of
separating sera and organizing field notes, we headed to the “Jokee Restaurant,” the
only such establishment in Oju. Here, there were two tables on a dirt floor, no elec-
tricity, and kitchen out back with open fire. We ordered cold beer and mango ice
cream every night, which never failed to send the proprietor into a fit of laughter as
his kerosene fridge hadn’t worked in years and no one had ever seen ice cream.
Pounded yam or rice was standard fare, but meat was generally unspecified and
mysterious. Kevin and I prided ourselves in a knowledge of comparative anatomy,
and we took turns identifying the entree rustled up each evening. Rodent meat was
376 T. P. Monath
Fig. 8 Patients waiting to be seen at a treatment facility, Oju, Nigeria, 1986 during the yellow
fever epidemic
Fig. 9 Peter Tukei, East African Institute of Virus Research inspecting fresh graves of yellow fever
victims outside Oju village, Nigeria, epicenter of the 1986 outbreak. (Acknowledgements
American Journal of Tropical Medicine & Hygiene)
Fig. 10 The author with a young man who had just succumbed to yellow fever, Oju, Nigeria,
1986, and had died in his compound. The family permitted a needle biopsy of the liver, from which
yellow fever virus was subsequently isolated
relatively easy to identify, especially the “porcupine” a large herbivorous rodent

(Threonomys swiderianus) and the smaller but also delicious pouched rat Cricetomys
gambianus. Chicken always provoked a roar of indignation since the head, feet, and
tail were served with no signs of the central, connecting anatomy. Our Ugandan and
Nigerian colleagues were happy with the peripheral parts and laughed at our annoy-
ance. Other bush meat was difficult to identify (Kevin and my insouciance was
rooted in prior exposures to “mystery meat” served in our prep school dining halls).
On the last night, poking around his plate, Kevin proudly announced a unique dis-
covery, the penis of a goat, served “bone-in.”
The 1986 outbreak was the cause of one of the largest yellow fever A. aegypti
epidemics in African history, which affected more than 120,000 in western Nigeria
378 T. P. Monath
in the following year (1987) and subsequently spread to the North and into Cameroun
between 1988 and 1992. This was a classic case of urbanization, the virus having
spread by the agency of viremic persons travelling west by road from the sylvatic
focus at Oju into densely populated towns in Oyo State (Nasidi et al. 1989). I spent
another 2 months investigating this outbreak, but this time, the approach focused on
hospitals, review of hospital records, in addition to the serosurveys and entomologi-
cal investigations.
I was in the fortunate position as Director in Ft Collins from 1973 to 1989 to
invite many of the colleagues with whom I worked in Africa (and elsewhere) to
spend time in the CDC lab. This was mutually beneficial and led to long-term
friendships and productive collaborations over many years. Visiting scientists would
come and work up samples from the field investigations and take primary responsi-
bility for the resulting publications concerning their home countries.
There were other opportunities to work in Africa on yellow fever. In 1982, I spent
a month in Dakar working at the Institut Pasteur with Jean-Francois Saluzzo and
Vincent Deubel on an outbreak in Senegal (Saluzzo et al. 1985). Vincent later came
to Ft Collins for a year. In 1984, Ted Tsai, Deputy Director CDC Ft Collins, and I
responded to a request from the Ministry of Health and WHO to investigate suspect
yellow fever cases in the far north of Cameroun. An extensive serological evaluation
of humans and nonhuman primates concluded that transmission had not occurred in
the far north but was prevalent in southern parts of the country (Tsai et al. 1987).
I am humbled by the many arbovirologists and investigators of viral hemorrhagic
fevers who have gone before me or have been contemporary colleagues in the quest
to unravel the mysteries of arbovirus persistence and emergence. My contributions
pale compared to many of them. I am ever grateful to the people and events that
enabled me to have these experiences in Africa and the many similar experiences I
have had in Ecuador, Argentina, and elsewhere. I am sometimes asked by young
scientists with an interest in field research how to find similar overseas opportunities
and orient their career in a similar fashion. It is harder today, since funding is
directed largely at hypothesis-driven research. But the opportunities are out there,
and answers to the question may be found from many of the authors and editors of
this book.
References
Calisher CH, Murphy FM, Monath TP (2014) In memoriam, Robert Emmons Kissling 1923–2013.
Carey DE et al (1972) Bull World Health Organ 46:645–651
Chamberlain RW (1980) History of St. Louis encephalitis. In: Monath TP (ed) Saint Louis enceph-
alitis. American Public Health Association, Washington, DC, pp 3–64
DeCock KM, Monath TP (1988) Yellow fever in eastern Nigeria. Lancet 2:39–40
DeCock KM, Monath TP, Nasidi A et al (1988) Epidemic yellow fever in Eastern Nigeria, 1986.
Lancet 1:630–633
Frame JD, Verbrugge GP, Gill RG, Pinneo L (1984) The use of Lassa fever convalescent plasma in
Nigeria. Trans R Soc Trop Med Hyg 78:319–324
Fraser DW, Campbell CC, Monath T et al (1974) Lassa fever in the Eastern Province, Sierra Leone,
1970–1972. I. Epidemiological observations. Am J Trop Med Hyg 23:1131–1139
Germain M, Francy DB, Monath TP et al (1980a) Yellow fever in the Gambia, 1978–1979: ento-
mological aspects and epidemiological correlations. Am J Trop Med Hyg 29:929–940
Germain M, Francy DB, Ferrara L, Sanyang Y, Monath TP, Adam C, Salaun J-J (1980b) Yellow
fever in the Gambia, 1978–1979: a complementary entomological survey done in October
1979. Cah ORSTOM ser Ent Med Parasitol 18:3–12
Gutierrez E, Monath TP, Alava A et al (1975) Epidemiologic investigations of the 1969 epidemic
of VEE in Ecuador. Am J Epidemiol 102:400–413
Lee VH, Monath TP, Tomori O et al (1974) Arbovirus studies in Nupeko Forest, a possible site
of endemic yellow fever transmission in Nigeria. II. Entomological studies. Trans R Soc Trop
Med Hyg 68:39
Mertens P, Patton R, Baum J, Monath TP (1973) Clinical presentation of Lassa fever cases during
an epidemic at Zorzor, Liberia, 1972. Am J Trop Med Hyg 22:781
Monath TP (ed) (1980) Saint Louis encephalitis. American Public Health Association,
Washington, DC
Monath TP, Wilson D (1973) The 1970 yellow fever epidemic at Okwoga District Nigeria.
II. Serologic survey to determine the extent and geographical origins of the epidemic. Bull
WHO 49:122
Monath TP, Nuckolls JG, Berall J et al (1970) Studies on California encephalitis in Minnesota.
Am J Epidemiol 92:40
Monath TP, Henderson BE, Kirya GB (1972a) Characterization of viruses (Witwatersrand and
Germiston) isolated from mosquitoes and rodents collected near Lunyo Forest, Uganda in
1968. Archiv ges Virus 38:125
Monath TP, Onejeme S, Okweke G et al (1972b) Surveillance of yellow fever in Nigeria,
1970–1971. Niger Med J 21:178–186
Monath T, Smith E, Wilson D et al (1973a) The 1970 yellow fever epidemic at Okwoga District,
Nigeria. I. Epidemiologic observations. Bull WHO 49:113
Monath TP, Wilson DC, Casals J (1973b) The 1970 yellow fever epidemic at Okwoga District,
Nigeria. III. Serological responses in persons with and without pre-existing heterologous
Group B immunity. Bull WHO 49:235–244
Monath TP, Mertens P, Patton R et al (1973c) A hospital epidemic of Lassa fever at Zorzor, Liberia,
March–April, 1972. Am J Trop Med Hyg 22:773–779
Monath TP, Lee VH, Wilson DC et al (1974a) Arbovirus studies in Nupeko Forest, a possible site
of endemic yellow fever transmission in Nigeria. I. Serosurvey of humans and non-human
primates. Trans R Soc Trop Med Hyg 68:30–38
Monath TP, Newhouse VF, Kemp GE et al (1974b) Lassa virus isolation from Mastomys natalensis
rodents during an epidemic in Sierra Leone, 1972. Science 185:263–265
Monath TP, Craven RB, Adjukiewicz A et al (1980) Yellow fever in the Gambia, 1978–1979: epi-
demiologic aspects with observations on the occurrence of Orungo virus infections. Am J Trop
Med Hyg 29:912–920
Moore DL, Causey OR, Carey DE et al (1975) Arthropod-borne viral infections of man in Nigeria,
1964–1970. Ann Trop Med Parasitol 69(1):49–64
Nasidi A, Monath TP, De Cock K et al (1989) Urban yellow fever epidemic in western Nigeria,
1987. Trans R Soc Trop Med Hyg 83:401–406
Nasidi A, Monath TP, Vandenberg J et al (1993) Yellow fever vaccination and pregnancy: a four-
year prospective study. Trans R Soc Trop Med Hyg 87:337–339
Saluzzo J-P, Monath TP, Cornet M, Deubel V, Digoutte JP (1985) Comparison de differentes tech-
niques virologiques pour la detection du virus de fiévre jaune dans les prélèvements humains
et des lôts de moustiques. Interêt d’une méthode rapide de diagnostic par ELISA. Ann Virol
(Inst Past) 136E:115–129
Tsai TF, Lazuick JS, Ngah RW, Mafiamba PC, Quicke G, Monath TP (1987) Investigation of a
possible yellow fever epidemic and serosurvey for flavivirus infections in northern Cameroon,
1984. Bull WHO 65:855–860
The “Golden Age” of Arbovirology,
1950–1969
David M. Morens
Abstract Although arbovirology’s origin might be considered to have been no later

than 1900, when the Walter Reed Commission established the mechanism of spread
of yellow fever, it was not until the mid-twentieth century that techniques for viral
isolation, serology, and vaccinology had become sufficiently advanced that a new
field of scientific endeavor could coalesce. The period from roughly 1950 to 1969
can be called arbovirology’s “golden age”: this was a time when, in the aftermath of
World War II, the US military and the globally oriented Rockefeller Foundation
reached out to “tropical disease” researchers around the world and drew them
together into a scientific community that quickly discovered many new viruses and
viral diseases, further developing scientific techniques to characterize them.
Excepting the work done in the Koch and Pasteur laboratories in the late nineteenth
century, arbovirology’s “golden age” is among the most productive times in the his-
tory of infectious diseases, and some of that era’s leaders are still working produc-
tively today. This chapter discusses how this remarkable era came together,
emphasizing discoveries made, challenges faced, the hard work involved, and many
of the men and women who brought the field into being.
1 Introduction
Reflecting on an earlier “golden age,” philosopher Søren Kierkegaard (1813–1855)

observed that life is lived forward – but understood backward. Arbovirology’s
golden age (said to have occurred during the 1950s and 1960s) was a period of virus
discovery and disease characterization unfolding at a breakneck pace, in the process
creating the new biomedical field of arbovirology. As scientific heirs, we should
look backward and try to understand this era and the men and women who made
it happen.
D. M. Morens (*)
National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD, USA
e-mail: dmorens@niaid.nih.gov; dm270q@nih.gov
382 D. M. Morens
Most who worked within this 1950–1969 era cohort have retired or, sadly, passed
away. But fortunately, several are still (in 2021) professionally active – Scott
Halstead, and Karl Johnson come readily to mind – to which we may add other
luminaries who, as young scientists at the time, barely made it into the era (e.g.,
Charlie Calisher, Tom Monath, and CJ Peters).
Before going further, readers, especially younger readers, are urged to put down
this book and actually talk to any – all – of the now-aging men and women whose
efforts 50 and more years ago carried the profession forward. It is my good fortune
to have worked with some of them and to have known others – some well, and oth-
ers just revered luminaries whose words I “hung on to” at scientific meetings. Most
of what I learned about arbovirology came not from textbooks but from listening to
what these scientists said and trying to follow their examples.
2 Background: Global Arbovirology Infrastructure

Before 1950
The early 1950s was, even at the time, being experienced as the dawn of a new era,
not just in biomedical science but in many other areas as well (Fig. 1). Elsewhere in
this book we learned the prior history of arbovirology, the long shadow of yellow
fever (YF), which had plagued the Americas since colonial times, the scientific
development of the field in the late 1800s, and the impact of the Spanish-American
War, which instantly made America a new tropical colonial power. We learned about
the explosion of viral discovery in the 1930s and 1940s that prominently featured
arboviruses (Table 1, Reeves 1987) and the subsequent rapid growth of tropical
medicine, under the ever-present shadow of yellow fever, of the Rockefeller
Foundation (RF) programs, of Max Theiler’s (1899–1972) yellow fever vaccine and
of the overly optimistic attempts to control yellow fever and malaria.
Arbovirology had long been dominated by one disease – yellow fever – and it
remained the most important disease throughout the 1950s and 1960s. In 1949, yel-
low fever reemerged violently in Central America; the RF tropical disease program
had to contend with it even as it sought to broaden arbovirology. Beginning in 1960,
an even more aggressive reemergence in Africa allowed YF to dominate that decade
too. Much important work was carried out, as described in Chap. 8. This chapter
will focus on the other arboviruses, only discussing YF in the context of other
efforts. Interested readers may also wish to consult an important/fascinating 1991
YF review by Tom Monath also containing numerous wonderful photographic
images of key scientists of the era (Monath 1991).
Despite WWII’s dengue epidemics – 90,000 US troops in the Pacific and North
Africa had been infected – the end of hostilities temporarily sidelined progress in
arboviral research. It took a while to regain momentum. In late 1945, the US Army
sent medical scientists to Japan, including infectious disease scientists. US-Japan
collaborations were gradually established, and scientists from other nations soon
joined in. Tissue culture techniques had been advanced, in the late 1940s, as a
The “Golden Age” of Arbovirology, 1950–1969 383
“McCarthy Era”, 1950-1956

Death of Charles Franklin Craig Korean War (1951-1953)
1950 Antibiotics and TB treatments commonly used
Rockerfeller Foundation Virus Program (1951-1970)
Korean hemorrhagic fever epidemic 1951 First hydrogen bomb
CDC’s Epidemic Intellignece Service begins
AJTMH begins publication 1952 Black and white televisions become affordable to many
Watson & Crick publish DNA “double helix” Death of Stalin

(Dengue) hemorrhagic fever epidemics in Southeast Asia 1953 Princess Elizabeth crowned as queen
Nobel Prize to Endlers/Weller/Robbins for tissue culture First transistor radio; First color TV; First nuclear power plant;
West Nile virus human challenges 1954 Segregation outlawed by U.S. Supreme Court
“Big Bang” theory accepted
First polio vaccine (”Salk” inactivated vaccine)
NIAID (NIH) established
1955
1956 Elvis Presley becomes international superstar

Hungarian revolution
H2N2 influenza pandemic 1957 Sputnick launch begins “space race”
Argentine hemorrhagic fever recognized 1958

NIH establishes MARU (Middle America Research Unit)
ACAV established
HIV epidemic remains undetected (until 1981) 1959 Castro conquers Cuba
Casals, Clarke, others - systematic classifcation of arbovirus Alaska becomes 49th State/Hawaii becomes 50th State
Arbovirus calolog and Arbovirus Information Exchange begins
NIH establishes ICMRT program 1960 Adolph Eichmann captured
First woman prime minister sworn in (Ceylon)
Kuru begins to be characterized
Berlin wall built
Hammon, Sathers and Rudnick try to identify DEN-5 and DEN-6 1961
(Future) President Obama born
NIH estabilshes Pacific Research Section 1962 Beatles begin recording career
Cuban misssile crisis
Karl Johnson et al. characterize Bolivian Hemorrhagic fever 1963 U.S. President John F Kennedy assassinated
DEN-3 epidemic in the Carribbean Martin Luther King Jr.’s “I Have a Dream” speech
US epidemic of congenital rubella (1964-1965)
AVAC Subcommittee on Arbovirus Laboratory Safety (SALS)
1964 Escalation into “Vietnam War” (1964-1975)
1965
1966
First detected filovirus epidemic - Marburg, Germany 1967 First heart transplant
H3N2 infuenza pandemic 1968 Assassinations of Martin Luther King Jr., and
Robert F Kennedy
Woodstock festival
First Lassa fever epidemic 1969 Humans land on the moon
1970
Fig. 1 Timeline of important scientific and other events, 1950–1969
by-product of developing polio vaccines; scientists were just then beginning to use
this promising new tool to open up a new era of viral disease discovery.
As the promise of a polio vaccine neared reality, around 1950, viral disease dis-
covery was among the hottest subjects in science. Not even Watson’s and Crick’s
1953 discovery of the “double helix” (Fig. 1) crowded viral disease discovery off
the scientific front burner. Some of the earliest (polio-like) enteroviruses were
384 D. M. Morens
Table 1 Selected viral isolations, made after the 1927 isolation of YF, through 1949
Year Virus/disease Discoverers
1930 WEE (first alphavirus) K. Meyer et al.
1931 RVF (first phlebovirus) R. Daubney and J. R. Hudson
1933 SLE R. S. Muckinfuss
1933 YF jungle cycle F. Soper et al.
1934 EEE M. Merrill and C. TenBroeck
1934 JE M. Hayashi et al.
1934 LCM (first arenavirus) C. Armstrong et al.
1937 TBE M. P. Chumakov and S. G. Gladkikh
1938 VEE V. Kubes
1940 WN K. C. Smithburn et al.
1942 SF K. Smithburn, A. Haddow
1945 CCHF (first nairovirus) M. P. Chumakov et al.a
1946 BUN (first bunyavirus) K. C. Smithburn et al.
Although isolations of important viruses prominently included arboviruses, arbovirology, as a
field, existed only tenuously. a(The Chumakov isolation of the Crimean hemorrhagic fever virus
was considered not definitive. The first definitive isolate was of Congo virus, by Woodall et al., in
1967. See text)
discovered by arbovirologists such as William McDowall Hammon (1904–1989),

Albert Bruce Sabin (1906–1993), and later by Leon Rosen (1926–2008), who would
go on to a career in arbovirology (see Chap. 3). By the early 1950s the US Army and
Navy had become fully involved in overseas collaborative tropical medicine
research, and the RF was planning a big new program designed as a “fishing expedi-
tion” in tropical virus discovery. The stage was set for important advances.
3 The Rockefeller Viral Disease Program
Theiler and Downs (Theiler and Downs 1973) and Downs (Downs 1982) have
described the thinking that led the RF, which had long been involved in yellow fever
research and control efforts (Strode 1951; Farley 2004; see Chap. 8), to begin, in
1951, a remarkable program in global tropical viral disease research. With only
moderate funding, about $1.5 million annually, on average, the RF identified exist-
ing laboratories, institutions, agencies, and individuals around the globe – resources
already on the ground, its own resources, and others – and added new laboratories
in partnership with governments and research institutions, drawing them together
into an international network to explode viral discovery (Table 2, Fig. 2).
Available resources in 1951 included the remnants of the RF International Health
programs focusing on yellow fever and the world-class Rockefeller Virology
Laboratory (RVL) in New York City: the US Army 406th laboratory in Tokyo, set
up in 1945 under the direction of civilian virologist-epidemiologist Joseph Edward
Smadel (1907–1963); the US Naval Medical Research Unit Number 2 (NAMRU-2),
Table 2 Rockefeller Foundation Laboratory Network for Arboviral Discovery, 1951–1970

Year Location Partner First RF director
1951 RVL Coordinating Lab Max Theiler
1952 Poona (Pune), India Indian Council Of Medical Research J. Austin Kerr
1953 Trinidad Trinidad Health Department Wilbur Downsa
1953 Johannesburg, South South Africa Institute for Medical J.H.S. Gear
Africa Research
1954 Belém, Brasil Brasil Special Service Public Health Ottis Causeyb
Department
1959 Cali, Colombia Colombia Government/Universidad de Robert H. Kokernotc
Valle
1964 Ibadan, Nigeria Nigerian Government Ottis Causeyd
Information abstracted from Theiler and Downs (1973) and Downs (1982)
a
Downs was assigned two RF scientists, Kenneth C. Smithburn and Robert H. Kokernot
b
Though not a formal member of the RF team, Ottis Causey’s scientist wife Calista was a produc-
tive researcher in her own right and a major player in RF research
c
Kokernot was assigned two RF scientists, Ronald F. MacKenzie and Vernon H. Lee
d
Causey was assigned 2 RF scientists, Graham E. Kemp and Vernon H. Lee
Rockefeller Foundation Arbovirus Program Staff, 1952-1970

Overseas
1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1968 1970
Charles R Anderson
Jordi Casals-Ariet
Ottis Robert Causey Retired
Delphine Harriet Clarke
Wilbur George Downs
Harald N Johnson
John Austin Kerr Reassigned
Kenneth C Smithburn Retired
Richard Moreland Taylor Retired
Max Theiler Retired
Loring Whitman Retired
Telford Hindley Work Resigned
Thomas HG Aitken
Robert H Kokernot Resigned
Ping-Yao Cheng Resigned
Harold Trapido Retired
Sonya M Buckley
Robert Ellis Shope
C Brooke Worth Resigned
Donald E Carey
Jorge Boshell Manrique Retired
Andreas H Jonkers Resigned

Vernon H Lee
Robert Wade Speir Reassigned
Gram E Kemp
John Payne Woodall
Ronald B MacKenzie
Totals 12 14 13 13 15 15 17 17 19 18 19 20 20 20 19 19 19 19 17 13
Fig. 2 Rockefeller Foundation Virus Program scientific personnel, 1951–1969. (Adapted from
Theiler and Downs 1973)
386 D. M. Morens
established at the RF in 1942 under the direction of Thomas Milton Rivers

(1888–1962), located in Guam from 1944 to 1954 and, beginning in 1955, relocated
to Taiwan, under the direction of clinician/cholera researcher Robert Allan Phillips
(1906–1976); NAMRU-3, established in 1942 under the direction of Phillips and in
1951 linked to the RF program in Cairo by Richard Moreland Taylor (1887–1981);
and cooperating scientists at NIAID’s Rocky Mountain Laboratory (in Hamilton,
Montana) and NIAID’s Bethesda Maryland-based International Centers for Medical
Research and Training (ICMRT) research grant program and the resources at the
Communicable Disease Center (CDC, later renamed the Centers for Disease Control
and Prevention) in Atlanta (which had been established only a few years earlier as
part of the US federal Malaria Control in War Areas program).
Drawn into this partnership in 1957 were the Panama-based Middle America
Research Unit (MARU), jointly run by NIAID and the Walter Reed Army Institute of
Research (WRAIR), first directed by Alexis Shelokov (1919–2016) and later by Henry
K. Beye (1912–1964) and others; in 1962 NIAID’s Hawai‘i-based Pacific Research
Section, directed by Rosen; also in 1962 the Armed Forces Research Institute for
Medical Sciences (AFRIMS; until 1958 the Southeast Asian Treaty Organization, or
SEATO, laboratory), under the direction, on the US side, of Oscar Felsenfeld
(1906–1978); and in 1968 Addis Ababa’s NAMRU-5, established in 1965 and in oper-
ation until 1977 as a NAMRU-3 detachment, under the direction of Raymond
H. Watten (1922–2013; Fig. 3). Other laboratories worked with the RF network
Fig. 3 Raymond H. Watten, commanding officer of NAMRU-3, presents an award to Navy virolo-
gist James M. Meegan for work on the 1977–1978 isolation and characterization of Rift Valley
fever virus in Egypt
collaboratively, e.g., the East African Virus Research Institute, Entebbe, the Instituto
Evandro Chagas, the Gorgas Memorial Laboratory, and the Instituto Oswaldo Cruz,
as well as laboratories in Russia, the Institut Pasteur Network, and elsewhere.
As a first Rockefeller effort, Smadel set up a new satellite laboratory in Kuala
Lumpur, Malaysia. Soon added to the RF system was the California State Health
Department virology laboratory (1954). From these initial resources, the RF quickly
assembled an international network that leveraged existing resources, wherever they
could be found and identified, hired, and frequently reposted its own talented scien-
tists around the globe (Fig. 2), emphasizing training, movement of scientists from site
to site, and investments that were designed to be quickly phased out and replaced by
national sources of funding. The program was “lean and mean,” catalytic, always on
the move, and capable of integrating the triad of clinical, field-based, and laboratory
research. It is doubtful that anyone would have imagined in 1951 how wildly success-
ful this program would soon become: it rapidly added to the viral disease map hun-
dreds of new infectious agents and human and animal diseases. With the sole exception
of the Koch and Pasteur laboratories between about 1876 and 1895, it can be argued
that no other program or entity has ever contributed so much new and important terri-
tory to the infectious disease landscape within such a short period of time.
Describing all of the significant events and important individuals of this remark-
able 20-year period is an impossibility. I will highlight only some of the efforts,
discoveries, and noteworthy accomplishments that come to mind, hoping that this
outline and the selected references will lead interested readers to more fully explore
the large body of published work from this remarkable era. I also add a few vignettes
of individual scientists who made important contributions which in some cases are
not well remembered today. Other important figures in this era, e.g., Max Theiler
and Jordi Casals-Ariet (1911–2002), are discussed in other chapters.
Richard Moreland Taylor (1887–1981) was a physician-epidemiologist-

microbiologist who left an academic post in pathology, fought in WWI, stayed
in Europe to work with the Red Cross, and in 1923 joined the RF’s International
Health Division. After receiving a PhD in public health, Taylor was posted in
the 1930s to Europe to study brucellosis. It was only in the 1940s, at which
time he was in his mid-50 s, that Taylor began to devote his energies to arbo-
virology, with RF postings first to Buenos Aires and then to Brasil as the RF
International health representative, at which time he began to elucidate mech-
anisms of jungle yellow fever. From 1945 to 1951, Taylor became director of
the RF International Health Division laboratory in New York City and, in
1952, at age 65, was sent to Cairo to set up a research program at NAMRU-3.
After retiring, he became a professor at Yale and later at UC Berkeley. Taylor
was instrumental in establishing, with Bill Reeves and others, the American
Committee on Arthropod-Borne Viruses (ACAV), the Arbovirus Catalog, and
the Arbovirus information Exchange. ACAV established a prestigious award
in his name and named Taylor its first recipient. Taylor was among the most
respected of the senior leaders in arbovirology in the “golden age.”
388 D. M. Morens
Before proceeding, it is also worth highlighting important general areas of

endeavor within which scientific work was organized during the period of
1950–1970:
1. Establishment of an international collaborative arbovirology network of institu-
tions, with a loosely bound cadre of dedicated communicating scientists
2. Isolation of a rapidly growing number of new arboviruses and characterization of
their vectors and reservoirs
3. Characterization of complex viral ecologic systems including reservoirs, verte-
brate and invertebrate hosts, vectors, transmission cycles, amplifying hosts, zoo-
notic “spillover,” environmental and climatic factors, and other aspects of viral
ecosystems now, in 2021, associated with the term “One Health”
4. Development of techniques to characterize new viruses and to distinguish them
from each other, including new viral isolation and serological techniques, and
development of standardized exchanges of information
5. Identification and characterization of arboviral and non- arboviral agents of hem-
orrhagic fevers
6. Pursuing the elusive possibility of disease control through vaccines, drugs, and
direct vector control, including renewed efforts at mosquito eradication (Soper
1963; Pinto 1955)
4 Perspectives on Arbovirology in 1950
At the beginning of the 1950s, arbovirologists had for decades been predominantly
concerned with yellow fever (Strode 1951) (yellow fever is discussed fully in Chap.
8). Diseases like dengue were important, but because dengue hemorrhagic fever had
not yet been recognized, dengue was considered largely a nuisance disease (during
all of World War II, the US Army’s 90,000 cases, undoubtedly greatly underdiag-
nosed, resulted in only four known deaths, none from what would later be described
as dengue hemorrhagic fever or dengue shock syndrome ― DHF and DSS).
The history of virology was at the time a short one, dating to the 1890s, and virol-
ogy was not a well-established field. In the 1930s and 1940s, several new viruses
had been isolated, including several arboviruses (Table 2), but work on character-
izing them had proceeded slowly. After the war, US military arbovirologists sta-
tioned in Japan were newly concerned about JE, which had been causing devastating
Southeast Asian/Asian epidemics and which in the late 1940s had extended to the
US-affiliated Trust Territory jurisdiction of (now the United States Territory of)
Guam, affecting protected peoples who would soon become US citizens.
As new arboviruses were being discovered, it was also becoming increasingly
clear why most – but not all – of them occurred in restricted geographic areas (e.g.,
MVE in Australia, RSSE in Eastern Russia, KFD in the Mysore region of India, and
so on): most such viruses existed in specific reservoirs associated with specific vec-
tor and usually vertebrate hosts, e.g., different mosquito species that fed on different
birds and/or mammals. The ecologic complexity of the situation grew as more and
more viruses were being discovered and their ecologies studied. Every new arbovi-
rus seemed to come with its own complicated set of (often inscrutable) rules.
While research on older, better-known diseases like SLE, JE, dengue, and others
was being invigorated in this era, the starting point of hundreds of different research
efforts was often either identification of a new disease or, more commonly, isola-
tion – from a human, animal, or vector, usually a mosquito or a tick – of a new virus.
In this “pre-genomics” era, it could take considerable time to determine whether the
virus was actually new, previously known, or serologically related to an existing
virus. Roughly well-defined viral taxonomic classifications in 1950 included the
Group A and Group B arboviruses (now known as the alphaviruses and flavivi-
ruses). In 1961, Casals and Whitman proposed a new arboviral “Group C” com-
prised of Brasilian viruses lacking antigenic relationships with either Groups A or B
viruses. These Brasilian viruses were generally related to each other and distantly
related to certain African isolates, but not to viruses elsewhere in the Caribbean or
in Southeast Asia (Casals and Whitman 1961). Other isolated viruses remained
unclassified or imperfectly classified. Complexity was getting more complex.
With more and more virus isolates, antigenic and taxonomic complexities con-
tinued to mount. After viral isolation, characterization, and development of sero-
logic tests, serosurveys were typically done in humans and animals to assess the
prevalence of past infections, i.e., to characterize the epidemiology and epizootiol-
ogy. Then there was the far more difficult task of characterizing the ecosystem in
which the virus existed – in some cases including multiple mosquito or avian or
mammalian species, as well as reservoirs, amplifying hosts, and bridging vectors.
As the challenges mounted, more and more energetic young scientists were drawn
to the excitement of this new and exotic field. Although space does not permit dis-
cussion of all of the many studies done by hundreds of investigators, Theiler and
Downs classical account (Theiler and Downs 1973) serves as a comprehensive start-
ing point.
5 Two Key Diseases: JE and VEE
Among the most important arboviral work in the early 1950s was the further char-
acterization of Japanese encephalitis or JE, first isolated in 1934 (Hayashi 1934) and
which had caused major epidemics in Okinawa between 1945 and 1949. American
scientists in Smadel’s group conducted important research: also involved in JE work
were a Who’s Who of arbovirologists including Hammon, Sabin, and his protégé
Edward L. Buescher (1925–1989), Trygve Obert Berge (1909–1995), Gordon
Meiklejohn (1911–1997), William Tigertt (1915–1992), William Carlisle Reeves
(1916–2004), Jordi Casals-Ariet, Medhat A. Darwish, Gladys E. Sather, and
William Franklin Scherer (1925–1982). After YF, JE was probably the most impor-
tant and exciting arboviral problem of the era. A massive multination epidemic of
JE in 1958 lent urgency to these efforts. Hammon started working on a vaccine, the
390 D. M. Morens
logical next step in arbovirology efforts to control human diseases. While effective
JE control throughout the region would not be achieved quickly, the groundwork
was well laid in the decade of the 1950s.
Venezuelan equine encephalitis, or VEE, first isolated in 1938 (Kubes and Ríos
1939), at that time never having been recognized as epizootic in the continental
United States, was a particular interest of Carlos Sanmartín-Barberi (1929–1996)
and later of the group led by Wilbur George (Wil) Downs (1913–1991) in Trinidad.
Downs had been a leading malariologist who had worked all over the Pacific,
Caribbean, and South America – and now joined the VEE work with colleagues
including Theiler; Ottis Robert Causey (1905–1988) and Causey’s scientist-wife
Calista Causey (1898–2000); Edwin H. Lennette (1908–2000), who had been work-
ing on VEE since the 1940s; Bill Tigertt; Roy William Chamberlain (1916–2013),
Berge; Robert Ellis (Bob) Shope (1929–2004); Karl Johnson; and Scherer, compris-
ing another Who’s Who of arbovirology. When VEE reemerged explosively in the
late 1960s – causing an astonishing 30,000 human and 50–100,000 equine cases –
definitive work was also done at MARU, especially work by Karl Johnson and
Nathaniel (Nat) Young (1938–1979) (Young and Johnson 1969). VEE, like SLE,
was proving to be more widespread than suspected, and less geographically
restricted than was true for most other arboviruses.
COL Edward L. Buescher (1925–1985) was a physician-scientist trained by

Albert Sabin in Cincinnati. He is today best remembered for his 1961 co-
discovery (with Malcolm Artenstein (1930–1976), Paul Parkman, and others)
of the rubella virus, which led rapidly to the development of the rubella vac-
cine used today. Stationed in Japan after the Korean War, Buescher conducted
seminal and influential work on all aspects of JE and had a significant influ-
ence on the field of arbovirology in the early years of arbovirology’s “golden
age” (Buescher and Scherer 1959). Later, as commander and director of the
Walter Reed Army Institute of Research (WRAIR), he influenced a genera-
tion of military scientists. A recipient of the Gorgas Medal, Buescher was also
awarded the bronze star during World War II and later the Legion of Merit
with Presidential Citation. Dying at a relatively young age, he was buried with
honors in Arlington National Cemetery.
Wilbur George Downs (1913–1991) was a physically and mentally energetic

“Renaissance Man” of arbovirology, who had already traveled the world and
become a leading malariologist by the time he retired from a long Army career
and joined the RF in malaria control (1946–1952). The RF then sent Downs
to Trinidad, where he had once worked, to set up a Trinidad Regional Virology
Laboratory (later the Caribbean Epidemiology Centre, or CAREC, which
later became a component of the Caribbean Public Health Agency). Under

Downs’ leadership, the lab quickly became an internationally acclaimed virus
discovery unit. Downs later headed the RF arbovirus program and shortly
after isolating, with colleagues, Tacaribe virus (Downs et al. 1963) became
director of the Yale Arbovirus Research Unit, or YARU (from 1963 to 1971).
Downs spoke at least five languages and was an avid sportsman, guitarist,
photographer, stamp collector, and experimenter with orchid hybridization
(the orchid genus Downsara bears his name). Near the end of his career, he
grew a beard which changed his appearance (favorably) and made him look
wonderfully avuncular. Although I never knew Downs personally, I remember
him well from scientific meetings; my most vivid memory is of a particular
meeting at which it was announced that he would be retiring, I think from his
academic position at Yale; he came up onto the stage and stood modestly as he
was vigorously applauded as one of the era’s greatest arbovirologists.
Work in Africa and the Middle East. British and especially Scottish investigators
in Entebbe and elsewhere in Africa and the Middle East were also making important
contributions to understanding geographically extensive arboviruses, including the
finding of geographic extension of viruses like the West Nile virus (WNV), long
before that virus came to the Western Hemisphere in 1999 to extend geographically
over much of the continent. Alexander John Haddow (1912–1978) worked through-
out the 1940s, 1950s, and 1960s to characterize major African arboviral diseases
including YF, WN, CHIK, Uganda S (Dick and Haddow 1952), Sindbis, and Usutu,
as well as, with Ken Smithburn and Jack Woodall, Zika (Dick et al. 1952),
O’nyong’nyong (Williams et al. 1965), and, with George Williamson Auchinvole
Dick (1914–1987), RVF (Dick 1953).
Ottis Robert (1905–1988) and Calista Post Eliot (1898–2000) Causey were an
American husband-wife research team – he an entomologist, she with a doc-
torate in hygiene – who married in 1939 (when he was 34 and she 41) and
headed directly to Brasil to work with the RF on Anopheles gambiae eradica-
tion. Dodging Nazi U-boats during the war, they survived to become key play-
ers in arboviral discovery. Emphasizing the role of mosquito vectors, the
Causeys had a powerful influence on the (then, in the 1950s and 1960s)
younger generation of RF arbovirologists, including Bob Shope and later, in
Nigeria, Don Carey, Graham E. Kemp (1927–2010), Vernon Harvey Lee, and
others. The Causeys’ early work focused on jungle cycles of YF and other
mosquito-transmitted viruses; they later broadened their interests to study
multiple important viruses of Brasil, Nigeria, and elsewhere, including blue-
tongue; CCHF; CHIK; DEN; Mayaro, which they co-discovered; Mokola; the
phlebotomus fever group including the discovery of Icoaraci; VEE; and vari-
ous “Group C” viruses. The Causeys are today regarded as pioneers who
greatly influenced the generation of the 1950s and 1960s.
392 D. M. Morens
Among important early work was the discovery and characterization by the
Entebbe group of chikungunya, following a large 1952 outbreak in Tanganyika
(now Tanzania). CHIK epidemiology was well characterized by WH Russell
Lumsden (1914–2002) and others, the virus being isolated by Ronald WNL Ross
(Lumsden 1955; Ross 1956). Characterization of the new disease was not only
important in its own right, or because chikungunya would spread pandemically
62 years later (in 2013–2014), but also because it shed light on 300 years of dengue-
like diseases that were not always clinically identical.
Examining anomalous historical epidemics of what had been called dengue, RF
physician Donald Edward Carey (1929–2022) (former director of the Ibadan labo-
ratory) made a convincing clinical historical argument that some of these past (so-
called) dengue epidemics, e.g., simultaneous 1789 epidemics in Batavia (now
Jakarta) and Cairo and even the 1827 Caribbean pandemic of dengue, had actually
been caused by chikungunya (Carey 1971). Carey’s scholarly efforts suggested that
more than two centuries ago, CHIK had spread pandemically, but then at some point
disappeared from the New World. The full history of chikungunya is still unresolved.
As arboviral disease discovery began to accelerate after 1951, it became clear
that the rapidly growing list of arboviruses was creating ever-more complicated
serologic and taxonomic challenges, including appearance of viral variants over
extreme geographic ranges. This was a serious problem that had to be addressed.
6 The Rockefeller Steps Up – Again – To Unravel

Arboviral Confusion
In the late 1950s, two important developments changed the virus isolation-
characterization picture considerably. First was the decision by the RF – roundly
supported by investigators – to form an informal committee (later called the
American Committee on Arthropod-Borne Viruses, or ACAV, and issuing an
Arbovirus Catalog that soon grew to be so big that only the strong could carry it
across the room – literally (Taylor 1962)), as well as a periodical Arbovirus
Information Exchange that was critical to communication between scientists around
the world (Fig. 4). These actions were intended to sort out many of the field’s grow-
ing problems by establishing a standardized format and forum for viral character-
ization and to emphasize sharing information and reagents.
The second development was improved viral identification and differentiation,
exemplified by improved serologic techniques, in this era predominately Nt, CF,
and HI, and especially the simplification of a reproducible HI test by Delphine
Harriet Clarke (1912–1985) and Casals (Clarke and Casals 1958; Casals 1963).
Also important were Sonja Buckley’s development of cell culture capacity at the RF
and her partnership with Telford (Tel) Work (1921–1995) and Wil Downs to use
mouse immune serums to improve viral identification. These efforts were instru-
mental in characterizing new viruses and virus groups, e.g., the work of Shope and
Ottis Causey in studying the relationship of Group C viruses (Shope and Causey
1962). At the same time, Oxford’s James Stuart Porterfield (1924–2010) was devel-
oping better arboviral plaquing techniques, including plaque-inhibition serologies.
Such improvements in Nt testing were of great importance.
In today’s (2021) genomics era, it may seem strange that organization/
information-sharing and simplified serologic techniques could be so important to
viral identification. But it must be realized that by the early 1960s, of 198 known or
suspected arboviruses, only 53 had been adequately placed within defined taxo-
nomic categories (Group A and Group B, eventually corresponding to alphaviruses
and flaviviruses, respectively), with three additional groups being worked on: 10–16
Bunyamwera-like, 9 provisional “Group C,” and 7–10 phlebotomus fever-like
viruses, leaving the remaining 119 (or so) viruses unclassified and in some cases
still unnamed.
Casals’ statement in the early 1960s that “there are between 17 and 19 viruses in
[Group B], depending upon the interpretation,” reflected uncertainties surrounding
classifications based on incomplete and often contradictory data, for even the best-
studied virus groups. Tissue culture was still a new technique; many viruses had not
been easily grown in tissue culture, and many could not be adapted to growth in
mice without extensive passaging, which was associated with mutational changes,
potentially compromising the relevance of the “model.” Serologic techniques were
cumbersome, and strain variants often gave confusing results and levels of cross-
reactivity – sometimes one way – that were not easily interpreted. Easier, cheaper,
more reliable, and standardized approaches were needed.
In retrospect, the new RF organizational effort was a turning point in harmoniz-
ing the work of a far-flung cadre of arbovirologists into a large, standardized, freely
shared, and continually updated database – the Arbovirus Catalog and the Arthropod-
Borne Virus Information Exchange (Fig. 4). Today’s ACAV, renamed the American
Committee on Arthropod-Borne and Zoonotic viruses in 2021, is the culmination of
these efforts.
7 Dengue Moves to the Front Burner
As noted in Chap. 3 and 6, dengue, recognized for centuries, had been linked to
Aedes aegypti transmission in the early 1900s. Two dengue serotypes, suspected
since the 1920s work of Army scientists Joseph Franklin Siler (1875–1960) and
James Stevens “Steve” Simmons, (1890–1954) were isolated during World War II
(DENV 1 and DENV 2), DEN-1 independently by Kyoto University-based Susumu
Hotta (1918–2011) and DENV 1 and DENV 2 by Sabin (Sabin’s success having
been supported by OSS interrogation of captured Japanese virologists, who had
provided information on Hotta’s work).
394 D. M. Morens
Susumu Hotta (1918–2011) was a Kyoto University-based physician and

infectious disease researcher (Konishi and Kuno, 2013) who, as a student,
investigated massive Aedes albopictus-borne wartime dengue epidemics in
Nagasaki. Making repeated field trips to that city, in summer 1943 he isolated
the first dengue virus using suckling mouse brain inoculation. This isolation
came several months before isolations of two dengue viruses in 1944 by
Albert Sabin. Arbovirologist John Aaskov relates that Hotta told him he had
proved that his isolation was a pathogenic dengue virus by inoculating his
own mother, then 57 years old, who developed clinical dengue. Hotta’s isola-
tion and Sabin’s first isolation were of the virus now known as DENV 1.
Sabin’s second isolation is now known as DENV 2. It is of some interest that
Sabin’s work was greatly influenced by OSS translations of Hotta’s publica-
tions and communications, by captured documents, and at least one OSS
interrogation of a captured Japanese dengue researcher. Hotta continued his
dengue research throughout the war, but his 1945 field trip to Nagasaki was
delayed because American planes had bombed the railroad line. Unable to
reach the city by August 9, Hotta survived while his research colleagues per-
ished in the atomic bomb blast. After the war, Hotta was among the first to
recognize the protective effects associated with dengue anamnestic antibody
responses associated with second infections. In the 1950s Hotta went to the
United States and received the PhD degree from the University of Washington.
Returning to Japan as a professor at Kobe University, Hotta had a long pro-
ductive career as an arbovirologist and researcher in other infectious diseases.
Another two serotypes (DEN-3 and DEN-4) were isolated by Hammon in the
1960s. DEN-5 and DEN-6 serotypes were proposed soon thereafter by Hammon,
but were eventually reclassified as strain variants. Sabin, who had isolated DENV 1
and DEN-2 protype strains in 1944, conducted human challenge studies and began
working on a vaccine (a DEN-1 version of which was tested in a 1963 field trial by
Charles L. Wisseman (1920–1998 (Wisseman et al. 1963)), but his vaccine was
never licensed.
William MacDowall Hammon (1904–1989) was a medical missionary and

ordained Methodist minister working in the Belgian Congo (now the
Democratic Republic of the Congo) who returned to the United States to
study medicine and then bacteriology under Hans Zinsser (1878–1940).
While conducting PhD research, he co-discovered, with future Nobel laureate
John Enders, the feline panleukemia virus and later in the 1940s conducted,
at the University of Pittsburgh, pioneering work on passive immunization
against polio, an approach that provided an important, if controversial, proof
of principle for polio vaccine developed soon thereafter by Hammon’s
Pittsburgh rival, Jonas Salk. Hammon’s polio work was, however, an interlude
in an important arbovirology career, working closely with Bill Reeves from
1943 onward to characterize SLE, WEE, and the California group arbovi-
ruses. When DHF appeared in the 1950s, Hammon turned his attention to
dengue, isolated the prototype DENV 3 and DENV 4 viruses, and made
important theoretical insights into the possible causes of DHF. His association
with Gladys Sather after about 1947 was especially productive.
Fig. 4 The Arthropod-Borne Virus Information Exchange

396 D. M. Morens
A lull in dengue research after the end of the war was soon overtaken by growing
interest. Rosen and others soon began to study transmission by alternative vectors,
including Aedes polynesiensis, which appeared to have been supporting dengue epi-
demics in various Pacific islands. With the appearance in the early 1950s of
Southeast Asian hemorrhagic fevers, Hammon and Sather turned their attention to
dengue as well, followed soon thereafter by Halstead and by Phillip King Russell
(1932–2021; later United States Army Major General Russell), both US Army offi-
cers stationed at Thailand’s AFRIMS. Additional dengue contributions were made
by British researchers like Scottish investigator Charles Edward “C. E.” Gordon
Smith (1924–1991), a mycologist/arbovirologist who also conducted important
work on JE, TBE, louping ill, and Langat and who infected terminally ill cancer
patients with Langat and Kyasanur Forest disease (KFD) in the hope of slowing the
course of their diseases (this vain hope had been based on promising data from
experimental animals (Webb et al. 1966)). In the United States, Chester M. Southam
(1919–2002) and Alice Elizabeth Moore (1906–?) conducted similar challenge
experiments, later to become controversial, with WNV and other arboviruses
(Southam and Moore 1954; Southam and Moore 1951).
8 Dengue and Other Hemorrhagic Fevers
As fatal DHF and DSS extended throughout Southeast Asia in the early-to-mid-
1950s, dengue was proving to be among the most fascinating and confusing of the
arboviral diseases. Until 1951, viral hemorrhagic fever was not a term in common
use and would have been thought of as largely synonymous with yellow fever, or
with atypical and complicated cases of smallpox, measles, Rocky Mountain spotted
fever, and other diseases. Crimean-Congo hemorrhagic fever virus had been iso-
lated in 1945 but was not then recognized as a major cause of epidemic disease. An
important “wake-up call” was the fatal 1951 epidemic of an allegedly new disease
called Korean hemorrhagic fever, which broke out during the Korean War (and was
shown decades later by Ho Wang Lee (1928–2022), Karl Johnson, and others to be
caused by a hantavirus (Lee et al. 1982)). Historical epidemics of hemorrhagic fever
associated with dengue-like illness had been published in the medical literature on
numerous occasions since the 1870s, including well-known epidemics in Athens-
Piraeus and in North Africa in 1928, but in the pre-virology era they had been
regarded as isolated “dengue-like” events of obscure origin.
When “Philippine hemorrhagic fever” first appeared in 1953, alarm bells
sounded. The appearance two years later of urban civilian hemorrhagic fever epi-
demics all over Southeast Asia indicated the emergence of an important new fatal
hemorrhagic disease syndrome. Hammon, Buescher, Halstead, Russell, and many
others begin studying this phenomenon in the late 1950s and early 1960s. Most of
these new hemorrhagic fevers were associated with dengue virus circulation.
Halstead and colleagues, working in Bangkok, distinguished dengue from chikun-
gunya as they co-circulated and caused clinically similar diseases, noting the strong
association between second dengue infections (determined by serologic patterns)

and greatly increased risk of DHF. Building upon the work of Hammon and others,
Halstead articulated and characterized a “second infection hypothesis” (Halstead
1988). The early stages of this important work are found in classical papers in the
American Journal of Tropical Medicine and Hygiene and in the Yale Journal of
Biology and Medicine (Halstead et al. 1969; Halstead 1970).
Looking further, Halstead focused on investigating why, in circumstances where
four dengue serotypes co-circulated and where all children got at least a first and a
second infection in early life, severe dengue disease seemed to occur predominantly
in association with a second infection. His work led him to examine and then study
in detail the phenomenon of antibody-dependent enhancement of viral infection, or
ADE (Halstead et al. 2010; Morens 1994), in which virus-IgG complexes infected
cells via Fc-receptor binding. Further epidemiologic work established and quanti-
fied the risk of second dengue infections and stratified risk by a sequence of sub-
types in the first and second infections, e.g., DENV 1 followed by DENV 2, DENV
3 followed by DENV 2, etc. (Sangkawibha et al., 1980). This important work is
explained in greater detail in Chap. 6.
In the beginning, Halstead’s theories were not universally credited. And it is curi-
ous that his data were less readily accepted by virologists than by epidemiologists –
Halstead was even invited by CDC to deliver 1981’s prestigious Alexander Duncan
Langmuir (1910–1993) Lecture on ADE (Halstead 1981) – at a time when many
virologists remained highly skeptical of a link between in vitro ADE and disease
pathogenesis. This may in part have been because virologists’ concepts of varying
viral virulence had been theorized about for many decades – both the increased
severity of the ostensible “second wave” of the 1918 influenza pandemic and of an
apparent second wave of severe dengue in Athens-Piraeus in 1928 – had long been
hypothesized to have resulted from increases in viral virulence during epidemic
viral circulation. Leon Rosen’s 1976 American Society of Tropical Medicine and
Hygiene Presidential Address on the subject (Rosen 1977) was viewed by some as
a skeptical point-by-point rebuttal of Halstead’s conclusions. Because Rosen, a pro-
tégé of Albert Sabin who had worked under another esteemed Sabin protégé, NIH’s
Robert Merritt Chanock (1924–2010), was a careful and accomplished virologist,
and rarely led astray, nor speculated without strong supporting data, Rosen’s nega-
tive views about Halstead’s interpretations persuaded many. Halstead only presented
a direct counter-argument to Rosen’s presidential address in 2022 – 46 years later.
9 Scientific Controversy: Scott Halstead and Leon Rosen
The differences of opinion between Rosen and Halstead – a viral virulence v. host
immunopathogenesis paradigm – was elevated by arbovirologists to mythical pro-
portions, reminiscent of the “Salk v. Sabin” controversy over polio vaccinology
15–20 years earlier. Like most myths, the facts never quite lived up, but arbovirolo-
gists became enthralled as publications and counter-publications appeared. It was
398 D. M. Morens
Fig. 5 Lē‘ahi Hospital, in the Kaimuki area of Honolulu, with the extinct Lē‘ahi volcano crater
(known to tourists as “Diamond Head”) in the background. Out of the picture, to the right, is
Waikīkī Beach, which I often gazed at as I looked out my third-floor office/lab window (upper left).
The Rosen lab began as the Pacific Research Section of the National Institute of Allergy and
Infectious Diseases, NIH. The Halstead lab represented the Department of Tropical Medicine and
Medical Microbiology of the University of Hawai‘i. The hospital had been built in 1902 in response
to important events such as the 1900 burning of Honolulu’s “Chinatown” during the plague epi-
demic, the need to isolate tuberculosis patients, and the national interest in tropical diseases that
occurred at the time of the Spanish-American War (1898). It later also housed patients with
Hansen’s disease
the most talked-about viral disease pathogenesis controversy of the era. Many who
were titillated by it seemed not to understand the complicated virologic, immuno-
logic, and epidemiologic issues at hand, but were nevertheless delighted to watch
the high-level dustup. It did not help that the labs of the two protagonists were in the
same building – Honolulu’s historic Lē‘ahi Hospital (Fig. 5), or that Scott’s labora-
tory was one floor up, its footprint directly over that of Leon’s.
I knew both men well. Ironically, neither sought to convince me of the other’s
“heresies.” And despite gentlemanly disagreements at meetings, I never heard either
say a truly unkind word about the other, even though each was sure the other was
wrong (gentlemanly restraint characterized the generation; I can think of few excep-
tions). For many years, working in the same building with the same cafeteria, exits
and entries, parking lots, etc., I do not know of anyone who ever saw Scott and Leon
having a conversation. At one point, perhaps around 1982 or so, I sat in the library
discussing the situation with Duane Gubler, who was then working closely with
Leon. We agreed that those of us in the “younger generation” needed to follow the
science and not get pushed into partisan disputes: in the end, we agreed; science
would reveal the truth, in all its complexities.
An amusing incident happened at the opening reception for the ASTMH annual
meeting in 1986, held in a crowded, dimly lit room with a large round floral center-
piece in the middle. Making the cocktail rounds, with a big floral obstruction in the
dim light, Scott and Leon were circling in opposite directions until each rounded the
corner and were suddenly face-to-face. Heads turned. Perhaps surprised, they
exchanged a few words of pleasantry which I did not overhear and then moved on.
It was the only time I ever saw them face-to-face.
A couple days later, Leon was speaking in an ACAV session and referred to his
1972 publication on an unusual epidemic of apparent primary DHF on the small,
virtually unknown Pacific island of Niuē (Barnes and Rosen 1974). He had made
the point that you do not always need a second infection to develop DHF; primary
dengue-associated DHF can occur too – arguably even at high frequency. When
Leon finished, Scott rose slowly to his feet, paused for an uncomfortably long time
― his standard theatrical flourish ― as all eyes turned his way. Softly and with
exquisite timing, Scott said: “Well…. I never believe anything I read from Niuē.”
Some years later, Scott and I and others published experiments suggesting that
both inherent viral virulence properties and ADE – together – were associated with
DHF (Morens et al. 1991), and later data were consistent with that notion. By 2021,
strong associations between DHF/DSS and secondary infections have been shown
countless times, as has an association with in vitro ADE. If not universal, the asso-
ciation has become so strong that few doubt it any longer. But much still remains to
be learned about the viral, immunologic, and epidemiologic variables associated
with severe dengue disease; a full reconciliation of Leon’s and Scott’s seemingly
opposing views may one day be agreed upon.
Working on dengue in the mid-1970s afforded me many opportunities to work
with, and get to know, some of the great arbovirologists of earlier generations. The
Caribbean epidemics beginning in late 1976 occupied much of my time at CDC
Atlanta and sent me packing on numerous trips to CDC’s San Juan Laboratory and
to sites in the Caribbean, particularly the US Virgin Islands (USVI). During the
USVI and Puerto Rico epidemics, I worked closely with CDC and other scientists,
most memorably Jack Woodall, Gladys Sather, Goro Kuno, Ernesto Ruiz-Tibén,
entomologists Chester G. (Chet) Moore and Donald A. Eliason, clinician Raúl
Hermanio López-Correa, and viral disease epidemiologists such as José Rigau-
Pérez (Morens et al. 1979; Morens et al. 1986).
The many trips and many days and weeks of field studies are a pleasant blur:
endless knocking on doors for questionnaires and blood draws, endless ice packing
and unpacking, endless afternoons and nights processing samples and running batch
serologies, and most of all the breakfasts with Jack and Chet and Raúl of eggs, cho-
rizos, tostones, and the garlicky vinegary hot sauce poured out of used catsup bot-
tles. Still sitting on my wet bar, to remind me of those days, is an unfinished bottle
of 151 proof Cruzan rum purchased in Charlotte-Amalie in early 1977 (Fig. 6). I
plan to finish it off when I grow up.
Those Caribbean nights involved dinners of local foods, most memorably an
ugly looking fish called oldwife that was fried in chili oil; rum-and-cokes that were
400 D. M. Morens
Fig. 6 Old St. Croix rum,

a memento of my
Caribbean pirate days with
Jack Woodall and friends,
during the dengue
epidemics of the late
1970s. A “fifth” of Cruzan
rum was then 79 cents a
bottle. Having forgotten to
parafilm the cap, this bottle
came unscrewed in my
briefcase during a flight
back to CDC Atlanta,
soaking and nearly
destroying all my
handwritten data and notes.
Fortunately, some of it
survived; 44 years later, a
few ccs still remain
dirt cheap because they were cut with Cruzan rum, which was much cheaper than
Coke; and wonderful boozy storytelling and joking. Jack was always the ringleader,
bemused, mischievous, bringing out the most extreme in the rest of us. When the
normally quiet Raúl, who lived with, and was devoted to, his mother, drank too
much, he got beet red and uncharacteristically loquacious. At one hilarious dinner
he got drunker and drunker and started excitedly telling a story about another time
he had drank too much, was talking with a woman, and somehow ended up in her
room to find her lathering whipped cream all over his bare body. We could never
decide what was funnier: the story itself, the shocked and horrified way in which he
told it, or our collective efforts to get him to stop telling it. We had additional comic
relief from public health adviser Alfredo Casta-Vélez, aka Freddy Casta, a can-do
guy who made everything happen and made sure it was also entertaining, an unfor-
gettable motley crew.
A similarly funny “Jack” story, involving Jack and myself at a Montego Bay
hotel, must remain untold, as Jack, a few years before his death, when his memory
was failing, felt sure it had never happened. Out of respect for Jack and because the
story is not exactly rated PG-13, the events must now remain only in my own mem-
ory. Too bad. Normally a happy, delightful, and usually delighted man, seemingly at
peace with himself and with the world, Jack, in quieter moments, could be intro-
spective. I remember sitting with him on a very long flight somewhere; he began
reminiscing about his childhood imprisonment in a Japanese internment camp in
China, where he had lived as a child of British missionaries. I remarked that it must
Fig. 7 Gladys E. Sather was an influential serologist who worked closely with Bill Hammon, Jack
Woodall, and others. In an era in which women were often marginalized, Gladys was a full co-
author on important papers and attended the major scientific meetings with the arbovirology lumi-
naries of the era, almost all of whom were men. They all respected her. In the above image,
probably from the 1960s, Gladys is seated among some of the movers and shakers, third from the
right, directly above Jordi Casals-Ariet, who is in the foreground
have been difficult, but he said no; it was OK; he was just a kid. Yet there was a
wistful, almost sad look on his face as he re-remembered it all. Many years later
when the film Empire of the Sun was released, I could not watch it without imagin-
ing Jack’s experiences. And now Jack is gone, very hard to accept. He was so life-
affirming, gentle, and unforgettable.
Gladys E. Sather (Fig. 7), who ran the CDC San Juan serology lab, also made a
lasting impression. She was then middle-aged and very “old school” and did not
hide her strong disapproval of a long-haired hippie-looking guy like me. Every time
she saw me, she gave me a look like a schoolmarm who could not wait to rap my
knuckles with a steel-edged ruler. And I had to see her at least once a day, when I
brought and unpacked the bloods and then stamped the accession numbers and
applied them to the tubes and questionnaires. But I took an interest in serology and
eventually got her to teach me the ins and outs of reading and interpreting HIs to
four different dengue antigens. As I spent more and more of my free time in the lab,
we gradually began to take a liking to each other. Finally, a breakthrough came,
when she entrusted me the keys to the lab so that she could go home, and I would
stay and finish the lab work. It was around midnight when I finished, locked the lab
door, and walked to the nearby hospital to find a cab. That was when I learned that
no cabs came out that way so late at night. I found a nearby park bench and spent
the night, peacefully, happily, under an approving moon.
402 D. M. Morens
William Carlisle Reeves (1916–2004). Few if any arbovirologist of the past

century has had as much of an impact on the field, via research, teaching, and
leadership, as Bill Reeves. Passionately interested in entomology since child-
hood, Bill got an undergraduate and then a PhD degree in entomology from
the University of California (UC) at Berkeley. Early in his career Bill was
mentored by ecologist-arbovirologist Karl Meyer at the Hooper Foundation
and by Meyer’s protégé Bill Hammon. Among the team that first isolated and
later characterized both WEE and SLE, Bill went on to a remarkably produc-
tive career in arbovirology, with myriad accomplishments that would take
pages to even list, and he became a key influence on three generations of
arboviral scientists. Like his early mentors Meyer and Hammon, Bill’s work
had a strong ecologic focus, i.e., understanding the complete ecosystem
within which arboviruses existed, not only insect and vertebrate hosts but also
reservoirs, amplifying hosts, bridging vectors, and the physical and climatic
environment. I have fond memories of Bill, including numerous conversations
about help he was eager to give me on this or that subject, as long as those
new-fangled computers and emails were not involved. Instead, he was happy
to exchange phone calls and extensive detailed hand-written letters to the end
of his life. He had a mischievous sense of humor and delighted telling stories
about the “good old days,” including many of his arbovirology friends who he
referred to as the “nutty buddies.” At one scientific meeting, he regaled the
audience with such stories, such as the time he coerced a very young Leon
Rosen to shinny dangerously high up a tree to collect arboreal mosquitoes
while he, the professor, yelled instructions from the ground. Bill’s seminal
role in starting ACAV is described in Chap. 3. Bill himself told the story with
an interesting anecdote about an argument between Albert Sabin and another
colleague. Finally, the normally mild-mannered Bill Reeves stepped in and
yelled “Albert, shut up!” Sabin did. Bill Reeves may have been the only per-
son to have ever shouted Albert Sabin into silence. Mycobacteriologist Jim
Douglas, who knew Bill in the 1960s, remembers how he revered his mentor
Karl Meyer, who was then in his 60 s and 70 s. Commenting on Bill’s incred-
ible depth and breadth of knowledge while still in his 20’s, Karl Johnson later
wrote that “It was as though a male Minerva had sprung from the forehead of
Zeus, fully formed and armored.” Johnson also offered a letter-perfect descrip-
tion of Bill’s infectiously loveable personality: “Staunch defender of princi-
ple, at times irascible, at times funny, always caring, forever… the pesky farm
child which refuses to enter the adult world.”
10 Other Group A and Group B Arboviruses
Also beginning in the 1950s, important previously identified arboviruses were better
characterized, including not only VEE in the Americas and EEE and WEE in the
Americas, but also WNV in the Middle East, MVE in Australia, and others. Several
Australian researchers worked on MVE, joined by Americans such as Reeves and

Anderson. In the mid-1950s, arbovirologists isolated and then characterized such new
viruses as Sindbis (Taylor, 1955, with Albert Rudnick [1922–2012], Hammon, and
Sather), Ilhéus (Taylor, Downs, and Pedro Galindo [1917–2007]), Oropouche
(Anderson, Downs, Leslie Percival Spence [1922–2021], and Thomas H. G. [Tommy]
Aitken [1913–2007]) (Anderson et al. 1961), Mayaro (Casals and Loring Whitman
[1904–1987] (Casals and Whitman 1957), Causey, Charles R. Anderson [1915–1984],
and Downs), and Bunyamwera (Robert Hutson Kokernot [1921–2016], Kenneth
C. Smithburn [1904–1974], Whitman, and many others).
Not well remembered today, Loring Whitman (1904–1987) and Robert Hutson
Kokernot (1921–2016) are nevertheless fascinating and important figures in
arbovirology. Loring Whitman was an adventurous ornithologist and medical
student who, in his 20 s, had had the good fortune to join one of the great
tropical adventures of the era, the legendary 1926–1927 Harvard Medical
African Expedition, a medical and biological survey of Liberia and the Belgian
Congo under the direction of tropical medicine expert Richard Pearson Strong
(1872–1948), accompanied by bacteriologist George Cheever Shattuck
(1879–1972), a 26-year-old Max Theiler, and others. Whitman served as
assistant ornithologist and photographer and wrote a two-volume diary of the
adventure. Although he worked in the 1930s on yellow fever jungle cycles and
in the 1940s on WEE, it was not until the 1950s that Whitman’s arbovirology
career bloomed upon joining the RF. Among his first efforts was isolation of
Mayaro virus with Casals (Casals and Whitman 1957). Over the next
10–15 years, he went on to work on isolating Guama virus and characterizing
various Group C viruses, including work with Bob Shope.
Kokernot was a charismatic veterinarian, a lifelong member of the Sons of

the Republic of Texas, who received an MD and, after WWII, signed up for a
10-year stint with the Rockefeller Foundation, posted to the Union of South
Africa and then to South America. He worked with Smithburn and others on
Germiston, BUN, SIN and RVF, WN, and most notably Wesselsbron, fol-
lowed by later work with RF naturalist Charles Brooke Worth (1908–1984),
Tel Work, and Lennette on viruses as diverse as SF, Spondweni, SLE, and
WEE. Later in his arbovirology career, while studying for a second degree in
Public Health (and his third doctorate), Kokernot worked with Cache Valley
and studied Mermet virus with a young Charlie Calisher, who along with
many others, have fond and often hilarious stories of this larger-than-life char-
acter. A playful, endlessly curious humanitarian, who idolized Albert
Schweizer, Kokernot was always motivated to help the weak and underprivi-
leged. After his time at the RF, he had a long academic career and in his spare
time compiled a five-volume opus of WWII love letters to his wife. Ever the
cheerful optimist, Kokernot died at age 94 years humming his favorite tune
“Oh What a Beautiful Mornin’” (from Rodgers and Hammerstein’s 1943
musical Oklahoma!).
404 D. M. Morens
The continental United States was not neglected in arbovirus discovery – new
interest in the long-problematic SLE led to a surge in research by Hammon and
Sather, Reeves, Lennette, and others and later extended by work of Causey and
Shope, Theiler, Aitken, Downs, engineer/epidemiologist Andries Hein (Dries)
Jonkers (1926–2008), Chamberlain, Work, Karl Johnson, and Galindo. The wide
geographic range of SLE was greatly extended across much of North, Central, and
South America and the Caribbean, and its complex ecology became better under-
stood. WEE was tackled even more energetically, first by Reeves and Sather and
then by Chamberlain and Lennette. At the same time, other investigators began to
work on EEE, probably prevalent in the United States since at least the 1830s, with
important contributions by Chamberlain and by Clarke. The alphavirus and flavivi-
rus encephalitides were shown to be spillover infections into dead-end hosts, associ-
ated with complex reservoirs, and often with amplifying hosts and bridging vectors.
Roy William Chamberlain (1916–2013) was a pioneer in medical entomology

who spent most of his professional life at CDC studying American arbovi-
ruses and also taking on various CDC leadership positions. His approach was
toward public health practicality, e.g., publication of a classical manual on
mosquito taxonomy designed to help vector control and other professionals,
studying the effects of insecticides on mosquitoes, and co-invention, with
entomologist William Daniel Sudia (1922–2020), of a small battery-powered
light trap. Roy was awarded the 1975 Richard Moreland Taylor Award. I knew
Roy when he was a Deputy Director under CDC’s Bureau of Laboratories
Director Roslyn Q. (Robbie) Robinson (?–1981) and I worked with him
closely in the 1970s. He was a wonderful man who helped introduce me to
arbovirology. I remember him as modest, generous, and quiet, but always
eager to help others and to teach. By then an upper-level manager, he was
nevertheless delighted to be directly involved in the Caribbean dengue epi-
demics that exploded in the mid−/late 1970s. As most of CDC’s vector experts
were then either in the Fort Collins or San Juan laboratories, Roy was the
point person at CDC Atlanta who, with CDC Director David Judson Sencer
(1924–2011), himself highly knowledgeable about arbovirology, coordinated
agency arbovirus efforts. He was the quiet man in the background who made
things happen without fuss.
11 Other Arboviruses
As all of the above events were unfolding, the Group C viruses were gradually being
better characterized, especially by the efforts of Casals, Causey, Whitman (Casals
and Whitman 1961), Shope, and Galindo. In early work, Shope, Whitman, Hammon,
Reeves, Sather, and poet-epidemiologist James O. Bond (1923–1999) began to char-
acterize another apparently new group or subgroup of viruses, the “California
group” (Hammon and Reeves 1952), discussed in Chap. 5. Haddow, Dick, and oth-
ers at Entebbe worked not only on yellow fever but also characterized such viruses
as Uganda S (Dick and Haddow 1952) and Zika (Dick et al. 1952). At the same
time, but independently, both Israeli scientists and Taylor, working in Egypt, were
conducting research on WNV (Taylor et al. 1956). By 1960, tick-borne arboviruses
were also being better characterized. Russian scientists of the era, including Mikhail
Petrovitch Chumakov (1909–1993), Anatoly Alexandrovich Smorodintsev
(1901–1986), and Dmitry Konstantinovich Lvov, were studying the still-confusing
“TBE complex,” including RSSE and Omsk hemorrhagic fever (first described in
1945). In India’s Mysore State, “monkey fever” outbreaks led to the isolation and
characterization of KFD by Work (Work et al. 1957) and others, including Harold
Trapido (1916–1991) and Jorge Boshell-Manrique (1903–1976). Beginning in
1959 in North America, Don McLean’s classic work characterized Powassan virus
and disease (McLean and Donohue 1959). Arbovirology just kept on expanding
in scope.
12 Hemorrhagic Fever Viruses
As noted, in 1950 the term “viral hemorrhagic fever” was not defined and would
have probably been used mostly to describe yellow fever. RVF had been known
since the early twentieth century but had not then been associated with hemorrhagic
fever. CCHF had been described in 1945 but was not a common epidemic disease.
(Chumakov et al. had ostensibly isolated the virus in 1944 (Chumakov et al. 1968),
but the first recognized isolate was the Congo virus isolation by Woodall et al., in
1967 (Woodall et al. 1967), reflecting Cold War controversy that spilled over into
the contentious naming of the virus as Crimean-Congo, rather than Congo-Crimean
hemorrhagic fever, a slight that Woodall never forgot). Although epidemics had
occurred earlier, KHF was not recognized as an entity until 1951. KHF was epide-
miologically characterized by US Army scientists, although the causative agent
remained unidentified until the late 1970s, when work by Lee, Johnson, and others
identified the etiologic Hantaan virus (Lee et al. 1982).
Robert Ellis Shope (1929–2004). Bob Shope was not only one of the most
influential arbovirologists of the last 50+ years, but also among the most
beloved. Humble, caring, unselfish, and inspirational, Bob had an almost mag-
ical gift of making you feel better about yourself, and the world, every time
you were in his presence. He seemed to live for the joy of giving to others.
Coming from an illustrious scientific family – in 1930 his RF-based father
Richard Shope (1901–1966) isolated the first influenza virus and became an
406 D. M. Morens
internationally recognized virologist, while his brother Tom became an impor-

tant clinical virologist – Bob started as a military scientist and then joined the
RF to train under Max Theiler. He was then posted to Belém to work with
Ottis and Calista Causey. There, Bob discovered and characterized over 50
arboviruses in 6 years (Lederberg et al. 1992). He then moved on to Yale
University and eventually to Galveston’s University of Texas Medical Branch
(UTMB), moving with the RF’s remarkable and important collection of
viruses, reagents, and information. He spent the rest of his life characterizing
arboviruses, was involved in the characterization of hemorrhagic fevers
including Lassa and Rift Valley fever, and with Alan Steere, he described
Lyme disease. With Nobel laureate Josh Lederberg and NIH scientists Stan
Oaks, Bob’s 1992 Institute of Medicine report on emerging infectious diseases
(Lederberg et al. 1992) was a game-changing look at infections that has
greatly influenced infectious disease science ever since, as well as research
and disease control programs of the disease CDC and the NIH. Bob was argu-
ably the sweetest, most selfless human being I ever met. He inspired more than
two generations of arbovirologists and had an enormous impact on the field.
Although, viewed in retrospect, what we now know as DHF had probably existed
since as early as the early 1870s, it had not been recognized as such. Following the
Korean War hantavirus epidemic, among the first to use the term “hemorrhagic
fever” in a generic sense was F. N. Quintos in 1954 to describe a new ongoing
Philippine hemorrhagic epidemic (Quintos et al. 1954), which turned out to be den-
gue. DHF epidemics were soon recognized over much of Southeast Asia, and the
term “hemorrhagic fever” was quickly adopted. When other new or newly appreci-
ated hemorrhagic fevers appeared, a new category of viral diseases was born, and it
would be globe-trotting arbovirologists who were best suited, and best positioned
geographically, and by existing collaborations, to characterize them, whether they
were arboviral or not.
In 1958, an outbreak of a new (since 1953) disease called Argentine hemorrhagic
fever led to isolation of Junin virus by Ignacio Pirosky (1901–1987) et al. and
Armando S. Parodi (1909–1969) et al. (Pirosky et al. 1959; Parodi et al. 1959). A
year later, an outbreak of another new disease called Bolivian hemorrhagic fever led
to isolation of a different agent by Karl Johnson and Patricia Ann Webb (1925–2005)
(Johnson et al. 1965; Webb et al. 1967), Shelokov, Ronald Boyce MacKenzie
(1924–1996), and Ned Harold Wiebenga (1925–1982). Johnson and Pat Webb
nearly died from the disease. In 1967, yet another new hemorrhagic fever, named
Marburg disease (Siegert et al. 1968), was related to importation of infected mon-
keys from Africa to the German university town, becoming the first filovirus identi-
fied (9 years before the first recognized Ebola outbreak). The final new important
hemorrhagic fever virus to be isolated in this decade was Lassa virus, associated
with an outbreak on Nigeria’s Jos Plateau, as described by John Davidson Frame
(1917–2008), Sonja M Buckley (1918–2005), Casals, Robert Wade Speir
(1928–1981), and Navy virologist Owen Wood (1936–2018) (Frame et al. 1970).
Buckley isolated the virus; Casals was infected by it in the laboratory but survived.
Other investigators had not been as lucky. RF arbovirologist Harald Norlin
Johnson (1907–1996), for example, developed a paralyzing neurologic disease
while working with rhabdoviruses. It was becoming clear that arbovirology, includ-
ing work with hemorrhagic fever and encephalitic viruses, was risky. ACAV’s
Subcommittee on Arbovirus Laboratory Safety (SALS) began looking more closely
at laboratory and specimen handling risks. BSL-4 laboratories came to be envi-
sioned even before safety classifications had been standardized.
Having worked on many hemorrhagic fever diseases, I had a chance to meet
some of the giants of the previous generation, including as mentioned Karl Johnson,
Scott Halstead, Jordi Casals-Ariet, and others. In 1976, during the first Ebola out-
breaks (simultaneously in Zaire and Sudan), Karl Johnson assembled a CDC team.
It was decided that in addition to Joe McCormick and Joel Breman, one EIS officer
would go as well. Only two of us volunteered; we flipped a coin and I lost. I was
devastated. But the coin toss-winning officer, who shall remain nameless here
(despite being identified in at least one book), had second thoughts about dying in
an East African jungle and bailed out upon landing with Karl in Genève. As a con-
solation prize, I got to be part of a small team, led by Pat Webb, that stayed in
Atlanta to “staff” the investigation, relaying information, sending items to the field,
taking notes from field calls, and so on.
It was not until 39 years later, in 2015, that I got on a plane to fly from NIH to an
Ebola epidemic, spending a month each in Guinée and Liberia. Those 2015 times
spent in West Africa brought back to mind my two years in CDC’s Lassa Fever
Research Station, in Kenema (Fig. 8), Sierra Leone. Although for me not an espe-
cially productive time scientifically, I had many formative, and more than a few
comical, experiences. The challenges associated with moving around daily to differ-
ent field sites, and to the missionary hospitals in Panguma and Segbwema – espe-
cially during the rainy season, when roads were washed out, rivers uncrossable, and
axles broken by deep potholes – were daunting. But the small expat community in
Kenema was fascinating, especially when pickled on scotch or the local Star Beer.
There were bankers, diamond dealers, a priest, a charming Mende woman of “spe-
cial talents,” a missionary doctor, a Vista worker, and an army of misfits who would
have been home in Europe had they been able to fit in there.
Retired British foreign service officer Reg Young plied us with the infamous ker-
osene-tasting Chinese firewater known as maotai – probably an excellent pesticide,
rodenticide, and larvicide, and definitely ulcerogenic – and claimed to have been on
the Long March with Mao Zedong, about whom he had many shocking tales. At one
point, our Lassa nurse and I decided to go the 200 miles to Freetown, where we
were “attached” to the US embassy and, while there, spend a night in the famous
City Hotel, where Graham Greene had lived while writing his 1948 classic The
Heart of the Matter. By 1979, the old Victorian hotel was in decadent disrepair: the
rooms were $3 a night, and the hotel girls $2 a night. No electricity, holes in the
walls, and rats. Even the Peace Corps kids would not stay there. Our room had a
circular 2-foot hole in the floor that looked down to the room below. It was beastly
hot, so we stripped down and climbed into bed with a torch.
408 D. M. Morens
Fig. 8 CDC’s Lassa Fever Research Station, Kenema, Sierra Leone, which is about 200 miles “up
country” from the capital of Freetown and, in those days, 200 miles away from the nearest phone.
Water and electricity were “iffy,” and we depended on a generator run on expensive diesel fuel to
keep the Revco’s going. My Volkswagen “beetle” is parked on the left, and one of the transport
vans on the right. Another vehicle, borrowed from a project of WHO scientist Arata Kochi, had
paintings on the body that read “Dr. Arata.” This caused hilarity among the kids, because arata is
the local word for “rat,” the very animal we were trapping, and because other rat species (i.e., not
Mastomys natalensis, the Lassa virus reservoir) were local food sources
As night fell, thousands of mosquitoes appeared. We pulled up and tucked in the

mosquito net, only to find it had hundreds of small holes. We spent almost 2 h tying
the little holes shut and around 10 PM fell asleep exhausted. Then we heard a noise
outside, then someone entered the room (of course, the doors had no locks – they
did not even close tightly). Turning on the torch we saw three young prostitutes.
Laughing and smiling, they approached us and started advertising their skills: ana-
tomically specific and hilariously X-rated. Afterward, we scratched our heads and
asked: what did they think was going to happen? Of course, we knew the answer:
just dash them so they would go away.
During our time there we often drove the 8 h down to Monrovia, Liberia, to visit
hepatitis virologist Alfred M. (Fred) Prince (1928–2011) at the Liberian Institute of
Biomedical research (LIBR). Fred had a chimpanzee colony and some roamed free,
while others, e.g., the juvenile males, had to be caged. One of the chimps went around
in diapers, and the head lab tech told us he often slept with her in her bed. Yikes. No, of
course we asked no questions. The main attraction, however, was the little offshore
peninsular beach where open-sided palm huts and hammocks had been set up. Small
groups of us would motor out for a few days with beer and provisions. The local Vai
kids would come out and sell us all varieties of seafood; a 2-foot-long lobster, for
example, cost about 25 cents. We would make a complicated bouillabaisse, eat until we
were stuffed, and drink, and when night fell, we would lie in the hammocks and listen
to the Atlantic waves crash on the sand only 10 feet away. Heaven.
Always with us in these adventures was Peace Corps volunteer lab tech Mariam
Boone. She loved Jack Daniels whiskey, and as a direct descendant of frontiersman
Daniel Boone, I suggested she write the Jack Daniels distillery in Lynchburg,
Tennessee, and tell them how much she loved their sour mash out here in the Heart
of Darkness. A couple months later, an enormous and shockingly expensive-to-ship
box of Jack Daniels swag arrived. I told Mariam she could have a month off to take
lorries up to Mali, but only if she brought me back a couple dung paintings. She did.
But when I later married, the new boss insisted the dung paintings go immediately
into long-term storage. When we divorced, some 20+ years later, the surviving one
came out of storage and now hangs in my room, reminding everyone that I do know
dung paintings from Shinola.
I had a Nikon camera set and took many photos of the local kids, who were joy-
ous and usually amazed to see “white people” or pumwes (Fig. 9). Throughout my
time in Kenema, I nursed our auto mechanic Nick T – through repeated bouts of
falciparum malaria, brought on by the inability to remember to take the chloroquine
I gave him. Nick was normally drinking from dawn to dusk, and I suppose that on
most days his blood alcohol level was high enough to kill all the parasites. Except
when they did not. When I left the country, I entrusted him with selling my car,
stereo, and household goods for a 20% commission. He stole everything and disap-
peared into the jungle.
Egypt’s 1977 Rift Valley fever outbreak also forever remains memorable. Initially
of unknown cause, I was sent from CDC to Cairo to attach to the Ministry of Health
and work with their scientists to characterize the epidemic. Going out of Atlanta,
none of us had any idea what the disease was. On the way to Egypt, I had to stop in
Washington, DC, to pick up my official passport (in those days, they were kept in
Washington). I phoned CDC and learned that literally moments before, someone
had received a call from Bob Shope at YARU: samples sent from Cairo by US Navy
virologist Jim Meegan indicated Rift Valley fever, a disease I had never seen and
knew less than nothing about.
The next six weeks were a whirl, and most of my work had to be censored. I
embedded with NAMRU-3 in Masr Al-Gedida, outside Cairo, and worked closely
with the Egyptian-American Navy team, including virologist Medhat Darwish, aca-
rologist Harry Hoogstraal (1917–1986), virologist Imam Zaghloul Imam, clinician
epidemiologist Larry Laughlin, virologist Jim Meegan, and Commanding Officer
Ray Watten (Fig. 3) who, with his delightful wife, Judy, supported us all like proud
parents. Harry’s field teams and Jim’s virology work were the key resources. Harry
himself was avuncular and quiet, directing his field team while sitting on a stool
puffing a cigar. One day he showed me a bound collection of all of his published
papers; I think there were over 800 of them, many with artistic drawings of ticks. He
was a proud collector, treasuring the papers and the memories they represented.
410 D. M. Morens
Fig. 9 Happy village kids, “up country,” Sierra Leone, 1979

I was housed in the Navy BAQ, an enormous luxurious house-sized hotel suite;
I had the place to myself until word came that USAMRIID was sending out an
Army major to bring existing doses of the experimental FRhL RVF vaccine. That
major was the even-then-legendary young, and even-then-larger-than-life, Major
Clarence J (CJ) Peters. He arrived at the BAQ on a Sunday afternoon: I opened the
door to meet a man in blue jeans under an official Army coat, an open can of cock-
tail nuts, an open bottle of Courvoisier brandy, and a painful case of epididymitis
that had come on during the long flight. Over the next few weeks, we had a tremen-
dous raucous time, much of it scatologically or otherwise unprintable. Throughout,
CJ had an endless collection of hilarious stories of almost-impossible-to-believe
escapades, mixed in with insightful knowledge of immunology and pathology. He
was a patient, if hilarious, teacher. We were both among the first to get the RVF shots.
An inveterate haggler, CJ and I went on multiday bargain hunts for things; after
his exhausting, theatrical bartering, each of us came home with two camel saddles,
supposedly from the historical World War II battle at Mersa Matruh (June 26–29,
1942). I still have mine, and I am sure CJ does too. I have spent the better part of
40 years trying to figure out what to do with those saddles, especially after my
then-wife banished them to the attic. My repeated trips to Egypt were capped off by
a memorable Thanksgiving feast at the home of Larry and Jane Laughlin, a magical
event that none of us present have ever forgotten, and which is often recalled these
decades later.
Shortly after I returned from Egypt and handed in my CDC epi-2 report, indicat-
ing that the vector was almost certainly Culex pipiens pipiens, based mostly on the
work of Harry’s field team plus my own epidemiology investigations, someone told
me that Bob Shope was visiting CDC and wanted to talk to me. Of course, I knew
who Bob Shope was; I had met his brother Tom Shope, a pediatrician in Michigan
where I had trained, I had read about his famous father Richard Shope, who had
isolated the first influenza virus in 1930, and I knew about Bob himself, already an
acknowledged leader in arbovirology. But I had no idea why he wanted to see me,
or how he even knew who I was. We met outside in a very small garden area in front
of the old CDC Building 1. Not knowing at the time that Bob was a kind and loving
soul, I was fairly intimidated. He had read my RVF report and wanted to know why
I thought the RVF vector was Culex pipiens pipiens and not Culicoides, as he had
been predicting. I barely knew what a Culicoides was, but carefully went through
my data, reasoning, and so on, including the fact that the village epidemics were so
explosive, with 90–95% human attack rates over periods of 1–3 weeks, and mos-
quito trappings and other efforts revealing almost no other vector than Culex pipiens
pipiens. Not exactly rocket science.
He listened politely, asked many questions, and then said perhaps I was right. I
was a bit embarrassed but, like almost anyone who ever met Bob, instantly fell
under his kindly spell. Decades later, when Bob was terminally ill with pulmonary
fibrosis, I spent a few days at UTMB. He was still coming into work at times, I guess
tying things up, knowing the end was approaching. Bob and I had half a day or so
together, just sitting in his office and chatting about things. Eventually, I had the
courage to say something like, “I know you won’t remember this, but when I came
412 D. M. Morens
Fig. 10 Two American Lassa fever researchers study the local language in the Kissy Mess Mess
area of Eastern Freetown, Sierra Leone, 1979
back from the Rift Valley outbreak you and I spoke….” He stopped me before I
could go any further and said yes (I paraphrase): “of course I remember. I was so
impressed with your work and what you said about it.” He then repeated that long-
ago conversation in startling detail. I was floored. Here I was with this great man,
who was near the end of his life, and he was talking not about his own life or his
many accomplishments, but only wanted to give me something to feel honored
about. Of course, there was nothing great or special about my work with Rift. It was
just that Bob believed that every human being was special in some way. That was
his gift. Later, I went back to my hotel room and cried. Soon thereafter Bob shape
was gone.
In short, there is much to be said for hanging around with the arbovirology and
the hemorrhagic fever crowd (Fig. 10).
13 Kuru
Beginning around 1955, Daniel Carleton Gajdusek (1923–2008), who had earlier
worked with VEE, started investigating a mysterious neurodegenerative disease,
called kuru, affecting only the Fore tribespeople in a remote area of Papua New
Guinée (Gajdusek et al. 1961). Early on, the epidemiology suggested an infection
and possibly an arboviral etiology. But Gajdusek’s work in the late 1950s and 1960s
identified a disease caused by a new class of infectious-like disease-causing agents:
prion proteins. These abnormally folded proteins induced other proteins to fold
abnormally, in a cascading fashion, causing tissue damage in a manner analogous

to, but distinct from, infectious agents such as viruses. For this work, Gajdusek was
awarded the 1976 Nobel Prize.
Carleton was an unforgettable character. If the adjective “cantankerous” had not
already been in the Oxford English Dictionary (OED), someone would have had to
invent it just to describe him. He was brilliant, opinionated, and charismatic, had a
bit of a wild and crazy look at times, loved a good fight, and picked the fights he was
best armed to win. For example, I once watched him bait students and young scien-
tists by challenging them to cite any data proving that human rabies vaccines (in
those days made in duck embryos) actually worked. It was of course, in a sense, a
parlor trick, because many people bit by rabid or allegedly rabid animals never got
rabies in the first place, but he delighted in tearing apart every study with criticisms
that could not be easily overcome. He relished it. After he died in exile, in Norway,
the niece of one of Carleton’s protégés wrote a mystical-fictional thinly veiled novel
based metaphorically on Carleton’s life. The protégé was virologist Ric Yanagihara;
Ric’s niece Hanya Yanagihara penned the Gajdusek-inspired 2013 novel The People
in the Trees and then followed it up with an international prize-winning A Little Life;
a new novel about a future viral pandemic will be published in 2022.
14 Other Important Efforts in Arbovirology, 1950–1970
14.1
Aedes aegypti Control and Eradication Efforts
In the background of investigation of new diseases and identifying/characterizing new

viruses, a can-do postwar optimism gripped American and much of the Western world.
In medical and scientific journals, the term “malaria eradication” was bandied about as
if it was right around the corner. It also seemed to many the right time to go back sev-
eral decades to successful programs in parts of South America and to extend them to
eradicate entirely Aedes aegypti mosquitoes from the Western Hemisphere.
After all, reasoning went, if Gorgas had eliminated Aedes aegypti from the
Panama Canal Zone and from Havana two generations earlier, without modern lar-
vicides or adulticides, it should be possible to do the same thing now, with modern
insecticides like DDT. An aggressive campaign, spearheaded by Fred L. Soper
(1893–1977) (Soper 1963), achieved significant early successes, but in the end
failed utterly, with Aedes aegypti eventually rebounding to previous levels. This
effort is discussed elsewhere, in Chap. 10. It is ironic that in the early twentieth
century, sanitarian George Albert Soper (1870–1948) had been similarly enthusias-
tic about eradicating hookworm, an effort that also failed completely. So too did
Aedes aegypti eradication, due to insecticide resistance, studied in the 1950s by
arbovirologists/entomologists such as Leigh Edward Chadwick (1904–1975), fail-
ure to sustain efforts, and eventually environmental damage caused by powerful
pesticides like DDT. In the 1950s and 1960s, optimism about mosquito eradication
faded and pessimism took over.
414 D. M. Morens
15 Viral Vectors, Reservoirs, and Ecology
Arbovirus research was making it clear that many newly discovered arboviruses
existed in rural or jungle cycles that sometimes spilled over into humans or other
mammals as dead-end hosts and that, with a few notable exceptions, humans were
neither reservoir nor amplifying hosts. Even dengue and yellow fever, which were
transmitted between humans by Aedes aegypti, had jungle cycles involving primates
and alternative vectors (Rudnick 1965), presumably representing the natural ecol-
ogy of these viruses before adaptation to Aedes aegypti. During the 1950s and
1960s, there was greater emphasis on insect vectors and ecologic characterization of
viral reservoirs, including the environment, avian and mammalian hosts, and ampli-
fying and bridging vectors. Important work in these areas were done by Karl Meyer,
the Causeys, Carpenter, Galindo, Trapido, Rudnick, and many others (Meyer 1953).
Karl Friedrich Meyer (1884–1974) and Jacques Meyer May (1896–1975)

were European-born/American-based scientists whose ideas about disease
ecology, zoonotic diseases, and environmental determinants of disease (Meyer
1953) were highly influential on arbovirologists of the era and were among
the first modern conceptualizations of what is now referred to as “One Health.”
The Swiss-born Meyer received a veterinary degree, then studied with Arnold
Theiler (1867–1936) in South Africa. At age 36 he came to the United States,
was appointed professor at UC Berkeley, and began working with Nobel lau-
reate George Hoyt Whipple (1878–1976) at the Hooper Foundation. In the
1930s, Meyer and colleagues isolated and were among those who character-
ized WEE, greatly influencing Meyer’s young protégé Bill Reeves. In an era
in which few top research scientists published as many as 100 papers in a
lifetime, Meyer published over 800 in virology, epidemiology, epizootiology,
and a host of other biomedical disciplines. His intellect and productivity
astounded all. He became among the most revered biomedical scientists of the
twentieth century. I never met Meyer, but Hawai‘i-based mycobacteriologist
Jim Douglas, who knew him in the 1960s, describes Meyer as tall and broad
shouldered, making a striking contrast to his protégé Bill Reeves, also tall,
who was at that time extraordinarily thin. The Switzerland-born Meyer had a
strong German accent, dressed expensively, belonged to an exclusive private
club, and drank “French 75s,” a cocktail mixture of champagne and brandy
named after a light field gun used by the French in WWI. Douglas also
remembers a story Meyer told about isolating WEE, in which he stalled off a
rancher with a dead horse in deep conversation after badgering Berkeley col-
league Stewart Harvey Madin (1918–2002; of MDCK cell line fame) to sneak
behind the barn and decapitate the horse in order to sneak the tissue back to
the lab. An old video recording of Meyer describing the same event survives,
but features altered details, recounted by Meyer as a swashbuckling striptease
adventure.
May was a French-born physician and tropical medicine researcher who

had worked in Thailand, Vietnam, Singapore, and Africa, before joining the
medical staff of exiled General Charles De Gaulle during WWII. He came to
the United States in 1948 as a medical geographer, a term that now roughly
corresponds to global health epidemiologist. Thereafter, his career focused on
disease ecology and nutrition. May’s 1959 book The Ecology of Human
Disease was an instant classic and remains so today. Though working entirely
independently, Meyer and May both promoted a “one health” focus, were
leading members of tropical medicine societies, and were regarded as impor-
tant thought leaders by arbovirologists of the era.
There was also renewed interest in “disease ecology,” as championed by vision-

aries like Karl Meyer and Jacques Meyer May (1896–1975). For example, examina-
tions of the role of deforestation in the epizootiology of Zika and other viruses were
notable, and the association of EEE with wetland management, forestry practices,
and suburban encroachment suggested that human arboviral diseases reflected the
relationship between man and the environment. In 1962, a remarkable aerial photo-
graphic examination of African forestry changes over a 30-year period – a forerun-
ner of today’s satellite imagery – suggested a possible link between human behavior
and arboviral diseases. All of these efforts congealed into an early conceptualization
of what in the modern era has been called “One Health.”
The modern idea of “One Health,” which CDC describes as “…a collaborative,
multisectoral, and transdisciplinary approach – working at the local, regional,
national, and global levels – with the goal of achieving optimal health outcomes rec-
ognizing the interconnection between people, animals, plants, and their shared envi-
ronment” and championed by figures like Karl Meyer, actually has a history that
stretches far back in time and is particularly associated with virologists and epidemi-
ologists such as Rudolf Virchow (1821–1902) and Theobald Smith (1859–1934). In
more recent times many other American scientists have shaped and expanded upon
the idea including parasitologist Calvin W. Schwabe (1927–2006), veterinary epide-
miologist James H. Steele (1913–2013), epidemiologist Myron G. (Mike) Schultz
(1935–2016) (Morens and Chitale 2019), and many practicing arbovirologists such
as Don Burke, Tom Monath, and Fred Murphy, to name only a few.
16 Women in Arbovirology
Before the 1940s, it was far less common than today for women to enter the work-
force. That began to change incrementally during WWI and even more so during
and after WWII. Women had long been a distinct minority in colleges, especially in
doctoral and medical programs. Although the road to professional attainment had in
theory been open to women since the 1870s, there were steep social barriers and
prejudices; advancement of women was usually strictly, if unofficially, limited in
virtually all male-dominated professions (i.e., the majority of professions). For
416 D. M. Morens
Fig. 11 Delphine Harriet Clarke (1912–1985; left); Sonja M. Buckley (1918–2005; right). Clarke
and Buckley were important laboratory-based scientists who helped establish the serologic and
tissue culture techniques that underlie arbovirology and, indeed, all of virology
example, women professors almost never became department chairs. Things had
finally begun to change around the time of World War I, the war coinciding in time
with women’s suffrage as well as an urgent need to backfill academic and scientific/
medical positions of men who went to war.
It is of interest that among the earliest champions of women scientists in this era had
been Simon Flexner (1863–1946) at the Rockefeller Foundation/University. Flexner
found and promoted the careers of talented women scientists such as bacteriologist
Martha Vollstein (1868–1939) and a decade later bacteriologist Rebecca Craighill
Lancefield (1895–1981), who is remembered today for having classified the human
streptococci (Lancefield was fortunate to have had Flexner as a mentor; her PhD advi-
sor, Hans Zinsser, would not allow her into the lab, on the grounds that women did not
belong there). Lancefield and many other women scientists became recognized as
being among America’s top biomedical scientists. Even in the earliest decades of the
American Society of Tropical Medicine and Hygiene, days when few women reached
professional scientific or leadership roles, women constituted about 5% of the society’s
membership, a number that seems far too low by today’s standards, but which never-
theless represented early events in the breaking of long-held obstacles.
When the Rockefeller Foundation virus program was established in 1951, accom-
plished women scientists were included from the beginning. Delphine Clarke and Sonja
Buckley (Fig. 11) focused on antigenic characterization of viruses, serology, and the new
technique of tissue culture. Their work was essential to progress in arbovirology. Calista
Causey, who worked as a coequal partner with her scientist-husband Ottis Causey,
though not independently funded by the RF, was also highly regarded as an
arbovirologist.
Fig. 12 Pat Webb, working at CDC’s Lass Fever Research Station in Kenema, Sierra Leone, 1979.
Pat was fearless, tenacious, hardworking, and extraordinarily well organized. She lived with two
very large hounds, smoked a pipe, and drank Johnny Walker Red whiskey straight from the bottle
Gladys Sather worked closely with Bill Hammon for over a decade and later
worked with Jack Woodall at CDC’s San Juan Laboratory. These men thought
highly enough of her work to include her as co-author on countless papers and to
make sure she had a prominent role at professional meetings (Fig. 7). Other impor-
tant women scientists include Nathalie Joan Schmidt (1928–1986) who worked
closely with Ed Lennette, and Pat Webb, mentioned previously with respect to her
work with hemorrhagic fevers.
It is difficult from the vantage point of 2021 to evaluate the role of women arbo-
virologists in the era of the 1950s and 1960s, a time when most women married
early and had children, making a professional career difficult. Despite the successes
of women like those mentioned above, it should also be noted that even at the end
of the 1960s, few women scientists were given leadership positions, and until the
time of Calista Causey and Pat Webb (Fig. 12), women arbovirologists were rele-
gated to the lab, being almost invisible in fieldwork. After Calista Causey, who
418 D. M. Morens
worked in partnership with her husband Ottis Causey, Pat Webb was perhaps arbo-
virology’s first independent field investigator of note. More about women scientists
in arbovirology can be found in Chap. 1.
As a teenager, Patricia Ann Webb (1925–2005), born in Cambridge,

England, to an American father and English mother, escaped the Nazi Blitz in
1940 to come to America. She graduated from Tulane medical school in 1950;
during pediatrics residency in California, she found herself in the middle of a
large highly fatal WEE epidemic. She saw the tragic results and was greatly
influenced by them. From 1955 to 1961, Pat worked on various infectious
diseases, with Bob Shope, Gordon Smith, and others, at Joe Smadel’s recently
established US Army Medical Research Unit in Kuala Lumpur. In 1961, Pat
entered the US Public Health Service, assigned to NIAID’s Laboratory of
Infectious Diseases, working with Bob Chanock and Karl Johnson (who
would later become Pat’s second husband). In 1962–1963 Pat and Karl were
posted to MARU where they studied multiple diseases, most notably Bolivian
hemorrhagic and the causative Machupo arenavirus, and contributed to the
establishment of the new viral taxonomic group. During their work, both Pat
and Karl survived potentially fatal cases of Bolivian hemorrhagic fever. In
1975 Pat and Karl transferred to CDC where Karl became founding director
of the Bureau of Laboratories’ Special Pathogens Branch, dealing with hem-
orrhagic fever viruses like Machupo and Lassa.
When Ebola first emerged in fall 1976, Karl led an outbreak investigation team
to Africa while Pat stayed in Atlanta to organize logistical and technical support
for the field work. I was part of this small support team, the first time I had worked
side by side with Pat. Three years later, by which time Pat had been posted to
CDC’s Lassa Fever Research Laboratory in Kenema, Sierra Leone, I joined her
for two years of epidemiologic and virologic work, as well as clinical trials of
ribavirin and of immune plasmas. Pat was an extremely talented field researcher
(a skill in short supply) capable of keeping a large complicated operation – with
33 employees, two remote field hospitals, and numerous village studies, all
200 miles from the nearest telephone – running. And this was in the face of end-
less research challenges, not to mention the daily challenges of broken Land
Rover axles, blown Revcos, washed-out roads, arranging air shipments of dry ice
from Europe, searches for diesel fuel to run the generators, etc. Pat was like an
orchestra director expertly conducting from a chaotic score. She was personally
fearless and relentless in her drive to accomplish all tasks large and small, and she
smoked a pipe (Fig. 12) like an expert. Her chief companions during those years
in a lonely remote outpost were two large dogs and Smithsonian research mam-
mologist John Krebs, with whom she enjoyed Johnny Walker Red scotch whiskey
(drunk straight out of the bottle), private meals, and banter about expatriate life.
When Pat died in 2005, Karl and Tom Monath penned a memorable obituary
(Monath and Johnson 2005) that spoke of her many accomplishments and
her important work over five decades. A detailed account of women in
arbovirology can be found in Chap. 1.
17 Final Thoughts
The world of arbovirology today, in 2021, is a vastly different world than it was in
1950, 73 years ago. Many new tools and approaches have revolutionized discovery
and characterization of viruses and their diseases. For example, in 1950, the struc-
tures of DNA and RNA were unknown; today, whole genomes of viruses can be
sequenced in almost any lab, at minimal cost. Genomics has allowed a whole new
way of looking at virus relationships and has both refined and complicated viral
taxonomy. Many vaccines against arboviruses have been used to control diseases
such as dengue, JE, TBE, Ebola, and others. Women scientists now play a promi-
nent role in arbovirology research and work as equals with their male counterparts.
The study of arboviruses has also had important spillover into other areas of medi-
cine and public health, with arbovirus researchers making important contributions
to the eradication of smallpox, the control of polio, measles, and many other human
and veterinary diseases.
I started my career in arboviral diseases when I entered CDC as an EIS officer in
1976 (Fig. 13). In addition to the few comments and anecdotes mentioned above, I
have had the pleasure of working in many other countries including Argentina,
Cambodia, Chile, France, Guinée, Indonesia, Liberia, Malaysia, Mexico, Israel,
The West Bank, Russia, Sierra Leone, Singapore, Thailand, Venezuela, Vietnam,
and probably adhere I have forgotten. All the US territories and commonwealths,
many Pacific island nations, and other places. I have worked with many wonderful
foreign scientists from whom I have learned much. Chile’s Pablo Vial taught me
about hantavirus pulmonary syndrome and Pablo Neruda; Indonesia’s Suharyono
helped me study dengue in Medan and Yogyakarta and took me on an around-the-
island culinary tour of Java that included delicacies fit to nauseate Gordon Ramsay.
The number two wife of Sierra Leone village Chief S--------, fed me rice chop for
4 months while I worked there, just before the chief was arrested for cannibalism;
Lev S. Sandakhchiev (1937–2006) helped me and a small team of Americans and
Europeans “pacify” Russia’s Koltsovo-based Vektor (former bioweapons)
Laboratory, at the end of the Cold War; José Cardier, who worked with Alan
Rothman on dengue vascular involvement in Venezuela, dragged me up into the
mountains to feast on chigüiro, a 100-pound rat, topped off with a sauce made from
decomposed fermented ants; and Jeremy David Kark (1943–2018) of the Israeli
Defense Force helped me study Rift Valley fever while taking me on historical,
archaeological, and cultural tours around Israel and the West Bank. And hun-
dreds more.
Over more than 5 decades I have had the fortune to know and to learn from many
great men and women scientists, public health workers, and unforgettable charac-
ters around the world. I cannot imagine a better road to have taken.
420 D. M. Morens
Fig. 13 The author at his CDC desk in the fall of 1976, a new EIS officer hoping to be able to
study dengue, a disease he had never seen. Sometimes wishes come true
References
Anderson CR, Spence L, Downs WG, Aitken TH (1961) Oropouche virus: a new human disease
agent from Trinidad, West Indies. Am J Trop Med Hyg 10:574–578
Barnes WJ, Rosen L (1974) Fatal hemorrhagic disease and shock associated with primary dengue
infection on a Pacific island. Am J Trop Med Hyg 23:495–506
Buescher EL, Scherer WF (1959) Ecologic studies of Japanese encephalitis virus in Japan.
IX. Epidemiologic correlations and conclusions. Am J Trop Med Hyg 8:719–722
Carey DE (1971) Chikungunya and dengue: a case of mistaken identity? J Hist Med Allied Sci
26:243–262
Casals J (1963) Relationships among arthropod-borne animal viruses determined by cross-
challenge tests. Am J Trop Med Hyg 12:587–596
Casals J, Whitman L (1957) Mayaro virus: a new human disease agent. I. Relationship to other
arbor viruses. Am J Trop Med Hyg 6:1004–1011
Casals J, Whitman L (1961) Group C, a new serological group of hitherto undescribed arthropod-
borne viruses. Immunological studies. Am J Trop Med Hyg 10:250–258
Chumakov MP, Butenko AM, Shalunova NV, Mart’ianova LI, Smirnova SE, Bashkirtsev IuN,
Zavodova TI, Rubin SG, Tkachenko EA, Karmysheva Via, Reĭngol’d VN, Popov GV,
Savinov AP (1968) [New data on the viral agent of Crimean hemorrhagic fever]. Voprosy
virusologiĭ 13:377
Clarke DH, Casals J (1958) Techniques for hemagglutination and hemagglutination-inhibition
with arthropod-borne viruses. Am J Trop Med Hyg 7:561–573
Dick GW (1953) Epidemiological notes on some viruses isolated in Uganda; Yellow fever, Rift
Valley fever, Bwamba fever, West Nile, Mengo, Semliki forest, Bunyamwera, Ntaya, Uganda
S and Zika viruses. Trans R Soc Trop Med Hyg 47:13–48
Dick GWA, Haddow AJ (1952) Uganda S virus. A hitherto unrecorded virus isolated from mos-
quitoes in Uganda. (1). Isolation and pathogenicity. Trans Roy Soc Trop Med Hyg 46:600–618
Dick GWA, Kitchen SF, Haddow AJ (1952) Zika virus. (1). Isolations and serological specificity.
Trans Roy Soc Trop Med Hyg 46:509–520
Downs WG (1982) The Rockefeller Foundation virus program: 1951–1971 with update to 1981.
Annu Rev Med 33:1–29
Downs WG, Anderson CR, Spence L, Aitken TH, Greenhall AH (1963) Tacaribe virus, a new
agent isolated from Artibeus bats and mosquitoes in Trinidad, West Indies. Am J Trop Med
Hyg 12:640–646
Farley J (2004) To cast out disease: a history of the international health division of the Rockefeller
Foundation (1913–1951). Oxford University Press, New York
Frame JD, Baldwin JM Jr, Gocke DJ, Troup JM (1970) Lassa fever, a new virus disease of man
from West Africa. I. Clinical description and pathological findings. Am J Trop Med Hyg
19:670–676
Gajdusek DC, Zigas V, Baker J (1961) Studies on kuru. III. Patterns of kuru incidence: demo-
graphic and geographic epidemiological analysis. Am J Trop Med Hyg 10:599–627
Halstead SB (1970) Observations related to pathogenesis of dengue hemorrhagic fever.
VI. Hypotheses and discussion. Yale J Biol Med 42:350–362
Halstead SB (1981) The Alexander D. Langmuir Lecture. The pathogenesis of dengue. Molecular
epidemiology in infectious diseases. Am J Epidemiol 114:632–648
Halstead SB (1988) Pathogenesis of dengue: challenges to molecular biology. Science 239:476–481
Halstead SB, Scanlon JE, Umpaivit P, Udomsakdi S (1969) Dengue and chikungunya virus infec-
tion in man in Thailand, 1962–1964. IV. Epidemiologic studies in the Bangkok metropolitan
area. Am J Trop Med Hyg 18:997–1021
Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM (2010) Intrinsic antibody-
dependent enhancement of microbial infection in macrophages: disease regulation by immune
complexes. Lancet Infect Dis 10:712–722
Hammon WM, Reeves WC (1952) California encephalitis virus. A newly described agent.
California Med 77:303–309
Hayashi VM (1934) Ubertragung des virus von encephalitis epidemica auf affen. Proc Imp Acad
Tokyo 10:41–44
Johnson KM, Wiebenga NH, Mackenzie RB, Kuns ML, Tauraso NM, Shelokov A, Webb PA,
Justines G, Beye HK (1965) Virus isolations from human cases of hemorrhagic fever in Bolivia.
Proc Soc Exp Biol Med 18:113–118
Konishi E, Kuno G (2013) In memorioam: Susumu Hotta (1918–2011), Emery Infect Dis
2013:843–844
Kubes V, Ríos FA (1939) The causative agent of infectious equine encephalomyelitis in Venezuela.
Science 90:20–21
Lederberg J, Shope RE, Oaks SC (1992) Emerging infections: microbial threats to health in the
United States. National Academies Press, Washington, DC
Lee HW, Baek LJ, Johnson KM (1982) Isolation of Hantaan virus, the etiologic agent of Korean
hemorrhagic fever, from wild urban rats. J Infect Dis 146:638–644
422 D. M. Morens
Lumsden WH (1955) An epidemic of virus disease in Southern Province, Tanganyika Territory, in

1952–53. II. General description and epidemiology. Trans Roy Soc Trop Med Hyg 49:33–57
McLean DM, Donohue WL (1959) Powassan virus. Isolation of a virus from a fatal case of
encephalitis. Can Med Assoc J 80:708–711
Meyer KF (1953) Vectors and reservoirs of virus diseases. Am J Trop Med Hyg 2:757–770
Monath TP (1991) Yellow fever: Victor, Victoria? Conqueror, conquest? Epidemics and research in
the last forty years and prospects for the future. Am J Trop Med Hyg 45:1–43
Monath TP, Johnson KM (2005) In memoriam. Patricia Ann Webb (1925–2005). Arch Virol
150:1268–1270
Morens DM (1994) Antibody-dependent enhancement of infection and the pathogenesis of viral
disease. Clin Infect Dis 19:500–512
Morens DM, Chitale R (2019) In memoriam: Myron Gilbert Schultz (1935–2016). Emerg Infect
Dis 25:1617–1619
Morens DM, Woodall JP, López-Correa RH, Sather GE, White ME, Chester TJ, Moore CG,
Kappus KD (1979) Dengue in the United States Virgin Islands and cases imported to the con-
tinental United States of America, 1977. In: Dengue in the Caribbean, 1977. Pan American
Health Organization, Washington, DC, pp 75–82
Morens DM, Rigau-Pérez JG, López-Correa RH, Moore CG, Ruiz-Tibén EE, Sather GE, Chiriboga
J, Eliason DA, Casta-Velez A, Woodall JP (1986) Dengue in Puerto Rico, 1977: public health
response to characterize and control an epidemic of multiple serotypes. Am J Trop Med Hyg
35:197–211
Morens DM, Marchette NJ, Chu MC, Halstead SB (1991) Growth of dengue type 2 virus isolates
in human peripheral blood leukocytes correlates with severe and mild dengue disease. Am J
Parodi AS, Rugerio HR, Greenway DJ, Mettler NE, Martinez A, Boxaca M, Barerra JM (1959)
Aislamiento del virus Junin (FHE) de los acaros de la zona epidemica (Echinolaelaps echidoni-
nus, Berlese). Presse med Argent 46:2242–2244
Pinto SO (1955) Paper 6. Eradication of the Aedes aegypti mosquito from the Americas. In:
Boshell-Manrique J (ed) Yellow fever, A Symposium in Commemoration of Carlos Juan
Finlay. The Jefferson Medical College of Philadelphia, Philadelphia, pp 39–59
Pirosky I, Zuccarini J, Molinelli EA, Di Pietro A, Barrera-Oro GB, Martini P, Copello AR
(1959) Virosis hemorragica del Noroeste Bonnaerense. Instituto Nacional de Microbiologica,
Buenos Aires
Quintos FN, Lim LE, Juliano L, Reyes A, Lacson P (1954) Hemorrhagic fever observed among
children in the Philippines. Philipp J Pediatr 1954(3):1–19
Reeves WC (1987) The discovery decade of arbovirus research in western North America,
1940–1949. Am J Trop Med Hyg 37(3 Suppl):94S–100S
Rosen L (1977) The Emperor’s New Clothes revisited, or reflections on the pathogenesis of dengue
hemorrhagic fever. Am J Trop Med Hyg 26:337–343
Ross RW (1956) The Newala epidemic. III. The virus: isolation, pathogenic properties and rela-
tionship to the epidemic. J Hyg (Lond) 54:177–191
Rudnick A (1965) Studies of the ecology of dengue in Malaysia: a preliminary report. J Med
Entomol 2:203–208
Sangkawibha N, Rojanasuphot S, Ahandrik S et al (1984) Risk factors in dengue shock syndrome:
a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol
120:653–669
Shope RE (1998) A history of arbovirology in Brasil: Belém, 1954–1965. In: Travassos de la Rosa
APA, Vasconcelos PFC, Travassos de la Rosa JFS (eds) An overview of Arbovirology in Brasil
and neighboring countries. Instituto Evandro Chagas, Belém, pp 13–17
Shope RE, Causey OR (1962) Further studies on the serological relationships of group C arthropod-
borne viruses and the application of these relationships to rapid identification of types. Am J
Siegert R, Shu HL, Slenczka W (1968) Isolierung und Identifizierung des, Marburg-Virus”. Dtsch
Med Wochenschr 93:604–612
Soper FL (1963) The elimination of urban yellow fever in the Americas through the eradication of
Aedes aegypti. Am J Public Health Nations Health 53:7–16
Southam CM, Moore AE (1951) West Nile, Ilhéus, and Bunyamwera virus infections in man. Am
Southam CM, Moore SE (1954) Induced viral infections in man by the Egypt isolates of West Nile
virus. Am J Trop Med Hyg 3:19–50. 18AA
Strode GK (ed) (1951) Yellow fever. McGraw-Hill
Taylor RM (1962) Purpose and progress in cataloguing and exchanging information on arthropod-
borne viruses. Am J Trop Med Hyg 11:169–174
Taylor RM, Work TH, Hurlbut HS, Rizk F (1956) A study of the ecology of West Nile virus in
Egypt. Am J Trop Med Hyg 5:579–620
Theiler M, Downs WG (1973) The arthropod-borne viruses of vertebrates: an account of the
Rockefeller Foundation Virus Program, 1951–1970. Yale University Press, New York
Webb HE, Wetherly-Mein G, Gordon Smith CE, McMahon D (1966) Leukemia and neoplastic
processes treated with Langat and KFD: a clinical and laboratory study of 28 patients. BMJ
29:258–266
Webb PA, Johnson KM, MacKenzie RB, Kuns ML (1967) Some characteristics of Machupo virus,
causative agent of Bolivian hemorrhagic fever. Am J Trop Med Hyg 16:531–537
Williams MC, Woodall JP, Gillet JD (1965) O’nyong-nyong fever: an epidemic virus disease in
East Africa. VII. Virus isolations from man and serological studies up to July 1961. Trans Roy
Soc Trop Med Hyg 59:186–197
Wisseman CL, Sweet BH, Rosenzweig EC, Eylar OR (1963) Attenuated type 1 dengue vaccines.
Woodall JP, Williams MC, Simpson DI (1967) Congo virus: a hitherto undescribed virus occurring
in Africa. II. Identification studies. East Afr Med J 44:93–98
Work TH, Trapido H, Murthy DP, Rao RL, Bhatt PN, Kulkarni KG (1957) Kyasanur forest disease.
III. A preliminary report on the nature of the infection and clinical manifestations in human
beings. Indian J Med Sci 11:619–645
Young NA, Johnson KM (1969) Antigenic variants of Venezuelan equine encephalitis virus: their
geographic distribution and epidemiologic significance. Am J Epidemiol 89:286–307
Twenty-Seven Years of Field Studies
on Dengue and Aedes aegypti in Latin
America
Amy C. Morrison
Abstract This chapter provides a chronology and partial memoir of the career of
Amy C. Morrison over the previous 27 years conducting research on dengue virus
transmission dynamics and its vector Aedes aegypti in Puerto Rico and Thailand and
22 years in Peruvian Amazon in Iquitos, Peru. She describes how the influences of
prominent arbovirologists and medical entomologists as well as good timing and
luck led her to focus on Ae. aegypti. For young scientists, it is an example of how
networking and prior experiences lead to new opportunities. After a PhD on the
bionomics of the sand fly vector of American visceral leishmaniasis at the Yale
Arbovirus Unit, she moved to the Centers for Disease Control and Prevention
Dengue Branch for her first postdoc, where she published “Exploratory space-time
analysis of reported dengue cases during an outbreak in Florida, Puerto Rico,
1991–1992,” the first use of spatial statistical analysis on the spatial distribution of
dengue cases and met Tom Scott with whom she would work with for 20 years in
Iquitos. This study and approach would become a key component of the first NIH
grant she and Tom wrote that initiated the Iquitos Project Dengue Research Program
and collaboration with the US Navy. From 1998 to 2019, Dr. Morrison has con-
ducted eight large-scale longitudinal cohort studies of which four were vector con-
trol intervention trials measuring the impact of the intervention on virus transmission.
Rather than focusing on all the scientific contributions of this productive program,
she describes some of the day-to-day activities of the Aedes aegypti survey team,
including some personal stories, which remained intact until 2019 when they com-
pleted their work on a cluster randomized control trial (cRCT) determining the pro-
tective efficacy of a spatial repellent product against Aedes-borne viral disease, the
largest and most complex project executed by the team. She describes many of the
challenges associated with working in the field including the fact that all her trials
have had to account for emergency vector control activities carried out by govern-
ment programs. The program has been able to develop a strong relationship with
their government counterparts to coordinate research and control activities. She
describes the integration of qualitative research and its value to the overall research
program. She concludes with a description of her role as an external advisor on
A. C. Morrison (*)
University of California, Davis, Davis, CA, USA
426 A. C. Morrison
dengue and Aedes aegypti and some of the key lessons learned from many years of
field work.
1 Introduction
In this chapter I hope to share some of the scientific and personal lessons I have
learned carrying out research on dengue and Aedes aegypti for over 27 years. I
started as an American Society of Microbiology (ASM) Postdoctoral Fellow for the
Centers for Disease Control and Prevention (CDC), Dengue Branch, located in San
Juan, Puerto, and then after a 2-year layover on the UC Davis campus, I moved to
Iquitos, Peru, in late 1998, living there full time ever since except for 14 months
during the COVID-19 pandemic when I found myself “trapped” outside of the city.
For my 22 years in Iquitos, I have been associated with the US Naval Medical
Research Unit No. 6 in one way or another. The Iquitos Research Program, which I
will discuss, has had many participating institutions, but the core has been a series
of National Institutes of Health, Bill and Melinda Gates Foundation, Wellcome
Trust, and WHO/TDR projects that complemented a strong base of Department of
Defense (DOD) funding streams that included the Military Infectious Disease
Program, Global Emerging Infectious Disease Surveillance (GEIS), Defense Threat
Reduction Agency (DTRA), and Deployed Warfighter Protection program. Our
projects were always large in scale and with few exceptions 3–7 years in duration.
Our outstanding Iquitos Field Team could manage multiple projects simultaneously,
and as the years moved on, they were consistently asked to accomplish more in less
time, and they always stepped up to any challenge I could throw their way. At the
time of this publication, Iquitos research activities have been suspended due to the
COVID-19 pandemic. As a field researcher I have been exceptionally fortunate to be
well funded and have an incredibly supportive supervisor/mentor in Thomas
W. Scott and a hard-working and productive Peruvian field team that became my
family. I believe my academic position is unique in that it allowed me to be based at
a field site for so long; for this reason, it is important to note that my personal expe-
rience and the Iquitos program may not be representative of other field-based
research programs. It is my impression that, especially for my Peruvian and other
international colleagues, they have had to conduct field studies with minimal
resources or at least less than I have typically had, and I would argue they
deserve more.
Before I begin, it is important that I acknowledge some important experiences
and people who contributed to my formation as a field researcher. I began my aca-
demic career as a determined premed who accidentally and fortunately landed in an
MPH program at the UCLA School of Public Health. My thesis advisor, A. Ralph
Barr, was a renowned medical entomologist, and I had numerous classes with
Telford Work, a pioneering arbovirologist. Both conducted field studies and brought
their activities to life. Growing up in southern California, I participated in multiple
outdoor activities (bagging peaks in the Sierras) but had never considered that I
Twenty-Seven Years of Field Studies on Dengue and Aedes aegypti in Latin America 427
could study diseases and be outside at the same time. My infectious disease epide-
miology program consisted of a tight-knit group of future Navy scientists with
whom I would work or interact professionally with in the future. We were the UCLA
Mafia: Mike Bangs, Stan Cope, Mike Medina, and Ellen Andersen, among others.
In 1984, the final year of my MSPH program, Los Angeles experienced a small
urban Saint Louis encephalitis (SLE) epidemic and our research group carried out a
series of studies on Culex quinquefasciatus and C. tarsalis in the San Joaquin marsh
during the summer of 1985 (Bangs et al. 1986; Barr et al. 1986; Cope et al. 1986). I
carried out nocturnal human-bait collections and learned to dissect ovaries out of
mosquitoes for parity determinations and drive an old land cruiser with a manual
transmission (the fence around the marsh still has not recovered). These were all
skills that served me well during field studies on mosquitos and sand flies that I
participated in as a Peace Corps volunteer in Honduras between 1986 and 1989.
With some colleagues from the National University, I drove a Peace Corps land
cruiser only available once a month during the weekend to Choluteca to collect
Lutzomyia longipalpis (Carrasco et al. 1998; Ponce et al. 1991). We were very short
on resources but had lots of enthusiasm, learning how to ask and answer questions
in the field despite making quite a few mistakes. From that point on, I was hooked.
I found a doctoral program and advisor (Robert Tesh) who facilitated my disserta-
tion work on Lu. longipalpis in Colombia (Ferro et al. 1995a, b, 1997; Morrison
et al. 1993b, 1995a, b; Pardo et al. 1996). This was my first full immersion into
another culture with minimal exposure to other North Americans. It was a crash
course in learning how to navigate the stickiness of international research. My
Colombian colleagues were skeptical at first, but relieved I spoke Spanish (albeit
badly and without embarrassment), would eat just about anything, and was willing
to work all night long in hot and stinky conditions in our favorite pigpen. My dear
colleague Cristina Ferro (Montoya Lerma 2016), who has also contributed to our
understanding of Venezuelan equine encephalitis in Colombia, taught me all I know
about mark-release-recapture studies and made it possible for me to just focus on
the project (Morrison et al. 1993a; Pardo et al. 1996). Many years later we laughed
about the fact that I had moved on in Peru to her role, dealing with all those prob-
lems that arise when doing fieldwork in international sites. For example, there were
rumors in our Colombian field site that our project was stealing chickens to put in
Disney traps, whereas in Peru I frequently had to address rumors that the small
blood samples collected in our studies were being pooled and sold for transfusions.
The lesson became clear after years in Iquitos – working in the field requires a hard-
earned relationship with the community where being present and visible is critical.
Without the trust of the community members and neighbors and with whom you are
collaborating, prospective research is not possible. Cultivating that relationship with
the community is the key to success. It has not been easy for me to articulate the
importance of my presence in our field sites, but as a resident in the community
where you are doing research, you gain credibility and I have always felt it has been
critical to our program.
428 A. C. Morrison
2 Yale University Arbovirus Unit
Although my doctoral dissertation at Yale University was on sand fly bionomics, I

had the joy of interacting with some arbovirus research legends: Bob Shope, Will
Downs, and Tommy Aikens. They had been featured in many stories about the
Rockefeller arbovirus programs told to me while at UCLA. I spent many hours in
their offices listening to them recount their experiences. I must also admit that Dr.
Aikens consistently beat me on the squash court. Interacting with these arbovirol-
ogy pioneers who were a part of the historic Rockefeller foundation where so much
of the research occurred in the field was inspiring. I also had met and worked with
Leonard Munstermann (Munstermann et al. 1998) who was a leading expert on
Aedes aegypti. He introduced me to population genetics, an important tool for
understanding the population dynamics of both mosquitoes and viruses. He also
demonstrated that a person could contribute significantly to science without follow-
ing a strict tenure tract, a path that I would eventually follow. As another aside, Bob
Tesh had worked with Leon Rosen and brought to life the scientific debate over the
causes of severe dengue between Dr. Rosen and Scott Halstead. Since then, I have
had many interactions with Scott Halstead who remains my last connection to that
bygone era of arbovirus field research. Another prominent scientist I met while at
Yale was Scott O’Neil who has successfully used the strategy of replacing wild-type
populations of Ae. aegypti with strains infected with Wolbachia to control dengue
(Utarini et al. 2021). I have gone on to serve as an external reviewer of the World
Mosquito Program activities in Latin America. Both Scotts made the trek to Iquitos
to serve as plenary speakers in our local but international tropical medicine meet-
ings, something I remain grateful for.
When looking for postdoctoral opportunities, it made sense to expand my scien-
tific portfolio from sand flies to a prominent arbovirus, dengue. It seemed like a
natural progression since Ae. aegypti and Lu. longipalpis were vectors that had
adapted to human activity. As an American Society of Microbiology (ASM) fellow-
ship recipient, I could not start my postdoc without a degree in hand which added
the pressure I needed to finish up my dissertation before the “real” submission dead-
line (about 3 months after the published deadline). My dissertation committee
(Mark Wilson, Ted Andreadis, and Bob Shope) cooperated, and Diane McMahon-
Pratt, my only female mentor, took it upon herself to convince three external review-
ers to turn my dissertation review around in about a week. Diane was a parasitologist
and laboratorian, but she always advocated for me.
3 Puerto Rico
Part of the reason I landed at the CDC Dengue Branch in Puerto Rico was the per-
ception that I would make a good CDC employee and in particular work in one of
their overseas laboratories. Both my doctoral work and the Peace Corps had me
convinced I wanted to do international work. My research did not go as originally

planned; instead, I was redirected to dengue case mapping using geographic infor-
mation system (GIS) technology with a wonderful USGS expert, Marilyn Santiago.
These were the early days of using GIS and Global Position System (GPS) technol-
ogy. We perfected the art of communication thorough Spanglish (a complete incom-
prehensible mix of English and Spanish) and spent many hours trying to get accurate
GPS reading with a large egg-shaped antenna on the top of our government vehi-
cle – it could take 20 min to get coordinates and we really stuck out like a sore
thumb. On a side note, my first presentation of this work was at the ASTMH meet-
ing during a government shutdown in San Antonio, TX, where I was grilled by Scott
Halstead for the first time in public. Finally, the spatial analysis used to eventually
analyze the cases we mapped from a local dengue outbreak was done in collabora-
tion with a prominent spatial statistician, Art Getis (Morrison et al. 1998). The col-
laboration with Art continued for more than two decades. When submitting, for the
second time, the R01 proposal that led to our first Iquitos cohort, it was Art’s exper-
tise and a much-improved discussion of our planned spatial analysis that moved us
across the funding line. He also was the person who suggested I dive into the
“Activity Space” literature that became the basis of our second NIH cohort in
Iquitos.
While at the Dengue Branch, I worked with a small cohesive group of scientists
(Jose Rigau, Vance Vordam, and Paul Reiter) led by Gary Clark and made some
other important connections that would guide my professional path. My UCLA
mafia colleague, Ellen Andersen, now a Navy Parasitologist was a member of a
small Navy deployment teaching an international tropical disease course and her
boss Greg Martin would both be important colleagues that helped facilitate my col-
laborations with the US Navy in Peru. I also met Scott Ritchie, a prominent Aedes
aegypti field biologist. Most important, however, was a personal interaction with
Thomas Scott who became my mentor for the next 27 years. With Tom, I conducted
two large mark-release-recapture studies, both in a rural community and in the
mountains a few hours from San Juan. This was the same community where I did
my mapping study; it was small with a pool hall that served food, bakery, and
Chinese restaurant and not much else. Our first study was designed to test the
hypothesis that Ae. aegypti dispersal could be driven by the search for oviposition
sites (Edman et al. 1998). We did mosquito releases inside homes twice: baseline
and after manipulating the number of larval habitat study households, removing the
habitats from one side of the neighborhood, and adding them to the other side. Tom
and John Edman flew in a group of their graduate students that included Laura
Harrington, John Gimnig, and Geoff Attardo along with Tom’s postdoc and my
officemate Adriana Costero. We all (n = 12) slept in the home of a local dentist, on
the floor, sharing a single bathroom, and conducted CDC backpack aspirations all
day every day for about 3 weeks. We developed nicknames for most of the houses
and were kindly allowed into all these homes every day to collect fluorescent mos-
quitoes. Some folks left us their keys, others awaited the visit, and one lovely family
became the restaurant house as they insisted on feeding the group. Conducting field
work in people’s homes is often challenging, but this study was an example of how
430 A. C. Morrison
with proper community engagement – Adriana had visited each home with the local
health department doctor to spend the time necessary to explain what we were
doing – so much is possible. There are too many funny stories to tell here, but I will
share one. We augmented oviposition sites by placing six small ovitraps with quite
a smelly hay infusion and two used tires in the yard of each household where we
were increasing the number of available oviposition sites. In one of our follow-up
visits, the homeowner had put the tires on his car and we found the children tasting
the hay infusion. Obviously, adjustments were made, but the research group still had
a good laugh. Overall, we became quite fond of the community and had a reciprocal
response from the community. In all my time in Latin America, I worked with a
range of social-economic situations, but most people are curious and want to help
their communities. If you take the time to talk and listen, a high proportion of com-
munity members are happy to collaborate with a research study. In the second mark-
release-recapture study, Adriana and I worked alone in fewer households examining
the potential role of sugar feeding on dispersal behavior (Morrison et al. 1999). This
time we slept on the floor of the local meeting house/field lab next to an evangelical
church that memorably had very loud singing going on all hours of the day. We had
to feed large numbers of mosquitos on our arms and conduct the releases, recap-
tures, and mosquito processing. It was hard work, but we were able to complete the
experiment successfully.
4 The Road to Iquitos (Two Years in Davis)
In another example of opportunities falling into place, the end of my ASM fellow-
ship coincided with Tom Scott’s move to the University of California, Davis to be
part of the Center for Vector-Borne Diseases (CVEC) established in 1996 with core
funding provided through the transfer of the Arbovirus Research Unit, previously
affiliated with the School of Public Health at UC Berkeley (see chapters by Laura
Kramer) to UC Davis. For me it was an opportunity to return to California (my
home) and continue working on Ae. aegypti. After arriving in California, Tom called
me in his office and told me about a trip he and Dana Focks had taken to Iquitos,
Peru, at the invitation of Doug Watts, the scientific director of the Naval Medical
Research Institute Detachment (NAMRID). Doug was looking to integrate a strong
entomological component into a strong dengue surveillance research program that
had been underway since the early 1990s. Tom and Dana had an idea to estimate
entomological transmission thresholds for dengue using simulation models (Focks
et al. 1993a, b, 1995) published a few years earlier. Tom wanted to know if I wanted
to be involved and to what degree. He gave me the option to develop the NIH pro-
posal and commit to working for a few years in Iquitos if we were funded. I agreed
to this option. As part of this process, Tom invited me to be a symposium speaker at
the 1997 International Congress of Society of Vector Ecologist in Orlando, Florida,
where I presented the talk “Entomological Assumptions of Dengue Control.” This
was my first presentation as an invited symposium speaker, and despite hours of
practice, I read my prepared talk, shaking rather violently through the whole talk. It
was rough, but the ideas and slides were clear, and several Latin American vector
control specialists talked to me afterwards, essentially thanking me for articulating
the key questions and issues that needed to be resolved for vector control programs
for dengue. This was the first time that I understood the power of ideas and properly
articulating a problem. I would get much better at giving presentations and have
enjoyed every invitation I have received since.
Tom and I later published the ideas from that presentation (Scott and Morrison
2003, 2008, 2010). Dengue vector control programs which had grown out of the
yellow fever eradication efforts aimed to eliminate Ae. aegypti and were highly suc-
cessful at the time. Ae. aegypti indices that were developed during this program
were qualitative and continue to be used by government programs, despite no strong
empirical evidence that reductions in these indices correspond to reductions in den-
gue transmission. We proposed four key questions that needed to be addressed: (1)
What is an acceptable level of dengue risk? (2) What are the mosquito densities
(thresholds) necessary to achieve this? (3) At what geographic scale are risk factors
for dengue important? (4) How do we measure entomological risk? As mentioned
above, on our second submission our R01-Entomological Assumptions of Dengue
Control was approved, and I arrived in Iquitos in September 1998 and quickly gen-
erated the data for our first publication on the spatial distribution Ae. aegypti in
Iquitos (Getis et al. 2003) that also started to answer questions 3 and 4 above
(Morrison et al. 2004a).
In addition to writing the grant that took me to Iquitos, I carried out some exten-
sive secondary data analysis from studies carried out by Tom in Thailand and Puerto
Rico. I spent more than a year cleaning these data sets, developing a strong appre-
ciation for the value of data management and how useful having someone at a field
site full time would be. Although data cleaning would be a major part of my job for
the next 22 years in Iquitos, it was done in real time, with the ability to interact with
field staff the same day data was collected. Having a senior scientist and data man-
agement at field sites is not always possible but is a unique characteristic of the
Iquitos dengue program. I would not claim perfection here, far from it, but you
develop an eye for issues and problems that can be resolved quickly and before they
endanger the integrity of a study. Protocol drift is a big risk with any large, messy
community-based study. Both these studies have contributed to our understanding
of Ae. aegypti population dynamics (Scott et al. 2000b) and blood feeding frequency
(Scott et al. 2000a) with the data set serving for additional analyses (Chaves et al.
2012, 2014).
5 Iquitos and the Proyecto Dengue Entomology Team
I was fortunate to arrive in Iquitos with the ability to hit the ground running. Doug
Watts had identified the perfect entomology field supervisor, a biologist named
Helvio Astete. He had been a contractor on many NAMRID projects in Iquitos and
432 A. C. Morrison
had a team of mosquito collectors that had been doing mosquito collections for a
series of classic arbovirology studies by USAMRID in Puerto Almendras (Jones
et al. 2004; Treangen et al. 2016; Turell et al. 2005, 2008), some malaria work
(Bautista et al. 2006), and some Ae. aegypti surveys in Iquitos. I still feel guilty for
hiring Helvio away from Adeline Chan, not an arbovirologist, but a fellow field
entomologist, who was a positive presence for me during my first year in Iquitos.
She knew how to work in the field providing important guidance navigating work
and life in the city. She would also bring me fresh baked apple pie and bailed me out
when I volunteered to make a “quinceanera” cake for one of my mosquito collec-
tors. Little did I know this was the equivalent to agreeing to make a wedding cake,
with multiple rings hidden in the cake and properly decorated. To make matters
worse, my oven ran out of gas mid-baking. We carried the pans to Adeline’s house
a few blocks away and she took over and saved the day. This whole episode was one
of those unplanned “bonding activities” with my nurse technician team who during
a project training realized I was trying to make the batter for this cake. My team
took over and helped me get the first layers in the oven before my later bail out.
Adeline also briefed me on the responsibilities of being a “madrina”; I am currently
a great godmother. I also watched our entomology team, a group of mostly single
men in their twenties (the only exception being Sr. Federico who had to retire in
2015 at age 70 years), start families and now their children are starting college. The
Proyecto dengue team are unrivaled at finding Ae. aegypti larval habitats in local
homes and highly skilled with Prokopack and CDC backpack aspirators. I claim
with confidence that this team has the best “search image” than any field team con-
ducting Ae. aegypti surveys in the world. Unless finding larval habitats is made an
Olympic event, I do not have the evidence to prove that statement, but with the
leadership of Helvio, we had a happy and productive workplace. As a team of 12
collectors, they surveyed an average of 120 houses per day and their efficiency con-
tinued to increase over time. We became a go-to team for large-scale vector control
trials testing the efficacy of insecticide curtains (Lenhart et al. 2020), lethal ovitraps
(Paz-Soldan et al. 2016), and most recently a spatial repellent product (Morrison
et al. 2022) to reduce dengue transmission as measured through epidemiological
endpoints. I will talk about these studies more later.
We had a field laboratory in the living room of a four-bedroom house I rented in
Iquitos (Fig. 1). We had one bedroom with an air-conditioner (window unit; central
air-conditioning units are extremely rare in the city) that became our data center.
Being heavily influenced by the literature, I was very concerned about quality con-
trol, keeping the team engaged and motivated and ensuring we were not missing any
larval habitats or adult mosquitos. Helvio carried out frequent spot-checks and I
accompanied the teams a few times a week in the field. It was not long before they
were all so much better than either of us at finding larval habitats that our ability to
identify sites they missed became impossible. As a strategy to keep team members
engaged, we would rotate each pair of collectors (they worked in teams of two – one
focused on adult mosquitoes and the other on the immature stages) through the
laboratory for a “rest” every six working days to support survey preparations and
data entry by helping to read the forms to the person at the computer. My rationale
Fig. 1 Original Proyecto Dengue team and field laboratory (1998–2005). (Panel A) Aedes aegypti
survey team in 2000 in front of field lab. From left, front row, Fernando Chota, Abner Varsallo,
Guillermo Inapi, Helvio Astete, Victor Elespuru, Rusbel Huinapi, Amy Morrison, Federico
Reategui, Maykol Castillo, Manuel Ruiz, second row, Tom Scott, Angel Puertas, Juan Luis
Sifuentes, Edson Pico, and Nestor Nonato. (Panel B) Federico Reategui and Juan Luis Sifuentes
counting pupae with Helvio Astete. (Panel C) Helvio processing samples from a productive day.
(Panel D) Jimmy Espinoza entering data in field data center/office. (Panel E) Team joined by proj-
ect physician Jorge and Amy Morrison
was that it would help the team members to see all the problems or inconsistencies
that we identified in their survey forms. The strategy was successful, resulting in a
white board of shame where team members happily posted any of their teammate’s
errors on the board. There was always some friendly competition among teammates
as to who found the most Ae. aegypti-positive containers, pupae, or adult mosqui-
toes. I asked one of my collectors during his day in the lab “Did spending a day in
the lab helped prevent boredom?” He looked at me as if the question was crazy and
emphatically insisted there was nothing boring about his job. I had a survey team
that took pride in their work and enjoyed their jobs. I believe this is one of the great-
est challenges for government programs. Finding Ae. aegypti will always be bad
news and an indication that government measures are not working or inadequate,
one of the many vicious cycles associated with public health programs. I believe
that this team of Ae. aegypti survey experts solely dedicated to research and continu-
ously working together for over 20 years is truly unique.
Although we added some members to the team over the years, the core group
stayed together until December 2019 just before the COVID-19 pandemic. Between
1998 and 2019, this team worked every day in the community mostly collecting Ae.
aegypti or alternatively making maps or painting study codes on houses. They moni-
tored Aedes populations in the context of the prospective studies described in
Table 1. We had enough flexibility, however, to conduct surveys for the Loreto
434 A. C. Morrison
Table 1 Summary of prospective cohort studies with an active febrile surveillance, longitudinal
sampling to detect seroconversion, and entomological monitoring between 1999 and March 2020
Active
Study Design Longitudinal surveillance Entomological monitoring
ECDC Geographically Jan 1999–Aug July 2000–Feb Sampling circuit of Η 6000
stratified sample of 2003 2005 households (range = 5721–
15 city blocks in 2400 w/ School-based 6466) from the same areas of
each of eight replacement 1100 w/ the city, divided into 17 groups.
regions of Iquitos replacement Within a circuit, each house
City, to include all 1800 total was sampled once. Except for
households on the first sampling circuit
blocks containing (9 months), the entire circuit
human cohort was sampled at 4-month
members intervals (13 consecutive times)
DVCS Twenty-six city April May 2004–Feb Vector control was one-time
blocks, 2004–October 2006 source reduction followed by
geographically 2005 Community- one-time residual spray with
distributed; 2400 w/ based Demand CS (lambda
preintervention replacement ~5000 cyhalotrin) and monthly
period April 2004– applications of BTI to water
January 2005, with containers. All blocks received
no vector control; three cycles of space sprays in
February 2005– December 2004, in MOH
February 2006, 12 campaign. All households on
received a vector study blocks were surveyed at
control intervention 2-month intervals
TDR Entomology only: Nonresidential sites were surveyed twice over a one-year period
(2006), including houses around a large open-air market and commercial areas in
Iquitos. Sites included markets, ports, factories, machine shops, lumberyards,
recreation areas, and government buildings
TDR2 Entomology only: Five city blocks from ten Ministry of Health (MOH) zones selected
at random. Study divided into pre- (Jan 2006–February 2007) and post-intervention
(after February 2007) periods. One block from each MOH zone was assigned to one of
the five following interventions: 92% targeted control with pyriproxyfen, 58% targeted
control with pyriproxyfen, nontargeted larval control with pyriproxyfen, temephos
control (2-month intervals), and temephos control (3-month intervals). Houses were
surveyed and then treated according to treatment starting in March–April 2007 at
approximately 2-month intervals. Ae. aegypti surveys carried throughout January 2008.
A large citywide ULV door-to-door space spray campaign was carried out in March
2008. An acceptability/cost-efficiency trial was conducted in 652 contiguous
households in the San Juan District of Iquitos. From July 27 to August 6, pre-
intervention surveys were carried out, followed by post-intervention (92% targeted
control versus standard MOH larviciding) from August 7 to 15, 2007
VEEV Post-Venezuelan Seroprevalence study: Single On-time survey carried out in
equine encephalitis blood sample from 1200, 400 October 2006. The
outbreak study residents each from three VEEV neighborhoods included in this
carried out in three neighborhoods and study were Bella Vista Nanay
areas of these city neighborhoods participating in located in the northernmost
based on the the DVCS study above (DENV section of the city; Belén,
residences of control) located in the eastern part of
VEEV-infected the city along the Itaya River;
patients and three sites in the Southern
San Juan District
(continued)
Table 1 (continued)
Active
Study Design Longitudinal surveillanceEntomological monitoring
PRED Geographically Aug 2006–Nov Oct 2006–MarEntomological surveys carried
stratified sample 2010 2011 out on the 92% and 58%
3000 w/ 3033–4399 targeted control blocks
replacement described in TDR2
corresponded to the cohort
blocks
AS Sampling of two Aug 2007–July Nov Entomological surveys carried
Iquitos 2015 2007–present out in open households at
neighborhoods 2400 w/ 4605–5860 approximately 4-month
(Maynas [~20 city replacement intervals. In addition to
blocks] and Tupac neighborhood houses, multiple
Amaru [14 city individual houses reported as
blocks]) of ~900 visited by neighborhood cohort
houses each. Effort members were surveyed at the
to work in same 4-month intervals. Study
self-contained area only received standard
neighborhoods MOH vector control
LIV Randomized cluster October No active The study area was in the
trial designed to 2009– surveillance district of San Juan in the
test the efficacy of November component southernmost region of urban
insecticide-treated 2010, 3400 w/ Iquitos. Entomological surveys
curtains placed in replacement in all households within each
10 of 20 clusters cluster prior to curtain
containing a deployment (October 2009),
minimum of 70 within 1–2 months after
households each. deployment (January 2010),
Clusters were with subsequent surveys in
contiguous May 2010, February 2011, and
May–June 2011. Curtain failure
was observed in May 2010 and
the curtains retreated with
deltamethrin in November 2010
ALOT One contiguous July 2011–July Aug An average of three ALOT
neighborhood, core 2015 2011–present traps per household in
area monitoring the 1200 w/ 3733 approximately 2000 houses.
human population replacement (approximately Entomological surveys were
and buffer area, 400 houses) carried out in 400 house core
both with lethal areas and 10% buffer area at
ovitraps (ALOT) 2-month intervals. The Maynas
area of AS study served as a
control
P01 Two contiguous January 2015 July 2015 to Entomological surveys were
neighborhoods to present present carried out a few times during
including (transition 5000 w/ the study but not at regular
individuals from period) replacement intervals
AS and ALOT Up to 1500 w/
cohorts replacement
(continued)
436 A. C. Morrison
Table 1 (continued)
Active
Study Design Longitudinal surveillance Entomological monitoring
SR Twenty-six clusters January 2015 July 2015 to Full pupal demographic surveys
of ~150 household to present present prior to and after removal of
each, (transition 5000 w/ spatial repellent products. Adult
noncontiguous, period) replacement Aedes aegypti surveys carried
across 13 MOH Up to 2400 w/ out 2× month with replacement
zones; 13 clusters replacement of SR products
receive spatial
repellent product
Regional Health Department (DIRESA) when asked. I provided periodic reports of

the Aedes indices from our surveys. Health department surveys and vector control
when we first started our studies were episodic at best. Teams were hired once or
twice a year, given a quick training course, and the data produced had many of the
problems commonly reported in the literature. I had several uncomfortable meet-
ings with the Regional Health Director trying to explain why my team’s house indi-
ces (HI) were an order of magnitude higher than for the DIRESA teams (30% versus
3%). In 2002, Iquitos experienced a large dengue-3 outbreak after about 5 years of
low transmission rates. Emergency control measures had been initiated and the sub-
director called Helvio and I into his office. He asked if we could evaluate their ongo-
ing intervention program. I said I thought we could but wanted to know if there was
any chance of a “control” area; the response was a quick and definitive NO! I had
ongoing surveys associated with our cohorts, recognizing we had a built-in step-
wedge design. All we needed were the larviciding and fumigation schedules. Our
cohort could be broken into 17 distinct neighborhood pupal demographic surveys
and adult mosquito aspirations carried out at 4-month intervals. This gave us the
ability to measure Ae. aegypti indices, both immature and adult within 1 week to
2 months before (eight surveys designated “control”) or after (nine surveys desig-
nated “treated”) control measures were deployed. We could then compare these
indices to historical and subsequent surveys carried out in the same neighborhoods
(Fig. 2). The intervention being employed was the application of temephos (1 ppm)
to all identified larval habitats 1–4 weeks prior to three cycles of indoor space spray
with deltamethrin (0.25%) using backpack sprayers (see Gunning et al. 2018 for a
description of the Ministry of Health spray methodology). The percent reduction or
increase between our designated control and treatment survey shown by the red bar
can be compared to surveys carried out in the same households 4 months (yellow
bars), 8 months (magenta bars), or 12 months (blue bars) earlier (Fig. 3). We
observed that for most surveys in the designated control areas (before the interven-
tion), Aedes populations were rising, whereas in the designated treatment areas,
population densities decreased as measured by adults/hectare, Breteau index, and
pupae/hectare (Fig. 3a, b). In the treatment areas, adult indices in seven out of nine
surveys decreased by >78% (Table 2). In the first area (PU01, Figs. 2 and 3), part of
the houses had received both larviciding and space sprays before the survey (pink
Fig. 2 Step-wedge evaluation of Ministry of Health emergency control program carried out in
Iquitos, Peru, between November 2002 and January 2003 in response to an outbreak caused by a
novel dengue serotype (serotype 3) into the city. Panel A shows the date that three cycles of indoor
ULV application of the pyrethroid deltamethrin were completed. The schedule is based on reported
dengue transmission rates, spraying areas of high transmission first (pink) and areas of lowest
transmission last (dark brown). Panel B shows the houses surveyed by neighborhood (n = 8) des-
ignated as control areas because our routine surveys were carried out before initiation of the inter-
ventions. Panel C shows the houses surveyed by neighborhood (n= 9) designated as treated areas
because our routine surveys were carried out after the initiation of the intervention
Fig. 3 Aedes aegypti indices from neighborhood surveys (n = 100–600 households) in eight “con-
trol” neighborhoods surveyed 1 week to 2 months before the interventions and nine “treated” area
surveys 1 week to 2 months after the intervention. The red bars represent the evaluation survey
(just before or after the intervention) compared to surveys conducted 4 (yellow bars), 8 (magenta
bars), or 12 (blue bars) months prior to the evaluation survey. Green bars represent surveys con-
ducted 4 months later. (Panel A) Adult Aedes aegypti collected per hectare in control (left) and
treated (right) neighborhoods. (Panel B) Breteau index (number of Aedes-positive containers per
100 houses surveyed) in control (left) and treated (right) neighborhoods. (Panel C) Neighborhood
PU01, where surveys were conducted as the intervention was being deployed; the area in pink had
received both larvicides and adulticides, and the other areas had only received larvicides
438 A. C. Morrison
Table 2 Percent increase (↑) or reduction (↓) between surveys conducted around the intervention
period (red bars in Figure 3b) with surveys in the same houses conducted 4 months earlier
(4-month) or the average of surveys conducted at 4, 8, and 12 months (YR average) in areas
designated as “control” areas (n = 8) because surveys were done before the intervention and
“treated” area (n = 9) surveys shortly after the intervention was carried out
Control areas Treatment areas
Index 4-month Year-average 4-month Year-average
Breteau 11–71% ↑ (8) 6% ↓ (1) 8–89% ↓ (9) 27–89% ↓ (8)
3–43% ↑ (7) 12% ↑ (1)
Pupae/hectare 38–89% ↑ (8) 40–41% ↓ (2) 8–97% ↓ (9) 10–96% ↓ (9)
2–66% ↑ (6)
Adults/hectare 10% ↓ (1) 2–58% ↓ (4) 4% ↓ (1) 16% ↓ (1)
17% ↑ (7) 16–64% ↑ (4) 78–98% ↓ (7) 83–93% ↓ (7)
0.2% ↑ (1) 4% ↑ (1)
Fig. 4 Laboratory-confirmed dengue cases from April 2001 to July 2003 identified through febrile
surveillance in 12 clinics and hospitals in Iquitos, Peru. Serotypes 1 (D1), 2 (D2-American strain
[AM]; D2-Asian strain [AS]), and 3 (D3) were identified by PCR or virus isolation. Individuals
showing a fourfold rise in IgM by ELISA between acute and convalescent samples were consid-
ered confirmed, whereas individuals with elevated IgM in an acute sample (IGM-pre) were consid-
ered presumptive. The red arrow shows when the citywide emergency vector control was
implemented
area, Figs. 3 and 4), whereas the remaining households had only received larvicides.
The area with both interventions showed a dramatic decrease in Aedes densities, but
areas with only larviciding did not (Figs. 3 and 4). Figure 3c shows the dramatic
decrease in dengue cases detected in an ongoing clinic-based febrile surveillance
study where only laboratory confirmed cases are shown. This was not the last
outbreak we would use for our ongoing studies to help confirm the operational out-
come of MOH interventions; using 10 years of data we demonstrated the public
health value of indoor ULV space sprays, mitigating transmission until the follow-
ing season (Reiner et al. 2019; Stoddard et al. 2014).
This was also not the first or last time we had evidence that larviciding on its own
was not sufficient to control dengue. When we presented these results to the sub-
director, we were accompanied by two representatives from the government agency
responsible for vector control (DIGESA), who looked worried. As I made my pre-
sentation, one started to object but his colleague tapped him on the arm saying,
“they are saying it worked, stop talking.” This report was passed on to superiors in
Lima and I was told later that it helps justify the funding for that and future interven-
tions. This was an example of the power of evidence, and it redefined our relation-
ship with the DIRESA/DIGESA moving forward. Sadly, as pyrethroid resistance
increased overtime (see figure in Baltzegar et al. 2021), these interventions slowly
became less effective. These opportunities to support the local health department
were particularly gratifying to me. We provided considerable information on con-
tainer productivity in Iquitos that translated into real changes in larviciding and
source reduction efforts. For example, we had shown that some cement porches and
floors were productive larval habitats; MOH personnel then started to apply larvi-
cides preemptively to some of these surfaces. We also provided evidence for shift-
ing their larviciding efforts away from water storage to unmanaged containers
(Morrison et al. 2004b). A favorite story, however, was being asked to accompany
my MOH colleagues to talk at a press conference. There was some backlash about
the ULV campaigns being ineffective. At the time I had many pairs of batik mos-
quito pants that had images of eggs and adult mosquitoes representing the genera
Aedes, Culex, and Anopheles. I could show in a very entertaining way that there
were different kinds of mosquitoes with different habits, and the ULV campaigns
did little to control the Culex mosquitoes that were biting people at night, but that
this did not mean the intervention was not killing Aedes mosquitos that transmitted
dengue. I wish I could still wear those pants (put on a few pounds) as they served
me many times to conduct an on-the-spot biology lesson.
6 Technology and Mapping Iquitos
I wanted to write a section on the impact of technology on my research program

because it has been profound. Data science as applied to medical entomology has
evolved very rapidly. We can do things that all my previous academic advisors could
not have dreamed of because of the limitations they had recording, storing, and
manipulating data. A small warning, I now rely on some outstanding statisticians
and modelers for hard-core data analysis but remain the data manager. I will also
argue that I always scrutinize any analyses and feel there is no substitute for having
eyes on the ground who can evaluate and sometimes criticize model output. Ae.
aegypti is very challenging statistically, and the principal solution is surveying a lot
440 A. C. Morrison
of houses as often as you can. When we wrote our first R01 (ECDC in Table 1), the
lack of empirical data measuring the relationship between commonly used Aedes
indices (i.e., premise/house, container, or Breteau) and dengue transmission was
unbelievable to me. Once on the ground attempting to collect this type of data, I
began to understand why there was so little empirical data available. Our study was
able to monitor humans and mosquitoes together in space and time because we used
a GIS to link viral, serological, and entomological data to individual households.
Both my entomology and human surveillance teams relied on maps we produced to
identify unique house codes that were central to our data management system. I
gave a presentation at the 2001 Entomological Society of America Meeting describ-
ing the development of our GIS that I will share here because I never published this
information. The methodology would now be considered obsolete but illustrates
how you can quickly and effectively develop a system from scratch that can become
operationally essential. In the field you must be creative and work with what you
have at the time, but once you have something like a functioning GIS for the city of
Iquitos, it becomes a major reason other researchers seek you out.
When I arrived in Iquitos, Helvio and his mosquito-collecting team had been
doing surveys in 600 houses in each of five neighborhoods for which they had hand-
drawn maps; these neighborhoods became the basis for dividing Iquitos into eight
large zones (Fig. 5a), each with a two-letter code (BG, TA, MC, IQ, PT, PU, MY,
SA). I had obtained Autocad files from the Peruvian Navy that were developed from
ortho-corrected aerial photographs that delineated city blocks. During a one-week
trip to the Focks’ Lab in Florida, we converted these line files using Arc/Info into
shape files with polygons representing the geolocation of each city block. Using
Arc/View, I used this shape file to build the GIS for Iquitos. I had some experience
working with GIS software from Puerto Rico, Arc/View recently loaded on to my
laptop, and a book on ArcView for dummies (Google was not a thing in those days)
before I returned to Iquitos. On a quick side note, I returned to Iquitos the same day
that major public protests were occurring in response to the signing of a peace treaty
between Peru and Ecuador by then president Fujimori. It remains a significant event
in the city and is a reminder of how residents in Iquitos feel neglected by the central
government.
While the social unrest settled, I attempted to modify my shape files using the
paper maps of TA and MY neighborhoods, but soon realized I needed measure-
ments of the front of each lot. The first maps of those two neighborhoods took me a
few months to complete, in large part because I had to learn the software through
trial and error. We also painted our codes on the electric boxes or walls of each
house and remained in a race with the electric company who periodically painted
over our codes (Fig. 6). Our coding syntax had to evolve over time. We started with
a two-digit neighborhood code combined with three digits in consecutive order
(e.g., TA001, TA002, …, TA999, as you circle the block), but we had to expand
three letters (Fig. 5h) and over time divided (TA200 became TA200A and TA200B)
or combined houses (TA405 and TA406 became TA405-406) (e.g., Fig. 6). In one
instance, we had a neighborhood ready to go but we experienced the phenomenon
of an entire neighborhood being demolished and then rebuilt and recoded.
Fig. 5 Summary of mapping strategy used to develop the Iquitos geographic information system
(GIS). (Panel A) Eight geographic zones originally designated by the research team (BG Bagazan,
TA Tupac Amaru, MC Moronacocha, IQ Iquitos, PT Putumayo, SA San Antonio, MY Maynas, and
PU Punchana). (Panel B) Paper maps were provided to research team. One map was at a scale that
showed multiple blocks which was accompanied by a single page for each block that the teams
would annotate. (Panel C) The team would then measure the front end of each lot and add this and
any visible address information to the map. (Panel D) The block corresponding to the paper map
was then visualized in Arc/View. (Panel E) After arbitrarily splitting the block along the length,
using the measuring tool, each lot was divided manually. (Panel F) All divisions were based on the
measurement of the lot front. (Panel G) Attribute table was populated with address numbers and
street names and assigned a project house code in chronological order. A map with the codes and
addresses would be printed and provided back to the team who would paint the codes on the house
and conduct Aedes aegypti surveys. (Panel H) Demonstrates how coding was developed for the SA
zone. Letters were added each time 999 houses were coded for a barrio code (e.g., SA, SAA, SAB,
SAC, SAD). All our human participants received a participant code that joins the location (house)
code with a P designation. For example, if there are five residents in house TA001, they would be
designated TA001P01, …., TA0001P05. This system has been critical for keeping track of
5000–20,000 residents at any given time
After a rough start mapping our first two neighborhoods (Getis et al. 2003;
Morrison et al. 2004a), we had to map the remainder of cohort houses and conduct
surveys simultaneously. Figure 5 describes this process in detail. We had one or two
teams mapping and the rest conducting surveys. My job was to keep up. It took us
9 months to develop the maps and conduct the baseline surveys for all the houses in
our first cohort (ECDC, Table 1). In early 1999, Tom Scott, Dana Focks, and Art
Getis were visiting and were a bit horrified at our labor-intensive mapping approach,
brainstorming for alternatives. The fact was there were no practical or timely alter-
natives, and while those alternatives were debated, we had maps we could use, and
that formed the base of our GIS. Figure 7 gives an example of the operational maps
provided to our field teams. Subsequently, I would find, and interchange other maps
(municipal sources) and we would obtain QuickBird satellite images that also
442 A. C. Morrison
Fig. 6 House codes painted on the front of all homes participating in cohort studies in Iquitos.
(Panel A) Code painted directly on the wall of the home. The “A” indicates the house had been
divided into two houses (TAD030A and TAD030B) from one larger house (TAD030). (Panel B)
House code MY574 is painted on the electric box, a location that is frequently painted over requir-
ing that codes be repainted frequently. (Panel C) In 2016 QR tags were implemented on the back
of doors with the house code to improve quality assurance
facilitated this mapping process. Overtime, we got faster and more efficient and had
several team members dividing up blocks in Arc/View and hired a full-time GIS
technician. We have mapped close to 70,000 lots representing >75% of the houses
in Iquitos. The shape files have been shared extensively. The GIS is now integrated
into our Iquitos relational database and is a critical quality assurance tool. No human
participant or entomological survey can be added to our database unless the location
exists in our GIS which is updated in real time. Our human participants link to loca-
tions, but our participants often live and spend time in more than one place. We
manage this phenomenon by allowing each unique participant to have multiple
“participant codes” as they have moved from one house to the next or are present in
multiple residences at the same time. Like our house coding syntax, tracking these
people in multiple places developed over time. My assistant and I have a favorite
participant who had five active participant codes, as he bounced between family
members’ homes from 2007 as a child too young to provide assent to 2019 as a
university student.
As a postscript to this section, it is important to note that I moved to Iquitos in
August 1998 before the explosion of social media and never having owned a cell
phone. My productivity has been tied to my Internet speed which started with a pay-
by-the-minute dial-up plan. We have a major technological upgrade every few years
for which I am first in line; each feels life altering, but it has only allowed us to keep
Fig. 7 Example of maps provided to the study teams. Legend designates the neighborhood code
MCF and the block numbers, and the house code is shown in numbers surrounded by squares. For
example, the lot MCF755-762
444 A. C. Morrison
up. Iquitos is the largest city in the world that is not connected by a road, so no fiber
optics; we are reliant on satellite options. With each new technological upgrade,
bandwidth eventually sells out. Despite demand, we do not believe the region has
economic resources to justify investment in Internet infrastructure. Access to the
Internet and smart phones, however, has had a profound effect on this community as
well as our research.
7 A Few Stories from the Field Laboratory
Combining my living quarters and laboratory seemed like a good idea at the time,
we could save money, visitors would have a place to stay, and there were other per-
ceived advantages. It was like living in a frat house with my 14-person entomology
team coming to the house each morning and afternoon. During those earlier years,
there was a lot of laughter, some late nights, and weekend sports days, but it felt like
being part of a family, and I have no regrets. I always seemed to have a lot of visitors
in that house and both my team members and random strangers often knocked on
my doors to ask a variety of favors, most of which involved borrowing money. In the
United States, the last person you would ask for a loan would be your boss, but here
in Peru, it was the first person you asked. It took me a little longer than most to man-
age these requests but is one reason I eventually separated the lab from my house.
Among many stories from that time, I will share three here. It is in the context of
these requests for loans and my less-than-perfect Spanish that I begin.
One Friday night I was watching TV and there was a frantic ring at my doorbell,
and Victor a member of the mosquito collection team was at the door. He seemed
agitated and had clearly been drinking (this was everybody’s favorite Friday night
activity) and wanted my help; I had to come now and meet his wife. He was not
making sense, but I kept hearing the word “luz” (light or electricity in Spanish).
How I concluded that he had spent the money meant for the electric bill on alcohol
and he was trying to get me to smooth it over with his wife. This seems like quite a
stretch now, but that was what I thought was going on. He was asking for money, so
I grabbed 50 soles and jumped in a motorcar (local taxi) with him thinking “how
crazy is this.” I recognized I was missing something, but when he turned to me in
the motorcar and blurted out that “this is the first time, I’m going to be a dad,” I
realized I had it all wrong. He was saying my wife is going to “dar la luz” which
translates to give birth. He wanted me to convince his wife to have the baby at the
hospital rather than with a midwife. I told him she was probably better off with a
midwife and but more importantly provided reassurance that he would rise to the
challenge of fatherhood. My words seemed to have a calming effect, then came the
important request – he needed me to contribute some beer for the ishpa, a local
tradition celebrating a birth. I soon learned that the ishpa is drinking beer all day
long with your buddies, and when I arrived the following afternoon, I found most of
my team passing a single glass of beer around a table as is custom in Iquitos. There
were a lot of empty bottles and I asked Victor about the baby. He replied he had not
seen it yet and I immediately insisted we go see the baby together. The next day, one
of the team, Fernando, said the doctora (me) “needed verification of the baby’s
existence.” We have been telling this story for 20 years and still laugh every time.
A unique characteristic of that first house/laboratory was a well-protected front
porch. Because it was well protected from the rain, a local man who I later learned
was a schizophrenic slept there each evening. He could be a bit intimidating and on
some occasions would defecate or masturbate, causing some nighttime shrieks from
unsuspecting neighbors and a very unpleasant mess in the morning for the first arriv-
ing team member. Nobody wants a homeless person living on your front porch, but
I would try to be polite, say hello and not engage further, and we never did much
more than exchange greetings. One day out of the blue, he spoke to Helvio like a
rather rational person. During this exchange, he mentioned CREMA, the local psy-
chiatric facility, and we offered to take him there if he wanted. It turned out that my
social worker neighbor had the commitment paperwork available and ready to go;
all we needed was to provide a few weeks’ worth of Haldol and transport him. He
mulled over our offer, but asked if he could make his rounds first, but he liked the
idea because CREMA had good soup. I said we really needed to go now, and he
agreed. Two team members, Juan Luis and Abner, were at the lab, and I asked that
they accompany him in the back of the project pickup truck hoping he would not
change his mind. We started the journey stopping at multiple pharmacies before we
were able to purchase the medication we needed for his admission. When they
dropped him off, we learned that he had been a resident many times but periodically
escaped. He came back to my porch again a few times, but eventually, a family
member showed up and took responsibility for his meds. Apparently, he had been a
talented law student before he started to exhibit symptoms. This was one of the first
examples of how, in Iquitos, the community would band together to take care of
someone less fortunate to the best of their ability. Most recently, seeing how people
came together to help the family, friends, and co-workers during the COVID-19
pandemic was amazing and heartbreaking at the same time.
One last story from the field laboratory was the day a canister of tear gas landed
in front of the house. It was in the afternoon, and we were finishing up the process-
ing of the day’s mosquito collections, and Helvio arrived early with the project
vehicle indicating there were protests at the university. Suddenly we heard the can-
ister land and gas started seeping through the windows. Armed with duct tape and
t-shirts, I thought I could seal the windows, but the guys knew better. I was over-
come by the gas quickly; Rusbel grabbed by arm and pulled me to a sink where he
pushed my head under running water. Everyone else had scattered to different loca-
tions in the house, either with running water or somehow isolated from the gas.
Helvio seemed to know that going to the office with the AC running was the best
place to be. A few of the team had started to assist the neighbors across the street
who were also overcome. Eventually, a large part of the neighborhood was huddled
in my office soaking wet but able to breathe easier. The gas eventually dissipated but
we had bonded with the neighbors.
446 A. C. Morrison
8 Giving a Talk on Dengue When You Have Dengue
Being a resident of Iquitos, I’ve been exposed to many tropical pathogens and in
22 years have only been hospitalized twice. The first time was in 2000 when I devel-
oped pneumonia, requiring a 2-day hospital stay in a public hospital (apoyo). Our
project physician used his personal connections to walk me straight onto ward to Dr.
Martin Casapia, one of the best infectious disease doctors in the city. He did a quick
exam, taking my blood pressure sitting up and then lying down, and calmly asking
if he could admit me. He asked if he could have permission to take an X-ray and do
some lab work but only if I wanted to. Suddenly, Helvio and a group of about five
other NAMRID personnel were arranging my lab testing for free, paying for my
X-ray and buying all the items I would need for my care. In hospitals in Iquitos,
doctors provide a list to family members of what is needed; in my case it was IV
tubing, needles, tape, and other stuff. Dr. Casapia gave me some antibiotic choices,
but recommended I opt for a more expensive drug, but seemed strangely apologetic
about it. I just kept saying give me the best and whatever you need. I do not think
this was typical of his other patients. My hospital room did not have running water
and I had to collect my urine in a bed pan and transfer it to a big brown bottle, using
one arm, since I had an IV. Dr. Casapia wanted to monitor my fluid input and output.
My care was great; I was well looked after and suspect received special treatment,
because I had no immediate family member to look after me, which is normal in
public hospitals. My social worker neighbor tried to get the hospital fee waived
which I emphasized was not necessary (about $5.00/day), but the whole adventure
cost me about $200 (mostly the antibiotic). I recovered after 2 days and Dr. Casapia
gave me permission to travel to Brazil a few days later where I have a talk; on the
condition I take it easy.
My second hospital stay was in 2014 when I developed a weird red streak on my
arm that looked like cellulitis. After about 4 days of auto-medication and a lack of
consensus among our medical research team, I decided to seek out Dr. Casapia
again, this time at his private clinic. My case was a bit of a mystery, with whatever
I had, migrating away from my heart via my lymphatic system – more red streaks
running down my arm to the tip of my thumb. I was convinced it was some sort of
migrating helminth, in part because on intake I had a very high eosinophilia but felt
fine otherwise. After 6 days of a wide variety of IV drugs and 4 additional days of
oral treatment, I recovered and traveled to the ASTMH meeting in New Orleans. On
the first day of the conference, I started receiving texts from several of my Iquitos
colleagues who had seen a presentation on gnathostomiasis that mirrored my symp-
toms exactly. Now that we had a likely diagnosis, I just needed a biopsy done in my
office at my desk and to take mebendazole for a month. I am not sure how I con-
tracted the disease but had eaten raw fish in both Iquitos and Ecuador during the
appropriate time frame. Costs were higher than my first hospitalization, but when
done I spent 6 days in the hospital for about $1000.
Since my research is focused on dengue, I carefully documented my two
laboratory-confirmed cases. My first infection was at the end of 2002 with serotype
3 during the outbreak described above. My second infection was in 2008 with den-
gue 4, again during a major outbreak. Both infections occurred after hosting site
visits with important visitors. In 2002, it was for an inspection from the Instituto
Nacional de Salud, IRB, whereas in 2008 it was a VIP visit from the CDC including
the director herself. Because of the high degree of transmission, we had lots of den-
gue cases and mosquitoes in our study areas for the visitors to see. The good news
was our guests used insect repellent and did not become infected and had been
impressed with our studies. The bad news is I was infected with the dengue virus
both times. The dengue 3 infection was impressive, nothing dangerous, but behaved
as dengue fever described in textbooks. The dengue 4 infection was milder, but I
needed a few days on the couch. My dengue 3 infection started while I was waiting
in the Iquitos airport on my way to the ASTMH meeting in Denver. I was drinking
a coke with Helvio and feeling a bit feverish, I jokingly said it might be dengue. As
I arrived in Lima after the 90-min flight, I felt bad. I popped an ibuprofen (it was all
I had with me), bought Gatorade, and carried it with me on the plane. As luck would
have it, I had the seat that did not recline, but felt so bad I did not care other than to
try and drink my Gatorade. Upon arrival I went straight (think death march) to a gift
shop and bought Tylenol and took one on my way to the restroom where I promptly
threw it up and had some light diarrhea. I was officially sick and increasingly suspi-
cious it was dengue! I walked for what felt like an eternity to the next gate, where I
just lied down on the floor, took the second Tylenol, and waited for the flight to be
called. I had prearranged a shuttle pickup and the driver let me lie down on the back
seat. Once in the hotel, my roommate bought me apple juice and Gatorade and I
crawled into bed and called Tom. The calls started coming in. You ought to take a
sample was the most common advice, but where? Claudio Rocha, study physician
and co-host of our recent IRB inspection, examined me and was convinced it was
dengue. We argued if I indeed had the dengue rash, but I wanted proof. I had a pre-
sentation to prepare for later in the week and attended a few sessions but spent most
of my time in bed. Never had I had such an aversion to food, another sign I had
dengue, but I was no closer to a laboratory test. Tylenol was my friend; my fever
was reasonably controlled with it, but by the time I was scheduled for my next dose,
the fever was very high. I was still with a fever when I gave my talk on our dengue
active surveillance study – so it was a talk on dengue while I had dengue. After the
session ended, I saw my old colleague and friend Jose Rigau from the dengue
branch was in the audience. I stopped for a quick chat and told him I thought I had
dengue. He snapped into action. He managed to find someone driving back to Fort
Collins that evening and agreed to spin down and freeze my serum. He knew to
contact the Colorado Health Department, which arranged for us to pick up a vacu-
tainer tube, needle, and rubber tourniquet at a hospital emergency room. After
retrieving our sample collection package, Jose drew my sample in my hotel room
and made it safely back to Puerto Rico where Vance Vordam confirmed my dengue
3 infection. The next morning, I felt better but was itching, and when I looked in the
mirror, I was completely red with a full-blown dengue rash, but my fever had bro-
ken. I am sure when Jose took that sample it was my last day of viremia. I had talked
448 A. C. Morrison
with so many participants during that outbreak that would tell me “Me puso rojo!
(I turned red!)” and it had happened to me just as they described.
9 The Best-Laid Plans
From 2004 to 2019, Project Dengue conducted multiple large-scale vector-control

intervention trials, four with epidemiological outcomes and four measuring impacts
solely on entomological parameters. I believe each was interrupted or had to account
for a Ministry of Health-led emergency fumigation campaigns. After working with
the Loreto Regional Health Department at the height of the 2002 dengue 3 outbreak
(Sect. 5, Iquitos and the Proyecto Dengue Entomology Team), our DIGESA col-
leagues would always inform us of any impending vector control campaigns and
were willing to coordinate their activities with us. What was most important was
documentation of any vector control activities in our study areas and, if possible,
ensuring that all the houses in our study areas received the MOH treatment equally
to reduce the impact on our trials. The real impacts varied. For the most part, we
were able to report that these campaigns had occurred during our well-planned stud-
ies, but the impacts were limited. There were two exceptions where the impacts
were great. An effective MOH ULV campaign at the end of 2004 dramatically
reduced both Ae. aegypti densities and dengue virus transmission in our study area
during the 2004–2006 DVCS trial (see Table 1 and Fig. 9), but in both control and
treated blocks, before the intervention was implemented. This is an example of how
trials need to last multiple years when measuring virus transmission. Furthermore,
the MOH intervention greatly reduced the statistical power of the trial; if we had
been able to apply our test intervention at the time of the MOH intervention and not
intervene in the control areas, I remain convinced we would have generated solid
evidence of protective efficacy, but not intervening during a public health emer-
gency is never an option (Fig. 9). In our 2014 study, another well-planned experi-
ment was interrupted by a MOH campaign; however, in this case, it was monitored
and evaluated as part of the final publication (Gunning et al. 2018) and provides
some useful insight on the difference between efficacy and effectiveness of the
intervention being evaluated.
Our research activities were interrupted or delayed for a variety of reasons. Rain
in Iquitos can be quite impressive, often an all-or-nothing affair. I found it very dif-
ficult to predict when we would lose a day or just need to have the team hang out for
a few hours. We looked to our senior collector; Sr. Federico would calmly say when
consulted, this is going to be all day. Iquitos also periodically has general strikes,
when the community organizes and shuts the city down by burning tires and placing
broken glass, and tree branches to block roads for 24–72 h. We have found Aedes
production in sites where tires are stored for this purpose. They are rarely violent
and in some ways were effective. As a kid who grew up in southern California, in
my imagination this must have been what a snow day felt like, a chance to catch up
with work for me, and play soccer or volleyball in the street for everybody else.
More than once, I had a flight out of Iquitos scheduled during one of these general
strikes. In some cases, the airport would be closed, but usually it was a challenge to
get to the airport. Usually, Helvio could give me a ride on the back of his motorcy-
cle, and we would need to find back routes; however, on one occasion my friend and
colleague Kacey Long, an expert on Mayaro virus, just walked 14 km to the airport.
Another cause of delays and challenges to our projects was obtaining emergency
use and/or importation permits and then coordinating the shipping of the products
we were evaluating, nothing like navigating a complicated legal process in a foreign
language. Fortunately, my assistant of 15 years had a unique ability to push through
these processes. We followed the rules, but my assistant’s superpower was the abil-
ity to politely and nicely follow up with every person through the process – in other
words, pester and cajole without ever seeming rude. Timing is key and you needed
to identify the appropriate person to check on your shipment every step of the way
and always be proactive. For two of our trials, the deployment of the intervention
was significantly delayed because of regulatory/shipping delays. We shipped 6000
insecticide-treated curtains from India and >10,000 lethal ovitraps from the United
States all arriving before the trials were initiated. Our last and most ambitious trial
required eight shipments (>200 boxes/shipment) of spatial repellent products.
During the trial we were replacing an average of 28,000 products twice a month and
the products had a 10-month window between their production and use. We had to
ensure that we used the oldest, but not expired, products for each product replace-
ment cycle on a cluster. The boxes were stored in my house and carefully arranged
by cluster number (Fig. 8). Of course, the number of required products was esti-
mated based on desired enrollment rates and house sizes. For a few clusters, those
Fig. 8 Storage of SR products in my house. Initial shipments were two layers thick (left panel),
but it was critical to use the oldest boxes first and ensure they had not passed their 10-month expi-
ration date. (Note each box had a cluster number)
450 A. C. Morrison
Fig. 9 Description of the Dengue Vector Control System (DVCS) study carried out from April
2004 to March 2006. (Panel A) DVCS intervention was a household assessment with a one-time
removal of disposable containers followed by indoor residual spray application of Demand CS
(lambda cyhalothrin) and treatment of permanent containers with BTI once per month. (Panel B)
Acute dengue cases were identified through active surveillance visits 3× per week with laboratory
confirmation by PCR and IgM ELISA. A Ministry of Health intervention was carried out during
the pre-intervention period that probably accounted for the dramatic decrease in dengue incidence
between the pre- and post-intervention period (89% and 94% decrease in the control and treatment
areas, respectively). During the pre-intervention period, dengue incidence was 50% less in the
treatment (yellow bar) compared to the control area (red bars), whereas in the post-intervention
period when dengue incidence was significantly lower overall, the treatment area has 72% of the
incidence of the control area. Panel C shows the Breteau index and Panel D shows the adult house
index (percentage of houses with at least one adult Ae. aegypti mosquito) over time comparing the
control (yellow) and treatment zones (green). The blue arrow indicates when the MOH interven-
tion was deployed, while the red arrows show when the DVCS intervention was initiated and when
the first post-intervention surveys were carried out. The DVCS demonstrated a short-term reduc-
tion in the BI and AI, but this decrease was a smaller magnitude than for the MOH intervention
which impacted our ability to effectively evaluate the DVCS intervention
initial estimates were off, and we had a few shipments that arrived just in the nick
of time to meet our ongoing product replacement schedule. Again, this is an exam-
ple of how fragile field protocols can be (Fig. 9).
Our research program has with a few exceptions met the definition of minimal
risk research. Our most invasive procedure has been taking blood samples which is
not trivial for the residents of Iquitos, but one of the IRB forms I have had to fill out
more than once was reporting an unexpected event. Below I provide examples and
how we dealt with them.
During the DVCS study described in the beginning of this section, we conducted
a one-time indoor residual spray (IRS) on the treated blocks. Iquitos has a wide
range of housing types, from wood houses with dirt floors to relatively well-finished
concrete houses with glossy painted walls. Although each of the clusters had the full
range of housing types, one block had a higher proportion of the well-off houses
with glossy paint. We were using a product called Demand CS (lambda cyhalothrin)
and were using a dose consistent with the upper range of the label instructions. We
had a 1-h wait period before residents could return to their homes after applying the
spray. We had discussed all the possible side effects associated with insecticide
applications and provided instructions on what to do if they occurred but had not
expected a high rate of these events. We were about halfway through our spray
schedule with no problems reported through our ongoing surveillance visits con-
ducted 3×/week when we sprayed the block with a lot of glossy paint on a Friday.
By Monday morning there were multiple reports of people experiencing skin irrita-
tion associated with the spray. The study PI, project physician, and I had to quickly
visit every house on the block, access the health status of each of our participants,
and apologize. We found a strong association of the adverse reactions with the
glossy paint, most likely because the solvent carrying the insecticide was not
absorbed by the wall and needed more than an hour to dry. Responses were varied;
in the houses without the glossy paint, participants were extremely grateful declar-
ing the product had made a dramatic difference in the number of mosquitos. In the
houses with the glossy paint, however, the kids had touched the walls with their
hands and then other parts of their bodies and described the experience as rubbing
hot peppers on their skin. Our instructions to wash the affected area with lots of soap
and water were not done in the heat of the moment. I learned the power of an apol-
ogy and the need to build in stronger surveillance protocols with any intervention
and ended up filing 120 unexpected adverse event reports. Nobody dropped out of
the trial in the end, but we reduced our dose to the lower range of label recommen-
dations, produced a simple pamphlet saying that these reactions were likely to
occur, emphasized what to do, and provided a contact number to reach our team.
The IRS applications for the remaining blocks were uneventful (less glossy paint),
but I did get a few calls in the evenings and made visits to reassure participants right
away. I am grateful that we had a responsive IRB that let us take immediate action
(approval for that pamphlet was immediate). Many residents on the other blocks
confided that they have had lots of previous experience with these kinds of reac-
tions, it was normal with fumigations, and in fact it was evidence we were using a
product that worked. The principal lesson here was every participant is a bit differ-
ent and you need to listen and answer their specific questions and concerns and be
available.
Our trial testing insecticide-treated curtains had a great start (Fig. 10). Over 4000
curtains were distributed smoothly and efficiently, and our participants in the treated
clusters (this was not a blinded RCT) were extremely happy as indicated by focus
groups conducted at the time. Initial entomological collections were also encourag-
ing. What was unique about this trial was the complete focus on the primary end-
point of seroconversion to the dengue virus. We would take blood samples just
before curtain deployment and at 9-month intervals after that. At the 6-month mark,
we carried out entomological surveys and pulled a sample of the curtains to verify
that the active ingredient deltamethrin was still effective. Results from those net
evaluations were alarming and highly variable; the curtains had lost their efficacy
452 A. C. Morrison
Fig. 10 Insecticide-treated curtains. Residents could request as many as they could use and the
colors they wanted
prematurely. Our plan B was to retreat the curtains with insecticide, but IRB
approval required a few months. After examining seroconversion rates between the
baseline and 9-month blood samples, the lack of impact on seroconversion rates and
mosquito densities resulted in difficult decision to end the trial. Because the trial did
not monitor active dengue disease, our team did not have a permanent presence
throughout the trial. I am convinced if we had we would have been aware of the
curtain problem earlier and been able to better correct course. As the medical ento-
mology community attempts to increase the rigor of intervention trials, especially
when measuring the public health impacts (transmission or disease), investigators
need to commit to the investment needed to conduct these trials. It is extremely dif-
ficult to power a dengue trial using disease as an outcome; the World Mosquito
Program was able to do so in a recent trial in Yogyakarta but using a unique test
negative design (Utarini et al. 2021), but after the experience with this trial, I decided
I would never do another trial where we did not have regular contact with the com-
munity. Another important lesson learned from the curtain trial was to never design
a cRCT for an Aedes-borne virus where the clusters were adjacent to one another.
We identified many extended families participating in the trial that had some mem-
bers in the control clusters and others in the treated clusters. One of the best exam-
ples of this was when a grandmother living in a treated cluster gifted her pink
curtains to her granddaughter in a control cluster for her “quinceanera.” In the field,
you will always have limited control.
Our lethal ovitrap study (see ALOT in Table 1), which we are still working on
publishing, had encouraging results but did not benefit of the cRCT design. The
logistics of the study were challenging. We deployed >8000 traps in a few months,
and honestly, it was a site to behold. We would stack the traps in the back of our
Toyota Hilux pickup and shuttle them to the field. Our surveillance team, who were
great at taking blood, learned to clean and monitor the traps, a labor-intensive effort
to say the least (Fig. 11). Our rationale was to ensure that the traps were properly
used and maintained; this was the first step to developing the evidence that they
worked. Real-world implementation would come later. The traps were placed both
inside and outside houses. We had consulted with the community on the design, but
many became garbage cans, a few urinals, knocked over by small children and pets,
and overall required regular attention. They were largely popular with the commu-
nity, especially when the team was taking care of them. We leveraged another ongo-
ing epidemiological study (see AS in Table 1) as a control area. Conducting cRCT
would have increased the trial at least tenfold. What I will say is it took all our
resources to conduct the study as it was. Both this and the curtain study provided
our team with the experience and lessons learned to conduct what was to become
our first trial with positive results using a spatial repellent (SR) product.
Fig. 11 Field surveillance teams cleaning and recharging attractant-based lethal ovitraps (ALOT).
Trap contains 500 mL of water, where lyophilized attractant beads and larvicide are added at 3–4-
week intervals, and is in line with DuraNet (alphacypermethrin)
454 A. C. Morrison
10 The Spatial Repellent Trial
In truth, after multiple intervention trials, our spatial repellent trial was the first to
show definitive positive results, but we had to work for it. We showed a 34% protec-
tive efficacy associated with the use of these products, but we got lucky (Morrison
et al. 2022). I thought we had logistics challenges in our earlier trials, but the SR
trial literally kicked my butt. The trial was a last-minute addition to a large BMGF
program conducting trials in Indonesia and three African countries testing the
impact of SR on malaria, at the suggestion of the project’s external scientific advi-
sory committee (ESAC). A budget and protocol were put together quickly where
our site followed the same cRCT design as the malaria sites, but did not receive
clinical trial monitoring. We were the only urban site, so we had the advantage of
easy accessibility of our clusters, but the disadvantage of diverse and complicated
houses that did not facilitate hanging the products on walls. We had to figure out
how to hang these products in a way that we could replace them rapidly. Proyecto
dengue developed a very clever and efficient approach, utilizing elastic lines and
photo check clips, placed at 3 m from one another (Fig. 12) which met the manufac-
turer requirement that the product cover an area of 9 m2. As we waited for the
Fig. 12 Deployment and replacement of the spatial repellent (SR) products in 2015–2019 cRCT
testing the protective efficacy for Aedes-borne virus disease, Zika and dengue virus. Panel A shows
the product, an 8.5 by 11 inch plastic sheet with 55 mg of transfluthrin. The products were shipped
folded in half and sealed. (Panel B) To deploy the sealed product was opened and rolled into a
cylinder with the chemical surface to the outside and clipped to elastic lines. (Panel C) Each rect-
angular space was measured. Most houses in Iquitos do not have ceilings so elastic lines were
attached to the top of walls and run the length of the household, with one line for every 3 m of
house width. For example, a house 4-m wide would have two lines at 1.33 and 2.66 m. On one line
the first product would be placed 1.5 m and the other 3 m from the wall and then products placed
every 3 m until reaching the opposite wall. For rooms with ceiling, the same process occurred but
treating the room as a unique space (Panel H). (Panel D) Team setting the lines and placing the
products. (Panel E) House with products deployed. (Panels F–G) Product replacement could be
done quickly by pulling down the elastic lines. (Panel H) Products in room with ceiling
products to arrive (about a year later than originally scheduled), our team placed the
hanging infrastructure in the homes, which had to be redone when the products did
arrive. It took a team of five 20–60 min to set up the lines and hang the products, but
replacement could be carried out in a few minutes. We received both active (trans-
fluthrin 55 mg) and blank products (solvent only) in boxes labeled by cluster num-
ber (Fig. 8). This was a double-blinded trial; nobody knew which clusters had the
active versus placebo products. The trial was powered for dengue virus transmis-
sion, and we were coming up short of our enrollment goals. We did not have the
bandwidth to add additional houses to increase our human enrollment. Zika virus
arrived in Iquitos a few months before we were able to deploy the intervention; I
remain frustrated that baseline blood samples could not have been timed just before
Zika’s arrival, but we did get them up in time to measure the impact on what was
very high Zika virus transmission rates. Unfortunately, our ability to measure dis-
ease was hampered by the time we needed to obtain IRB approval to change our
inclusion criteria – many Zika infections do not result in fever and many partici-
pants did not experience symptoms. We did however have permission to test for
Zika prior to the initiation of the trial. It was our experience with previous trials, and
significant help from two individuals (Will Elson and Ania Kaweicki who both
spent about a year in Iquitos), that allowed us to use mobile phone technology to
monitor the products, adverse events in the human populations (mild and limited but
well documented), and the presence of our human participants in the study area. I
was lucky; this trial pushed our capacity to the limits, but we managed. I am not the
first to say this, but if the scientific community, as led by the Vector Control Advisory
Group (VCAG), wants to elevate the rigor and quality of vector control intervention
trials, especially for dengue, resources will need to be increased. Every group doing
these kinds of trials is struggling with these issues.
11 GPS, Direct Mosquito Feeds, and Home-Based

Rapid Diagnostics
Working in medical entomology for so many years, one is used to the research com-
munity thinking you do unusual stuff. Mark-release-recapture studies often seem
unusual to the public and some IRBs. Conducting human landing collections can be
controversial. Starting with our Activity Space project, however, our research group
started to propose studies that pushed normal conventions. In 2007 I began a profes-
sional (and personal friendship) collaboration with Valerie Paz Soldan, a Peruvian
behavioral scientist with Tulane University who made it possible to push these
research boundaries. We first met in Iquitos when she was conducting interviewers
with sex workers in Iquitos. I suggested her to Nicole Achee to conduct some focus
groups on vector control practices. She knocked out ten focus group discussions in
a week, while I listened from my office holding her 3-month-old son, Diego, in my
lap. Diego is now 14 years old, and Valerie has two more boys and can only be
456 A. C. Morrison
described as a force of nature. She has been a key member of our dengue studies
ever since. We have done a series of studies together where we start with qualitative
studies followed by subsequent quantitative studies. Below I describe three exam-
ples of this approach, when we had significant concerns about the community’s
willingness to participate, and in each case those initial focus group discussions
provided us with the information we needed to inform and carry out those “unusual”
studies moving forward.
When we proposed our Activity Space study (see AS in Table 1), there had been
sufficient improvements in GPS technology to propose tracking human movements.
I wrote the sections of the NIH proposal about how we would track participants with
GPS but dreaded getting IRB permission and was skeptical about our ability to
effectively implement GPS in our studies. I had visions of our participants putting
the devices on their dogs and having a good laugh. The focus groups on the topic
were once again fun to listen too (I was not yet an active participant), with the par-
ticipants giggling as they put different-colored pieces of Plato on table size satellite
images of their community. People’s concerns were not what we expected and not
related to potential confidentiality breaches (Paz-Soldan et al. 2010). What sur-
prised us was how much people liked the idea – “Finally! Someone is investigating
where people are getting dengue.” Discussions revealed the community was tired of
being blamed for the problem and this line of investigation made sense. Our partici-
pants expressed concerns about people taking good care of the devices and what we
would do to make sure they did not lose them, even suggesting we had participants
sign a contract. In addition to ensuring that people took responsibility for the
devices, it would add legitimacy to the study, especially for women who could prove
the device was not a gift from a lover. Participants were satisfied that we would not
use the information for nefarious purposes (“bad”). The subsequent quantitative
studies where participants carried the GPS units were some of our most popular
ever. Of course some folks did forget to put them on and there were some technical
challenges, but combined with self-reporting of locations visited by our partici-
pants, we were able to characterize movement patterns of this community, the first
study of this kind in a lower- or middle-income country like Peru (Paz-Soldan et al.
2014; Vazquez-Prokopec et al. 2013). These studies were the basis for comparing
movement patterns of people across the spectrum of dengue illnesses (Elson et al.
2020; Schaber et al. 2019, 2021) and future modeling efforts (Perkins et al. 2014,
2016, 2019; Ten Bosch et al. 2018).
Next, we attempted to address the role of human movement in dengue transmis-
sion dynamics, by asking more questions about the role of inapparent infections for
dengue virus transmission dynamics and the ability of viremic individuals to infect
Ae. aegypti mosquitos. Having laboratory-reared mosquitoes feed on dengue vire-
mic individuals, we captured through our surveillance activities was the best way to
address these questions, but the perception among the research team was it would
not be feasible. We developed a pilot study to compare direct and indirect feeding
protocols with the intention of developing the data to support doing indirect feeds.
We had to work slowly and incrementally with our local IRB. We developed a con-
sent video that clearly showed the direct feeding process and conducted focus
groups with Valerie on the topic, including one group with participants who had
already participated in a direct feeding experiment. This was the first time I partici-
pated directly in the focus groups; we combined the topic of direct mosquito feeding
with some other topics (see below). Valerie and I did 22 focus groups in 10 days.
One of the other topics was the interest of community members to use rapid diag-
nostics for dengue on themselves; I became a “designated patient” for our FG par-
ticipants to try out the rapid diagnostics on. The findings from the focus groups have
been published and I encourage those who are interested to read them (Morrison
et al. 2019; Paz-Soldan et al. 2019). We were initially surprised that most of the FG
participants were not that bothered at the prospect of feeding mosquitoes, even
though it was strange (there was quite a bit of giggling when they were shown the
feeding section of the consent video). When the rationale for the study was explained,
most found the study perfectly reasonable. One woman looked at me, declaring
“why would she not feed mosquitoes if it would help us learn more about dengue?”
I was moved by the expression of civic duty expressed by the majority of mostly
woman participants and the willingness to trust us as investigators. There were
plenty of interesting questions and concerns, which we discussed. Being a focus
group rookie and with a propensity to talk too much, I found myself giving detailed
answers to some of these questions and Valerie had to impose a small gag order on
me. She emphasized our jobs were to listen and learn. Participants were often less
interested in the scientific details but our word that what we are doing was of value
and being done safely.
I was a very active participant in a pilot feeding study being present for most of
the direct feeds. We brought people to our lab and the process was always monitored
by one of our project physicians. My job was usually to put the two containers of 25
hungry mosquitoes on the persons’ arms or legs (we had one participant opt for his
stomach) and did my best to distract them during the 10-min feeding period. We had
a comfortable lounge chair, provided beverages and cookies, and drove the partici-
pants to and from the procedure. We had itch cream on hand and made sure it was
applied before we drove each participant home with the rest of the tube. We moni-
tored our participants until their illness had resolved and none were bothered by the
bites including our child participants. Since they all had dengue, most were not
feeling that well, and one participant, an 18-year-old girl, was glided through the
feeds but started to feel dizzy while waiting for her ride home. Our physician opted
to put her under observation at the Navy clinic where the field laboratory is located.
I accompanied the participant and her mother for a few hours while she was under
observation. As we drove them home, I apologized for the process taking so long
and for any inconveniences she had experienced. Her response was emphatic, “why,
I felt like a princess.” I guess I would get a good YELP review. Our pilot eventually
concluded that we would continue using direct feeds rather than using an indirect
feeding method (Long et al. 2019). We went on to do an additional 150 feeds on
dengue viremic participants for our NIH program project (see P01 in Table 1).
The last study I will describe was our FGDs asking participants their views on
“home rapid diagnostics” for dengue, which included the participants practicing
with some prototype devices on their own after viewing an instructional video and
458 A. C. Morrison
with pictorial instructions prepared by our team (Paz-Soldan et al. 2019). We made
video instructions and let residents try it out on themselves, their child, or friends in
a community-based study where we observed residents’ ability to carry out the test
appropriately (Morrison et al. 2021b). The results were mixed, we tested two
devices, and competency was good with the simplest device. What was clear was
the sense of empowerment that conducting the test gave many of the residents. They
expressed some fear initially but proud they could do it. When COVID-19 hit, I
wished we could have provided dengue kits to our participants since there was con-
siderable dengue-1 transmission during the first wave of the pandemic. If people
had had the ability to distinguish between the two infections at home, it might have
helped with patient management.
12 Scientific Diplomacy
Although not directly related to my work in the field, as the Iquitos program started
to yield results, I began to be invited to give talks in conferences, evaluate other
projects, and sit on advisory panels. I will say this is one of the most gratifying
aspects of my work in arbovirology. Below I highlight just a few of these experiences.
12.1 WHO/TDR Multicounty Study on the Pupal Survey

Technique for the Dengue Vector Aedes aegypti
When we applied for what was a small grant designed to deploy and evaluate the
pupal demographic survey developed by Dana Focks, as component of our first R01
grant in Iquitos, we had conducted >60,000 of these surveys between 1998 and
2002. We proposed using the methodology to survey nonresidential sites and the
grant was funded (Morrison et al. 2006). More important, however, was the oppor-
tunity to be part of a working group of young scientists from all over the world. We
met a few times in Puerto Rico and Cuba during the International Dengue Course.
At the Cuba meeting, I roomed with Audrey Lenhart and Claudia Romero, also
members of the group. I only mention this because doing so just about broke the
system at the Hotel Palco. We became the talk of the hotel, three people in one
room, and had to make multiple visits to a scary basement office to explain that we
wanted to split the bill three ways. People would point at us in the elevator declaring
we were the three women sharing a room; we would counter with “No, somos las
chicas de la pupas (No we are the Pupae Girls)”. Audrey and I were also guilty of
putting more than the legal limit of people in a cab to the center of town; we were
quite the rebels. This meeting was also the site of one of my favorite stories about
Scott Halstead. He was an institution at the dengue course, given every other year
and for some reason decided to join our session. During a presentation by Dana
Focks, Dr. Halstead challenged him about his presentation and a slightly heated
debate ensued. Dana asked him what evidence for his position was and Dr. Halstead
just paused and said, “Because I’m Scott Halstead.” Audrey and I were in the back
of the room; dumbstruck because the statement was made cold and without hesita-
tion, we started to giggle under our breaths rather uncontrollably. I had yet to make
friends with Dr. Halstead but now look back on that moment fondly. It would be at
a later dengue course where I was on the instructor list, that Dawn Wesson and
would join Dr. Halstead for dinner at the Palco restaurant each night to listen to his
research tales. He had great stories and genuine interest in my research, especially
our cohort in Iquitos. We continue to cross paths at meetings throughout Latin
America and he graciously gave a talk and joined a panel in one of our meetings in
Iquitos. After so many conversations about our cohort PRNT data (he once said we
were heroes, for doing the serotype-specific neutralization testing on our longitudi-
nal cohort participants), it meant a lot for him to see our research team in action.
This multicounty pupal survey group went on to test the efficacy of targeted control
of highly productive containers in their respective countries (Alexander et al. 2006;
Tun-Lin et al. 2009). I continue to collaborate with many of the people I met through
this group.
12.2 Camino Verde (the Green Way) to the PAHO New

Technologies Group
I first met Eva Harris while I was still on the UC Davis Campus when we applied
for a California Mosquito Research Program grant. After that, Eva periodically
reached out for advice on collecting Ae. aegypti and implemented some of our pro-
tocols in Nicaragua. I became an external reviewer on both a pilot and cRCT trial
evaluating a unique social mobilization methodology to control dengue. This was
my first experience as an external reviewer, and it was a serious job. It was a few
weeks each of preparation time, site visits, and an extensive report of my findings.
This was the first time I realized I was considered an expert. Their program, which
I would describe as “grassroots epidemiology” included a well-developed and tested
methodology for working and organizing communities and the first example of a
properly done vector control intervention trial (Andersson et al. 2015). Since then,
I have been a member of several advisory groups, including guideline development
for the WHO and most recently the PAHO – External Evaluation Group of New
Technologies. We are a group of scientists from or who have worked extensively in
Latin America. In addition to developing PAHO guidelines on rolling out novel vec-
tor control technologies, we have conducted site visits in Medellin, Colombia, and
Rio de Janeiro, Brazil, to evaluate the World Mosquito Program Wolbachia release
programs in both sites. These were intense evaluations that included visiting the
release areas and meeting with community members and government and program
officials. Evenings were spent reviewing our findings and developing
460 A. C. Morrison
recommendations that were presented before leaving each country and then our
return preparation of a formal report. Again, it is during the activities where you feel
as though you are making an immediate contribution to addressing dengue. One of
my favorite aspects of working with PAHO is it is truly a trilingual activity. Listening
to a Zoom call with myself, Haroldo Bezzera, and Giovanini Coelho, both from the
Regional Public Health Entomology Section and both Brazilians, is quite entertain-
ing. We speak in Portunol, a tortured mixture of Spanish and Portuguese, but man-
age to communicate. The dynamic is the same among the members of the group, an
interesting mix of English, Spanish, and Portuguese.
13 Lessons Learned and Recommendations
To work effectively in the field, you need strong relationships with your team and
the community where you are doing the research. For me the most effective way to
do this is as a member of the community you work in. For my research activities in
Iquitos, the team was comprised of lifelong Iquitos residents and a few transplants
from other parts of Peru. We have had considerable input from the large number of
collaborators and scientists who contributed to the program. There were a few
US-European scientists who spent a year or more living and working in Iquitos:
Greg Devine, Brett Forshey, Kanya Long, Claudine Kocher, and Will Elson. Each
benefited tremendously from their time living in the field site and have gone on
to make significant contributions to either arbovirology, tropical medicine, or medi-
cal entomology. A few were able to contribute significantly from the United States,
like Steve Stoddard and Gonzalo Vasquez, who visited Iquitos often enough to
establish relationships with the teams and who could use Internet-based communi-
cations in Spanish. The contact cluster investigation and GPS studies that they coor-
dinated from outside Iquitos are some of our most influential contributions to the
understanding of dengue virus transmission (Stoddard et al. 2013; Vazquez-
Prokopec et al. 2009, 2013).
At the time of writing this chapter, our research program has been greatly affected
by the COVID-19 pandemic at the same time as some funding gaps. Cohort activi-
ties have been scheduled to restart in October 2021 after ceasing on March 12, 2020.
During the pandemic, I started work on a U01 grant, along with new UC Davis col-
leagues, we became grant was a member of the Center for Research in Emerging
Infectious Diseases (CREID, https://creid-network.org) where we are focusing on
other arboviruses circulating in the region and will conduct significant wildlife sam-
pling, which represents a new challenge for me. For our dengue work, I believe the
next step is to implement much of the lessons we have learned and am committed to
continue operational research studies. Below are three recommendations I have
been making to my health department counterparts and anyone else who will listen.
Scientifically, as a community, we need to (1) improve ongoing government sur-
veillance programs so that information can be used to constantly evaluate these
programs, (2) utilize step-wedge study designs to evaluate vector control
interventions, and (3) develop protocols that are preapproved and ready to imple-
ment when emergency situations develop.
References
Alexander N, Lenhart AE, Romero-Vivas CM et al (2006) Sample sizes for identifying the key
types of container occupied by dengue-vector pupae: the use of entropy in analyses of compo-
sitional data. Ann Trop Med Parasitol 100(Suppl 1):S5–S16
Andersson N, Nava-Aguilera E, Arostegui J et al (2015) Evidence based community mobilization
for dengue prevention in Nicaragua and Mexico (Camino Verde, the Green Way): cluster ran-
domized controlled trial. BMJ 351:h3267
Baltzegar J, Vella M, Gunning C et al (2021) Rapid evolution of knockdown resistance haplotypes
in response to pyrethroid selection in Aedes aegypti. Evol Appl 14(8). Wiley:2098–2113
Bangs MJ, Barr AR, Cope SE, et al (1986) Assessment of adult mosquito populations in a fresh-
water marsh in southern California by various trapping methods. In Proceedings and papers of
the annual conference of the California Mosquito and Vector Control Association (USA), vol
54, pp 113–116
Barr AR, Morrison AC, Guptavanij P et al (1986) Parity rates of mosquitoes collected in the San
Joaquin marsh. In Proceedings and papers of the annual conference of the California Mosquito
and Vector Control Association (USA), vol 54, pp 117–118
Bautista CT, Chan AST, Ryan JR et al (2006) Epidemiology and spatial analysis of malaria in
the Northern Peruvian Amazon. Am J Trop Med Hyg 75(6). American Society of Tropical
Medicine and Hygiene:1216–1222
Carrasco J, Morrison A, Ponce C (1998) Behaviour of Lutzomyia longipalpis in an area of south-
ern Honduras endemic for visceral/atypical cutaneous leishmaniasis. Ann Trop Med Parasitol
92(8):869–876
Chaves LF, Morrison AC, Kitron UD et al (2012) Nonlinear impacts of climatic variability on
the density-dependent regulation of an insect vector of disease. Glob Chang Biol 18(2).
Wiley: 457–468
Chaves LF, Scott TW, Morrison AC et al (2014) Hot temperatures can force delayed mosquito
outbreaks via sequential changes in Aedes aegypti demographic parameters in autocorrelated
environments. Acta Trop 129:15–24
Cope SE, Barr AR, Bangs MJ et al (1986) Human bait collections of mosquitoes in a south-
ern California freshwater marsh. In Proceedings and papers of the annual conference of the
California Mosquito and Vector Control Association (USA) 54. agris.fao.org: 110–112
Edman JD, Scott TW, Costero A et al (1998) Aedes aegypti (Diptera: Culicidae) movement influ-
enced by availability of oviposition sites. J Med Entomol 35(4):578–583
Elson WH, Riley-Powell AR, Morrison AC et al (2020) Measuring health related quality of life
for dengue patients in Iquitos, Peru. PLoS Negl Trop Dis 14(7). Public Library of Science
(PLoS):e0008477
Ferro C, Morrison AC, Torres M et al (1995a) Age structure, blood-feeding behavior, and
Leishmania chagasi infection in Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic
focus of visceral leishmaniasis in Colombia. J Med Entomol 32(5):618–629
Ferro C, Morrison AC, Torres M et al (1995b) Species composition and relative abundance of sand
flies of the genus Lutzomyia (Diptera: Psychodidae) at an endemic focus of visceral leishmani-
asis in Colombia. J Med Entomol 32(4):527–537
Ferro C, Pardo R, Torres M et al (1997) Larval microhabitats of Lutzomyia longipalpis (Diptera:
Psychodidae) in an endemic focus of visceral leishmaniasis in Colombia. J Med Entomol
34(6):719–728
462 A. C. Morrison
Focks DA, Haile DG, Daniels E et al (1993a) Dynamic life table model for Aedes aegypti (Diptera:
Culicidae): analysis of the literature and model development. J Med Entomol 30(6):1003–1017
Focks DA, Haile DG, Daniels E et al (1993b) Dynamic life table model for Aedes aegypti (Diptera:
Culicidae): simulation results and validation. J Med Entomol 30(6):1018–1028
Focks DA, Daniels E, Haile DG et al (1995) A simulation model of the epidemiology of urban
dengue fever: literature analysis, model development, preliminary validation, and samples of
simulation results. Am J Trop Med Hyg 53(5):489–506
Getis A, Morrison AC, Gray K et al (2003) Characteristics of the spatial pattern of the dengue vec-
tor, Aedes aegypti, in Iquitos, Peru. Am J Trop Med Hyg 69(5):494–505
Gunning CE, Okamoto K, Astete H et al (2018) Efficacy of Aedes aegypti control by indoor Ultra
Low Volume (ULV) insecticide spraying in Iquitos, Peru. PLoS Negl Trop Dis 12(4):e0006378
Jones JW, Turell MJ, Sardelis MR et al (2004) Seasonal distribution, biology, and human attraction
patterns of culicine mosquitoes (Diptera: Culicidae) in a forest near Puerto Almendras, Iquitos,
Peru. J Med Entomol 41(3). Oxford University Press (OUP):349–360
Lenhart A, Morrison AC, Paz-Soldan VA et al (2020) The impact of insecticide treated curtains
on dengue virus transmission: a cluster randomized trial in Iquitos, Peru. PLoS Negl Trop Dis
14(4):e0008097
Long KC, Sulca J, Bazan I et al (2019) Feasibility of feeding Aedes aegypti mosquitoes on dengue
virus-infected human volunteers for vector competence studies in Iquitos, Peru. PLoS Negl
Trop Dis 13(2):e0007116
Montoya Lerma J (2016) Tributo a María Cristina Ferro (q.e.p.d.). Rev Colomb Entomol 42(1).
Universidad del Valle:97
Morrison AC, Ferro C, Morales A et al (1993a) Dispersal of the sand fly Lutzomyia longipal-
pis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in Colombia. J Med
Entomol 30(2):427–435
Morrison AC, Ferro C, Tesh RB (1993b) Host preferences of the sand fly Lutzomyia longipalpis
at an endemic focus of American visceral leishmaniasis in Colombia. Am J Trop Med Hyg
49(1):68–75
Morrison AC, Ferro C, Pardo R, Torres M, Wilson ML et al (1995a) Nocturnal activity patterns of
Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in
Colombia. J Med Entomol 32(5):605–617
Morrison AC, Ferro C, Pardo R, Torres M, Devlin B et al (1995b) Seasonal abundance of Lutzomyia
longipalpis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in Colombia.
J Med Entomol 32(4):538–548
Morrison AC, Getis A, Santiago M et al (1998) Exploratory space-time analysis of reported
dengue cases during an outbreak in Florida, Puerto Rico, 1991–1992. Am J Trop Med Hyg
58(3):287–298
Morrison AC, Costero A, Edman JD et al (1999) Increased fecundity of Aedes aegypti fed human
blood before release in a mark-recapture study in Puerto Rico. J Am Mosq Control Assoc
15(2):98–104
Morrison AC, Astete H, Chapilliquen F et al (2004a) Evaluation of a sampling methodology
for rapid assessment of Aedes aegypti infestation levels in Iquitos, Peru. J Med Entomol
41(3):502–510
Morrison AC, Gray K, Getis A et al (2004b) Temporal and geographic patterns of Aedes aegypti
(Diptera: Culicidae) production in Iquitos, Peru. J Med Entomol 41(6):1123–1142
Morrison AC, Sihuincha M, Stancil JD et al (2006) Aedes aegypti (Diptera: Culicidae) produc-
tion from non-residential sites in the Amazonian city of Iquitos, Peru. Ann Trop Med Parasitol
100(Suppl 1):S73–S86
Morrison AC, Schwarz J, Long KC et al (2019) Acceptability of Aedes aegypti blood feeding on
dengue virus-infected human volunteers for vector competence studies in Iquitos, Peru. PLoS
Negl Trop Dis 13(2):e0007090
Morrison AC, Reiner RC Jr, Elson WH et al (2022) Efficacy of a spatial repellent for control of
Aedes-borne virus transmission: a cluster randomized trial in Iquitos, Peru. Proc Natl Acad Sci
U S A 119(26):e2118283119
Morrison AC, Schwarz J, Mckenney JL et al (2021b) Potential for community based surveillance
of febrile diseases: feasibility of self-administered rapid diagnostic tests in Iquitos, Peru and
Phnom Penh, Cambodia. PLoS Negl Trop Dis 15(4):e0009307
Munstermann LE, Morrison AC, Ferro C et al (1998) Genetic structure of local populations
of Lutzomyia longipalpis (Diptera: Psychodidae) in Central Colombia. J Med Entomol
35(1):82–89
Pardo RH, Torres M, Morrison AC et al (1996) Effect of fluorescent powder on Lutzomyia longi-
palpis (Diptera: Psychodidae) and a simple device for marking sand flies. J Am Mosq Control
Assoc 12(2 Pt 1):235–242
Paz-Soldan VA, Stoddard ST, Vazquez-Prokopec G et al (2010) Assessing and maximizing the
acceptability of global positioning system device use for studying the role of human movement
in dengue virus transmission in Iquitos, Peru. Am J Trop Med Hyg 82(4):723–730
Paz-Soldan VA, Reiner RC Jr, Morrison AC et al (2014) Strengths and weaknesses of Global
Positioning System (GPS) data-loggers and semi-structured interviews for capturing fine-scale
human mobility: findings from Iquitos, Peru. PLoS Negl Trop Dis 8(6):e2888
Paz-Soldan VA, Yukich J, Soonthorndhada A et al (2016) Design and testing of novel lethal ovitrap
to reduce populations of Aedes mosquitoes: community-based participatory research between
industry, academia and communities in Peru and Thailand. PLoS One 11(8):e0160386
Paz-Soldan VA, Morrison AC, Sopheab H et al (2019) Potential use of community-based rapid
diagnostic tests for febrile illnesses: formative research in Peru and Cambodia. PLoS Negl Trop
Dis 13(10):e0007773
Perkins TA, Garcia AJ, Paz-Soldan VA et al (2014) Theory and data for simulating fine-scale
human movement in an urban environment. J R Soc Interface/R Soc 11(99):20140642. https://
doi.org/10.1098/rsif.2014.0642
Perkins TA, Paz-Soldan VA, Stoddard ST et al (2016) Calling in sick: impacts of fever on intra-
urban human mobility. Proc Biol Sci/R Soc 283(1834). https://doi.org/10.1098/rspb.2016.0390
Perkins TA, Reiner RC Jr, España G et al (2019) An agent-based model of dengue virus trans-
mission shows how uncertainty about breakthrough infections influences vaccination impact
projections. PLoS Comput Biol 15(3):e1006710
Ponce C, Ponce E, Morrison A et al (1991) Leishmania donovani chagasi: new clinical variant of
cutaneous leishmaniasis in Honduras. Lancet 337(8733):67–70
Reiner RC Jr, Stoddard ST, Vazquez-Prokopec GM et al (2019) Estimating the impact of city-wide
Aedes aegypti population control: an observational study in Iquitos, Peru. PLoS Negl Trop Dis
13(5):e0007255
Schaber KL, Paz-Soldan VA, Morrison AC et al (2019) Dengue illness impacts daily human mobil-
ity patterns in Iquitos, Peru. PLoS Negl Trop Dis 13(9):e0007756
Schaber KL, Perkins TA, Lloyd AL et al (2021) Disease-driven reduction in human mobility influ-
ences human-mosquito contacts and dengue transmission dynamics. PLoS Comput Biol 17(1).
Public Library of Science (PLoS):e1008627
Scott TW, Morrison AC (2003) Aedes aegypti density and the risk of dengue virus transmission.
In: Takken W, Scott Thomas W (eds) Ecological aspects for application of genetically modified
mosquitoes. Kluwer Academic Publishers, Dordrecht/Boston, pp 187–206
Scott TW, Morrison AC (2008) Longitudinal field studies will guide a paradigm shift in dengue
prevention. In: Lemon SM, Institute of Medicine (U.S.). Forum on Microbial Threats (eds)
Vector-borne diseases: understanding the environmental, human health, and ecological connec-
tions: workshop summary. National Academies Press, Washington, DC, pp 32–149
Scott TW, Morrison AC (2010) Vector dynamics and transmission of dengue virus: implications
for dengue surveillance and prevention strategies: vector dynamics and dengue prevention.
Curr Top Microbiol Immunol 338:115–128
464 A. C. Morrison
Scott TW, Amerasinghe PH, Morrison AC et al (2000a) Longitudinal studies of Aedes aegypti
(Diptera: Culicidae) in Thailand and Puerto Rico: blood feeding frequency. J Med Entomol
37(1):89–101
Scott TW, Morrison AC, Lorenz LH et al (2000b) Longitudinal studies of Aedes aegypti (Diptera:
Culicidae) in Thailand and Puerto Rico: population dynamics. J Med Entomol 37(1):77–88
Stoddard ST, Forshey BM, Morrison AC et al (2013) House-to-house human movement drives
dengue virus transmission. Proc Natl Acad Sci U S A 110(3):994–999
Stoddard ST, Wearing HJ, Reiner RC Jr et al (2014) Long-term and seasonal dynamics of dengue
in Iquitos, Peru. PLoS Negl Trop Dis 8(7):e3003
Ten Bosch QA, Clapham HE, Lambrechts L et al (2018) Contributions from the silent majority
dominate dengue virus transmission. PLoS Pathog 14(5):e1006965
Treangen TJ, Schoeler G, Phillippy AM et al (2016) Identification and genomic analysis of a novel
Group C orthobunyavirus isolated from a mosquito captured near Iquitos, Peru. PLoS Negl
Trop Dis 10(4):e0004440
Tun-Lin W, Lenhart A, Nam VS et al (2009) Reducing costs and operational constraints of dengue
vector control by targeting productive breeding places: a multi-country non-inferiority cluster
randomized trial. Trop Med Int Health TM & IH 14(9):1143–1153
Turell MJ, O’guinn ML, Jones JW et al (2005) Isolation of viruses from mosquitoes (Diptera:
Culicidae) collected in the Amazon basin region of Peru. J Med Entomol 42(5). Oxford
University Press (OUP):891–898
Turell MJ, Sardelis MR, Jones JW et al (2008) Seasonal distribution, biology, and human attraction
patterns of mosquitoes (Diptera: Culicidae) in a rural village and adjacent forested site near
Iquitos, Peru. J Med Entomol 45(6):1165–1172
Utarini A, Indriani C, Ahmad RA, Tantowijoyo W, Arguni E, Ansari MR, Supriyati E, Wardana
DS, Meitika Y, Ernesia I, Nurhayati I, Prabowo E, Andari B, Green BR, Hodgson L, Cutcher Z,
Rancès E, Ryan PA, O’Neill SL, Dufault SM, Tanamas SK, Jewell NP, Anders KL, Simmons
CP (2021) Efficacy of Wolbachia-infected mosquito deployments for the control of dengue. N
Engl J Med 384(23). Massachusetts Medical Society: 2177–2186
Vazquez-Prokopec GM, Stoddard ST, Paz-Soldan V et al (2009) Usefulness of commercially
available GPS data-loggers for tracking human movement and exposure to dengue virus. Int J
Health Geogr 8:68
Vazquez-Prokopec GM, Bisanzio D, Stoddard ST et al (2013) Using GPS technology to quantify
human mobility, dynamic contacts and infectious disease dynamics in a resource-poor urban
environment. PLoS One 8(4):e58802
50 Years of Medical Entomology:
Miscellaneous Interesting Findings
William K. Reisen
Abstract The following account describes what I felt were unexpected results and
observations and some methods developed to expedite our research projects. Most
have been published and the short descriptions I have included have been annotated.
The chapter starts with a little of my personal background and provides a road map
to times and places.
A thought from the field: “What you see is based on opportunity, how you see
it and use it is based on training and experience….”
1 Background
Although many of my colleagues carefully focused their training towards a career

in arbovirology, my background was diverse, the journey convoluted, and the arrival
at arboviruses serendipitous. I originally was interested in a career in marine biol-
ogy after having worked during the summer of 1963 at the Sandy Hook Marine
Laboratory in New Jersey; however, I was denied admittance into the rather elite
University of Delaware marine biology program and was advised to seek admit-
tance into the Entomology Department within the School of Agriculture. Classes
here and within the associated Plant Pathology Department introduced me to agri-
culture, insects, and hands-on microbiology (thankfully you could not infect your-
self studying plant pathogens!). Being a pragmatist, I quickly realized that there
were many more job opportunities in entomology after graduation than in marine
biology, so I stuck with this program. Through my course work, I became interested
in pursuing graduate work and in 1967 was accepted into a master’s program in the
Department of Entomology and Zoology at Clemson University. The Piedmont
region of South Carolina was filled with interesting streams and Clemson was adja-
cent to the Hartwell Reservoir, so I became interested in limnology and developed a
W. K. Reisen (*)
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine,
University of California, Davis, CA, USA
466 W. K. Reisen
research project investigating how fish mouth position determined prey selection
within the aquatic insect community.
During my studies at Clemson in 1968, the Nixon administration invented a lot-
tery system to populate our troop needs in Vietnam and my draft registration num-
ber was drawn, as was my roommate. He (a forestry entomology student) found out
that the US Air Force had openings for two medical entomologists, so based upon
having taken an undergraduate class in medical entomology, we both applied and
were awarded direct commissions as second Lieutenant Medical Entomologists.
The USAF gave us 6 months deferred enlistment to finish our MS degrees and in
January 1969 we wound up sharing an office at Lackland AFB in Texas. I was soon
reassigned to the Epidemiological Laboratory in Manila, Republic of the Philippines,
a unit created to support disease prevention and control programs on USAF bases in
the Pacific. Without much training, little hands-on experience with vectors, and a lot
of enthusiasm, our entomology group provided support for programs on about 40
installations in eight countries throughout the Pacific Air Forces (PACAF) from
Hawaii west through Thailand. This was my introduction to Asia and “real” medical
entomology. After a year in Manila, the “unit” was deemed a failure and closed, and
most personnel were sent back to the USA. I was reassigned to Clark Air Base in
central Luzon, where I was the only medical entomologist in PACAF. This period,
where we did insect and rodent surveys on various installations, investigated out-
breaks of malaria, scrub typhus and murine typhus, and assisted various agencies
with logistical support in the Philippines, kindled my interest in medical entomol-
ogy and international health. During this period, I also met and married my life’s
partner, Norma Reyes, providing a unique and very personal insight into Philippine
society and culture.
After completing my service requirements in 1971, I used the “GI bill” to help
fund my PhD program. The University of Oklahoma in Norman, OK, offered me a
teaching assistantship, and Cluff Hopla agreed to be my advisor. He thought the
PhD should be more than just research and should focus on course work, so my
major in medical entomology was augmented with supporting fields in ecology,
medical microbiology, and experimental statistics. With Norma’s full support, I was
able to finish in 1974, but found it difficult to land a job. We finally accepted a posi-
tion with the International Health Program at the University of Maryland’s School
of Medicine in Baltimore working at the Pakistan Medical Research Center in
Lahore under the direction of Richard H. Baker. I was the field ecologist assigned to
conduct and evaluate releases of sterile or genetically modified Culex tritaeniorhyn-
chus and Anopheles culicifacies. Work with Cx. tritaeniorhynchus led to my col-
laboration with our virologist, Curtis Hayes, on the epidemiology of West Nile virus
(WNV) – a preview for things to come. My primary job was to determine when,
where, and how many males were necessary to release and to measure the impact of
these releases on target populations. This required a lot of fieldwork in rural villages
and a lot of fundamental studies on mosquito ecology and biology. The work was
fascinating, the local Islamic culture interesting, and my colleagues/technicians
great to work with. Norma organized a great home life for me and our two young
children. However, political trouble was brewing in Pakistan concurrent with the
50 Years of Medical Entomology: Miscellaneous Interesting Findings 467
hostage crisis at the American Embassy in Iran and the rise of Wahhabi Islamic
fundamentalism. This eventually led to an attempted takeover of the US Embassy in
Islamabad and NIH to withdraw our funding.
Concurrently in the late 1970s, William C. Reeves at the School of Public Health
at the University of California, Berkeley, with Monica Asman and James Hardy,
was investigating similar genetic technology to control Culex tarsalis in California.
During one home leave, I stopped in California on my way to the Philippines and
visited the Berkeley program. Bill and I had a couple of beers at a local bar,
exchanged ideas, and departed our ways. Soon afterwards, he was informed that the
director of his field station in Bakersfield was ill and requested early retirement. He
wrote asking if I was interested to come back to the USA and would consider apply-
ing for this job in Bakersfield. As the politics in Pakistan were strained and our
funding entering crisis mode, I was left with trying to develop a self-funded position
at the International Health Program in Baltimore or accepting a fully funded faculty
job with the University of California; we showed up in California within a month.
The Arbovirus Field Station of the University of California, Berkeley, was an
“unofficial” University of California field station established in the 1940s and was
dedicated to the study of mosquitoes and arboviruses, mostly St. Louis (SLEV) and
western equine encephalitis (WEEV) viruses. The principal thrust when I arrived in
1980 was the use of genetics to control mosquitoes; however, the epidemiology of
arboviruses remained an important undercurrent theme. Several genetic release
experiments with negative findings (quite similar to the results of studies in Pakistan)
sounded the death knoll for the genetic control research thrust, resulting in a renewed
focus on arboviruses in the Bakersfield area. With waning activity of SLEV and
WEEV in the Central Valley, we developed new programs on the enzootic activity
of these viruses in the desert regions of southeastern California. In 1999 WNV
emerged in New York and within five short years invaded California. This allowed
us to study how the invading WNV displaced closely related endemic SLEV and
then allowed its resurgence. Increased WNV amplification and epidemic activity
enhanced our ability to revisit long-studied basic concepts of overwintering, disper-
sal, amplification, and intervention, but with more virus activity and new sampling
and diagnostic methods.
2 Discoveries
Discoveries are often based on how you interpret and then develop casual observa-
tions. Frequently, these do not appear significant when you first observe them, but
maybe the foundation of the pyramid of learning that is important in the long term.
468 W. K. Reisen
3 Some Interesting Entomological Findings
Colonization: Mosquito Population Structure and Selection During

Colonization Not all aspects of mosquito biology and virus transmission can be
studied in nature, requiring the colonization and intentional/unintentional selection
of field populations to mate in cages. Eurygamous Anopheles and Culex species
typically mate in large swarms and therefore are notoriously difficult to colonize.
Colonization frequently requires taking large collections from the field and getting
them to mate voluntarily in large and then progressively smaller cages until an
apparently vigorous “wild-type” colony emerges that theoretically is representative
of the source field population. Although it was known for years that these popula-
tions accumulate mutations and change while in colony (Munstermann 1994), it
was less clear what biological characters were selected/altered during
colonization.
My first disappointing discovery was that individuals within field populations do
not mate randomly and that colonization selects for a deem from within the field
population that can mate within small cages (Reisen 1985). These colonized deems
do not mate competitively when released back into the source population. Assortative
mating was documented during our assessments of the mating competitiveness of
sterilized mosquitoes, first with Culex tritaeniorhynchus (Baker et al. 1979; Reisen
et al. 1980) and Anopheles culicifacies (Baker et al. 1980; Reisen et al. 1981b) in
Pakistan and then with Cx. tarsalis (Reisen et al. 1981a, b) in California. Colonized
males sterilized either chemically or by irradiation would mate competitively
against fertile colonized wild-type males for colonized wild-type females within
laboratory and even outdoor cages, but not against field males for field females in
natural settings. This outcome remained the same, even when populations of Cx.
tarsalis were colonized in the fall, amplified during winter, and then the F3 genera-
tion irradiated and released the following spring into the source foothill canyon,
allowing us to inundate the target population (Reisen et al. 1982b). Interestingly,
these released sterilized males did mate competitively in nature with marked and
released colonized wild-type females, indicating that the released males were able
to fly and mate, but they did not mate competitively with all field females, indicating
that mating was assortative (Reisen et al. 1982b). Statistically, the released sterile
males were supercompetitive for marked and released colonized females, but were
uncompetitive for unmarked field females. In support that this was due to assortative
mating related to colonization, when males emerging from field-collected pupae
were sterilized and released without colonization, these males were equally com-
petitive for both concurrently emerging females and those from the target popula-
tion (Reisen et al. 1981a). Assortative mating of colonized males for colonized
females also was documented when males carrying an autosomal recessive eye
color mutant only visible in the early larval instars (Asman 1975) were released into
a relatively closed target field population (Reisen et al. 1985).
Although low competitiveness of released and sterilized males seemed originally
to be just a disappointing finding, the discovery that it was related to assortative
mating was highly important because it showed that colonization selected for only
a portion of the field population. This recently became important as genetic control
or population replacement for Aedes aegypti control gained impetus in the wake of
dengue virus increases and recently the chikungunya and Zika virus pandemics.
Although body size seems critical in stenogamous Aedes populations (Callahan
et al. 2018), less is known about the behavioral or morphological phenotypes that
lead to assortment in eurygamous mosquito genera and taxa, although both pheno-
typic similarity and dissimilarity can produce assortative differences (Kopp et al.
2018). The ability of future genetic control programs to address this issue may be
critical for reduction or replacement of target vector populations.
Colonization: Importance in the Study of Overwintering How arboviruses persist
during unfavorable periods when the vectors are not active has been a mystery since
arboviruses were discovered in the previous century (Reeves 1961, 1987; Rosen
1987). Among the possible mechanisms considered, persistent infection within the
vector population seems highly plausible and has been investigated in the laboratory
and field. Temperate Culex are vectors of several important viruses in North
America, including SLEV and WNV. In North America the Culex pipiens complex
consisting of Cx. pipiens var. pipiens and var. molestus and Cx. quinquefasciatus are
important urban vectors. Early studies with colonized populations showed that
shortening day length and cool temperature during larval development induced fac-
ultative reproductive diapause in Cx. pipiens v. pipiens, but not in Cx. pipiens v.
molestus that produce their first eggs autogenously without a bloodmeal (Spielman
1974; Spielman and Wong 1973a, b) or in the southern Cx. quinquefasciatus
(Eldridge 1987). The induction of reproductive diapause indicated that these females
would not take blood meals prior to overwintering, eliminating or at least drastically
reducing the possibility that they would serve as overwintering hosts for arbovi-
ruses, unless they were infected through the vertical passage of the virus.
Culex tarsalis is the primary vector of WEE, SLE, and WN viruses in the western
USA, has been difficult to colonize, and exhibits autogeny (Bellamy and Kardos
1958). When I came to California in 1980, little had been done experimentally on
the overwintering of this species, although field studies indicated they most likely
entered reproductive diapause (Bellamy and Reeves 1963; Nelson 1964). Copying
diapause induction protocols used by Eldridge for studying the Cx. pipiens complex
(Eldridge and Bailey 1979), we were unsuccessful in inducing diapause in colo-
nized Cx. tarsalis, even when these colonies were selected for anautogeny (Eberle
and Reisen 1986; Reisen 1986b). However, when the F1 progeny of field-collected
females were reared under diapause induction protocols of cool water temperature
(18 °C) and short photoperiod (10 L:14D), the emerging females readily entered
reproductive diapause based on ovariole morphology (Reisen 1986a; Reisen et al.
1986a). Quite different from autogenous Cx. pipiens molestus, genetically autoge-
nous Cx. tarsalis subjected to diapause induction conditions would arrest ovarian
development and enter diapause, but their progeny reared under summer conditions
would produce eggs autogenously (Reisen 1986b). Collectively, these data indi-
cated that the deme of Cx. tarsalis that typically was selected during colonization
470 W. K. Reisen
and would mate in small cages lacked the ability to enter diapause and that this was
not related to autogeny. The fact that field populations consisted of diapausing and
nondiapausing females was implied from the collection of reproductively active
females (albeit in low numbers) throughout the year (Bellamy and Reeves 1963;
Reisen et al. 1983), especially at warmer latitudes (Reisen et al. 1995). Genetic
studies subsequently indicated spatial structuring within Cx. tarsalis populations
(Venkatesan et al. 2007), but this has not been related to either mating types or the
ability to enter diapause.
How Do Autogenous Cx. tarsalis Females Overwinter? Autogenous Culex mos-
quitoes lay their initial egg batch without taking a blood meal (Spielman 1971).
Early studies on the Cx. pipiens complex found that although anautogenous female
Cx. p. pipiens spent the winter in facultative diapause, autogenous Cx. p. molestus
spent the winter in underground habitats as larvae and females and would not
undergo diapause (Spielman 2001; Vinogradova 2000). Culex tarsalis populations
also contain autogenous individuals (Bellamy and Kardos 1958). However, autog-
enous and anautogenous individuals are found within the same populations and do
not appear to be reproductively isolated. Autogeny was found to be a dominant
genetic trait (Eberle and Reisen 1986; Moore 1966), with phenotypic expression
determined by larval rearing conditions. Experimental proof of principal studies
showed that the F1 progeny of genetically autogenous females would enter faculta-
tive ovarian diapause, remain in ovarian developmental arrest through a simulated
winter, terminate diapause during simulated spring, and then imbibe a blood meal
(Reisen 1986b). When the progeny of these overwintering phenotypically anautog-
enous females were reared under suitable larval conditions, the emerging females
again expressed autogeny. Therefore, unlike Cx. p. molestus, genetically autoge-
nous Cx. tarsalis arrest ovarian development, enter diapause, and then must take a
blood meal for initial ovarian development. These studies show the complexity of
understanding the overwintering biology in mosquito populations.
Are There Cx. pipiens in California? Based on the morphology of male termina-
lia, Cx. pipiens in California were thought to consist of Cx. quinquefasciatus in the
south and Cx. pipiens in the north, with intergrades in the Central Valley (Barr 1957;
Iltis 1966). Discrepancies between population genetics and male terminalia mor-
phology questioned these distributions (McAbee et al. 2008). Here, above-ground
populations were found to express autogeny (Iltis 1966; Strickman and Fonseca
2012), although autogenous subterranean populations were described that were
similar to Cx. p. molestus (Cornel et al. 2012). Genetic studies have shown that
Central Valley populations were a complex admixture of all three species (Cornel
et al. 2012; Kothera et al. 2013). Interestingly, none of the field or laboratory popu-
lations from California evaluated to date were able to enter reproductive arrest char-
acteristic of diapause, although concurrently tested Cx. tarsalis and Cx. stigmatosoma
from California and Cx. pipiens from Washington State readily entered diapause
under the same experimental conditions (Nelms et al. 2013). These data indicated
that true Cx. p. pipiens are not found in California and may be restricted to more
northern latitudes on the west coast. Further study is needed to carefully map the
distribution of this species complex and understand the impact of these findings on
arbovirus overwintering and transmission.
Seasonal Change in Vector Competence Vector competence or the ability of a spe-

cies to be infected with and transmit a pathogen typically is investigated by collect-
ing a large number of vectors in the field or rearing them in the laboratory, feeding
them a high concentration of pathogen, incubating the exposed vector at a suitable
temperature for a suitable period of time, and then testing them for infection and
their ability to transmit. In mosquitoes, the primary barrier to infection with arbovi-
ruses is the midgut barrier (Hardy et al. 1983), and this is best measured by feeding
females on a dilution series of virus and then determining what percentage became
infected after incubation at a suitable temperature. The best metric to express these
data is the ID50, or the concentration of virus needed to infect 50% of the target
population. During genetic release experiments in Kern County, the ID50 values of
Cx. tarsalis females emerging from field-collected larvae/pupae were measured for
both WEEV and SLEV each summer to determine if the releases of genetically
altered mosquitoes were changing the susceptibility of the target population over
time (Hardy et al. 1990). Interestingly, susceptibility to WEEV, but not SLEV,
seemed to decrease in summer when transmission activity was greatest. This was
unexpected and counterintuitive, but was supported by laboratory observations
(Kramer et al. 1983), but not by testing female Cx. tarsalis collected host-seeking at
several valley locations in Kern County, CA (Reisen et al. 1990a).
We decided to revisit this discovery for Cx. tarsalis from near the Salton Sea in
the Coachella Valley, a warmer area of California where both WEEV and SLEV
were actively transmitted each summer. Similar to Kern County, the ID50 for WEEV,
but not SLEV, increased markedly during summer and was threefold higher in July
than in January (Reisen et al. 1996), indicating the population was 1000 times less
susceptible to infection during mid-summer when WEEV was most frequently
detected by testing Cx. tarsalis pools and sentinel chicken sera (Reisen et al. 1992a,
b). These findings led to a series of unsuccessful experiments measuring the WEEV
ID50 for Cx. tarsalis reared in different larval habitats (Reisen et al. 1997) and at
different semi-natural temperature regimens (Hardy and Reeves 1990a).
The summer of 2007 was the last time that WEEV was detected in California,
despite the testing of over 30,000 pools of mosquitoes during each subsequent sum-
mer (Reisen and Wheeler 2016). This disappearance coincided with similar declines
throughout North America, without a clear explanation (Bergren et al. 2014).
Evaluations of avian host and vector competence (Reisen et al. 2008b) and growth
patterns in cell culture (Zhang et al. 2011) failed to delineate a loss in replicative
effectiveness, although one recent strain seemed to exhibit a loss of neurovirulence
in experimentally infected mice (Mossel et al. 2013). Early speculation on the
impact of global warming on WEEV seemed to provide the only possible clue to its
disappearance (Reeves et al. 1994).
472 W. K. Reisen
Vectors Rarely Bite Humans Infectious bites per human per night or the entomo-
logical inoculation rate is the most important epidemiological metric in the trans-
mission of vector-borne pathogens. It forms the core for estimates of vectorial
capacity (Garrett-Jones 1964). As a young medical entomologist, it seemed logical
to presume that important malaria vectors should feed frequently and repeatedly on
humans. The malaria eradication program in Pakistan was all but discontinued by
the time I arrived in Punjab in 1976. The primary vectors, Anopheles culicifacies
and An. stephensi, were largely resistant to organochloride insecticides (Rathor
et al. 1980) and the major donors had all but discontinued funding the indoor resid-
ual spray (IRS) program. Upon arrival in the country and seeking to develop effec-
tive sampling methods, we conducted some preliminary all-night biting catches of
mosquitoes using both human and buffalo bait, only to find that almost no mosqui-
toes would bite humans, including me (a foreign Caucasian) or our local Pakistani
staff (Reisen et al. 1976a). This prompted an extension of these observations (Aslam
et al. 1977; Reisen and Aslamkhan 1978), with essentially the same findings.
Mosquitoes included were primary and secondary malaria vectors, West Nile virus
vectors, and other species incriminated as being important in the transmission of
other human pathogens. These unexpected observations prompted an extensive
study on the blood-feeding patterns of multiple mosquito species, only to find that
few bit humans and that most frequently selected buffalo or cattle (Reisen and
Boreham 1979). This was unexpected, to say the least, but agreed with our observa-
tion that all species exited indoor resting sites at dusk to bite outdoors (Reisen et al.
1976b). Search of the older literature revealed that this behavior seemed common
enough to suggest that cyclic human malaria outbreaks were weakly associated with
the decline of “zooprophylaxis” due to hoof and mouth disease outbreaks among
the Punjab buffalo population (Yacob and Swaroop 1944). Assortative host selec-
tion also was observed among Nepal anophelines (Reisen et al. 1993b).
Surprisingly similar results were found during studies of Culex arbovirus vectors
in California. Collections of blood-fed resting Culex females throughout California
showed that chickens and passeriform birds were the most frequent blood meal
source and that humans were rarely fed upon (Thiemann et al. 2012). Likewise,
historical studies found that although Cx. tarsalis frequently fed on mammals dur-
ing late summer, none fed on humans, even when blood-fed females were collected
at restrooms in a large park on Monday morning after Sunday’s evening barbecues
and picnics (Tempelis et al. 1965). Similar attempts to detect human feeding by Cx.
pipiens complex females were equally disappointing, even when females were col-
lected in backyards in Los Angeles (Reisen et al. 1990b) or under bridges near
homeless encampments near Sacramento (Montgomery et al. 2011). Careful studies
of Cx. tarsalis flight paths found that host-seeking females were collected most
frequently along vegetative ecotones, perhaps increasing their chances of contacting
nesting and/or roosting birds (Lothrop and Reisen 2001). Collectively, these and
related studies indicated that only a low portion of some vector populations feed on
humans and that tangential transmission of zoonotic arboviruses to humans must be
a relatively rare event.
3.1 Sampling Methods
Aspirators Quantitative collection of indoor or outdoor resting females is impor-

tant to assess resting behavior as well as to monitor abundance and collect blood-
engorged females to determine host selection patterns. Oral aspirators with a
¾-inch-diameter glass or plastic tube used with a flashlight were standard equip-
ment to collect indoor resting anophelines in Asia. These worked well in the hands
of a trained collector when house/animal shed roof construction was fairly low, but
became compromised when roofs were “out of reach.” There also was the problem
of inhalation of dust and insect parts that can lead to serious allergies. A first alterna-
tive was a modification of a handheld military-type flashlight into a mechanical
aspirator (https://www.clarke.com/mechanical-aspirator). These were soon modi-
fied with a variety of different collection tubes for higher roof collections or for use
in transferring dead or alive mosquitoes in the laboratory.
Collection of mosquitoes outdoors required different equipment with stronger
suction. In Pakistan, we constructed the Pakistan Medical Research Center (PMRC)
sweeper, a handheld aspirator made from galvanized ducting, a 6-volt Volkswagen
blower motor, and a 6-volt motorcycle battery (Fig. 1a). Outdoor PMRC sweeper
collections in selected habitats for timed periods became a quantifiable metric of
mosquito resting abundance comparable to indoor hand catches (Reisen 1978). As
the mosquitoes were captured alive, they could be used for blood meal analysis
(Reisen and Boreham 1979) as well as virus testing (Reisen et al. 1982a).
In California, we were faced with the same problems when sampling outdoor
resting mosquitoes, but we had access to better hardware stores. To solve this prob-
lem, my colleague Richard Meyer constructed the Arbovirus Field Station backpack
aspirator (Fig. 1b) from a Dayton blower motor, flexible ducting, PVC drainage
pipe, and a 12-volt gel cell battery (Meyer et al. 1983). By changing the length of
the drainage pipe, this could be used for confined spaces under bridges as well as
sampling the roofs/eaves of higher structures and formed the foundation for our
residential sampling (Reisen et al. 1990b) as well as collections of mosquitoes for
overwintering studies (Reisen et al. 1986b). We loaned an AFS aspirator to Bruce
Francy of the US CDC for use in some fieldwork in Sweden. The CDC modified
Meyer’s design, which was then renamed the CDC backpack aspirator and mar-
keted by John W. Hock (https://johnwhock.com/products/aspirators/modified-cdc-
backpack-aspirator/) and Bioquip (https://www.bioquipinc.com/catalog/
collecting-equipment-supplies/aspirators-vacs/backpack-aspirator/) for well over
$1200 each, somewhat more than the $125 it cost us in parts! This mechanical aspi-
rator and its variants now have become a standard tool for the collection of indoor
resting Aedes aegypti in the Neotropics (Chadee 2013; Dzul-Manzanilla et al. 2017).
Fly Nap and the Rise of Trimethylamine A recurring problem for mosquito biolo-
gists and arbovirologists has been, how do you immobilize groups of mosquitoes for
long periods without them recovering and flying about or dying, while preserving
infections with pathogens? In Pakistan, we used a piece of cotton dampened with
474 W. K. Reisen
Fig. 1 Outdoor resting collections. (a) PMRC sweeper used in Pakistan during 1976–1980. (b)
AFS backpack aspirator used in California during the 1980s
ether to first “knock down” mosquitoes within collection cartons and then keep
them anesthetized in glass Petri dishes for sorting under the microscope. This
worked well and the mosquitoes usually remained alive; however, for large collec-
tions you had to keep adding ether to the cotton. This posed an inhalation health risk
for those working with these collections repeatedly (<https://www.cdc.gov/niosh/
ipcsneng/neng0355.html>), not to mention the risk of fire and/or explosion during
transport, especially when summer temperatures were >100 °F and the ether would
boil after the bottle cap was removed! In California, ether was replaced by chloro-
form using the same protocol, but this compound required more careful administra-
tion to keep the mosquitoes alive and had even more serious potential health effects
from long-term exposure (https://www.atsdr.cdc.gov/substances/toxsubstance.
asp?toxid=16). This left mosquito immobilization by either chilling or CO2 gas.
Both approaches required more equipment (chill tables, tanks of gas) and mosqui-
toes would recover rapidly, creating the risk of having potentially infected mosqui-
toes loose in the laboratory. Clearly an alternative method was needed.
FlyNap® (https://www.carolina.com/drosophila-fruit-fly-genetics/flynap-
anesthetic-kit/173010.pr) was developed by Carolina Biological Supply Company
to anesthetize Drosophila for genetic studies and consisted of triethylamine (TEA)
mixed with a strong perfume. I bought some to try on mosquitoes and found that it
would permanently immobilize, but not kill, mosquitoes and other Nematocera, but
not other insects including cyclorrhaphid flies. Although FlyNap was expensive
($70/100 mL), the active ingredient TEA was cheap ($50/500 mL) and a single
bottle would last a very long time. A small cotton Q-Tip wetted with TEA would
permanently anesthetize a large collection of mosquitoes in a 1 gallon collecting
carton without killing them. Although direct contact and inhalation should be
avoided, TEA did not seem to have long-term harmful health effects comparable to
ether or chloroform (https://www.cdc.gov/niosh/ipcsneng/neng0203.html) and did
not have to be continually reapplied. The best thing about TEA was that the
mosquitoes would remain immobilized and alive for more than a day and could be
used for arbovirus testing. TEA did not kill arboviruses or reduce virus titers within
anesthetized and infected female mosquitoes (Kramer et al. 1990; O’Guinn and
Turell 2002).
As we processed large collections of mosquitoes, we observed that females
seemed to remain alive and were able to move their limbs, and under the microscope
you could observe peristalsis of the gut. This led us to try using TEA to immobilize
mosquitoes for saliva collection by the capillary tube method (Aitken 1977) during
vector competence experiments (Goddard et al. 2002; Mahmood et al. 2004b).
Direct comparison of cold and CO2 gas with TEA revealed no differences in trans-
mission rates or virus titers. Use of TEA now allows the safe and rapid processing
of large numbers of females for saliva collection without concern of recovery or loss
of virus titer and has become a standard method in vector competence, especially
useful for large experiments (Reisen et al. 2008a).
4 Some Interesting Virus-Related Discoveries
Our studies on WEEV and SLEV ecology led us to revisit the role of avian infection
in the persistence and amplification of these viruses. Our early work focused on
antibody surveys to update earlier studies (Milby and Reeves 1990) to determine
which bird species were most frequently infected (Reisen et al. 2000) and on experi-
mental infections (Hardy and Reeves 1990b) to determine their viremia response or
host competence (Reisen et al. 2003b). Low antibody prevalence in areas with rela-
tively frequent seroconversions in sentinel chickens (Reisen and Wheeler 2016) led
us to investigate antibody persistence in experimentally infected birds that were
held in an outdoor aviary and then tested for antibody and virus the following spring
(Reisen et al. 2004a, 2001). Discovery of viral RNA in these birds at necropsy again
raised the issue of the importance of chronic infections in virus persistence. The
arrival of WNV in California and the ensuing epidemics allowed us to revisit these
same issues, but at a time when the virus was much more prevalent within the
environment.
How to Bleed Nestling Birds Our host competence studies were done mostly on
adult birds, because they were easily captured and maintained. However, many
adult bird species failed to produce sufficient viremias to infect mosquitoes (Reisen
et al. 2003b, 2005). This led to our colonization of house finches and mourning
doves and the hand rearing of their nestlings. Three- to 5-day-old house finch nest-
lings were too small to collect blood samples by needle, so we decided to let mos-
quitoes collect the blood samples for us (Mahmood et al. 2004a). Nestlings were
placed on the mesh tops of pint cartons in which we caged previously starved Cx.
tarsalis females. Immediately after engorgement, females were frozen at −80 °C
until tested for virus using the Vero cell plaque assay. These data showed that even
as nestlings, house finches were a better host for SLEV than mourning doves and
that viremias in nestlings were higher in both species than in adults.
476 W. K. Reisen
Where Have the Viruses Gone? The arrival of WNV in California during 2003 was
followed by the disappearance of SLEV for the next 12 years. Although we initially
suspected that avian immunity and innate resistance to SLEV would prevent the
invasion of WNV, this was not the case and WNV seemed to rapidly displace SLEV
in California and North America in general. The reemergence of SLEV in 2015 was
equally surprising as the emergent strain was most similar genetically to strains
active in Arizona in 2014 and an isolate previously made in Argentina in 2006
(White et al. 2016). The exact timing and the mechanism(s) for this recent SLEV
introduction remain unknown.
Even more unexpected was the disappearance of WEEV in 2007 (Bergren et al.
2014; Reisen and Wheeler 2016). WEEV is an alphavirus and therefore has no
cross-immunity with SLE or WNV, can replicate at colder temperatures than SLEV
or WNV (Reisen et al. 2006b, 1993a), and uses the same mosquito and avian hosts
as WNV that was transmitted effectively during the period of WEEV disappearance.
There were no changes in virus infectiousness for mosquito or avian hosts when
examined in vivo (Forrester et al. 2008; Reisen et al. 2008b) or in vitro (Zhang et al.
2011). When a small amount of WEEV and a standard dose of WNV were inocu-
lated concurrently into several icterids, WEEV viremia peaked earlier than WNV
(Reisen and Hahn 2007). The only possible clue to WEEV disappearance may be
related to climate change. Early studies at UC, Berkeley, found that certain strains
of Cx. tarsalis were able to clear their infections with WEEV at elevated incubation
temperatures (Kramer et al. 1983), leading Reeves to hypothesize that global warm-
ing could result in the disappearance of WEEV (Reeves et al. 1994). Interestingly,
field studies found that the vector competence of Cx. tarsalis for WEEV markedly
declined during midsummer in California (Hardy et al. 1990; Reisen et al. 1996),
although mechanisms for this change have yet to be found (Hardy and Reeves 1990a).
Antibody Cross-Protection We initially felt that enzootic SLEV transmission in
California might prevent or inhibit the invasion by WNV, based on the hypothesis
that similar flaviviruses cannot amplify concurrently within the same host systems.
This raised the question: would previous infection of passerines with SLEV provide
protective immunity against WNV? To investigate this question we infected hatch-
ing year house finches with either WNV or SLEV and then challenged the surviving
birds with either WNV or SLEV (Fang and Reisen 2006). In summary, previous
infection with either WNV or SLEV provided protective immunity when the birds
were challenged with the same viruses; however, although previous WNV infection
provided complete protective immunity against SLEV, SLEV did not prevent a vire-
mia response when birds were infected with WNV. Although the WNV viremia was
several orders of magnitude lower in birds previously infected with SLEV, it still
was sufficiently elevated in some birds (>5 log10 PFU/mL) to be able to infect mos-
quitoes. After the viremia period, all challenges produced an elevated antibody
response that far exceeded antibody titers produced during the initial infection, with
heterospecific challenges producing the greatest antibody response. Interestingly,
the invasion of California by WNV (Reisen et al. 2004b) was followed by the virtual
disappearance of SLEV for the next 12 years, despite the fact that >30,000 mosquito
pools, sentinel chicken, and avian sera were tested annually for both viruses (Reisen
and Wheeler 2016).
Avian Flock Immunity Limits WNV Vernal Amplification As WNV invaded dif-
ferent areas of the USA, a 3-year epidemiological pattern emerged, with silent intro-
duction during year 1, rapid amplification to outbreak levels during year 2, and then
subsidence during year 3 (Hayes et al. 2005). Los Angeles provided an excellent
venue to address the hypothesis that avian immunity elevated during year 2 pre-
vented amplification during year 3 and general subsidence over the next few years.
Avian fauna in Los Angeles was dominated by competent house finches, house spar-
rows, and American crows, and these birds were fed upon frequently by the primary
vector species, Cx. quinquefasciatus (Molaei et al. 2010; Thiemann et al. 2012).
Following its introduction during 2003, the WNV epidemic of 2004 in Los Angeles
was preceded by a large die-off of American crows, with >90% of the dead birds
reported by the public WNV positive, and was followed by a seroprevalence increase
to >45% in surviving house finches and house sparrows during November/December
(Kwan et al. 2010). Considering that many of these passerines also died from infec-
tion, we estimated that as many as 85% of the passerine population may have been
infected. The importance of the epiornitic among American crows in WNV amplifi-
cation was striking and spatially delineated the greatest mosquito infection rates and
the location of most human cases (Reisen et al. 2006a). The repopulation of
American crows, the decrease in WNV seroprevalence to <10% in passerines, and
the repopulation of house finches and house sparrows by the spring of 2008 were
followed by the resurgence of WNV and a subsequent outbreak of human cases
(Kwan et al. 2012). Antibody levels in passerine populations during late winter
seemed predictive of human cases during the succeeding summer.
Adulticiding Protects Avian Populations Although detailed guidelines were in

place describing how to respond to amplification of WNV to limit human cases in
California (Kramer 2014), local conditions and funding varied, and therefore, the
control response by mosquito control agencies differed markedly. The Greater Los
Angeles Mosquito and Vector Control District (GLAMVCD) was not able to apply
adulticides over wide areas because of traffic problems for ground applications and
limitations to air space with multiple international airports. In contrast, the
Sacramento-Yolo Mosquito and Vector Control District (SYMVCD) was able to
rapidly respond to surveillance data and apply aerial adulticides, closely complying
with guideline recommendations. Comparing the annual estimates of mosquito
infection rates and the numbers of human cases between these two areas (Reisen
2013), it seemed that in Los Angeles limited adulticiding allowed WNV to amplify
to outbreak levels, increase avian immunity, and thereby limit amplification during
several subsequent years. In contrast, effective adulticiding in the Sacramento area
478 W. K. Reisen
seemed to effectively interrupt transmission, limit the numbers of human cases,

protect the avian population precluding high levels of avian immunity and depopu-
lation, and thereby permit WNV amplification each year.
Chronic Avian Infections: True or False Close examination of qRT-PCR test

results for WNV RNA taken from tissue samples from dead birds throughout
California seemed to indicate a bimodal pattern, with peaks at ca. 20 and 36 Ct
(Reisen et al. 2013). The low values (more virus) were found in corvids that suc-
cumbed to infection and the high values (less virus) were found in passerines such
as house finches, house sparrows, and American robins that succumbed less fre-
quently from infection than crows. Based on experimental infections, the high Ct
values were unexpected for birds that died during infection and indicated that these
birds may have survived peak viremias and later died either from infection sequelae
or from other causes. House finches and house sparrows that were infected experi-
mentally and then held until the following spring before necropsy were found to
retain detectable antibody as well as low levels of WNV RNA in selected tissues,
especially spleens and kidneys (Wheeler et al. 2012a). Long-term persistence of
flaviviruses had been shown previously in birds (Nemeth et al. 2009a; Reisen et al.
2003a; Semenov et al. 1973), rodents (Appler et al. 2010; Tesh et al. 2005), and
primates (Pogodina et al. 1983).
The finding of long-term WNV persistence in tissues, infrequent recrudescence
of viremia, and persistence of neutralizing antibody in experimentally infected
house sparrows (Nemeth et al. 2009a, b; Wheeler et al. 2012c) complicated the
interpretation of the role of chronic infections in virus overwintering. In addition,
experimental blood meals of mosquitoes showed that WNV-antibody complexes
were protective against mosquito viral infection; however, if antibody titers were
insufficient to bind all virus in these experimental mosquito blood meals, then a low
percentage of female mosquitoes could become infected and later would transmit
the virus (Wheeler et al. 2012b). These data were important because they showed
that factors that would compromise avian immunocompetence could allow viral
relapse and mosquito infection.
5 Summary
The current chapter summarized a series of disjunct serendipitous observations that

resulted in a series of discoveries that produced both positive and negative findings.
Negative findings can be extremely informative because they prove that your view
of nature was incorrect or that you cannot develop a protocol worthy of investigat-
ing the phenomenon you observed. Many of the topics covered were methodologi-
cal improvements that may have made life easier and experiments more efficient
and facilitated future research.
References
Aitken THG (1977) An in vitro feeding technique for artificially demonstrating virus transmission
by mosquitoes. Mosq News 37:130–133
Appler KK, Brown AN, Stewart BS, Behr MJ, Demarest VL, Wong SJ, Bernard KA (2010)
Persistence of West Nile virus in the central nervous system and periphery of mice. PLoS One
5:e10649
Aslam Y, Reisen WK, Aslamkhan M (1977) The influence of physiological age on the biting
rhythm of Culex tritaeniorhynchus Giles (Diptera : Culcidae). Southeast Asian J Trop Med
Public Health 8:364–367
Asman SM (1975) The genetics of two new eye color mutants in Culex tarsalis. J Hered 66:297–300
Baker RH, Reisen WK, Sakai RH, Hayes CG, Aslamkhan M, Saifuddin UT, Mahmood F, Perveen
R, Javed S (1979) Field assessment of mating competitiveness of male Culex tritaeniorhynchus
carrying a complex chromosomal aberration. Ann Entomol Soc Am 72:751–758
Baker RH, Reisen WK, Sakai RK, Rathor HR, Raana K, Azra A, Niaz S (1980) Anopheles culicifa-
cies: mating behavior and competitiveness in nature of males carrying a complex chromosomal
aberration. Ann Entomol Soc Am 73:581–588
Barr AR (1957) The distribution of Culex p. pipiens and C.p. quinquefasciatus in North America.
Bellamy RE, Kardos EH (1958) A strain of Culex tarsalis Coq. Reproducing without blood meals.
Mosq News 18:132–134
Bellamy RE, Reeves WC (1963) The winter biology of Culex tarsalis (Diptera:Culicidae) in Kern
County, California. Ann Entomol Soc Am 56:314–323
Bergren NA, Auguste AJ, Forrester NL, Negi SS, Braun WA, Weaver SC (2014) Western equine
encephalitis virus: evolutionary analysis of a declining alphavirus based on complete genome
sequences. J Virol 88:9260–9267
Callahan AG, Ross PA, Hoffmann AA (2018) Small females prefer small males: size assortative
mating in Aedes aegypti mosquitoes. Parasit Vectors 11:445
Chadee DD (2013) Resting behaviour of Aedes aegypti in Trinidad: with evidence for the re-
introduction of indoor residual spraying (IRS) for dengue control. Parasit Vectors 6:255
Cornel A, Lee Y, Fryxell RT, Siefert S, Nieman C, Lanzaro G (2012) Culex pipiens sensu lato in
California: a complex within a complex? J Am Mosq Control Assoc 28:113–121
Dzul-Manzanilla F, Ibarra-Lopez J, Bibiano MW, Martini-Jaimes A, Leyva JT, Correa-Morales F,
Huerta H, Manrique-Saide P, Vazquez-Prokopec GM (2017) Indoor resting behavior of Aedes
aegypti (Diptera: Culicidae) in Acapulco. Mexico J Med Entomol 54:501–504
Eberle MW, Reisen WK (1986) Studies on autogeny in Culex tarsalis: selection and genetic exper-
iments. J Am Mosq Control Assoc 2:38–43
Eldridge BF (1987) Diapause and related phenomena in Culex mosquitoes: their relation to arbo-
virus disease ecology. Curr Topics Vector Res 4:1–28
Eldridge BF, Bailey CL (1979) Experimental hibernation studies in Culex pipiens (Diptera:
Culicidae): reactivation of ovarian development and blood-feeding in prehibernating females.
J Med Entomol 15:462–467
Fang Y, Reisen WK (2006) Previous infection with West Nile or St. Louis encephalitis viruses
provides cross protection during reinfection in house finches. Am J Trop Med Hyg 75:480–485
Forrester NL, Kenney JL, Deardorff E, Wang E, Weaver SC (2008) Western Equine Encephalitis
submergence: lack of evidence for a decline in virus virulence. Virology 380:170–172
Garrett-Jones C (1964) Prognosis for interruption of malaria transmission through assessment of
the mosquito’s vectorial capacity. Nature 204:1173–1175
Goddard LB, Roth AE, Reisen WK, Scott TW (2002) Vector competence of California mosquitoes
for West Nile virus. Emerg Infect Dis 8:1385–1391
Hardy JL, Houk EJ, Kramer LD, Reeves WC (1983) Intrinsic factors affecting vector competence
of mosquitoes for arboviruses. Annu Rev Entomol 28:229–262
480 W. K. Reisen
Hardy JL, Meyer RP, Presser SB, Milby MM (1990) Temporal variations in the susceptibility of a
semiisolated population of Culex tarsalis to peroral infection with western equine encephalo-
myelitis and St. Louis encephalitis viruses. Am J Trop Med Hyg 42:500–511
Hardy JL, Reeves WC (1990a) Experimental studies on infection in vectors. In: Reeves WC
(ed) Epidemiology and control of mosquito-borne arboviruses in California, 1943–1987
Sacramento, Calif. Calif Mosq Vector Control Assoc, pp 145–250
Hardy JL, Reeves WC (1990b) Experimental studies on infection in vertebrate hosts. In: Reeves
WC (ed) Epidemiology and control of mosquito-borne arboviruses in California, 1943–1987
Sacramento, Calif. Calif. Mosq Vector Control Assoc, pp 66–127
Hayes EB, Komar N, Nasci RS, Montgomery SP, O’Leary DR, Campbell GL (2005) Epidemiology
and transmission dynamics of West Nile virus disease. Emerg Infect Dis 11:1167–1173
Iltis WG (1966) Biosystematics of the Culex pipiens complex in northern California. Ph.D. Diss.
Univ. Calif., Davis
Kopp M, Servedio MR, Mendelson TC, Safran RJ, Rodriguez RL, Hauber ME, Scordato EC,
Symes LB, Balakrishnan CN, Zonana DM, van Doorn GS (2018) Mechanisms of assorta-
tive mating in speciation with gene flow: connecting theory and empirical research. Am Nat
191:1–20
Kothera L, Nelms BM, Reisen WK, Savage HM (2013) Population genetic and admixture analyses
of Culex pipiens complex (Diptera: Culicidae) populations in California, United States. Am J
Kramer LD, Hardy JL, Presser SB (1983) Effect of temperature of extrinsic incubation on the vec-
tor competence of Culex tarsalis for western equine encephalomyelitis virus. Am J Trop Med
Hyg 32:1130–1139
Kramer LD, Presser SB, Houk EJ, Hardy JL (1990) Effect of the anesthetizing agent triethylamine
on western equine encephalomyelitis and St. Louis encephalitis viral titers in mosquitoes
(Diptera:Culicidae). J Med Entomol 27:1008–1010
Kramer VL (2014) California Mosquito-borne Virus Surveillance and Response Plan. http://west-
nile.ca.gov/resources.php
Kwan JL, Kluh S, Madon MB, Reisen WK (2010) West Nile virus emergence and persistence in
Los Angeles, California, 2003-2008. Am J Trop Med Hyg 83:400–412
Kwan JL, Kluh S, Reisen WK (2012) Antecedent avian immunity limits tangential transmission of
West Nile virus to humans. PLoS One 7:e34127
Lothrop HD, Reisen WK (2001) Landscape affects the host-seeking patterns of Culex tarsalis
(Diptera: Culicidae) in the Coachella Valley of California. J Med Entomol 38:325–332
Mahmood F, Chiles RE, Fang Y, Barker CM, Reisen WK (2004a) Role of nestling mourning
doves and house finches as amplifying hosts of St. Louis encephalitis virus. J Med Entomol
41:965–972
Mahmood F, Fang Y, Chiles RE, Reisen WK (2004b) Methods for studying the vector compe-
tence of Culex tarsalis for western equine encephalomyelitis virus. J Am Mosq Control Assoc
20:277–282
McAbee RD, Green EN, Holeman J, Christiansen J, Frye N, Dealey K, Mulligan FS III, Brault
AC, Cornel AJ (2008) Identification of Culex pipiens complex mosquitoes in a hybrid zone of
West Nile virus transmission in Fresno County, California. Am J Trop Med Hyg 78:303–310
Meyer RP, Reisen WK, Hill BR, Martinez VM (1983) The “AFS sweeper”, a battery-powered
backpack mechanical aspirator for collecting adult mosquitoes. Mosq News 43:346–350
Milby MM, Reeves WC (1990) Natural infection in vertebrate hosts other than man. In: Reeves
WC (ed) Epidemiology and control of mosquito-borne arboviruses in California, 1943–1987
Sacramento, Calif. Calif. Mosq. Vector Control Assoc, pp 26–65
Molaei G, Cummings RF, Su T, Armstrong PM, Williams GA, Cheng ML, Webb JP, Andreadis
TG (2010) Vector-host interactions governing epidemiology of West Nile virus in southern
California. Am J Trop Med Hyg 83:1269–1282
Montgomery MJ, Thiemann T, Macedo P, Brown DA, Scott TW (2011) Blood-feeding patterns
of the Culex pipiens complex in Sacramento and Yolo Counties, California. J Med Entomol
48:398–404
Moore CG (1966) Environmental factors influencing the proportion of autogenous ovarian devel-
opment in populations of the mosquito Culex tarsalis Coq. PhD Diss. Univ. Calif., Davis
Mossel EC, Ledermann JP, Phillips AT, Borland EM, Powers AM, Olson KE (2013) Molecular
determinants of mouse neurovirulence and mosquito infection for Western equine encephalitis
virus. PLoS One 8:e60427
Munstermann LE (1994) Unexpected consequences of colonization and inbreeding: allozyme
tracking in Culicidae (Diptera). Ann Entomol Soc Am 87:157–164
Nelms BM, Kothera L, Thiemann T, Macedo PA, Savage HM, Reisen WK (2013) Phenotypic
variation among Culex pipiens complex (Diptera: Culicidae) populations from the Sacramento
Valley, California: horizontal and vertical transmission of West Nile virus, diapause potential,
autogeny, and host selection. Am J Trop Med Hyg 89:1168–1178
Nelson RL (1964) Parity in winter populations of Culex tarsalis Coquillett in Kern County,
California. Am J Hyg 80:242–253
Nemeth N, Young G, Ndaluka C, Bielefeldt-Ohmann H, Komar N, Bowen R (2009a) Persistent
West Nile virus infection in the house sparrow (Passer domesticus). Arch Virol 154:783–789
Nemeth NM, Oesterle PT, Bowen RA (2009b) Humoral immunity to West Nile virus is long-lasting
and protective in the house sparrow (Passer domesticus). Am J Trop Med Hyg 80:864–869
O’Guinn ML, Turell MJ (2002) Effect of triethylamine on the recovery of selected South American
alphaviruses, flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools. J Med
Entomol 39:806–808
Pogodina VV, Frolova MP, Malenko GV, Fokina GI, Koreshkova GV, Kiseleva LL, Bochkova
NG, Ralph NM (1983) Study on West Nile virus persistence in monkeys. Arch Virol 75:71–86
Rathor HR, Toqir G, Reisen WK (1980) Status of insecticide resistance in anopheline mosquitoes
of Punjab Province, Pakistan. Southeast Asian J Trop Med Public Health 11:332–340
Reeves WC (1961) Overwintering of anthropod-borne viruses. Progr Med Virol 3:59–78
Reeves WC (1987) Importance of vector overwintering to disease maintenance. Bull Soc Vector
Ecol 12:561–563
Reeves WC, Hardy JL, Reisen WK, Milby MM (1994) Potential effect of global warming on
mosquito-borne arboviruses. J Med Entomol 31:323–332
Reisen WK (1978) A quantitative mosquito survey of 7 villages in Punjab Province, Pakistan, with
notes on bionomics, sampling methodology and the effects of insecticides. SE Asian J Trop
Med Publ Hlth 9:587–601
Reisen WK (1985) Male mating competitiveness: the key to some problems associated with the
genetic control of mosquitoes. In: Louniboset LP (ed) Ecology of mosquitoes: proceedings of
a workshop. Florida Med. Entomol. Lab, Vero Beach, Florida, pp 345–358
Reisen WK (1986a) Owerwintering studies on Culex tarsalis (Diptera:Culicidae) in Kern County,
California: life stages sensitive to diapause induction cues. Ann Entomol Soc Am 79:674–676
Reisen WK (1986b) Studies on autogeny in Culex tarsalis: simulated diapause induction and ter-
mination in genetically autogenous females. J Am Mosq Control Assoc 2:44–47
Reisen WK (2013) Ecology of West Nile virus in North America. Viruses 5:2079–2105
Reisen WK, Aslamkhan M (1978) Biting rhythms of some Pakistan mosquitoes (Diptera:
Culicidae). Bull Entomol Res 68:313–330
Reisen WK, Aslamkhan M, Suleman M, Naqvi ZA (1976a) Observations on the diel activity pat-
terns of some Punjab mosquitoes (Diptera: Culicidae). Biololgia 22:67–77
Reisen WK, Aslamkhan M, Suleman M, Naqvi ZA (1976b) Observations on the resting habits
and diel changes in the ovarian condition of some Punjab mosquitoes (Diptera: Culicidae).
Biololgia 22:79–88
Reisen WK, Asman SM, Milby MM, Bock ME, Stoddard PJ, Meyer RP, Reeves WC (1981a)
Attempted suppression of a semi-isolated population of Culex tarsalis by release of irradiated
males. Mosq News 41:736–744
482 W. K. Reisen
Reisen WK, Baker RH, Sakai RH, Mahmood F, Rathor HR, Raana K, Toqir G (1981b) Anopheles
culicifacies Giles: mating behavior and competitiveness in nature of chemosterilized males
carrying a genetic sexing system. Ann Entomol Soc Am 74:395–401
Reisen WK, Barker CM, Carney R, Lothrop HD, Wheeler SS, Wilson JL, Madon MB, Takahashi
R, Carroll B, Garcia S, Fang Y, Shafii M, Kahl N, Ashtari S, Kramer V, Glaser C, Jean C
(2006a) Role of corvids in epidemiology of West Nile virus in southern California. J Med
Entomol 43:356–367
Reisen WK, Barker CM, Fang Y, Martinez VM (2008a) Does variation in Culex (Diptera:
Culicidae) vector competence enable outbreaks of West Nile virus in California? J Med
Entomol 45:1126–1138
Reisen WK, Bock ME, Milby MM, Reeves WC (1985) Attempted insertion of a recessive autoso-
mal gene into a semi-isolated population of Culex tarsalis (Diptera: Culicidae). J Med Entomol
22:250–260
Reisen WK, Boreham PF (1979) Host selection patterns of some Pakistan mosquitoes. Am J Trop
Med Hyg 28:408–421
Reisen WK, Chiles RE, Green EN, Fang Y, Mahmood F (2003a) Previous infection protects finches
from re-infection with St. Louis encephalitis virus. J Med Entomol 40:300–305
Reisen WK, Chiles RE, Martinez VM, Fang Y, Green EN (2003b) Experimental infection of
California birds with western equine encephalomyelitis and St. Louis encephalitis viruses. J
Reisen WK, Chiles RE, Martinez VM, Fang Y, Green EN (2004a) Encephalitis virus persistence
in California birds: experimental infections in mourning doves (Zenaidura macroura ). J Med
Entomol 3:462–466
Reisen WK, Fang Y, Brault AC (2008b) Limited interdecadal variation in mosquito (Diptera:
Culicidae) and avian host competence for Western equine encephalomyelitis virus (Togaviridae:
Alphavirus). Am J Trop Med Hyg 78:681–686
Reisen WK, Fang Y, Martinez VM (2005) Avian host and mosquito (Diptera: Culicidae) vector
competence determine the efficiency of West Nile and St. Louis encephalitis virus transmis-
sion. J Med Entomol 42:367–375
Reisen WK, Fang Y, Martinez VM (2006b) Effects of temperature on the transmission of West Nile
virus by Culex tarsalis (Diptera: Culicidae). J Med Entomol 43:309–317
Reisen WK, Hahn DC (2007) Comparison of immune responses of brown-headed cowbird and
related blackbirds to West Nile and other mosquito-borne encephalitis viruses. J Wildl Dis
43:439–449
Reisen WK, Hardy JL, Presser SB (1997) Effects of water quality on the vector competence of
Culex tarsalis (Diptera: Culicidae) for western equine encephalomyelitis (Togaviridae) and St.
Louis encephalitis (Flaviviridae) viruses. J Med Entomol 34:631–643
Reisen WK, Hardy JL, Presser SB, Chiles RE (1996) Seasonal variation in the vector compe-
tence of Culex tarsalis (Diptera:Culicidae) from the Coachella Valley of California for western
equine encephalomyelitis and St. Louis encephalitis viruses. J Med Entomol 33:433–437
Reisen WK, Hardy JL, Presser SB, Milby MM, Meyer RP, Durso SL, Wargo MJ, Gordon EW
(1992a) Mosquito and arbovirus ecology in southeastern California, 1986-1990. J Med
Entomol 29:512–524
Reisen WK, Hardy JL, Reeves WC, Presser SB, Milby MM, Meyer RP (1990a) Persistence
of mosquito-borne viruses in Kern County, California, 1983-1988. Am J Trop Med Hyg
43:419–437
Reisen WK, Hayes CG, Azra K, Niaz S, Mahmood F, Parveen T, Boreham PFL (1982a) West
Nile virus in Pakistan. II. Entomological studies at Changa Manga National Forest, Punjab
Province. Trans Roy Soc Trop Med Hyg 76:437–448
Reisen WK, Kramer LD, Chiles RE, Green E-GN, Martinez VM (2001) Encephalitis virus persis-
tence in California birds: preliminary studies with house finches (Carpodacus mexicanus). J
Reisen WK, Lothrop H, Milby MM, Presser SB, Hardy JL, Wargo MJ (1992b) Landscape ecology
of encephalitis virus transmission in the Coachella Valley: temporal patterns among mosquito
abundance and virus infection rates, and seroconversions among sentinel chickens. Proc Calif
Mosq Vector Control Assoc 60:71–75
Reisen WK, Lothrop HD, Chiles RE, Madon MB, Cossen C, Woods L, Husted S, Kramer VL,
Edman JD (2004b) West Nile Virus in California. Emerg Infect Dis 10:1369–1378
Reisen WK, Lothrop HD, Hardy JL (1995) Bionomics of Culex tarsalis (Diptera:Culicidae) in
relation to arbovirus transmission in southeastern California. J Med Entomol 32:316–327
Reisen WK, Lundstrom JO, Scott TW, Eldridge BF, Chiles RE, Cusack R, Martinez VM, Lothrop
HD, Gutierrez D, Wright S, Boyce K, Hill BR (2000) Patterns of avian seroprevalence to
western equine encephalomyelitis and St. Louis encephalitis viruses in California, USA. J Med
Entomol 37:507–527
Reisen WK, Meyer RP, Milby MM (1986a) Overwintering studies on Culex tarsalis
(Diptera:Culicidae) in Kern County, California: survival and the experimental induction and
termination of diapause. Ann Entomol Soc Am 79:664–673
Reisen WK, Meyer RP, Milby MM (1986b) Overwintering studies on Culex tarsalis
(Diptera:Culicidae) in Kern County, California: temporal changes in abundance and repro-
ductive status with comparative observations on C. quinquefasciatus (Diptera:Culicidae). Ann
Entomol Soc Am 79:677–685
Reisen WK, Meyer RP, Presser SB, Hardy JL (1993a) Effect of temperature on the transmission of
western equine encephalomyelitis and St. Louis encephalitis viruses by Culex tarsalis (Diptera:
Culicidae). J Med Entomol 30:151–160
Reisen WK, Meyer RP, Tempelis CH, Spoehel JJ (1990b) Mosquito abundance and bionomics
in residential communities in Orange and Los Angeles Counties, California. J Med Entomol
27:356–367
Reisen WK, Milby MM, Asman SM, Bock ME, Meyer RP, McDonald PT, Reeves WC (1982b)
Attempted suppression of a semi-isolated Culex tarsalis population by the release of irradi-
ated males:a second experiment using males from a recently colonized strain. Mosq News
42:565–575
Reisen WK, Milby MM, Reeves WC, Meyer RP, Bock ME (1983) Population ecology of Culex
tarsalis (Diptera:Culicidae) in a foothill environment of Kern County, California: temporal
changes in female relative abundance, reproductive status, and survivorship. Ann Entomol Soc
Am 76:800–808
Reisen WK, Padgett K, Fang Y, Woods L, Foss L, Anderson J, Kramer V (2013) Chronic infections
of West Nile virus detected in California dead birds. Vector Borne Zoonotic Dis 13:401–405
Reisen WK, Pradhan SP, Shrestha JP, Shrestha SL, Vaidya RG, Shrestha JD (1993b) Anopheline
mosquito (Diptera: Culicidae) ecology in relation to malaria transmission in the inner and outer
terai of Nepal, 1987-1989. J Med Entomol 30:664–682
Reisen WK, Sakai RH, Baker RH, Rathor HR, Raana K, Azra A, Niaz S (1980) Field competitive-
ness of Culex tritaeniorhynchus Giles males carrying a complex chromosomal aberration: a
second experiment. Ann Entomol Soc Am 73:479–484
Reisen WK, Wheeler SS (2016) Surveys for antibodies against mosquitoborne encephalitis viruses
in California Birds, 1996-2013. Vector Borne Zoonotic Dis 16:264–282
Rosen L (1987) Overwintering mechanisms of mosquito-borne arboviruses in temperate climates.
Am J Trop Med Hyg 37:695s–765s
Semenov BF, Chunikhin SP, Karmysheva VI, Iakovleva NI (1973) [Study of chronic forms of
arbovirus infections in birds. 1. Experiments with West Nile, Sindbis, Bhandja and Sicilian
mosquito fever viruses]. Vestn Akad Med Nauk SSSR 28: 79–83
Spielman A (1971) Bionomics of autogenous mosquitoes. Annu Rev Entomol 16:231–248
Spielman A (1974) Effect of synthetic juvenile hormone on ovarian diapause of Culex pipiens
mosquitoes. J Med Entomol 11:223–225
Spielman A (2001) Structure and seasonality of nearctic Culex pipiens populations. Ann N Y Acad
Sci 951:220–234
484 W. K. Reisen
Spielman A, Wong J (1973a) Environmental control of ovarian diapause in Culex pipiens. Ann
Entomol Soc Am 66:905–907
Spielman A, Wong J (1973b) Studies of autogeny in natural populations of Culex pipiens.
III. Midsummer preparation for hibernation in anautogenous populations. J Med Entomol
10:319–324
Strickman D, Fonseca DM (2012) Autogeny in Culex pipiens complex mosquitoes from the San
Francisco Bay Area. Am J Trop Med Hyg 87:719–726
Tempelis CH, Reeves WC, Bellamy RE, Lofy MF (1965) A three-year study of the feeding habits
of Culex tarsalis in Kern County, California. Am J Trop Med Hyg 14:170–177
Tesh RB, Siirin M, Guzman H, Travassos da Rosa AP, Wu X, Duan T, Lei H, Nunes MR, Xiao SY
(2005) Persistent West Nile virus infection in the golden hamster: studies on its mechanism and
possible implications for other flavivirus infections. J Infect Dis 192:287–295
Thiemann TC, Lemenager DA, Kluh S, Carroll BD, Lothrop HD, Reisen WK (2012) Spatial
variation in host feeding patterns of Culex tarsalis and the Culex pipiens complex (Diptera:
Culicidae) in California. J Med Entomol 49:903–916
Venkatesan M, Westbrook CJ, Hauer MC, Rasgon JL (2007) Evidence for a population expansion
in the West Nile virus vector Culex tarsalis. Mol Biol Evol 24:1208–1218
Vinogradova EB (2000) Culex pipiens pipiens mosquitoes: taxnomy, distribution, ecology, phjysi-
ology, genetics, applied importance and control. Pensoft, Sofia, Bulgaria
Wheeler SS, Langevin SA, Brault AC, Woods L, Carroll BD, Reisen WK (2012a) Detection of
persistent West Nile virus RNA in experimentally and naturally infected avian hosts. Am J Trop
Med Hyg 87:559–564
Wheeler SS, Vineyard MP, Barker CM, Reisen WK (2012b) Importance of recrudescent avian
infection in West Nile virus overwintering: incomplete antibody neutralization of virus allows
infrequent vector infection. J Med Entomol 49:895–902
Wheeler SS, Vineyard MP, Woods LW, Reisen WK (2012c) Dynamics of West Nile virus persis-
tence in house sparrows (Passer domesticus). PLoS Negl Trop Dis 6:e1860
White GS, Symmes K, Sun P, Fang Y, Garcia S, Steiner C, Smith K, Reisen WK, Coffey LL (2016)
Reemergence of St. Louis encephalitis virus, California, 2015. Emerg Infect Dis 22:2185–2188
Yacob M, Swaroop S (1944) The forecasting of epidemic malaria in Punjab. J Mal Inst India
5:319–335
Zhang M, Fang Y, Brault AC, Reisen WK (2011) Variation in Western equine encephalomyelitis
viral strain growth in mammalian, avian, and mosquito cells fails to explain temporal changes
in enzootic and epidemic activity in California. Vector Borne Zoonotic Dis 11:269–275
Remembrances of Virology Past
Sondra Schlesinger
Abstract This chapter is a look back in time and a reflection on how the fields of
virology and immunology have changed. And the subject of arboviruses represents
a specific example of the change. My first introduction to virology was the study of
bacteriophage as a graduate student, but it was not until I established my own
research laboratory that I ventured into the field of animal virology and I came to
recognize that field through teaching of medical students. I was interested in the
value of viruses as a probe of cellular functions, to learn what cellular functions the
viruses required for replicating and packaging and what mechanisms cells had to
inhibit virus replication. But the role of viruses as agents of disease became more
important in my thinking, not only because of lecturing to medical students but even
more so because of the pandemics, first of HIV (the cause of AIDS) and now SARS-
CoV-2 (responsible for COVID-19). My interest in science began when I was in
high school but I begin this history starting in graduate school and continuing into
the twenty-first century.
1 Introduction
I am writing this during the period of sheltering in place and the quarantine because
of the coronavirus pandemic (March 2020–2021). I hope by the time you read this,
the pandemic will no longer be with us and we can think about other issues and
breathe without masks. But the present pandemic is a reminder that viruses, includ-
ing arboviruses, are and continue to be the cause of disease leading to epidemics and
pandemics.
I and Milton Schlesinger (my husband) had a special interest in science history,
and when we edited the book, The Togaviridae and Flaviviridae, we had asked
James Porterfield to write the first chapter on the historical aspects of these viruses.
James Porterfield (1924–2010) is no longer alive but had been an important
S. Schlesinger (*)
Department of Molecular Microbiology, School of Medicine, Washington University in Saint
Louis, Saint Louis, MO, USA
e-mail: sondra@wustl.edu
486 S. Schlesinger
contributor to the field of arbovirology and it seemed appropriate to begin my essay

with a quote from him.
He wrote,
The first clearly recognizable accounts of yellow fever come not from Africa, but from the
New World, probably having been introduced there by early sailing ships. Carter1 covers
this early history and cites the Yucatan epidemic of 1648 as the first recorded outbreak of
the disease in the Americas. Throughout the 18th and 19th centuries, epidemics raged in the
Caribbean and Central America, extending as far north as Philadelphia, New York and
Baltimore.
The threat of yellow fever epidemics was essentially eliminated in the late 1930s
when Max Theiler and his associates working at what was then known as the
Rockefeller Foundation developed an attenuated strain of the virus as a vaccine that
is still in use today. But arboviruses continue to be a threat to human health as infec-
tions with viruses such as dengue, chikungunya, and Zika virus continually
remind us.
Many scientists, I included, prefer writing about their research and their data
rather than providing a historical context for the work being done today although
that is what we are supposed to do here. But I hope not to neglect viruses and more
recent research. My history in the field of virology starts with the following.
2 Graduate School
My interest in viruses began when I was a graduate student in Biological Chemistry

at the University of Michigan. My research involved the T-even bacteriophage –
viruses that infect E. coli. I have rather recently been thinking again about bacterio-
phage, perhaps not surprisingly as there are now more and more studies about these
bacterial viruses. They can make up a significant part of the microbiome and there
is research on the potential use of bacteriophage as antibiotics. I only learned a few
years ago that bacteriophage had actually been used as antibiotics soon after their
discovery by d’Herelle and by Twort in the early years of the twentieth century
(Summers 1999).
I have often been asked about what it was like being a woman in the fields of
molecular biology and biochemistry in the early 1960s. I do not remember feeling
discrimination during my graduate training. I had applied for and was given a fel-
lowship from the NIH for the 4 years of my graduate training. I was not the only
woman in the graduate school program. There were, however, almost no women on
the faculty who could serve as role models for us.
1
His reference to Carter is Carter, H. R. 1931 Yellow Fever: An Epidemiological and Historical
Study of its Place of Origin, Williams and Wilkins, Baltimore.
Remembrances of Virology Past 487
3 Postdoctoral Training at MIT
My postdoctoral training at MIT was in the laboratory of Boris Magasanik. Research

in his laboratory focused on microbial physiology and the regulation of gene expres-
sion in bacteria. Viruses were no longer in my scientific thoughts.
One subject of my postdoctoral research had been the role of amino acyl tRNAs
in regulating the biosynthetic pathway for the synthesis of amino acids – a far cry
from viruses. When I started my own laboratory at Washington University, some of
this postdoctoral research became part of my initial grants from the NIH. I also do
not remember any particular discrimination as a woman during my 3 years at MIT;
there were no women faculty in the Biology Department and I never heard the word
mentoring being mentioned. When it came time to look for more permanent posi-
tions, it was taken for granted by everyone, including me, that Milt would be the one
applying for tenure-track faculty positions and then I would find out what was avail-
able for me.
4 Washington University School of Medicine
When Milt was asked to come to the Microbiology Department at Washington

University in Saint Louis to be interviewed for a faculty position, I came along and
was rather surprised and pleased that I was also asked to give a more informal semi-
nar. We were very happy when we were both offered positions as assistant profes-
sors. At that time in 1964, the Microbiology and Immunology Department (as it was
then called) had very few faculty. The previous chairman of the department, Arthur
Kornberg, had moved to Stanford University in 1958 taking most of the faculty with
him. The next chairman, Herman Eisen, an immunologist, is the person who hired
us (and see the end of this essay for my later interactions with Herman).
In addition to devoting most of our efforts to research, the Microbiology
Department did teach medical students microbiology and immunology. I thought
that it would be interesting for me to lecture to the medical students about animal
virology. At that time in the 1960s, there was not much known about animal viruses
except perhaps for poliovirus and influenza virus. But medical students were not
very interested in these viruses – vaccines could protect against a few viruses (small-
pox, polio, yellow fever), but there were no antivirals available. There was not much
a physician could do for a patient who had a virus infection.
By the end of the 1960s, molecular tools and the growing of cells in culture were
making animal virology more accessible to analysis, and the more I learned, the
more interested I became in this field. I wanted to spend a few months learning more
about this field in a virology laboratory. Fortunately for me, Milt was also interested
in moving into studies using cultured cells and viruses. So we returned to MIT for
the summer in the early 1970s to the laboratory shared by Phillips Robbins and
Boyce Birge who were studying Sindbis virus. Their interest was not in the virus as
488 S. Schlesinger
an infectious agent, but as a tool for analyzing cellular functions and membrane
glycoproteins.
During that summer at MIT, Milt and I worked together and one of the first new
techniques we learned was to prepare and use acrylamide slab gels to separate
radioactively labeled proteins of the virus particles and infected cells by electropho-
resis. Previously radioactively labeled proteins had been separated on acrylamide
gels that had been poured into narrow glass tubes. The gels that had the shape of the
tube were then removed from the glass tube and sliced into small pieces, and the
radioactivity in each gel slice was determined. With these tube gels, only two pro-
teins had been identified associated with Sindbis and Semliki Forest virus particles:
the capsid protein that formed a shell around the virus RNA and a single glycopro-
tein that constituted the surrounding envelope. We discovered using slab gel electro-
phoresis that the virus contained two glycoproteins and that in infected cells one of
these (designated E2) was first synthesized as a precursor (PE2) and was cleaved to
form the E2 protein before the virus particle was released from the infected cell.
When Sindbis virus infected chicken or baby hamster kidney cell monolayers, pro-
tein synthesis was inhibited and the only proteins that were detected by labeling
with a radioactive amino acid were those coded by viral RNA. At that time the
synthesis of larger-molecular-weight proteins that were then cleaved to form the
final protein products had not been a well-established observation among the differ-
ent virus families.
The data establishing that a viral mRNA could be translated into a polyprotein
and then be cleaved to the individual virus proteins was first demonstrated with
poliovirus by Jacobson and Baltimore (1968). Most of you will be aware of David
Baltimore’s contributions to virology but may not know that after receiving his
PhD, Michael Jacobson in 1971 went on to become the co-founder and long-time
executive director of the Center for Science in the Public Interest. The center pub-
lishes the Nutrition Action Health letter, on food safety and nutrition information.
My initial interest in arboviruses like Sindbis virus was not because of their abil-
ity to replicate in both animals and mosquitoes and their transmission to animals
and humans via mosquitoes, but because they offered a way to learn more about the
steps in RNA virus replication, about the structure and synthesis of virus proteins
and about cellular responses to virus infection. I was, however, teaching medical
students. Most of these students were not interested in molecular details. They were
interested in disease! My initial interest in alpha- and flaviviruses as agents causing
disease did come from lecturing. Teaching did lead to my thinking about disease.
During the time that Milt and I were spending the summer at MIT learning about
animal viruses, we did follow the research of Alice Huang and David Baltimore also
at MIT. They were studying defective interfering virus particles generated by vesic-
ular stomatitis virus (VSV) by high multiplicity passaging in cultured cells (Huang
and Baltimore 1970). These defective RNAs were able to suppress the replication of
the wild-type virus. John Holland exploited the phenomenon by using defective
particles to damp down the replication of wild-type VSV. This led to the establish-
ment of persistent infections in cultured BHK cells. In other cultured cells that
produce type 1 interferon upon infection, the cells were usually cured of the infec-
tion rather than establishing a persistent infection.
During that summer at MIT, Milt and I generated defective interfering particles
of Sindbis virus and that became a major interest of my laboratory for many years.
We were able to establish persistent infections of BHK cells and isolated mutants
of the virus that were less cytopathic or essentially not cytopathic in BHK cells even
in the absence of defective RNAs (Weiss et al. 1983; Schlesinger and Weiss 1986).
My research with defective genomes had allowed us to identify noncytopathic
Sindbis virus mutants and determine the viral gene that led to this phenotype.
Initially, they also were important in identifying the sequences essential for the
replication and packaging of the genome (Levis et al. 1986).
Some years later, in 2020, after I had closed my laboratory, Carolina Lopez
joined my department (now the Department of Molecular Microbiology). Carolina
is a virologist who studies negative-strand respiratory viruses. One of the major
programs in her laboratory involves how defective viral genomes affect the out-
comes of experimental and natural infections in animals and humans (Genoyer and
Lopez 2019). Her research has demonstrated that defective genomes can have an
adverse effect on the outcome of infections with negative-strand viruses such as
Sendai and respiratory syncytial viruses. It has been very gratifying for me to learn
that defective virus genomes may play a critical role in natural infections in humans.
What we could not have imagined initially is the prospect of being able to
sequence these viral RNAs and to modify their sequences. How I learned to sequence
is tangential to this essay but perhaps worth including here. When sequencing
became feasible as a technique that could be performed in most laboratories, Jim
and Ellen Strauss invited me to spend a couple of weeks in their laboratory at the
California Institute of Technology to learn how. I, along with my student (Stephan
Monroe, who is now the Associate Director for Laboratory Science and Safety at the
CDC), learned to sequence using the Maxam-Gilbert and the Sanger methods. Both
of these methods have now been replaced by other procedures but the chemistry
involved might have some historical interest.
Our teacher was Charlie Rice who was a graduate student and later joined the
faculty of my department at Washington University. In 2020 he was awarded the
Nobel Prize in Physiology or Medicine for his research on hepatitis C virus.
Those sequencing efforts led to our first publication on the sequences of defec-
tive Sindbis RNAs (Monroe et al. 1982). Sequencing of the defective RNAs became
an important tool for identifying noncoding sequences important for replication
before the cloning and analysis of the entire functional virus genome. They also
became part of the toolbox that we used to create Sindbis virus as a vector for the
expression of heterologous proteins.
The initial idea of engineering Sindbis virus to express heterologous proteins
originated with Charles Rice and Henry Huang, both of whom came from the
California Institute of Technology to join the faculty of the Microbiology
Department. My research on developing alphaviruses as expression vectors was
often in collaboration with Charlie and Henry.
490 S. Schlesinger
5 Togaviruses: Alphaviruses and Flaviviruses
Originally, alphaviruses and flaviviruses were grouped together in the same family
referred to as Togaviridae. As the structure and replication strategy of these two
virus groups became known, it was clear that they were distinct and were divided
into two different families. The book that Milt and I edited, Togaviridae and
Flaviviridae, was one of the first times flaviviruses were designated as a separate
family. Scientists who were introduced to the family before the split still retained an
interest in both families. My research, however, continued to focus on alphaviruses.
I wrote above that I had not felt any discrimination as a woman in graduate
school or as a postdoctoral fellow. As a faculty member at Washington University
there were no obvious or specific instances of overt discrimination. About eight to
ten years after we arrived at Washington University, however, it became obvious
that there was a lack of women in positions of authority in universities, in busi-
nesses, and in governments. As far as I remember until the early 1970s, I had not
been on any university or national committees. I had, however, always been suc-
cessful in receiving funding of my NIH grants! With the recognition of discrimina-
tion against women, there was a noticeable effort made in some areas to change this
so that I found myself on various university committees as well as serving on NIH
virology study sections. I also learned that there had been some discrimination in
my salary that was corrected.
During the last decade of my tenure as a professor at Washington University, I
did see a significant increase in the number of women faculty and I was very pleased
to be able to interact with them and perhaps even provide some support. In 1990, I
was involved in the founding of the Academic Women’s Network at Washington
University, an organization, now well-established, that has served as an advocate for
women and as a source of information and mentoring at Washington University.
6 Berkeley, California
I had closed my research laboratory in 2001 and Milt and I decided to move to
Berkeley, California, in 2003. I still maintained my interest in virology and inter-
acted with some of my virology colleagues at the University of California. For a
couple of years, I even gave a few lectures in the virology course at the University
of California in Berkeley. I had contacted Eva Harris at the University, and for about
the next 10 years, I closely followed the research in her laboratory on dengue virus.
My last publication in the field of arbovirology was in collaboration with Claire
Quiner, Eva Harris, and Laura Kramer titled Increased Replicative Fitness of a
Dengue Virus 2 Clade in Native Mosquitoes (Quiner et al. 2014). It also was my first
publication focusing on the importance of mosquitoes in the epidemiology of
arboviruses.
I had really appreciated the interactions with Eva and the people in her labora-
tory. In 1998, Eva had also been responsible for establishing a nonprofit organiza-
tion, the Sustainable Science Institute. SSI works with local scientists and health
officials in developing countries around the world to solve global health problems
related to infectious diseases; the major focus has been on the flaviviruses, dengue,
zika, and chikungunya and also on hepatitis C virus. During the time I was involved
with Eva’s laboratory, I was also on the Board of SSI. These connections continued
until Milt became ill and I wanted to spend more time at home.
7 Oral Histories: Virology and Immunology
My involvement in virology history stems from the time I was President of the
American Society for Virology and realized that the members had an interest in
learning more about the history of our field. The financial contribution of two of the
ASV members, David and Evelyne Lennette, made it possible to establish an annual
lecture to be given by distinguished and older virologists to tell us about their expe-
riences. That was the first step. Then I learned from Bill Summers (Yale University)
that the Alfred P. Sloan Foundation was interested in providing grants for obtaining
science history that would be available on the web. My last grant was from the Sloan
Foundation and I was able to establish a website on the History of Structural
Virology (http://virologyhistory.wustl.edu).
I eventually conducted a number of oral histories with structural virologists that
can be found on that website. This led to more oral histories but also to diversifying
the scientific fields to include, among others, immunology. The two immunologists
that I interviewed and wrote about were Emil Unanue and Herman Eisen. Emil
Unanue is a very distinguished immunologist whose research included the discov-
ery of antigen processing and MHC-peptide binding essential for T-cell recognition.
The interviews with Emil Unanue were privately published in a book.
The last oral history was with Herman Eisen, the Chairman of Microbiology who
had hired me in my first position as an assistant professor at Washington University.
This history was published in the Annual Review of Immunology. The title is
Remembrances of Immunology Past: Conversations with Herman Eisen (Schlesinger
2015). It is one that seemed appropriate for this chapter as well and for which I
thank the initial translators of A la recherché du temps perdu.
References
Genoyer E, Lopez C (2019) The impact of defective viruses on infection and immunity. Annu Rev
Virol 6:547–566
Huang AS, Baltimore D (1970) Defective viral particles and viral disease processes 1970. Nature
226:325–327
492 S. Schlesinger
Jacobson MF, Baltimore D (1968) Polypeptide cleavages in the formation of poliovirus proteins.
Proc Natl Acad Sci U S A 61:77–84
Levis R, Weiss BG, Tsiang M, Huang H, Schlesinger S (1986) Deletion mapping of Sindbis virus
DI RNAs derived from cDNAs defines the sequences essential for replication and packaging.
Cell 44:137–145
Monroe SS, Ou J-H, Rice CM, Schlesinger S, Strauss EG, Strauss JH (1982) Sequence analysis
of cDNA’s derived from the RNA of Sindbis Virions and of defective interfering particles. J
Virol 41:153–162
Quiner CA, Parameswaran P, Ciota AT, Ehrbar DJ, Dodson BL, Schlesinger S, Kramer LD, Harris
E (2014) Increased replicative fitness of a dengue virus 2 clade in native mosquitoes. J Virol
88:13125–13134
Schlesinger S (2015) Remembrance of immunology past: conversations with Herman Eisen. Ann
Rev Immunol 33:1–28
Schlesinger S, Weiss B (1986) Defective RNAs of alphaviruses. In: Schlesinger S, Schlesinger MJ
(eds) Togaviridae and Flaviviridae. Plenum Press
Summers WC (1999) Felix d’Herelle and the origins of molecular biology. Yale University Press,
New Haven
Weiss B, Levis R, Schlesinger S (1983) Evolution of virus and defective interfering RNAs in BHK
cells persistently infected with Sindbis virus. J Virol 48:676–684
An Overview of Arbovirology in São Paulo
State, Southeastern Brazil, Highlighting
the Virus Research Center, in Ribeirão
Preto City
Luiz Tadeu Moraes Figueiredo
Abstract This chapter presents an overview of arbovirology in São Paulo State,

Brazil, starting on outbreaks of yellow fever at the end of nineteenth century, includ-
ing experiments performed in 1903 to show that the disease was transmitted by the
mosquito Aedes aegypti and not by contact with secretions of patients. Later, in
1960, the work in Adolfo Lutz Institute on the isolation of new arboviruses is men-
tioned. Following, aspects of the outbreak of meningoencephalitis by the Rocio
virus 1973–1978 are shown. Starting in 1982, I talk about my career studying den-
gue, other Brazilian arboviruses, and their diseases, especially those affecting the
central nervous system. The creation of the Virus Research Center in Ribeirão Preto
is described as well as some research performed there. Finally, I mention on many
works performed by using high-throughput nucleotide sequencing in the Virus
Research Center after 2010.
Yellow fever virus (YFV) was the first arbovirus to produce large outbreaks and
thousands of deaths in Brazil since the seventeenth century. YFV was probably
introduced into the Americas in the sixteenth century from Africa, probably as part
of the slave trade in ships bringing sick viremic individuals, in addition to the vector
Aedes aegypti also introduced. In the Brazilian northeast, YFV produced large out-
breaks probably transmitted by the Aedes vector. At some moment between the
sixteenth and twentieth century, presumably as a consequence of more than one
spillover, YFV began to infect sylvatic American primates and be transmitted by
Haemagogus mosquitoes from tree canopies. This sylvatic cycle presently main-
tains YFV in South America and is the causative agent in human outbreaks
(Figueiredo 2019; Soper 1936).
São Paulo is a state in the southeastern Brazil, bordering the Atlantic Ocean. São
Paulo is Brazil’s most economically productive and populous in the country
(approximately 45,000,000 inhabitants), accounting for more than one-fifth of the
national population. However, at the end of nineteenth century, the countryside of
L. T. M. Figueiredo (*)
School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil
e-mail: ltmfigue@fmrp.usp.br
494 L. T. M. Figueiredo
São Paulo State was an agricultural frontier where many new villages were created
motivated by coffee farming and the arrival of dozens of thousands of European and
Asian immigrants, mostly Italians, Portuguese, Spanish, Syrians, and Japanese.
New railroads were built for the transport of coffee production from the countryside
to the harbor of Santos City, to be exported. Unfortunately, São Paulo State, at that
moment, had suffered outbreaks of yellow fever that killed thousands of local peo-
ple and immigrants. YFV and the vector Aedes aegypti spread toward the country-
side thru the new railroads. Starting in Santos City, it is possible to follow the
movement northward of the yellow fever wave. Yellow fever outbreaks occurred in
Santos since 1850, in the city of Campinas in 1889, in the city of Casa Branca in the
next years, and in the villages of São Simão and Ribeirão Preto in 1896 to 1903, as
shown in Fig. 1. The village of São Simão, with 4000 inhabitants, suffered three
outbreaks of yellow fever, in 1886, 1888, and 1902, having about 800 deaths by yel-
low fever in 1896. Recommendations by public health authorities to control out-
breaks of yellow fever had radically changed from 1896 to 1902. In 1896, the real
mechanism of transmission of yellow fever was unknown, and trying to avoid trans-
mission from the corpses, public health authorities recommended to open a ditch
around the cemetery and fill it up with lime. Besides, sheets of zinc were put in the
ditch, in order to avoid infiltration. In each coffin of individuals dead by yellow
fever, their corpses were covered by 30 liters of lime. Differently, in 1902, after
being informed on yellow fever transmission by Stegomyia fasciata (Aedes aegypti)
by Walter Reed’s publication on the disease in Cuba, Emilio Ribas, the public health
Fig. 1 The train carrying yellow fever from the harbor of Santos City to the countryside of the
State of São Paulo reaching in sequence the cities of Campinas, Casa Branca, and São Simão.
(Luiz and Figueiredo. 1996)
An Overview of Arbovirology in São Paulo State, Southeastern Brazil, Highlighting… 495
minister of São Paulo State, recommended: avoid stagnant water in homes and their
surroundings, protect sick people through curtains, extinguish by all means the
mosquitoes found in households, protect homes from mosquitoes with screens on
doors and windows, close and prohibit the entry of people into the sick house before
local treatment with insecticides, and protect yellow fever patients from mosquitoes
in hospitals. These measures were successful and yellow fever was eradicated of
São Simão as well as Ribeirão Preto and other cities in the state (Figueiredo 1996;
Reed 1902; Franco 1976).
However, in 1902, after many yellow fever outbreaks, it was difficult to convince
Brazilian physicians that yellow fever was transmitted by a mosquito. It was neces-
sary that three prominent physicians, Emilio Ribas, Adolfo Lutz, and Luiz Pereira
Barreto, shown in Fig. 2, conceive experiments in order to prove transmission by
mosquitoes. The work began at the end of 1902 when larvae of Aedes aegypti were
sent to São Simão city, and the encloded mosquitoes bite individuals with yellow
fever. Later, these mosquitoes were taken by train to the city of São Paulo, where
there was no yellow fever. Sixteen days later, at the isolation hospital (presently
named Hospital Emílio Ribas), six people were bitten by the same mosquitoes. The
physicians Emílio Ribas himself and Adolfo Lutz allowed themselves to be bitten
by the mosquitoes. However, they did not manifest any disease symptoms which
was attributed to previous immunity, as Ribas and Lutz had had previous contact
with epidemics of the disease. Domingos Vaz, André Ramos, and the Italian Januário
Fiori, who had never been in contact with the disease, showed symptoms of yellow
fever. Two of them had benign forms of the infection and Fiori had a more severe
condition. His illness appeared 3 days after the bite, with high fever, hemorrhage,
albuminuria, and jaundice, and he eventually recovered. The successful experiment
was divulged in order to clearly show the vector mediated transmission of yellow
fever. However, many physicians remained sceptical and it was necessary to prove
Fig. 2 Physicians that performed experiments on the transmission of yellow fever in the state of
São Paulo, 1903. (a) Dr. Luiz Pereira Barreto, (b) Dr. Emílio Ribas, and (c) Dr. Adolfo Lutz. (Luiz
and Figueiredo. 1996)
that the disease was not transmitted by other means. A new series of experiments
were carried out in the isolation hospital of São Paulo City (Fig. 3). An isolation
room was prepared, and on April 20, 1903, the Italian Giuseppe Malagutti was
admitted in this room dressed with the clothes of a recently diseased yellow fever
patient. His bed was prepared with pillowcases and sheets stained with blood and
black vomit from patients who died of yellow fever in São Simão. In the following
days, Angelo Paroletti and Giovanni Siniscalchi, both Italian volunteers, joined
Giuseppe Malagutti. These Italians never had contact with yellow fever. Once a
week, in the isolation room, vomited blood, feces, and urine from yellow fever
patients were spread over their clothes and on the floor of the room. Adolfo Lutz,
visiting the room, found that although the air was impregnated, all three confined
volunteers were healthy and in good spirits. The experiment was interrupted on May
12 and a medical committee informed that everyone was doing well. The volunteers
remained another 10 days in the hospital and were discharged in perfect health. The
results of this experiment helped convince the Brazilian medical community that the
transmission of YFV was only by mosquito bite and not by contact or inhalation. It
is also important to highlight that these crude and primitive experiments were the
first in clinical virology made in Brazil (Figueiredo 1996; Franco 1976).
Many years later, in 1961, the section of arboviruses of Adolfo Lutz Institute
(IAL) of the Health Ministry of São Paulo State (Fig. 4), under the direction of Dr.
Oscar de Souza Lopes, started ecological and epidemiological studies on arbovi-
ruses. Virus isolation was obtained from sentinel mice and hamsters as well as from
arthropods, birds, small mammals (rodents, marsupials, and chiroptera), and occa-
sionally humans. Most of the isolated arboviruses were new to the world. Cotia
virus was reported in 1965; Boracéia in 1974; Bertioga and Anhembi in 1975; Rocio
in 1978; Bruconha, Cananeia, Enseada, Guaratuba, and Itimirim in 1983; Iguape in
1993; and Sabiá arenavirus and some hantaviruses in 1994. Other IAL studies
showed easter equine encephalitis virus (EEEV) infecting birds, arthropods, and
mammals. Mucambo (Venezuelan encephalitis virus [VEEV]) virus was isolated
from a sentinel mouse in 1970. Serologic evidence of human infection by these
Fig. 3 Isolation hospital of the city of São Paulo in 1903 showing a nursery. (Luiz and
Figueiredo. 1996)
Fig. 4 Adolfo Lutz Institute in 1940
alphaviruses were observed in surveys. IAL scientists also isolated Saint Louis
encephalitis virus (SLEV), Caraparu and Tacaiuma Orthobunyaviruses, and Iguape,
a new flavivirus. Therefore, the research performed by the scientists of IAL was
very important for arbovirology in the southeast and south of Brazil and showed a
large diversity of arboviruses, especially in the Atlantic rainforest (Coimbra
et al. 1998).
In 1973, a meningoencephalitis outbreak started in the Ribeira Valley, southern
coast of São Paulo State. This outbreak lasted from 1973 to 1978. The disease struck
mostly young men after a 7–14-day incubation period. Patients presented acutely
with fever, headache, anorexia, nausea, vomiting, myalgia, and malaise. Encephalitis
signs appeared later, including confusion, reflex disturbances, motor impairment,
meningeal irritation, and cerebellar syndrome. Some patients presented convul-
sions. Other symptoms included abdominal distension and urinary retention. The
disease produced serious sequelae such as visual, olfactory, and auditory distur-
bances, lack of motor coordination, equilibrium disturbance, swallowing difficul-
ties, incontinence, and defective memory (Fig. 5). During this epidemic, 1021 cases
were reported, approximately 100 deaths were observed, and more than 200 surviv-
ing patients presented with long term sequelae. It was discovered that the causative
agent of the outbreak was a new flavivirus, Rocio virus (ROCV), originally isolated
from nerve tissues of a fatal case of encephalitis. However, many aspects of ROCV
epidemiology still remain unknown and the factors responsible for its appearance
and disappearance in the Ribeira Valley remain a mystery. The virus was isolated
Fig. 5 Patients with infection of the central nervous system by the Rocio virus showing palpebral
ptosis, nuchal rigidity, coma, and gait sequelae. (Tiriba et al. 1976)
from a wild bird, Zenotrichia capensis, and serologic studies suggested that wild
birds could be the virus reservoirs. ROCV was also isolated from the mosquito
Psorophora ferox. Aedes scapularis is another mosquito that could be involved in
ROCV transmission (Lopes et al. 1978; Tiriba et al. 1976; Iversson and Tiriba 1997;
Figueiredo 2000).
The author of this chapter became interested on arbovirology in 1982. At that
time, I was a young physician looking for a research subject for my PhD thesis and
casually attended lectures during the Congress of the Brazilian Society of Tropical
Medicine by Dr. Amelia Travassos da Rosa and Dr. Lygia Iversson talking on arbo-
viruses and aspects of the Rocio encephalitis epidemic (Fig. 6). Watching those
lectures, I discovered that arbovirology includes a large variety of subjects besides
virology, such as internal medicine, epidemiology, ecology, and zoology. All this
new perspective fascinated the young scientist and this love at first sight showed me
the way forward – the study of arboviruses. In 1985, I concluded a study on arbovi-
rus infections in the region of Ribeirão Preto, state of São Paulo. It consisted of
serological surveys to detect levels of antibodies to arboviruses in humans and in
domestic and wild animal populations in the Ribeirão Preto region. A total of 320
individuals living close to a remnant forest, living in a rural environment, already
deforested, or living in the urban perimeter were studied. Inhibition of hemaggluti-
nation and neutralization tests were used, looking for antibodies to members of the
families Togaviridae, Flaviviridae, Peribunyaviridae and Rhabdoviridae (specifi-
cally members of the genus Vesiculovirus). Further, 19.6% of the studied individu-
als had antibodies to these viruses. Higher levels were observed among residents
living close to the natural forest (38.5%), intermediate levels in rural areas, and low
Fig. 6 Dr. Luiz Tadeu M Figueiredo, Dr. Amélia Travassos da Rosa, and Dr. Lygia B Iversson
Fig. 7 Dr. Robert Shope (1999) and Dr. Akira Igarashi (1995) with Dr. Luiz Tadeu Moraes
Figueiredo and his wife Ana Lucia
levels in urban areas. The higher positivity was observed in individuals over 40 years
of age suggesting a previous circulation of these viruses in the region, which prob-
ably was interrupted by a large deforestation, which occurred in the 1970s
(Figueiredo et al. 1986). Interestingly, a 12.1% positivity for Piry virus, a vesiculo-
virus isolated in the Amazon region and with an unknown mechanism of transmis-
sion, was observed (Figueiredo et al. 1985). After that, during 1985 thru 1987, I
have visited the Yale Arbovirus Research Unit in the USA, as a postdoctoral fellow
of Dr. Robert Shope, and, in 1982, I visited the Virus Laboratory of the Tropical
Medicine Institute of the University of Nagasaki in Japan, as a postdoctoral fellow
of Dr. Akira Igarashi (Fig. 7). Both training periods were very important for my
future career as an infectologist clinician and a virus researcher in the School of
Medicine of the University of São Paulo in Ribeirão Preto City.
In 1990, an outbreak of dengue virus type 1 started in Ribeirao Preto City. I per-
formed studies on this dengue outbreak developing methods for serological diagno-
sis by immunoenzymatic and Western blot assays, performing dengue isolation and
typing the virus (Figueiredo et al. 1992). I also made observations on the clinical
picture of dengue, particularly in pregnant women and following up their children
that did not show any disease (Figueiredo et al. 1994). Finally, I carried out a sero-
logical survey for dengue in the city of Ribeirão Preto, assessing antibody levels of
the local population to the virus. The dengue outbreak affected the districts of the
city differently and we have estimated that 23,000 inhabitants had the disease
(Figueiredo et al. 1995). At that time, PAHO highlighted this work as one of the few
works that clearly show good results of fighting Aedes aegypti mosquito and that
allowed to block or abort an established dengue epidemic.
In 1995, in collaboration with Prof. Akira Igarashi from Nagasaki University,
Japan, I performed the first Brazilian studies with RT-PCR for the diagnosis of
dengue. This molecular biology technique was also enlarged to other Brazilian fla-
viviruses such as yellow fever, Rocio virus, Saint Louis encephalitis virus, and
Ilheus virus. We showed the practicality and high sensitivity and specificity of
RT-PCR on the diagnosis of these viruses (Figueiredo et al. 1997, 1998).
In 1998, after taking care of one of two fatal cases of hantavirus cardiopulmo-
nary syndrome (HCPS), I was impressed by the extreme severity of the acute illness
that suddenly struck and killed a young and previously healthy man (Fig. 8). These
HCPS cases were clinically studied including complementary exams and necropsy
data (Figueiredo et al. 1999). New cases of HCPS observed in the following years
were the subject of new studies. The levels of antibodies to hantavirus in the popula-
tion of the region of Ribeirão Preto were studied and we found that 14.3% of the
inhabitants of Jardinopolis City, a neighboring town, had IgG antibodies to hantavi-
rus (Holmes et al. 2000). We studied different aspects of hantavirus epidemiology in
the region of Ribeirão Preto showing that the Araraquara virus (ARAQV) was the
causative agent of HCPS in the region. A rapid diagnostic method for this virus, by
RT-PCR, was developed, and we studied the clinical evolution of patients with
HCPS (Campos et al. 2009)). With my doctorate student Alessandra Borges, I par-
ticipated on studies describing the physiopathology and the myocardial aggression
in fatal cases of hantavirus cardiopulmonary syndrome (Borges et al. 2006, 2008;
Saggioro et al. 2007).
In 1999, supported by the University of São Paulo and by the São Paulo Research
Council (FAPESP) and helped by my colleague virologists in Ribeirão Preto, we
were able to plan and build the Virology Research Center (VRC) of the School of
Medicine of the University of São Paulo in Ribeirão Preto. However, we only moved
to the VRC in 2005. This facility, with 2100 m2, includes laboratories of molecular
biology, serology, cell biology, animal facilities for rodents and bats, and BSL2 and
BSL-3 laboratories. It also includes workspace, offices for graduate students and
postdoctoral fellows, and a conference room. For fieldwork we have a pull truck and
Fig. 8 House for corn storage where two agricultural workers from the region of Ribeirão Preto
became infected by hantavirus also showing feces of rodents in a corner of the house (Pereira LE,
1998). Radiographs from both fatal cases showing severe bilateral pneumonitis by hantavirus
a trailer-laboratory that allows the capture, identification, and sampling of small

mammals (rodents and bats) following biosafety procedures (Fig. 9). Four principal
researchers, Luiz Tadeu Moraes Figueiredo, Eurico Arruda, Benedito Antonio
Lopes da Fonseca, and Luis Lamberti, and their technicians, graduate students,
postdoctoral fellows, and other interns, about 50 people, work in the VRC. This new
facility made it possible to expand our research activity significantly.
After amplification of flavivirus gene segments, we started to sequence the
amplicons for selection of primers looking for a virus-specific diagnosis. We also
performed phylogenetic studies on Brazilian flaviviruses, based on the nucleotide
sequence of the 3′ noncoding region and the NS5 gene. Therefore, we developed
multiplex RT-PCRs for the diagnosis of arboviruses at the genus level (Alphavirus,
Flavivirus and Orthobunyavirus) and using nested PCR, the agents could be identi-
fied at the species level (Baleotti et al. 2003; Batista et al. 2001; Bronzoni et al.
2005; Moreli et al. 2002). In 2006, in cooperation with my postdoctoral fellow
Roberta Bronzone and Prof. Mauricio Lacerda that had just created a virus labora-
tory in the School of Medicine of São José do Rio Preto City, using multiplex
RT-PCR, we were able to detect, for the first time in Brazil, an outbreak of Saint
Louis encephalitis, and it occurred in the middle of a dengue epidemic (Mondini
et al. 2007).
Serological evidence of circulation of the Rocio virus (ROCV) in the original
area as well as in other parts of Brazil has been reported after the outbreak in Ribeira
Valley. Public health authorities are always concerned about the reappearance of
Fig. 9 The Virology Research Center of the School of Medicine of the University of São Paulo in
Ribeirão Preto, 2005
ROCV outbreaks in Brazil. A serological survey of horses from various Brazilian

regions showed that 415 (55.1%) of the 753 studied horses were seropositive for
flavivirus, and among them, a monotypic reaction to ROCV was found in 46 ani-
mals (6.1%) (Figueiredo and Figueiredo 2014). Besides that, in 2010, testing 23
cerebrospinal fluid (CSF) samples from human patients from the city of Manaus by
RT-PCR, we found amplicons of the ROCV genome in two patients that were con-
firmed by nucleotide sequencing. These were a 53-year-old man with seizures and
abnormal conscientiousness and a 30-year-old woman with headache, vomiting,
and signs of intracranial hypertension. Curiously, both had AIDS and the woman
also had tuberculosis. Both survived after 20 days of hospitalization. Interestingly,
evidence of horse infections and these human cases occurred in the north of Brazil,
more than 2000 km away from where the virus was originally isolated. Thus, it is
possible that ROCV circulates unrecognized in Brazil and possibly in other South
American countries, producing human infections including those of the CNS, par-
ticularly affecting immunodeficient individuals, such as AIDS patients (Figueiredo
and Figueiredo 2014, 2018).
Our studies on hantavirus had a serious difficulty for serologic diagnosis due to
a lack of antigens. In order to solve this problem, helped by my graduate student
Marcos Moreli, we produced and purified a recombinant N protein from Araraquara
hantavirus (ARQV). An ELISA was developed using ARQV N recombinant protein

as antigen. The results obtained were highly encouraging, and the recombinant N
protein as well as protocols for ELISA tests was provided to the government labo-
ratories that work in the diagnosis of the hantavirus including Adolfo Lutz Institute
in São Paulo, Fiocruz in Rio de Janeiro, Evandro Chagas Institute in Belém, and
Carlos Malbrán Institute in Buenos Aires, Argentina (Figueiredo et al. 2009a). We
also obtained a patent for the N recombinant protein of the hantavirus Araraquara.
We have studied the clinical presentation of Araraquara (ARQV) hantavirus car-
diopulmonary syndrome and its high case fatality rate (Figueiredo et al. 2009b). We
also studied the immune response to the disease in patients having distinct clinical
presentations including fatal cases. We found that respiratory failure and shock in
ARQV cardiopulmonary syndrome are consequent to a capillary leaking syndrome
produced by a cytokine storm and hantavirus myocarditis (Borges et al. 2006;
Saggioro et al. 2007). We also performed field studies capturing small mammals
and confirming, as previously reported, that Necromys lasiurus (Sigmodontinae)
rodent is the main reservoir of ARQV (de Souza 2008). Recently, our research, with
postdoctoral fellow Gilberto Sabino, showed that the common vampire bat
(Chiroptera: Desmodus rotundus) is a potential natural reservoir of ARQV (Fig. 10)
(Sabino-Santos Jr et al. 2018).
We also started to sequence complete virus genomes of Oropouche virus
(OROV) (Family Peribunyaviridae, genus Orthobunyavirus). My doctoral student
Victor Hugo Aquino sequenced the three RNA segments of OROV using a laborious
step-by-step process and dozens of primers (Aquino et al. 2003; Aquino and
Figueiredo 2004). High-throughput nucleotide sequencing of virus genomes started
Bats ?
Viral particles in aerosols
Rodents
Hantavirus disease
Humans
Fig. 10 A schematic figure illustrating the transmission dynamics of hantavirus, in nature, in the
study region, southeastern Brazil. (Sabino-Santos Jr et al. 2018)
to be performed in VRC after 2010. In collaboration with colleagues from the

University of São Paulo, Evandro Chagas Institute from the Amazon region, and the
University of Glasgow, UK, my fellow William de Souza obtained complete
genomes of known viruses and unknown viruses, in a large variety of clinical sam-
ples. We also carried out sophisticated studies of phylogeny and molecular epidemi-
ology. These studies included Capim Orthobunyavirus complete characterization
(de Souza et al. 2016a), as well as viruses of the sandfly fever group viruses belong-
ing to Bujaru, Frijoles, and Taquara antigenic groups (Nunes-Neto et al. 2017). The
complete genome of Piry virus was also sequenced (de Souza et al. 2016). We have
studied Chapparvoviruses in at least three vertebrate classes of a broad biogeo-
graphic distribution (de Souza et al. 2016b). We discovered novel anellovirus spe-
cies in wild small mammals and proposed two new genera into the Anelloviridae
family (Marciel et al. 2018). We studied the genomic evolution of Tacaiuma
Orthobunyavirus (Peribunyaviridae family) isolated in Brazil (de Souza et al. n.d.).
We characterized genome viruses of ticks from southern Brazil and studied novel
parvoviruses from wild animals (de Souza et al. 2018a). We described a novel
Orthohepevirus in wild rodents (de Souza et al. 2018b). We performed studies that
helped the revalidation and the genetic characterization of new members of group C
(Orthobunyavirus genus, Peribunyaviridae family) (Nunes et al. 2018). The viral
diversity of Rhipicephalus microplus parasitizing cattle in southern Brazil was stud-
ied (de Souza et al. 2018c). A novel polyomavirus in Sigmodontinae rodents, novel
astrovirus and calicivirus, and novel hepacivirus in rodents from South America
were discovered (de Souza et al. 2019a). We described novel astrovirus and calici-
virus identified in ruddy turnstones of Brazil (de Souza et al. 2019b). Finally, we
described Pingu virus: a new picornavirus in penguins from Antarctica, as shown in
Fig. 11 (de Souza et al. 2019c).
We have also performed studies on arboviruses infecting the CNS. It is known
that special capabilities are necessary for an arbovirus to infect the CNS. The patho-
gen, besides being virulent, has to defeat general cellular and humoral immune
response, be able to cross the blood-brain barrier (BBB) or the choroid plexus, and
when in nervous tissue, defeat the local host defense mechanisms (Barros et al.
2011). We found that genomic mutations can increase the virulence of flaviviruses,
alphaviruses and orthobunyaviruses that produce infection in the CNS. An example
of that was a study on ROCV and WNV where a chimeric WNV containing prM-E
genes of ROCV replicated in mammalian cells more efficiently than WNV or chi-
meric ROCV containing WNV prM-E genes. WNV containing ROCV prM-E genes
was as virulent as ROCV in adult mice. Proteins prM and E of ROCV inhibited type
I interferon response which could potentially enhance neurovirulence (Amarilla
et al. 2017). The local inflammation of CNS produces brain edema and ischemia
and can damage the nervous tissues. Blood cell migration induced by chemokines
produced after viral presence in the CNS produces and aggravates inflammation.
The importance of the impact of macrophages on the severity of encephalitis has
been clearly shown in our study on macrophage chemokines, CCR5 (CC chemokine
receptor 5) and MIP-1 (chemokine receptor that binds to the macrophage inflamma-
tory protein), using knockout mice to genes of these chemokines. The knockout
Pingu picornavirus - Sample 22

0.1 substitutions per site
100 Pingu picornavirus - Sample 44
NC_036588 Passerivirus sp
91
GU182406 Turdivirus 1
NC_018400 Gallivirus A
100
100 NC_024770 Chicken gallivirus 1
NC_023861 Sicinivirus A
Fig. 11 Phylogenetic tree showing the evolutionary relationships of four partial genomes of
PingPcV with related avian picornaviruses. The phylogeny was inferred with IQ-TREE
(TPM3uþFþI nucleotide substitution model) using an alignment of 417 nucleotides derived from
the RdRp gene. Phylogenies are midpoint rooted for clarity of presentation. The scale bar indicates
evolutionary distance in numbers of substitutions per nucleotide site. Bootstrap values of 1000
replicates are shown in principal nodes. PingPcV sequences are shown in red with a penguin sil-
houette. (de Souza et al. 2019c)
animals, after infected with ROCV, survived longer in meningoencephalitis and had
reduced inflammation in the brain than wild-type (WT) mice infected with
ROCV. Knockout mice also required a higher lethal dose of ROCV for CNS infec-
tion than wild-type mice (Chávez 2013).
We used RT-PCR to look for virus in the cerebrospinal fluid (CSF) of patients
with lymphocytic meningoencephalitis. In a study performed from 2005 to 2010 in
Manaus, a city in the north region of Brazil, CSF samples of 110 patients were sub-
mitted for RT-PCR, and the Oropouche virus (OROV) was found in three samples.
Patients were a 20-year-old agricultural worker man, a 54-year-old fisherman, and a
37-year-old domestic worker woman. All patients referred headache; one patient
had dizziness, cloud vision, and Romberg sign; the second had fever, chills, and
malaise; and the third had nausea, vomiting, and paraplegia. All patients survived
after hospitalization. Interestingly, two of these patients had other diseases affecting
the CNS or immune system; the first had neurocysticercosis and the last had AIDS
(de Souza Bastos et al. 2012). In another study including 49 patients from Manaus
City, four had dengue virus in the CSF (DENV-2 and DENV-1). These patients
presented with headache, myalgias, and arthralgias that evolved to neck stiffness
and impaired consciousness that healed without sequelae (Bastos et al. 2014). In
2016, we reported a 2-year-old girl that became ill during a ZIKV epidemic. The
girl had fever (up to 38.5 °C) for 8 days and a macular rash on the extremities for
the past 2 days. She also presented with irritability, weakness, myalgias, and dys-
uria. Finally, 9 days after the onset of illness, she developed ataxic gait with normal
reflexes and a positive Babinski sign. RT-PCR for ZIKV was positive in plasma and
CSF. Magnetic resonance imaging (MRI) demonstrated rhombencephalitis with
lesions in the vermix and left cerebellar hemisphere. The ataxic gait disappeared
after 3 days, and the patient recovered without sequelae (Figueiredo and Figueiredo
2018). Another case was a 36-year-old heart transplant recipient from Ribeirão
Preto that also had ZIKV encephalitis. He presented with a 2-day history of high-
grade fever, malaise, headache, and seizures that evolved with progressive hemody-
namic instability, mental deterioration, and finally death. MRI revealed extensive
cortical encephalitis with image suggestive of necrosis of the brain parenchyma and
vasogenic edema (Fig. 12). The ZIKV genome was detected in the CSF by RT-PCR
followed by nucleotide sequencing and the infection was also confirmed by immu-
nohistochemistry, immunofluorescence, and electron microscopy of brain tissue
(Schwartzmann et al. 2017).
Fig. 12 Magnetic resonance images from an immunocompromised patient with Zika virus infec-
tion showing hypointense and hyperintense lesions with cortical and subcortical involvement of
the cingulate and superior frontal gyrus by a meningoencephalitis focus. (a) Axial T1-weighted
spin-echo image showing hypointense lesion (arrow). Axial T2-weighted turbo spin-echo image
(b) and axial fluid-attenuated inversion recovery image (c) show cytotoxic cortical edema with
diffusion restriction (arrows). (d) Diffusion imaging shows low apparent diffusion coefficient
(arrow). (e–g) Orthogonal reconstruction of three-dimensional magnetization-prepared rapid
acquisition and multiple gradient echo images T1-weighted after gadolinium injection showing no
gadolinium enhancement (arrows). (h) Cerebral blood volume perfusion map showing hypoperfu-
sion (arrow). (Schwartzmann et al. 2017)
In the last years, we have studied human urban arboviruses infecting wild
animals that thus could jump into sylvatic maintenance cycles. We suppose that
the most prominent human arboviruses worldwide (dengue viruses 1, 2, 3, and
4, chikungunya virus, and Zika virus) can infect wild animals and transfer from
urban to sylvatic maintenance cycles, as did the yellow fever virus (YFV) in the
past. All these viruses are transmitted by the anthropophilic mosquito Aedes
aegypti and cause epidemics throughout Brazil. YFV is the oldest example of an
urban arbovirus that became sylvatic in South America. Currently, the disease is
a zoonosis of nonhuman primates that moves like a wave through the forests of
the Brazilian countryside, traveling thousands of kilometers, killing many ani-
mals, and eventually infecting humans. However, since 2016, this zoonotic
wave has reached the highly populated areas of southeast Brazil, producing the
largest human outbreak in the past 60 years (Figueiredo 2019). As with the YFV,
sylvatic cycles may occur with dengue, Chikungunya, and Zika. In order to
become sylvatic, arboviruses require an apparently unlikely conjunction of fac-
tors to unexpectedly take place. These arboviruses could start to infect sylvatic
primates and be transmitted by Haemagogus mosquitoes that inhabit tree cano-
pies. Evidence of sylvatic cycles of dengue, chikungunya, and Zika viruses in
South America has been reported. Indeed, it is almost unfeasible to control these
cycles of arboviruses since it is impossible to know where, when, or why an
arboviral spillover would occur in wild animals (Fig. 13). The sylvatic mainte-
nance cycle could preclude the eradication of an arbovirus. Moreover, an arbo-
virus in a sylvatic cycle could reemerge anytime, infecting humans and producing
outbreaks. In case of the reemergence of an arbovirus, it is crucial to prevent
the occurrence of an urban cycle as a spill-back from the sylvatic cycle
(Figueiredo 2019).
Finally, I want to highlight that many of my studies resulted from important
scientific collaborations with Brazilian colleagues from the Instituto Evandro
Chagas (Amélia Travassos da Rosa, Pedro Vasconcelos, Cecilia Cruz, and
Marcio Nunes), Oswaldo Cruz Institute (Hermann Schatzmayr, Edson da Silva,
and Elba Lemos), Adolfo Lutz Institute (Luis Eloy Pereira, Akemi Suzuki, and
Elza Nassar), and University of São Paulo (Edson Durigon and Paolo Zannotto).
I also performed studies in collaboration with scientists from other countries,
such as Argentina (Paula Padulla, Delia Enria, and Mario Lozano), Chile
(Marcela Ferrer), Colombia (Salim Mattar, Camilo Guzman, and Alfonso
Calderon), Finland (Olli Vahpalatti), France (Jean-Louis Camicas and Francisco
Viegas), Japan (Akira Igarashi), United Kingdom (Pablo Murcia), and USA
(Robert Shope, Gregory Tignor, Colleen Jonsson, Charles Fullhorst, and Jorge
Salazar).
508
Aedes Hormogogus
Urban cycle Sylvatic cycle
Humans Humans Monkeys Monkeys
Aedes Hormogogus
Fig. 13 Arbovirus in the urban cycle jumping to the wild maintenance cycle due to the Aedes aegypti vector infecting nonhuman primates or viremic individu-
als infecting the wild mosquito. (Figueiredo 2019)
L. T. M. Figueiredo
References
Amarilla AA, Setoh YX, Periasamy P, Pali G, Figueiredo LT, Khromykh AA, Aquino VH (2017)
Chimeras between Rocio and West Nile viruses reveal the role for Rocio virus prM and E
proteins in virulence and inhibition of type I interferon signaling. Sci Rep 7:44642. https://doi.
org/10.1038/srep44642
Aquino VH, Figueiredo LTM (2004) Linear amplification followed by single primer poly-
merase chain reaction to amplify unknown DNA fragments: complete nucleotide sequence of
Oropouche virus M RNA segment. J Virol Methods 115:51–57
Aquino VH, Moreli ML, Moraes Figueiredo LT (2003) Analysis of Oropouche virus L protein
amino acid sequence showed the presence of an additional conserved region that could harbor
an important role in the polymerase activity. Arch Virol 148:19–28
Baleotti FG, Moreli ML, Figueiredo LTM (2003) Brazilian Flavivirus phylogeny based on NS5,
vol 98. Memórias do Instituto Oswaldo Cruz, Rio de Janeiro, pp 379–382
Barros VD, Penharvel S, Forjaz J, Saggioro FP, Neder L, Figueiredo LTM (2011) An experimental
model of meningoencephalomyelitis by Rocio virus in Balb-C mice: histopathology, inflam-
matory response and cytokine production. Am J Trop Med Hyg 85:363–373
Bastos MS, Lessa N, Naveca F, Alves VR, Figueiredo RMP, João Bosco L, Gimaque RL, Monte SF,
Nogueira ML, Braga WS, Figueiredo LTM, Ramasawmy R, Mourão MPG (2014) Detection
of herpesvirus, enterovirus and arbovirus infection in patients with suspected central nervous
system viral infection in the western Brazilian Amazon. J Med Virol 86:1522–1527. https://doi.
org/10.1002/jmv.23953
Batista WC, Kashima S, Marques AC, Figueiredo LTM (2001) Phylogenetic analysis of Brazilian
Flavivirus using nucleotide sequences of parts of NS5 gene and 3′ non-coding regions. Virus
Res 75:35–42
Borges AA, Campos GM, Moreli ML, Souza RLM, Figueiredo LTM (2006) Pathogenesis of hanta-
virus cardiopulmonary Syndrome and the human immune responses to hantavirus. Microbes
& Infection 8:2324–2330
Borges AA, Campos GM, Moreli ML, Souza RLM, Saggioro FP, Figueiredo GG, Livonesi MC,
Figueiredo LTM (2008) Pathophysiologic and prognostic role of cytokines in hantavirus car-
diopulmonary syndrome. Microbes & Infection 10:1150–1157. PMID: 18606242, 2008
Bronzoni RV d M, Nogueira RMR, Nunes M, Figueiredo LTM (2005) Detection and identification
of Brazilian alphaviruses and flaviviruses by multiplex RT-PCR. J Clin Microbiol 43:696–702
Campos GM, Borges AA, Moreli ML, Badra SJ, Souza RLM, Figueiredo GG, Figueiredo LTM
(2009) Síndrome Pulmonar e Cardiovascular por Hantavírus: aspectos clínicos de uma doença
emergente no sudeste brasileiro. Rev Soc Bras Med Trop 42:282–289
Chávez JH, de Oliveira França RF, Oliveira CJ, de Aquino MTP, Farias KJ, Machado PR, Yokosawa
J, da Silva JS, da Fonseca BAL, Figueiredo LTM (2013) CCR-5/MIP-1 alpha affect the patho-
genesis of Rocio virus encephalitis in a mouse model. Am J Trop Med Hyg 89:1013–1018
Coimbra TLM, Rocco IM, Suzuki A, Pereira LE, de Souza LTM, Nassar ES, Ferreira IB, Chamelet
E (1998) Arthropod- and rodent-borne viruses detected in São Paulo State, Brazil. In: Travassos
da Rosa AP, Vasconcelos PC, Travassos da Rosa JF (eds) An overview of arbovirology in Brazil
and neighboring countries. Instituto Evandro Chagas, pp 168–176
de Figueiredo MLG, Figueiredo LTM (2014) Review on infections of the central nervous sys-
tem by St Louis Encephalitis, Rocio and West Nile Flaviviruses in Brazil, 2004–2014. J Adv
Microbiol 4:955–961. https://doi.org/10.4236/aim.2014.413106
de Figueiredo MLG, Figueiredo LTM (2018. ISBN 978-3-319-92678-0) Emerging causes
of encephalitis: Zika, dengue, chikungunya and beyond. In: Hasbun R (ed) Meningitis and
encephalitis: management and prevention challenges, vol Chapter 15. Springer, pp 217–227.
https://doi.org/10.1007/978-3-319-92678-0_15
de Souza Bastos M, Figueiredo LT, Naveca F, Figueiredo R, Oliveira CM, Gimaque JB, Assis
K, Monte RL, Mourão MP (2012) Infection of central nervous system by Oropouche
Orthobunyavirus in three Brazilian patients. Am J Trop Med Hyg 86:732–735
de Souza RLM, Moreli ML, Borges AA, Campos GM, Livonesi MC, Figueiredo LTM, Pinto
AA (2008) Natural host relationships and genetic diversity of rodent-associated hantavirus in
Southeastern Brazil. Intervirology 51:299–310
de Souza WM, Acrani GO, Romeiro MF, Júnior OR, Tolardo AL, de Almeida Medeiros DB, Nunes
MRT, Murcia PR, Figueiredo LTM (2016) Complete genome sequence of Piry vesiculovirus.
Arch Virol 161:2325–2328. https://doi.org/10.1007/s00705-016-2905-9
de Souza WM, Acrani GO, Romeiro MF, Reis Jr O, Tolardo AL, Patroca S, da Silva D, de Almeida
B, Medeiros MV, Nunes MRT, Figueiredo LTM (2016a) Molecular characterization of Capim
and Enseada orthobunyaviruses. Infect Genet Evol 40:47–53. https://doi.org/10.1016/j.
meegid.2016.02.024
de Souza WM, Romeiro MF, Fumagalli MJ, Modha S, de Araujo J, Queiroz LH, Durigon EL,
Figueiredo LTM, Murcia PR, Gifford R (2016b) Chapparvoviruses occur in at least three ver-
tebrate classes and have a broad biogeographic distribution. J Gen Virol 4:1–5. https://doi.
org/10.1099/jgv.0.000671. [Epub ahead of print]
de Souza WM, Fumagalli MJ, de Araujo J, Sabino-Santos Jr G, Maia FGM, Romeiro MF, Modha
S, Hughes J, Queiroz LH, Durigon EL, Nunes MRT, Murcia PR, Figueiredo LTM (2018)
Discovery of novel anelloviruses species in wild small mammals expands the host diver-
sity and proposes two new genera into Anelloviridae family. Virology 514:9–17. https://doi.
org/10.1016/j.virol.2017.11.001
de Souza WM, Dennis TPW, Fumagalli MJ, de Araujo J, Sabino-Santos Jr G, Maia FGM, Acrani
GO, de Oliveira A, Carrasco T, Romeiro MF, Modha S, Vieira LC, Queiroz LH, Durigon EL,
Nunes MRT, Murcia PR, Figueiredo LTM, Gifford RJ (2018a) Novel parvoviruses from wild
and domestic animals in Brazil provide new insights into parvovirus distribution and diversity.
Viruses 10:143. https://doi.org/10.3390/v10040143
de Souza WM, Romeiro MF, Sabino-Santos Jr G, Maia FGM, Fumagalli MJ, Modha S, Nunes
MRT, Murcia PR, Figueiredo LTM (2018b) Novel orthohepeviruses in wild rodents from São
Paulo state, Brazil. Virology 519:12–16. https://doi.org/10.1016/j.virol.2018.03.025
de Souza WM, Fumagalli MJ, de Oliveira A, Carrasco T, Romeiro MF, Modha S, Seki MC, Gheller
JM, Daffre S, Nunes MRT, Murcia PR, Acrani GO, Figueiredo LTM (2018c) Viral diversity of
Rhipicephalus microplus parasitizing cattle in southern Brazil. Sci Rep 8:16315. https://doi.
org/10.1038/s41598-018-34630-1
de Souza WM, Fumagalli MJ, Sabino-Santos Jr G, Maia FGM, Romeiro MF, Modha S, Nunes
MRT, Murcia PR, Figueiredo LTM (2019a) Identification of a novel hepacivirus in wild rodents
from South America. Viruses 11:297. https://doi.org/10.3390/v11030297
de Souza WM, Fumagalli MJ, de Araujo J, Ometto T, Modha S, Thomazelli LM, Durigon EL,
Murcia PR, Figueiredo LTM (2019b) Discovery of novel astrovirus and calicivirus identified in
ruddy turnstones in Brazil. Sci Rep. https://doi.org/10.1038/s41598-019-42110-3
de Souza WM, Fumagalli MJ, Martini M, de Araujo J, Orsi MA, Sanfilippo LF, Modha S, Durigon
E, Proença-Módena JL, Arns C, Murcia P, Figueiredo LT (2019c) Pingu virus: a new picorna-
virus in penguins from Antarctica. Virus Evol 5(2):vez047. https://doi.org/10.1093/ve/vez047.
eCollection 2019 Jul
de Souza WM, Melo Junior AB, Acrani GO, Carvalho VL, Romeiro MF, Tolardo AL, Silva SP,
Cardoso JF, Chiang JO, Vianez Júnior JLS, Azevedo RS, Figueiredo LTM, Vasconcelos PFC,
Nunes MRT, Medeiros DBA (n.d.) Genomic characterization and evolution of Tacaiuma
orthobunyavirus (Peribunyaviridae family) isolated in Brazil. Infect Genet Evol 60:71–76.
https://doi.org/10.1016/j.meegid.2018.02.027
Figueiredo LTM (1996) A febre amarela na Região de Ribeirão Preto durante a virada do
século XX: importância científica e repercussões econômicas. Rev Soc Bras Med Trop
29:63–76
Figueiredo LTM (2000) The Brazilian Flaviviruses. Microbes Infect 2:1643–1649
Figueiredo LTM (2019) Human urban arboviruses can infect wild animals and jump to sylvatic
maintenance cycles in tropical countries. Front Cell Infect Microbiol 9(art. 259):1–6. https://
doi.org/10.3389/fcimb.2019.00259
Figueiredo LTM, Amélia PA, da Rosa T, Fiorillo AM (1985) Prevalência de anticorpos neutrali-
zantes para o arbovírus Piry em indivíduos da Região de Ribeirão Preto, Estado de São Paulo,
vol 27. Revista do Instituto de Medicina Tropical, São Paulo, pp 157–161
Figueiredo LTM, Amélia PA, da Rosa T, Fiorillo AM (1986) Níveis de anticorpos para arbovírus
em indivíduos da Região de Ribeirão Preto, SP (Brasil), vol 20. Revista de Saúde publica, São
Paulo, pp 204–211
Figueiredo LTM, Owa MA, Carlucci RH, de Oliveira L (1992) Estudo sobre diagnóstico laborato-
rial e sintomas do dengue, durante epidemia ocorrida em Ribeirão Preto, SP, Brasil, vol 34.
Revista do Instituto de Medicina Tropical, São Paulo, pp 121–130
Figueiredo LTM, Carlucci RH, Duarte G (1994) Estudo prospectivo com crianças cujas mães
tiveram dengue na gravidez, vol 36. Revista do Instituto de Medicina Tropical, São Paulo,
pp 417–421
Figueiredo LTM, Owa MA, Carlucci RH, Vieira N, de Mello A, dal Fabbro L, Capuano DM,
Santili MB (1995) Dengue serologic survey in Ribeirão Preto, SP, Brazil. Bull Pan Am Health
Organ 29:59–69
Figueiredo LTM, Batista WC, Igarashi A (1997) Detection and identification of dengue virus iso-
lates from Brazil by a simplified reverse transcription – polymerase chain reaction (RT-PCR)
method, vol 39. Revista do Instituto de Medicina Tropical, São Paulo, pp 95–99
Figueiredo LTM, Batista WC, Kashima S, da Silva Nassar E (1998) Identification of Brazilian
flaviviruses by using Flavivirus-universal primers in a simplified reverse transcriptase – poly-
merase chain reaction (RT-PCR) method. Am J Trop Med Hyg 59(3):357–362
Luiz T. M. Figueiredo, Marcos L. Moreli, Simone Kashima, Vanessa S.O. Almeida, Paulo R. Félix,
Júlio C. Bruno, Ivani B. Ferreira, Francisco D. Mançano (1999) Hantavirus Pulmonary
Syndrome (HPS) in Guariba, SP, Brazil. Report of 2 cases. Revista do Instituto de Medicina
Tropical, São Paulo, 41(2): 131–137
Figueiredo LTM, Moreli ML, Borges AA, Garcia G, de Figueiredo S, Badra J, Bisordi I, Suzuki
A, Capria S, Padula P (2009a) Evaluation of a solid-phase enzyme immunoassay based on
Araraquara hantavirus recombinant nucleoprotein. Am J Trop Med Hyg 81:273–276
Figueiredo LTM, Moreli ML, de Sousa RLM, Borges AA, de Figueiredo GG, Machado AM,
Bisordi I, Nagasse TK, Sugahara AS, Pereira LE, de Souza RP, de Souza LTM, Braconi CT,
Harsi CM, de Andrade Zanotto PM, the VGDN Consortium (2009b) Hantavirus pulmonary
syndrome, central plateau, Southeastern, and Southern Brazil. Emerg Infect Dis 15:561–567
Franco O (1976) História da febre amarela no Brasil. Ministério da Saúde, Rio de Janeiro
Holmes RR, Boccanera R, Luiz TM, Figueiredo SR, Mançano CP (2000) Seroprevalence of
human hantavirus infection in the Ribeirão Preto region of São Paulo state, Brazil. Emerg
Infect Dis 6:560–561. (Letters)
Iversson LB, Tiriba AC (1997) Encefalite por arbovírus Rocio. In: Veronesi R, Focaccia R (eds)
Tratado de infectologia. Atheneu, São Paulo, pp 233–239
Lopes OS, Coimbra TLM, Sacchetta LA, Calisher CH (1978) Emergence of a new arbovirus
disease in Brazil. 1. Isolation and characterization of the etiologic agent, Rocio virus. Am J
Epidemiol 107:444–449
Mondini A, Lázaro E, Cardeal ILS, Nunes SH, Moreira CC, Rahal P, Figueiredo LTM, Bronzoni
RVM, Neto FC, Nogueira ML (2007) Saint Louis encephalitis virus, Brazil. Emerg Infect Dis
13(1):176–178
Moreli ML, Aquino VH, Ana CR, Cruz LT, Figueiredo M (2002) Diagnosis of Oropouche virus
infection by RT-nested-PCR. J Med Virol 66:139–142
Nunes MRT, de Souza WM, Acrani GO, Cardoso JF, da Silva SP, Badra SJ, Figueiredo LTM,
Vasconcelos PF d C (2018) Revalidation and genetic characterization of new members of
Group C (Orthobunyavirus genus, Peribunyaviridae family) isolated in the Americas. Plos One
24. https://doi.org/10.1371/journal.pone.0197294
Nunes-Neto JP, Marciel W, de Souza G, Acrani O, Romeiro MF, Fumagalli MJ, Vieira LC, de
Almeida Medeiros DB, Lima JA, de Lima CPS, Cardoso JF, Figueiredo LTM, da Silva SP, Tesh
R, Nunes MRT, da Costa Vasconcelos PF (2017) Characterization of the Bujaru, Frijoles and
Tapara antigenic complexes into the Sandfly Fever group and two unclassified phleboviruses
from Brazil. J Gen Virol 98:585–594. https://doi.org/10.1099/jgv.0.000724
Reed W (1902) Recent researches concerning the etiology, propagation, and prevention of yellow
fever, by the United States Army Commission. J Hyg 2:102–119
Sabino-Santos G Jr, Maia FGM, Martins RB Jr, Gagliardi TB, Marciel W, de Souza N, da Silva
B, de Lara R, Muylaert MC, Pontelli L, Luna d KS, Melo DM, de Souza Cardoso R, Zapana
PRM, Vieira TM, Melo NM, Jonsson CB, Goodin D, Salazar-Bravo J, da Silva LLP, Neto
EA, Figueiredo LTM (2018) Common vampire bat (Chiroptera: Desmodus rotundus) as
potential natural-reservoir of Araraquara hantavirus. Sci Rep 8:9018. https://doi.org/10.1038/
s41598-018-27442-w
Saggioro FP, Rossi MA, Maria IS, Duarte CC, Martin S, Alves VAF, Moreli ML, Luiz TM,
Figueiredo JE, Moreira AA, Borges LN (2007) Hantavirus infection induces a typical myo-
carditis that may be responsible for myocardial depression and shock in hantavirus pulmonary
Syndrome. J Infect Dis 145:1541–1549
Schwartzmann PV, Vilar FC, Takayanagui OM, dos Santos AC, Ayub-Ferreira SM, Ramalho LNZ,
Neder L, Romeiro MF, Maia FGM, Tollardo AL, Zapata PM, Figueiredo LTM (2017) André
Schmidt, Marcus Vinicius Simões. Zika virus associated encephalitis in immunocompromised
patient. Mayo Clin Proc 92:460–466. https://doi.org/10.1016/j.mayocp.2016.12.019
Soper FL (1936) Febre amarela silvestre. Novo aspecto epidemiológico da doença, Revista de
Hygiene e Saúde Pública 10:107–144
Tiriba AC, Miziara AM, Lourenço R, Costa CRB, Cota CS, Pinto GH (1976) Encefalite humana
primária epidêmica por arbovirus observada no litoral sul do Estado de São Paulo. Rev Assoc
Med Bras 22:415–420
An Unplanned Career in Arbovirology
Robert B. Tesh
Abstract The following account describes my unplanned and unexpected career in

tropical medicine, the epidemiology of vector-borne and zoonotic diseases, vector
biology, and arbovirology. It also illustrates the vicissitude of research priorities and
funding and the need to be flexible and to change research directions as priorities
and technology changes.
1 Background
My route to a career in virus research was unplanned and serendipitous. After grad-
uation from a public high school in Wilmington, Delaware, I enrolled in Franklin
and Marshall College, a small liberal arts college in Lancaster, Pennsylvania, with
the goal of becoming a physician or veterinarian. In my junior year, I decided to
apply to medical school and was accepted at Jefferson Medical College (now
Thomas Jefferson University) in Philadelphia. During my time at Jefferson, I devel-
oped an interest in infectious diseases and considered specializing in that field.
Following a rotating internship at San Francisco General Hospital in California, I
applied for several residency programs in pediatrics. On a lark, I also applied and
was accepted for a position as first-year pediatric resident at Gorgas Hospital in the
old Panama Canal Zone. This turned out to be a fortuitous and life-changing
decision.
Gorgas Hospital was located next to the Middle America Research Unit (MARU),
a research laboratory in the Canal Zone, run jointly by the National Institute of
Allergy and Infectious Disease (NIAID/NIH) and the US Army. Because of its
proximity, I got to know some of the scientists there and their work. One of the
research projects at MARU at that time was on the epidemiology of histoplasmosis,
a fungal infection highly endemic in Panama. In fact, the first human cases of histo-
plasmosis were described by Samuel T. Darling, an American pathologist at Gorgas
Hospital during construction of the Panama Canal. In 1906, Darling published the
R. B. Tesh (*)
Adjunct Professor of Pathology and of Microbiology & Immunology, University of Texas
Medical Branch, Galveston, TX, USA
e-mail: rtesh@utmb.edu
514 R. B. Tesh
first description of four fatal human cases of the disease in foreign laborers who had
come to work in construction of the canal (Darling 1906). Darling originally thought
the men had died of kala azar because of the histological similarity of the tissue
forms of Leishmania (amastigotes) with those of Histoplasma capsulatum (yeast
phase). Later, Darling realized his mistake and described the newly recognized fun-
gal disease (Darling 1908). For this reason, I began working with scientists at
MARU, looking for cases of acute histoplasmosis among children on the pediatric
ward at Gorgas Hospital. Four pediatric cases were identified, and a subsequent
report of our findings was my first scientific publication (Tesh et al. 1964).
However, the American involvement in Vietnam interrupted my residency, as the
US military needed doctors. Consequently, residents as well as interns were getting
draft notices. So in 1963, I enlisted in the commissioned corps of the US Public
Health Service for 2 years to fulfill my selective service obligation. My assignment
was to the Peace Corps, and after a brief total immersion course in Portuguese, I was
sent to Recife, Brazil, as the Peace Corps physician for American volunteers in
Northeast Brazil. This was another fortuitous assignment; because for the next two
years, I traveled widely in Brazil, learning about the history, culture, and geography
of this diverse and fascinating country. On a trip to Belem, I accidently met Robert
E. Shope, who was then the director of the Rockefeller Foundation’s Arbovirus Unit
at the Instituto Evandro Chagas (IEC) (Travassos da Rosa 2016), a large field and
diagnostic reference laboratory maintained by the Brazilian Ministry of Health in
Para state, the eastern Amazon region. This was another fortuitous meeting, since
Shope later became my mentor and longtime collaborator in arbovirology. The IEC
and its arbovirology group also would later play an important role in my career.
In 1965 after completing my Peace Corps assignment, I planned to continue my
training in pediatrics and obtained an NIH Postdoctoral Fellowship in the Department
of Pediatrics at Tulane University School of Medicine in New Orleans under
Margaret H.D. Smith, a renowned infectious disease specialist. Under Dr. Smith’s
tutelage, fellows were encouraged to develop a research project and to also enter in
a graduate program. I entered a master’s program in epidemiology in the School of
Tropical Medicine and Public Health and developed a research project on histoplas-
mosis with two faculty mycologists, John Schneidau and Lorraine Friedman. At the
time, workers at MARU had just reported finding H. capsulatum in bats and bat
feces, suggesting that Chiroptera might serve as vectors and reservoirs of the fungus
(Klite and Diercks 1965). Thus, we began collecting bats (Tadarida brasiliensis) in
old abandoned buildings in the French Quarter of New Orleans and later in bat caves
in the southeastern United States, to study the pathogenesis of the fungus in bats and
their potential role in the ecology of H. capsulatum (Tesh and Schneidau 1966,
1967). Tulane at that time had a large NIH-funded International Collaboration in
Medical Research and Training (ICMRT) grant with the Universidad del Valle in
Cali, Colombia. I obtained a 4-month travel grant to go to Cali to work with a
Tulane biologist studying bat ecology in Colombia. We did not find much H. capsu-
latum in Colombia, because most of my cultures were contaminated with airborne
fungi, as we had no air filters or biosafety hoods in the lab. However, the Rockefeller
Foundation also had an arbovirus group at the Universidad del Valle, and I spent
An Unplanned Career in Arbovirology 515
many hours with Dr. Harold Trapido (Aitken 1992), one of the American scientists
there, who instructed me on the importance of climate in the ecology and geo-
graphic distribution of pathogens and their vectors. During this visit, I also learned
much about Colombia and its unique geography and fauna. This experience and
knowledge also would prove invaluable for future research in that country.
During my time at Tulane, it became apparent to me that research on infectious
diseases was more to my liking than clinical pediatrics. So upon completion of the
fellowship and course work at Tulane, I began looking for a job. Karl M. Johnson,
who I had met previously while at Gorgas Hospital and who was then Scientific
Director of MARU, offered me a position in Panama as an NIAID junior scientist.
The offer was readily accepted.
My first assignment at YARU was to elucidate the epidemiology of vesicular
stomatitis virus (VSV) in Panama. Previous collaborative studies between scientists
at YARU and at Gorgas Memorial Laboratory (GML) in Panama City had demon-
strated that humans living in rural areas of Panama and Central America had a high
prevalence of specific neutralizing antibodies to both the Indiana and New Jersey
serotypes of VSV (VSIV and VSNJV, respectively). In addition, isolations of VSIV
had recently been made from field-collected phlebotomine sandflies (Lutzomyia sp.)
in Almirante, Bocas del Toro province, in western Panama (Shelokov and Peralta
1967; Galindo et al. 1966). At the time, the ecology of VSV in the Americas was a
puzzle. Animal to animal transmission by direct contact was well documented dur-
ing epizootics of vesicular stomatitis among bovines, equines, and swine, but the
mechanisms of transmission and maintenance of VSIV and VSNJV in nature were
unknown. Hanson (Hanson 1952) and others had suggested that an arthropod vector
might be involved, but definitive proof was lacking until the Panama isolations of
VSIV from sandflies.
Thus in 1968, together with Brian Chaniotis, a recent medical entomology grad-
uate from UC/Davis who had experience working with sandflies, we began field and
laboratory studies on various aspects of the epidemiology and ecology of VSV. These
studies included (a) serosurveys for VSV antibodies in local human and animal
populations to determine the vertebrate host range of the virus (Tesh et al. 1969;
Tesh and Johnson 1975); (b) experimental infection of a wide variety of wild ani-
mals with VSIV and VSNJV in hope of identifying possible vertebrate reservoirs of
the two viruses (Tesh et al. 1970); (c) collection of sandflies from diverse habitats in
Panama to determine their species distribution, seasonal abundance, and infection
rates with VSV and other arboviruses (Tesh et al. 1971a; a, 1974, 1975a; Chaniotis
et al. 1971, 1972, 1975); and (d) establishment of laboratory colonies of several
local sandfly species for experimental infection and transmission studies. As a result
of this work, I developed a lifelong interest in phlebotomine sandflies and the patho-
gens that they transmit, namely, Leishmania, rhabdoviruses, and phleboviruses.
In reading earlier reports on the epidemiology of sandfly (pappataci) fever, I was
stuck by reports of the isolation of sandfly fever group viruses (phleboviruses) from
male sandflies and evidence from other scientists that these viruses were vertically
(transovarially) transmitted in their phlebotomine hosts (Barnett and Suyemoto
1961; Whittingham 1924; Mochkovski et al. 1937; Petrischeva and Alymov 1938).
516 R. B. Tesh
Consequently, we attempted to demonstrate vertical transmission of VSIV in our

laboratory sandfly colonies. To our surprise and exhilaration, VSIV was vertically
transmitted by experimentally infected sandflies (Lutzomyia trapidoi and Lu. ylephi-
lator) to their progeny (Tesh et al. 1971b; b). The infected F1 adult females in turn
transmitted the virus by bite to susceptible animals and transovarially to their off-
spring (F2 generation). These results suggested a possible mechanism for transmis-
sion and maintenance of VSIV in nature without a vertebrate. This experimental
model was used subsequently by me with other collaborators to demonstrate verti-
cal or transovarial transmission (TOT) of a number of different rhabdoviruses, bun-
yaviruses, and flaviviruses in their natural insect vectors (Rosen et al. 1978; Aitken
et al. 1979; Beaty et al. 1980; Tesh and Cornet 1981; Tesh and Modi 1983a; Endris
et al. 1983; Rosen et al. 1983; Comer et al. 1990; Tesh et al. 1992; Thangamani et al.
2016; Contreras-Gutierrez et al. 2017a). TOT offers an explanation of how some
arboviruses can survive during adverse periods (i.e., cold or dry seasons), when
their adult vectors are absent or present in very low numbers.
During the MARU years, I also published several reports on the natural history,
biology, and laboratory rearing of Panamanian rodents that we had captured during
our fieldwork, including two about Diplomys darlingi, a relatively rare arboreal
rodent named for Samuel T. Darling (Tesh 1970a; b; Tesh and Cameron 1970; Noble
and Tesh 1974).
In 1972, NIH made a decision to close MARU. American employees were
offered three choices: (1) resign and return to the United States; (2) remain with
NIH, but be transferred to another NIAID facility; or (3) remain in Panama as a
contract employee at GML. I chose option #2 and was moved to NIAID’s Pacific
Research Section (PRS) in Honolulu. The director, Leon Rosen, and most of the
laboratory staff were involved in field and laboratory studies on the epidemiology of
dengue. In the 1970s, dengue was absent from Hawaii, and since there were no
other endemic arboviruses, most of our studies were laboratory-based or in the
South Pacific or Asia. Consequently, I had an opportunity to travel and work in
American Samoa, Taiwan, Malaysia, Iran, and the Yale Arbovirus Research Unit
(YARU) with a variety of alphaviruses, flaviviruses, and phleboviruses (Rosen et al.
1978; Aitken et al. 1979; Tesh et al. 1977a; b, 1981; Javadian et al. 1977; Saidi et al.
1977). Serosurveys were also done to determine the prevalence of some of these
agents in human populations living in the South Pacific, the Middle East, and Africa
(Tesh et al. 1975b, 1976a, b; Saidi et al. 1976; Tesh 1978). Laboratory studies were
done with Duane Gubler, Leon, and others on chikungunya, dengue, encephalo-
myocarditis, and California encephalitis group viruses (Tesh and Wallace 1978;
Tesh 1979; Tesh and Shroyer 1980; Tesh et al. 1976c).
In 1979, NIH decided to close the PRS, and the scientific staff were again offered
the three options noted before. At this time, Robert Shope offered me a position as
Associate Professor at MARU in the Dept. of Epidemiology and Public Health, Yale
University School of Medicine. I took his offer and in 1980 moved to New Haven,
where I remained for the next 15 years.
Being at YARU opened up many new research opportunities that were not avail-
able in Hawaii, where there were state restrictions of what viruses and vectors we
were permitted to introduce and to work with. One of my first activities at MARU
was to establish sandfly colonies, with the help of Govind Modi, an Indian graduate
student (Modi and Tesh 1983). The availability of the sandfly colonies allowed us to
develop two continuous sandfly cell lines (Tesh and Modi 1983b) and to collaborate
with Jose Ribeiro and other investigators at Harvard, who were interested in the
components and properties of sandfly saliva (Ribeiro et al. 1989a; b; Samuelson
et al. 1991).
I initially received a contract from the US Army to study the behavior of several
phleboviruses in their presumed and potential sandfly vectors (Tesh et al. 1992;
Tesh and Modi 1984a, 1987; Tesh 1988). But the early 1980s was a difficult period
for funding in arbovirus research. Dengue had not yet returned to Central or most of
South America, and the concept of emerging infectious diseases had not yet gained
popularity. Consequently, classical arbovirus research had a low priority for exter-
nal funding at NIH; in contrast, there was more interest in parasitology and vector
biology. Unlike at NIH, research faculty at Yale Medical School were expected to
cover most of their salary and that of their technical staff, graduate students, and
postdocs as well as laboratory expenses from grants or other external sources, so I
was forced to look for other sources of funding. One of my colleagues in the depart-
ment, Diane McMahon Pratt, had an active research program on leishmaniasis.
Since Govind and I had sandfly colonies, we decided to work with her to study the
development of various Leishmania species in their sandfly vectors (McMahon-
Pratt et al. 1983; Tesh and Modi 1984b; Grimaldi et al. 1989; Warburg et al. 1989;
Grimaldi and Tesh 1993; Tesh 1995). Laurel Walters, one of my postdocs, began
learning electron microscopy and soon produced a series of excellent publications
on the ultrastructural development of Leishmania promastigotes in the alimentary
track of sandflies (Walters et al. 1989a; b, 1992, 1993a, b, 1995; Guzman et al. 1994).
Because of our work with parasites and vectors, in 1989 we were invited to
become part of the MacArthur Foundation’s Network on the Biology of Parasite
Vectors (Beaty et al. 2009). This led to other collaborative studies with Yale col-
leagues Serap Aksoy, Scott O’Neill, and Ben Beard on bacterial endosymbionts in
arthropod vectors and their effects on vector competence (Beard et al. 1992, 1993a,
b; Braig et al. 1994; O’Neill et al. 1997), a subject I would pursue later with insect-
specific viruses.
Based on our previous work, we successfully competed for a large ICMRT pro-
gram project to study the epidemiology of leishmaniasis and sandfly-transmitted
viruses in Colombia in collaboration with scientists at the Colombian National
Institute of Health (Instituto Nacional de Salud) in Bogota. This collaborative proj-
ect and a subsequent NIH RO-1 grant resulted in the discovery of new Leishmania
species, novel sandfly-associated viruses, and new data on the epidemiology of
cutaneous and visceral leishmaniasis in the country (Young et al. 1987; Corredor
et al. 1989a, b, 1990; Kreutzer et al. 1991; Grimaldi et al. 1992; Tesh et al. 1986a,
1987, 1989; Morrison et al. 1993a, b, 1995a, b; Ferro et al. 1995a; b; Alexander
et al. 1992) (Fig. 1). As part of the program project, David G. Young, a sandfly tax-
onomist and co-investigator at the University of Florida/Gainesville, completed his
518 R. B. Tesh
Fig. 1 Collecting sandflies at night from a chicken house in a leishmaniasis-endemic region in

Colombia
classic book on sandfly distribution, taxonomy, and ecology in the tropical America
(Young and Duncan 1994).
Meanwhile, my work at YARU continued with other collaborators on the iden-
tification and antigenic characterization of new viruses (Knudson et al. 1984;
Travassos da Rosa et al. 1984a; b; Tesh et al. 1986b, 1994; Calisher et al. 1989;
Chen et al. 1992; Gligic et al. 1983; Lisieux et al. 1994). In 1986, Bob Shope des-
ignated me as director/curator of the World Reference Center for Arboviruses
(WRCA), a position that I held for the next 28 years (Vasilakis et al. 2019a). The
WRCA had its origins at the Rockefeller Foundation Virus Laboratory in New York
City (Vasilakis et al. 2019a). Initially, arbovirus laboratories throughout the world
sent samples to the New York laboratory for identification and phenotypic charac-
terization. These samples were then added to the growing reference collection. In
1964, the Rockefeller Foundation ended its overseas field and laboratory arbovirus
program and moved its entire arbovirus collection and most of the remaining staff
to Yale University School of Medicine and YARU (Vasilakis et al. 2019a).
Additional virus isolates were subsequently sent by collaborating overseas labora-
tories for identification, by foreign visitors coming to Yale for training, and by
exchanges with other arbovirus laboratories. In the past, exchange of virus samples
between arbovirus laboratories was common, and most arbovirologists shared
samples. The situation is now much different for a variety of reasons such as pro-
prietary concerns, restrictive biosafety and biosecurity regulations which vary
from country to country, the Nagoya protocol, and national and international rules
for shipping infectious agents as well as issues of ownership and use. In addition
to arboviruses, the WRCA collection also includes a number of other zoonotic
viruses such as arenaviruses, hantaviruses, paramyxoviruses, orthomyxoviruses,
rhabdoviruses, and picornaviruses as well as insect-specific viruses (ISVs), as

many of these agents were originally isolated by arbovirologists during field stud-
ies or outbreak investigations.
In 1989, cases of hemorrhagic fever were occurring in the central plains (llanos)
of Venezuela, which local physicians were reporting as “dengue hemorrhagic fever.”
However, an astute epidemiologist in Portuguesa state, Nuris Manzione, suspected
it was something else and sent tissue samples from two fatal cases to Rosa Alba
Salas, a virologist at the National Institute of Hygiene in Caracas. Dr. Salas inocu-
lated the tissue homogenates into cultures of HeLa cells and observed a cytopathic
effect (CPE). She then sent the culture material to YARU, where Bob Shope and I
subsequently identified the agent as a new arenavirus, designated Guanarito virus
(GTOV) for the town where the fatal cases lived (Tesh et al. 1994).
We subsequently obtained an NIH grant to study the epidemiology of Venezuelan
hemorrhagic fever (VHF) in collaboration with staff at the National Institute of
Hygiene and biologists at two local universities. Assuming that Guanarito virus was
rodent-borne, for the next 5 years we captured, dissected, and cultured liver and
spleen samples from wild and peridomestic rodents in the VHF-endemic region in
an attempt to identify the rodent reservoir(s) and to better understand how and
where humans became infected (Fig. 2) (Tesh et al. 1993; Fulhorst et al. 1997a, b,
1999a; Weaver et al. 2000; Utrera et al. 2000; Weaver et al. 2001). Clinical informa-
tion was also collected on confirmed human cases of VHF treated at the regional
hospital in Guanare (de Manzione et al. 1998). During these studies several other
novel arenaviruses and hantaviruses were also isolated and characterized (Fulhorst
et al. 1997a, b). James Mills, a rodent expert from CDC/Atlanta, and Charles
Fulhorst, a Yale postdoc and later UTMB faculty member, participated in the field-
work as well.
Once we realized that GTVO was the cause of VHF, it was classified as a BSL-4
agent, and we could no longer work with it at YARU. Consequently, C.J. Peters and
Tom Ksiazek invited us to continue experimental studies and virus isolations in the
high-security (BSL-4) labs of the CDC Special Pathogens Branch. Fulhorst tempo-
rarily moved to Atlanta and did much of the experimental work with Ksiazek
(Fulhorst et al. 1999b), while the fieldwork continued in Venezuela. This was a truly
collaborative project which involved many different people and institutions, but it is
also noteworthy that such a collaborative project would be very difficult now for
some of the restrictions and rules alluded to before.
In addition to the Rockefeller Foundation’s overseas virus laboratories, other
important sources of new virus strains for the WRCA collection came from the vari-
ous overseas research labs of the Institute Pasteur and the US military. However, for
a variety of reasons (some noted above), it is now almost impossible to get clinical
samples or virus isolates from most of these laboratories.
By 1994, it became apparent that Yale Medical School had lost interest in YARU
and arbovirology (Vasilakis et al. 2019a). Consequently, when David Walker, chair
of the Department of Pathology at the University of Texas Medical Branch (UTMB),
obtained funding for a new Center for Tropical Diseases from a local foundation,
Bob Shope and I applied for positions there. In 1995, we both moved to Galveston,
520 R. B. Tesh
Fig. 2 Dissecting and collecting tissues from wild rodents in the VHF-endemic region in
Venezuela
taking most of the WRCA viruses and reagents with us. As with previous moves,
this one offered new research opportunities and colleagues to work with.
By this time (1995), there was worldwide recognition of the threat of emerging
infectious diseases. The 1992 publication of the Institute of Medicine’s seminal
report, entitled Emerging Infections. Microbial Threats to Health in the United
States (Lederberg et al. 1992), edited by Joshua Lederberg, Robert Shope, and
Stanley Oaks, was largely responsible for the renewed interest in emerging infec-
tions by biomedical funding agencies of the US government. Since a significant
number of the identified emerging infections were caused by arthropod-borne and
zoonotic viruses, financial support for research on these agents suddenly increased.
Accordingly, NIAID substantially increased funding for World Reference Center
for Arboviruses (now at UTMB) and changed the name of the center to World
Reference Center for Emerging Viruses and Arboviruses (WRCEVA). In 2001, fol-
lowing the terrorist attacks in New York and Washington and the anthrax scare, the
USA Patriot Act was passed by Congress. It established strict new regulations on
work with bioterrorist threat agents. This law resulted in new funds for research
with select agents as well as the construction of new high-security biocontainment

facilities at a number of US academic institutions. UTMB received NIH and state
funds to construct the Galveston National Laboratory (GNL), one of two university-
based high-containment facilities in the United States with BSL-3 and BSL-4 labo-
ratories for research with select agents in cell cultures, animals, and arthropods.
These state-of-the art facilities at UTMB also attracted new investigators and addi-
tional research funding.
The availability of these new biocontainment and animal facilities allowed me to
begin work with a number of high-containment viruses. In collaboration with Shu-
Yuan Xiao, a surgical pathologist in the department, and others, we began studies on
the pathogenesis of yellow fever, West Nile, monkey pox, St. Louis encephalitis,
and several phleboviruses, arenaviruses, and orthomyxoviruses in rodents and non-
human primates (Tesh et al. 2001, 2002a, 2004, 2005; Xiao et al. 2001a, b, c, 2003,
2005; Solomon et al. 2003; Fisher et al. 2003; Ratterree et al. 2004; Mutebi et al.
2004; Sbrana et al. 2004, 2005, 2006a, b, 2007a, b; Tonry et al. 2005; Ding et al.
2005; Mateo et al. 2006, 2007a; Siirin et al. 2007; Li et al. 2008a, b; Wu et al. 2008).
Results of these studies allowed us to develop animal models for human infection
with some of the pathogens and to test specific therapeutic agents (Tesh et al. 2002b;
Watts et al. 2007; Mateo et al. 2007b; Ziegler et al. 2008; Widman et al. 2010).
For a period of about 20 years, when Douglas Watts was scientific director of the
US Navy Medical Research Detachment Number 6 (NAMRU-6) and afterward, we
had a highly productive collaborative research program in Peru. UTMB students,
postdocs, and faculty had an opportunity to do fieldwork in Peru, and NAMRU-6
personnel came to Galveston for training. Reagents from the WRCEVA repository
were also supplied to NAMRU-6 as needed. A number of new viruses and signifi-
cant publications resulted from this consortium (Tesh et al. 1999; Powers et al.
1999, 2006; Bunnell et al. 2000; Travassos da Rosa et al. 2001; Moncayo et al.
2001; Russell et al. 2003; Mercer et al. 2003; Aguilar et al. 2004, 2009, 2010; Attoui
et al. 2009; Long et al. 2011; Vasilakis et al. 2014a; Amarasinghe et al. 2017).
Unfortunately, this program eventually ended, because of the new restrictions on
sharing viruses and the biosecurity issues noted earlier.
In 1999, West Nile virus (WNV) appeared in New York and then rapidly spread
across North America (Kramer et al. 2019). At the time, we had about 12 strains of
WNV in the WRCEVA collection from various localities in Africa, the Middle East,
and Central Asia, so there was great interest by investigators in the United States
and abroad to study these viruses in order to determine the geographic origin and
possible source of the New York introduction. I began collecting representative
WNV isolates from various hosts, geographic regions, and time periods as the virus
moved across the United States and into Canada, Mexico, and the Caribbean. Many
persons shared their WNV isolates, which were added to the WRCEVA collection;
these isolates were valuable for later studies on the evolution of the virus, as it
moved into new regions (Beasley et al. 2003; Davis et al. 2003, 2004, 2005, 2007;
May et al. 2011; McMullen et al. 2011; Mann et al. 2013; Swetnam et al. 2018).
By the summer of 1991, WNV had reached Louisiana in its march Westward in
the United States, so I met with Ray Parsons, the director of the Harris County
522 R. B. Tesh
Mosquito and Vector Control Division (HCMVCD) in Houston. We agreed to begin

surveillance for WNV activity in the Houston metropolitan area. St. Louis encepha-
litis virus (SLEV) had long been endemic in East Texas, and both Houston and
Dallas had suffered serious epidemics of SLEV encephalitis in the past (Lillibridge
et al. 2004). Consequently, the HCMCD already had a good SLEV surveillance and
mosquito control program in place. We agreed to expand the program to
include WNV.
With additional funding from NIH, we began testing sera of wild birds netted,
bled, and released by HCMVCD personnel for antibodies to WNV and SLEV. Dead
birds collected in Houston and in Harris County by HCMVCD were also sent to
UTMB for culture for the presence of WNV. In addition, a portion of their mosquito
collections (mainly Culex species) were also cultured for virus. No WNV activity
was detected in 1991, but in June of 1992, we made the first isolation of WNV in
Texas from a dead blue jay collected in Harris County (Lillibridge et al. 2004).
From 2002 to 2014, a total of 9047 dead birds were submitted to UTMB for culture;
1644 of the birds (18.2%) yielded WNV (Walker et al. 2019). Other viruses isolated
from brain cultures of dead birds from Harris County included Newcastle disease
(NDV), eastern equine encephalitis (EEEV), Mermet (MERV), Flanders (FLAV),
San Jacinto (SJCV), Buffalo Bayou (BBAV), and Mason Creek (MCRV) viruses
(Walker et al. 2019).
With the development of novel high-throughput sequencing (NGS) and metage-
nomics, I became interested in trying to identify and characterize some of the older
unidentified virus isolates in the WRCEVA collection. Initial studies were done
with my long-time colleague Amelia Travassos da Rosa at UTMB and with Gustavo
Palacios, Ian Lipkin, David Wang, Hideki Ebihara, Elodie Ghedin, and others at
their respective institutions. Later, when UTMB established its own sequencing
center, these studies were done locally with UTMB colleagues Nikos Vasilakis,
Scott Weaver, Vsevolod Popov, Alan Barrett, Steve Widen, and Thomas Wood, with
help from Peter J. Walker and Edward C. Holmes (Australia) in the phylogenetic
analyses. Many known and previously unknown viruses were identified and charac-
terized (Walker et al. 2015, 2019; Mihindukulasuriya et al. 2009; Presti et al. 2009;
Palacios et al. 2010, 2011a, b, 2013a, b, c, 2014, 2015; Loh et al. 2011; Zhao et al.
2011; Bussetti et al. 2012; Forrester et al. 2012, 2013; Blasdell et al. 2012, 2013,
2014, 2015; Chowdhary et al. 2012; Nasar et al. 2012; Vasilakis et al. 2013a, b,
2014b, 2019b; Ghedin et al. 2013; Kapoor et al. 2013; Matsuno et al. 2013, 2015;
Allison et al. 2014; Rogers et al. 2014, 2017; Briese et al. 2014; Ladner et al. 2014,
2016; Groseth et al. 2014; Auguste et al. 2014, 2015; Carrera et al. 2015; Shi et al.
2015; Contreras-Gutierrez et al. 2017b, 2018; Navarro et al. 2016; Diagne et al.
2019; Sadeghi et al. 2017; Guzman et al. 2018).
Some of the unidentified agents from the WRCEVA collection turned out to be
insect-specific viruses (ISVs), so this turned our attention to searching for new
viruses in mosquitoes. Our initial plan was to process pools of field-collected mos-
quitoes and occasionally other types of arthropods in cultures of Vero and C6/36
(Ae. albopictus) cell cultures to observe for evidence of CPE. We found that many
mosquito pools processed in this manner produced CPE in mosquito cell cultures,
but none in vertebrate cell lines. Since most of the mosquito cell isolates also did not
cause illness when inoculated intracranially into newborn mice or react in serologic
tests with arbovirus grouping antisera, our next step was to attempt to visualize their
morphology by electron microscopy (Popov et al. 2019) and, if unusual, to obtain
their sequence by NGS for phylogenetic studies. Not infrequently, some mosquito
pools contained two or three different viruses, sometimes a known arbovirus and
one or more insect-specific viruses (ISVs). Additional investigators have reported
similar findings, doing NGS and metagenomic studies with mosquitoes from other
regions of the world (Jungler and Drosten 2013; Li et al. 2015; Sadeghi et al. 2018;
Pettersson et al. 2019; Zirkel et al. 2013; Blitvich and Firth 2015; Bolling et al.
2015a). Despite being more labor intensive, our methods usually yielded a virus
isolate (Walker et al. 2019), whereas NGS simply yielded a partial or full genomic
sequence.
As more and more novel ISVs were isolated and/or characterized, it became
increasingly apparent that most biting arthropods have their own unique virome.
Since some of these ISVs are closely related genetically to important arbovirus
pathogens, it raised the question of how prior infection with an ISV affects a mos-
quito’s subsequent vector competence if it encountered a related arbovirus patho-
gen. (Blitvich and Firth 2015; Bolling et al. 2015a; b; Goenaga et al. 2015;
Hall-Mendelin et al. 2016; Ohlund et al. 2019) In addition, what effect do ISVs have
on an insect’s behavior, longevity, fecundity, and resistance or susceptibility to other
insect pathogens?
One of my long-term international collaborations has been with Brazilian scien-
tists at the IEC in Belem. This relationship began with my chance meeting with Bob
Shope in 1963, when he was working at the IEC. In addition to Bob Shope, the col-
laboration has included Francisco Pinheiro, Pedro Vasconcelos, and Amelia
Travassos da Rosa, as well as several Brazilian students and postdoctoral fellows
who spent time in my lab. From 2014 to 2017 (Goenaga et al. 2015; Hall-Mendelin
et al. 2016; Ohlund et al. 2019; Vasconcelos et al. 2004; Nunes et al. 2005, 2015a;
b; Dinitz et al. 2006; Silva et al. 2014; Azevedo et al. 2016, 2018; Nunes Neto et al.
2017; Chiang et al. 2018), I was a visiting scientist at the IEC and work there
included collaborative studies on yellow fever, chikungunya, and Zika virus infec-
tions and their epidemiology in Brazil. This experience again illustrates the seren-
dipitous nature of my career and the unexpected events that can occur if one takes
advantage of opportunities, is willing to try new things, develops collaborations,
and travels to new places.
Acknowledgments I am indebted to Dora Salinas for help in preparing the manuscript for publi-
cation and to my wife Hilda Guzman, who helped curate the WRCEVA virus collection with me
for many years.
524 R. B. Tesh
References
Aguilar PV, Greene IP, Coffey LL, Medina G, Moncayo AC, Anishchenko M, Ludwig GV, Turell
MJ, O’Guinn ML, Lee J, Tesh RB, Watts DM, Russell KL, Hice C, Yanoviak S, Morrison
AC, Klein TA, Dohm DJ, Guzman H, Travassos da Rosa APA, Guevara C, Kochel T, Olson
J, Cabezas C, Weaver SC (2004) Endemic Venezuelan equine encephalitis in Northern Peru.
Emerg Infect Dis 10:880–888
Aguilar PV, Camargo W, Vargas J, Guevara C, Roca Y, Felices V, Laguna-Torres A, Tesh RB,
Ksiazek TG, Kochel TJ (2009) Reemergence of Bolivian hemorrhagic fever, 2007-2008.
Aguilar PV, Morrison AC, Watts DM, Cruz C, Rocha C, Beingolea L, Suarez V, Vargas J, Roca Y,
Tesh RB, Kochel TJ (2010) Guaroa virus infection among humans in Peru and Bolivia. Am J
Aitken TH (1992) Harold Trapido 1916–1991. J Am Mosq Control Assoc 8:111–114
Aitken THG, Tesh RB, Beaty BJ, Rosen L (1979) Transovarial transmission of yellow fever virus
by mosquitoes (Aedes aegypti). Am J Trop Med Hyg 28:119–121
Alexander B, Ferro C, Young DG, Morales A, Tesh RB (1992) Ecology of phlebotomine sand
flies (Diptera: Psychodidae) in a focus of Leishmania (Viannia) braziliensis in northeastern
Colombia. Mem Inst Oswaldo Cruz 87:387–395
Allison AB, Mead DG, Palacios GF, Tesh RB, Holmes EC (2014) Gene duplication and phylo-
geography of North American members of the Hart Park serogroup of avian rhabdoviruses.
Virology 448:284–292
Amarasinghe GK, Bao Y, Basler CF, Bavari S, Beer M, Bejerman N, Blasdell KR, Bochhnowski
A, Briese T, Bukreyev A, Calisher CH, Chandran K, Collins PL, Dietzgen RG, Dolnik O,
Dürrwald R, Dye JM, Easton AJ, Ebihara H, Fang Q, Formenty P, Fouchier RA, Ghedin E,
Harding RM, Hewson R, Higgins CM, Hong J, Horie M, James AP, Jiāng D, Kobinger GP,
Kondo H, Kurath G, Lamb RA, Lee B, Leroy EM, Li M, Maisner A, Mühlberger E, Netesov
SV, Nowotny N, Patterson JL, Payne SL, Paweska JT, Pearson MN, Randall RE, Revill PA,
Rima BK, Rota P, Rubbenstroth D, Schwemmle M, Smither SJ, Song Q, Stone DM, Takada
A, Terregino C, Tesh RB, Tomonaga K, Tordo N, Towner JS, Vasilakis N, Volchkov VE, Wahl-
Jensen V, Walker PJ, Wang B, Wang D, Wang F, Wang LF, Werren JH, Whitfield AE, Yan
Z, Ye G, Kuhn JH (2017) Taxonomy of the order Mononegavirales: update 2017. Arch Virol
162:2493–2504.
Attoui H, Mendez-Lopez MR, Rao S, Hurtado-Alendez A, Lizaraso-Caparo L, Mohd Jaafar F,
Samuel AR, Belhouchet M, Pritchard LI, Melville L, Weir RP, Hyatt AD, Davis SS, Lunt
R, Calisher CH, Tesh RB, Fujita R, Mertens PP (2009) Peruvian horse sickness virus and
Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia. Virology
394:298–310
Auguste AJ, Carrington CVF, Forreser NL, Popov VL, Guzman H, Widen SG, Wood TG, Weaver
SC, Tesh RB (2014) Characterization of a novel Negevirus and a novel Bunyavirus isolated
from Culex declarator mosquitoes in Trinidad. J Gen Virol 95:481–485
Auguste AJ, Kaelber JT, Fokam EB, Guzman H, Carrington CVF, Erasmus JH, Kamgang B,
Popov VL, Jakana J, Liu X, Wood TG, Widen SG, Vasilakis N, Tesh RB, Chiu W, Weaver SC
(2015) A newly-isolated reovirus has the simplest genomic and structural organization of any
reovirus. J Virol 89:676–687
Azevedo RS, Araujo MT, Martins Filho AJ, Oliveira CS, Nunes BT, Cruz AC, Nascimento AG,
Medeiros RC, Caldas CA, Araujo FC, Quaresma JA, Vasconcelos BC, Queiroz MG, da Rosa
ES, Henriques DF, Silva EV, Chiang JO, Martins LC, Medeiros DB, Lima JA, Nunes MR,
Cardoso JF, Silva SP, Shi PY, Tesh RB, Rodrigues SG, Vasconcelos PF (2016) Zika virus
epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects. J Clin Virol
85:56–64
Azevedo RSS, Araujo MT, Oliveira CS, Martins Filho AJ, Nunes BTD, Henriques DF, Silva EVP,
Carvalho VL, Chiang JO, Martins LC, Vasconcelos BCB, Sousa JR, Montenegro Araujo F,
Ribeiro EM, Castro ARP, de Queiroz MGL, Verotti MP, Nunes MRT, Cruz ACR, Rodrigues
SG, Shi PY, Quaresma JAS, Tesh RB, Vasconcelos PFC (2018) Zika virus epidemic in Brazil.
II. Post-mortem analyses of neonates with microcephaly, stillbirths and miscarriage. J Clin
Med 7(12):E496. https://doi.org/10.3390/jcm7120496
Barnett HC, Suyemoto W (1961) Field studies on sandfly fever and kala-azar in Pakistan, in Iran,
and in Baltistan (little Tibet) Kashmir. Trans NY Acad Sci 23(7):609–617
Beard CB, Mason PW, Tesh RB, Aksoy S, Richards FF (1992) Transformation of an insect symbi-
ont and expression of a foreign gene in a Chagas’ disease vector Rhodnius prolixus. Am J Trop
Med Hyg 46:195–200
Beard CB, O’Neill S, Mason PW, Mandelco L, Woese CR, Tesh RB, Richards FF, Aksoy S (1993a)
Genetic transformation and phylogeny of bacterial symbionts from tsetse. Insect Mol Biol
1:123–131
Beard CB, O’Neill SL, Tesh RB, Richards FF, Aksoy S (1993b) Modification of arthropod vector
competence via symbiotic bacteria. Parasitol Today 9:179–183
Beasley DW, Davis CT, Guzman H, Vanlandingham DL, Travassos da Rosa APA, Parsons RE,
Higgs S, Tesh RB, Barrett ADT (2003) Limited evolution of West Nile virus has occurred dur-
ing its southwesterly spread in the United States. Virology 309:190–195
Beaty BJ, Tesh RB, Aitken THG (1980) Transovarial transmission of yellow fever virus in
Stegomyia mosquitoes. Am J Trop Med Hyg 29:125–132
Beaty BJ, Prager DJ, James AA, Jacobs-Lorena M, Miller LH, Law JH, Collins FH, Kafotos
FC (2009) From Tucson to genomics and transgenics: the Vector Biology Network and the
emergence of modern vector biology. PLOS Neg Trop Dis 3(3):e343. https://doi.org/10.1371/
journal.prtd.0000343
Blasdell KR, Voysey R, Bulach D, Joubert DA, Tesh RB, Boyle DB, Walker PJ (2012) Kotonkan
and Obodhiang viruses: African ephemeroviruses, with large and complex genomes. Virology
425:143–153
Blasdell KR, Adams MM, Davis SS, Walsh SJ, Aziz-Boaron O, Klement E, Tesh RB, Walker PJ
(2013) A reverse-transcription PCR method for detecting all known ephemeroviruses in clini-
cal samples. J Virol Methods 191:128–135
Blasdell KR, Widen SG, Diviney S, Firth C, Wood TG, Guzman H, Holmes EC, Tesh RB, Vasilakis
N, Walker PJ (2014) Koolpinyah and Yata viruses: two newly recognized ephemeroviruses
from tropical regions of Australia and Africa. Vet Microbiol 174(3-4):547–553
Blasdell KR, Guzman H, Widen SG, Firth C, Wood TG, Holmes EC, Tesh RB, Vasilakis N, Walker
PJ (2015) Ledantevirus: a proposed new genus in the Rhabdoviridae has a strong ecological
association with bats. Am J Trop Med Hyg 92:405–410
Blitvich BJ, Firth AE (2015) Insect-specific flaviviruses: a systemic review of their discovery,
host range, mode of transmission, superinfection exclusion potential and genomic organiza-
tion. Viruses 7:4911–4928
Bolling BG, Vasilakis N, Guzman H, Widen SG, Wood TG, Popov VL, Thangamani S, Tesh RB
(2015a) Insect-specific viruses detected in laboratory mosquito colonies and their potential
implications for experiments evaluating arbovirus vector competence. Am J Trop Med Hyg
92:422–428
Bolling BG, Weaver SC, Tesh RB, Vasilakis N (2015b) Insect-specific virus discovery: signifi-
cance for the arbovirus community. Viruses 7:4911–4928
Braig HR, Guzman H, Tesh RB, O’Neill SL (1994) Replacement of the natural Wolbachia symbi-
ont of Drosophila simulans with a mosquito counterpart. Nature 367:453–455
Briese T, Chowdhary R, Travassos da Rosa A, Hutchison SK, Popov V, Street C, Tesh RB, Lipkin
WI (2014) Upolu virus and Aransas Bay virus, two presumptive bunyaviruses, are novel mem-
bers of the family Orthomyxoviridae. J Virol 88:5298–5309
Bunnell JE, Hice CL, Watts DM, Montrueil V, Tesh RB, Vinetz JM (2000) Detection of pathogenic
Leptospira spp. infections among mammals captured in the Peruvian Amazon basin region.
526 R. B. Tesh
Bussetti AV, Palacios G, Travassos da Rosa A, Savji N, Miller C, Guzman H, Hutchison S, Popov
VL, Tesh RB, Lipkin WI (2012) Genomic and antigenic characterization of Jos virus. J Gen
Virol 93:293–298
Calisher CH, Karabatsos N, Zeller H, Digoutte JP, Tesh RB, Shope RE, Travassos da Rosa AP, St.
George TD (1989) Antigenic relationships among rhabdoviruses from vertebrates and hema-
tophagous arthropods. Intervirology 30:241–257
Carrera JP, Guzman H, Beltran D, Diaz Y, Lopez-Vargas S, Torres R, Popov V, Widen S, Wood TG,
Weaver SC, Caceres L, Vasilakis N, Tesh RB (2015) Mercadeo virus: a novel mosquito-specific
flavivirus from Panama. Am J Trop Med Hyg 93:1014–1019
Chaniotis BN, Neely JM, Correa MA, Tesh RB, Johnson KM (1971) Natural population dynamics
of phlebotomine sandflies in Panama. J Med Entomol 8:339–352
Chaniotis BN, Tesh RB, Correa MA, Johnson KM (1972) Diurnal resting sites of phlebotomine
sand flies in a Panamanian tropical forest. J Med Entomol 9:91–98
Chaniotis BN, Correa MA, Tesh RB, Johnson KM (1975) Horizontal and vertical movements of
phlebotomine sandflies in a Panamanian rain forest. J Med Entomol 11:369–375
Chen WR, Rico-Hesse R, Tesh RB (1992) A new genotype of Japanese encephalitis virus isolated
in Indonesia. Am J Trop Med Hyg 47:61–69
Chiang JO, Souza WM, Nunes MRT, Acrani GO, Travassos da Rosa APA, de Freitas NM, da
Silva SP, de Silva PHD, de Sousa AW, Rodrigues SG, Quaresma JAS, Dutary B, Guzman H,
Vasilakis N, Tesh RB, Vasconcelos PFC (2018) Characterization of the Gamboa virus sero-
group (Orthobunyavirus Genus, Peribunyaviridae Family). Am J Trop Med Hyg 98:1502–1511
Chowdhary R, Street C, Travassos da Rosa A, Nunes MRT, Tee KK, Hutchison SK, Vasconcelos
PF, Tesh RB, Lipkin WI, Briese T (2012) Genetic characterization of the Wyeomyia group of
orthobunyaviruses and their phylogenetic relationships. J Gen Virol 93:1023–1034
Comer JA, Tesh RB, Modi GB, Corn JL, Nettles VF (1990) Vesicular stomatitis virus New Jersey
serotype: Growth in and transmission by Lutzomyia shannoni (Diptera: Psychodidae). Am J
Contreras-Gutierrez MA, Guzman H, Thangamani S, Vasilakis N, Tesh RB (2017a) Experimental
infection with and maintenance of cell fusing agent virus (Flavivirus) in Aedes aegypti. Am J
Contreras-Gutierrez MA, Eastwood G, Guzman H, Popov V, Savit C, Uribe S, Kramer LD, Wood
TG, Widen SG, Fish D, Tesh RB, Vasilakis N, Walker PJ (2017b) Almendravirus: a proposed
new genus of rhabdoviruses isolated from mosquitoes in tropical regions of the Americas. Am
Contreras-Gutierrez MA, Guzman H, Cardosa JF, Popov VL, Nunes MRT, Uribe S, Widen SG,
Wood TG, Vasilakis N, Tesh RB (2018) Genome sequence of Chiqui virus, a novel reovirus
isolated from mosquitoes collected in Colombia. Microbiol Resour Annouc 7:e0081–e0016.
https://doi.org/10.1128/MRA.0081-18
Corredor A, Gallego JF, Tesh RB, Peláez D, Diaz A, Montilla M, Paláu MT (1989a) Didelphis
marsupialis, an apparent wild reservoir of Leishmania donovani chagasi in Colombia, South
America. Trans R Soc Trop Med Hyg 83:195
Corredor A, Gallego JF, Tesh RB, Morales A, Ferro de Carrassquilla C, Young DG, Kreutzer RD,
Boshell J, Palau MT, Caceres E, Pelaez D (1989b) Epidemiology of visceral leishmaniasis in
Colombia. Am J Trop Med Hyg 40:480–486
Corredor A, Kreutzer RD, Tesh RB, Boshell J, Palau MT, Caceres E, Duque S, Palaez D,
Rodriguez G, Nichols S, Hernandez CA, Morales A, Young DG, Ferro de Carrasquilla C (1990)
Distribution and etiology of leishmaniasis in Colombia. Am J Trop Med Hyg 42:206–214
Da Silva EV, Travassos da Rosa AP, Nunes MR, Diniz JA, Tesh RB, Cruz AC, Viera CM,
Vasconcelos PF (2005) Araguari virus, a new member of the family Orthomyxoviridae: sero-
logic ultrastructural, and molecular characterization. Am J Trop Med Hyg 73:1050–1058
Darling ST (1906) A protozoan general infection producing pseudotuberculosis in the lungs and
focal necrosis in the liver, spleen and lymph nodes. J Am Med Assoc 46:1283–1285
Darling ST (1908) Histoplasmosis: a fatal infectious disease resembling kala-azar found among
natives of tropical America. Arch Int Med 2:107–123
Davis CT, Beasley DW, Guzman H, Raj R, D’Anton M, Novak RJ, Unnasch TR, Tesh RB, Barrett
AD (2003) Genetic variation among temporally and geographically distinct West Nile virus
isolates, United States, 2001, 2002. Emerg Infect Dis 9:1423–1429. Erratum in: Emerg Infect
Dis. 10:160, 2004
Davis CT, Beasley DW, Guzman H, Siirin M, Parsons RE, Tesh RB, Barrett ADT (2004) Emergence
of attenuated West Nile virus variants in Texas, 2003. Virology 330:342–350
Davis CT, Ebel GC, Lanciotti RS, Brault AC, Guzman H, Siirin M, Parsons RE, Beasley DWC,
Novak RJ, Elizondo-Quiroga D, Green EN, Young DS, Stark LM, Drebot MA, Artsob H, Tesh
RB, Kramer LD, Barrett ADT (2005) Phylogenetic analysis of North American West Nile
virus isolates, 2001-2004: Evidence for the Emergence of a Dominant Genotype. Virology
342:252–265
Davis CT, Li L, May FJ, Bueno R Jr, Dennett JA, Bala AA, Guzman H, Elizondo-Quiroga D, Tesh
RB, Barrett AD (2007) Genetic stasis of dominant West Nile virus genotype, Houston, Texas.
de Manzione N, Salas RA, Paredes H, Godoy O, Rojas L, Araoz F, Fulhorst CF, Ksiazek TG, Mills
JN, Ellis BA, Peters CJ, Tesh RB (1998) Venezuelan hemorrhagic fever: clinical and epidemio-
logic studies of 165 cases. Clin Infect Dis 26:308–313
Diagne MM, Gaye A, Ndione MHD, Faye M, Fall G, Widen SG, Wood TG, Popov V, Guzman
H, Granjon L, Handschumacher P, Ba Y, Weaver SC, Diallo M, Tesh RB, Faye O, Vasialkis N,
Sall AA (2019) Dianke virus: a new mesonivirus species isolated from mosquitoes in eastern
Senegal. Virus Res. https://doi.org/10.1016/j.viruses.2019.197802
Ding X, Wu X, Duan T, Siirn M, Guzman H, Yang Z, Tesh RB, Xiao SY (2005) Nucleotide and
amino acid changes in West Nile virus strains exhibiting renal tropism in hamsters. Am J Trop
Med Hyg 73:803–807
Dinitz JAP, Travassos da Rosa APA, Guzman H, Xu F, Xiao SY, Popov VL, Vasconcelos PFC, Tesh
RB (2006) West Nile virus infection of primary mouse neuronal and neuroglial cells. The role
of astrocytes in chronic infection. Am J Trop Med Hyg 75:691–696
Endris RG, Tesh RB, Young DG (1983) Transovarial transmission of Rio Grande virus
(Bunyaviridae: Phlebovirus) by the sand fly, Lutzomyia anthophora. Am J Trop Med
Hyg:862–864
Faria NR, Azevedo RS, Kraemer MU, Souza R, Cunha MS, Hill SC, Thézé J, Bonsall MB,
Bowden TA, Rissanen I, Rocco IM, Nogueira JS, Maeda AY, Vasami FG, Macedo FL, Suzuki
A, Rodrigues SG, Cruz AC, Nunes BT, Medeiros DB, Rodrigues DS, Nunes Queiroz AL, da
Silva EV, Henriques DF, Travassos da Rosa ES, de Oliveira CS, Martins LC, Vasconcelos HB,
Casseb LM, Simith Dde B, Messina JP, Abade L, Lourenço J, Carlos Junior Alcantara L, de
Lima MM, Giovanetti M, Hay SI, de Oliveira RS, Lemos Pda S, de Oliveira LF, de Lima CP,
da Silva SP, de Vasconcelos JM, Franco L, Cardoso JF, Vianez-Júnior JL, Mir D, Bello G,
Delatorre E, Khan K, Creatore M, Coelho GE, de Oliveira WK, Tesh RB, Pybus OG, Nunes
MR, Vasconcelos PF (2016) Zika virus in the Americas: early epidemiological and genetic
findings. Science 352(6283):345–349
Ferro C, Morrison AC, Torres M, Pardo R, Wilson ML, Tesh RB (1995a) Species composition
and relative abundance of sand flies from the genus Lutzomyia (Diptera: Psychodidae) at an
endemic focus of visceral leishmaniasis in Colombia. J Med Entomol 32:527–537
Ferro C, Morrison AC, Torres M, Pardo R, Wilson ML, Tesh RB (1995b) Age structure, blood
feeding behavior, and Leishmania chagasi infection in a population of the sand fly Lutzomyia
longipalpis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in Colombia.
Fisher AF, Tesh RB, Tonry J, Guzman H, Liu D, Xiao S-Y (2003) Induction of severe disease
in hamsters by two sandfly fever group viruses, Punta Toro and Gabek Forest (Phlebovirus:
Bunyaviridae), similar to that caused by Rift Valley fever virus. Am J Trop Med Hyg 69:269–276
528 R. B. Tesh
Forrester NL, Palacios G, Tesh RB, Savji N, Guzman H, Sherman M, Weaver SC, Lipkin WI
(2012) A genome-scale phylogeny of the Alphavirus genus suggests a marine origin. J Virol
86:2729–2738
Forrester NL, Widen SG, Wood TG, Travassos da Rosa APA, Ksiazek TG, Vasilakis N, Tesh RB
(2013) Identification of a new Newcastle disease virus isolate from Indonesia represents an
ancestral lineage of class II genotype XII. Virus Genes 47(1):168–172. https://doi.org/10.1007/
s11262-013-0900-8
Fulhorst CF, Bowen MD, Salas RA, de Manzione NMC, Duno G, Utrera A, Ksiazek TG, Peters CJ,
Nichol ST, de Miller E, Tovar D, Ramos B, Vasquez C, Tesh RB (1997a) Isolation and charac-
terization of Pirital virus, a novel South American arenavirus. Am J Trop Med Hyg 56:548–553
Fulhorst CF, Monroe MC, Salas RA, Duno G, Utrera A, Ksiazek TG, Nichol ST, Manzione NMC,
Tovar D, Tesh RB (1997b) Isolation, characterization and geographic distribution of Cano
Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae). Virus
Res 51:159–171
Fulhorst CF, Bowen MD, Salas RA, Duno G, Utrera A, Ksiazek TG, Manzione NMC, Miller E,
Vasquez C, Peters CJ, Tesh RB (1999a) Natural rodent host associations of Guanarito and
Pirital viruses (family Arenaviridae) in central Venezuela. Am J Trop Med Hyg 61:325–330
Fulhorst CF, Ksiazek TG, Peters CJ, Tesh RB (1999b) Experimental infection of the cane mouse
Zygodontomys brevicauda (family Muridae) with Guanarito virus (Arenaviridae), the etiologic
agent of Venezuelan hemorrhagic fever. J Infect Dis 180:966–969
Galindo P, Srihongse S, de Rodaniche E, Grayson MA (1966) An ecologic survey for arboviruses
in Almirante, Panama, 1959–1962. Am J Trop Med Hyg 15:385–400
Ghedin E, Rogers MB, Widen SG, Guzman H, Travassos da Rosa AP, Wood TG, Fitch A, Popov
V, Holmes EC, Walker PJ, Vasilakis N, Tesh RB (2013) Kolente virus, a rhabdovirus species
isolated from ticks and bats in the Republic of Guinea. J Gen Virol 94:2609–2615
Gligic A, Tesh RB, Miscevic Z, Travassos da Rosa APA, Zivkovic V (1983) Jug Bogdanovac virus –
a new member of the vesicular stomatitis virus serogroup (Rhabdoviridae: Vesiculovirus) iso-
lated from phlebotomine sand flies in Yugoslavia. Mikrobiologija 20:97–105
Goenaga S, Kenney JL, Duggal NK, Delorey M, Ebel GD, Zhang B, Levis SC, Enria DA, Brault
AC (2015) Potential for co-infection of a mosquito-specific flavivirus, Nhumirim virus, to block
West Nile virus transmission in mosquitoes. Viruses 7:5801–5812. https://doi.org/10.3390/
v7112911
Grimaldi G, Tesh RB (1993) Leishmaniases of the New World: current concepts and implications
for future research. Clin Microbiol Rev 6:230–250
Grimaldi GG, Tesh RB, McMahon-Pratt D (1989) A review of the geographic distribution and
epidemiology of leishmaniasis in the New World. Am J Trop Med Hyg 41:687–725
Grimaldi GG, Kreutzer RD, Hashiguchi Y, Gomez EA, Mimori T, Tesh RB (1992) Description of
Leishmania equatorensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting
arboreal animals in Ecuador. Mem Inst Oswaldo Cruz 87:221–228
Groseth A, Mampilli V, Weisend C, Dahlstrom E, Porcella SF, Russell BJ, Tesh RB, Ebihara H
(2014) Molecular characterization of human pathogenic bunyaviruses of the Nyando and
Bwamba/Pongola virus groups leads to the genetic identification of Mojuí dos Campos
and Kaeng Khoi virus. PLoS Negl Trop Dis 8(9):e3147. https://doi.org/10.1371/journal.
pntd.0003147
Guzman H, Walters LL, Tesh RB (1994) Histologic detection of multiple blood meals in
Phlebotomus duboscqi (Diptera: Psychodidae). J Med Entomol 31:890–897
Guzman H, Contreras-Gutierrez MA, Travassos da Rosa APA, Nunes MRT, Cardos JF, Guevara
C, Popov VL, Young K, Savit C, Wood TG, Widen SG, Watts DM, Hanley K, Perera D, Fish
D, Vasilakis N, Tesh RB (2018) Characterization of three new insect-specific flaviviruses: their
relationship to the mosquito-borne flavivirus pathogens. Am J Trop Med Hyg 98:410–419
Hall-Mendelin S, McLean BJ, Bielefeldt-Ohmann H, Hobson-Peters J, Hall RA (2016) The insect-
specific Palm Creak virus modulates West Nile virus infection in and transmission by Australian
mosquitoes. Parasites and Vectors 9:414. https://doi.org/10.1186/s13071-016-1683-2
Hanson RP (1952) The natural history of vesicular stomatitis. Bacterial Rev 16:179–204
Javadian E, Tesh R, Saidi S, Nadim A (1977) Studies on the epidemiology of sandfly fever in Iran.
III. Host feeding patterns of Phlebotomus papatasi in an endemic area of the disease. Am J
Jungler S, Drosten C (2013) Virus discovery and recent insights into virus discovery in arthropods.
Cur Opinion Microbiol 16:1–7
Kapoor A, Tesh RB, Duraisamy R, Popov VL, Travassos da Rosa AP, Lipkin WI (2013) A novel
mosquito-borne Orbivirus species found in South-east Asia. J Gen Virol 94:1051–1057
Klite PD, Diercks FH (1965) Histoplasma capsulatum in fecal contents and organs of bats in the
Panama Canal Zone. Am J Trop Med Hyg 14:433–439
Knudson DL, Tesh RB, Main AJ, St. George TD, Digoutte JP (1984) Characterization of the
Palyam virus serogroup (Reoviridae: Orbivirus). Intervirology 22:41–49
Kramer LD, Ciota AT, Kilpatrick AM (2019) Introduction, spread and establishment of West Nile
virus in the Americas. J Med Entom 56:1448–1455
Kreutzer RD, Corredor A, Grimaldi G Jr, Grogl M, Rowton ED, Young DG, Morales A, McMahon-
Pratt D, Guzman H, Tesh RB (1991) Characterization of Leishmania colombiensis sp.n.
(Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals and phleboto-
mine sand flies in Colombia and Panama. Am J Trop Med Hyg 44:662–675
Ladner JT, Savji N, Lofts L, Travassos da Rosa A, Wiley MR, Gestole MC, Rosen GE, Guzman H,
Vasconcelos PF, Nunes MR, Kochel T, Lipkin WI, Tesh RB, Palacios G (2014) Genomic and
phylogenetic characterization of viruses included in the Manzanilla and Oropouche species
complexes of the genus Orthobunyavirus, family Bunyaviridae. J Gen Virol 95:1055–1066
Ladner JT, Wiley MR, Beitzel B, Auguste AJ, Dupuis AP, Lindquist ME, Sibley SD, Kota KP,
Fetterer D, Eastwood G, Kimmel D, Prieto K, Guzman H, Aliota MT, Reyes D, Brueggemann
EE, St John L, Hyeroba D, Lauck M, Friedrich TC, O’Connor DH, Gestole MC, Cazares LH,
Popov VL, Castro-Llanos F, Kochel TJ, Kenny T, White B, Ward MD, Loaiza JR, Goldberg TL,
Weaver SC, Kramer LD, Tesh RB, Palacios G (2016) A multicomponent animal virus isolated
from mosquitoes. Cell Host Microbe 20(3):357–367
Lederberg J, Shope RE, Oaks SC (eds) (1992) Emerging infections. Microbial threats to health in
the United States. Institute of Medicine. National Academy Press, Washington, DC
Li G, Wang N, Guzman H, Sbrana E, Yoshikawa T, Tseng C, Tesh RB, Xiao S-Y (2008a) Dhori
virus (Orthomyxoviridae: Thogotovirus) infection of mice produces a disease and cytokine
response pattern similar to that of highly virulent influenza A (H5N1) virus infection in
humans. Am J Trop Med Hyg 78:675–680
Li G, Duan T, Wu X, Tesh RB, Soong L, Xiao SY (2008b) Yellow fever virus infection in Syrian
golden hamsters: relationship between cytokine expression and pathologic changes. Int J Clin
Exp Pathol 1:169–179
Li C-X, Shi M, Tian JH, Lin XD, Kang YJ, Chen LJ, Qin XC, Xu J, Holmes EC, Zhang YZ
(2015) Unprecedented genome diversity of RNA viruses in arthropods reveals the ancestry of
negative-senses RNA viruses. eLIFE 4:e05378. https://doi.org/10.7754/eLife.05378
Lillibridge KM, Parsons R, Randle Y, Travassos da Rosa APA, Guzman H, Siirin M, Wuithiranyagool
T, Hailey C, Higgs S, Bala A, Pascual R, Meyer T, Vanlandingham DL, Tesh RB (2004) The
2002 introduction of West Nile virus into Harris County, Texas, an area historically endemic
for St. Louis encephalitis. Am J Trop Med Hyg 70:676–681
Lima JA, Nunes Neto JP, Castro KS, Travassos da Rosa APA, Tesh RB, Vasilakis N, Guzman H,
Widen S, da Silva SP, Madeiros DBA, Cardosa JF, Martins LC, Azevedo RSS, Vasconcelos
PFC, Chiang JO (2019) Characterization of Triniti virus supports its reclassification in the fam-
ily Peribunyaviridae. J Gen Virol 100:137–144
Lisieux T, Coimbra M, Nassar ES, Burattini MN, de Souza LT, Ferreira IB, Rocco IM, Travassos
da Rosa AP, Vasconcelos PF, Pinheiro FP, JW LD, Rico-Hesse R, Gonzalez J-P, Jahrling PB,
Tesh RB (1994) New arenavirus isolated in Brazil. Lancet 343:391–392
Loh J, Zhao G, Tesh RB, Barrett AD (2011) Phylogeography of West Nile virus: From the cradle
of evolution in Africa to Eurasia and the Americas. J Virol 85:2964–2974
530 R. B. Tesh
Long KC, Ziegler SA, Thangamani S, Hausser NL, Kochel TJ, Higgs S, Tesh RB (2011)
Experimental transmission of Mayaro virus by Aedes aegypti. Am J Trop Med Hyg 85:750–757
Mann BR, McMullen AR, Guzman H, Tesh RB, Barrett AD (2013) Dynamic transmission of West
Nile virus across the United States-Mexican border. Virology 436:75–80
Mateo R, Xiao SY, Guzman H, Lei H, Travassos da Rosa APA, Tesh RB (2006) The effects of
immunosuppression on West Nile virus infection in hamsters. Am J Trop Med Hyg 75:356–362
Mateo RI, Xiao SY, Travassos da Rosa AP, Lei H, Guzman H, Lu L, Tesh RB (2007a) Yellow fever
17-D vaccine is neurotropic and produces encephalitis in immunosuppressed hamsters. Am J
Mateo RI, Xiao SY, Lei H, Travassos da Rosa AP, Tesh RB (2007b) Dhori virus (Orthomyxoviridae:
Thogotovirus) infection in mice: a model of the pathogenesis of severe orthomyxovirus infec-
tion. Am J Trop Med Hyg 76:785–790
Matsuno K, Weisend C, Travassos da Rosa AP, Anzick SL, Dahlstrom E, Porcella SF, Dorward
DW, Yu XJ, Tesh RB, Ebihara H (2013) Characterization of the Bhanja serogroup viruses
(Bunyaviridae): a novel species of the genus Phlebovirus and its relationship with other emerg-
ing tick-borne phleboviruses. J Virol 87:3719–3728
Matsuno K, Weisend C, Kajihara M, Matysiak C, Williamson B, Simuunza M, Mwoene A, Takada
A, Tesh RB, Ebihara H (2015) Comprehensive molecular detection of tick-borne phleboviruses
leads to the retrospective identification of taxonomically unassigned bunyaviruses and the dis-
covery of a novel member of the genus Phlebovirus. J Virol 89:594–604
May FJ, Davis CT, Tesh RB, Barrett AD (2011) Phylogeography of West Nile virus: From the
cradle of evolution in Africa to Eurasia and the Americas. J Virol 85:2964–2974
McMahon-Pratt D, Modi G, Tesh RB (1983) Detection of promastigote stage- specific antigens on
Leishmania mexicana amazonensis developing in the midgut of Lutzomyia longipalpis. Am J
McMullen AR, May FJ, Li L, Guzman H, Bueno R, Dennett JA, Tesh RB, Barrett ADT (2011)
Evolution of a new genotype of West Nile virus in North America. Emerg Infect Dis 17:785–789
Mercer DR, Spinelli GR, Watts DM, Tesh RB (2003) Biting rates and developmental substrates for
biting midges (Diptera: Ceratopogonidae) in Iquitos, Peru. J Med Entomol 40:807–812
Mihindukulasuriya KA, Nguyen NL, Wu G, Huang HV, Travassos da Rosa APA, Popov VL, Tesh
RB, Wang D (2009) Nyamanini and Midway viruses define a novel taxon of RNA viruses in
the order Mononegavirales. J Virol 83:5109–5116
Mochkovski SD, Diomina NA, Nossina VD, Pavlova EA, Livchitz JL, Pels HJ, Roubtzova VP
(1937) Researches on sandfly fever. Part VIII. Transmission of sandfly fever virus by sand-
flies hatched from eggs laid by infected females. Med Parasitol Parasit Bolesn 6:922–937. (in
Russian)
Modi GB, Tesh RB (1983) A simple technique for mass-rearing Lutzomyia longipalpis and
Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory. J Med Entomol 20:568–569
Moncayo A, Hice CL, Watts DM, Travassos da Rosa APA, Guzman H, Russell KL, Calampa C,
Gonzalo A, Popov VL, Weaver SC, Tesh RB (2001) Allpahuayo virus, a new arenavirus from
the Peruvian Amazon. Virology 284:277–286
Morrison AC, Ferro C, Morales A, Tesh RB, Wilson ML (1993a) Dispersal of the sand fly
Lutzomyia longipalpis (Diptera: Psychodidae) in an endemic focus of visceral leishmaniasis in
Colombia. J Med Entomol 30:427–435
Morrison AC, Ferro C, Tesh RB (1993b) Host preferences of the sand fly Lutzomyia longipalpis
at an endemic focus of American visceral leishmaniasis in Colombia. Am J Trop Med Hyg
49:68–75
Morrison AC, Ferro C, Pardo R, Torres M, Devlin B, Wilson ML, Tesh RB (1995a) Seasonal
abundance of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic focus
of visceral leishmaniasis in Colombia. J Med Entomol 32:538–548
Morrison AC, Ferro C, Pardo R, Torres M, Wilson ML, Tesh RB (1995b) Nocturnal biting activity
of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic focus of visceral
leishmaniasis in Colombia. J Med Entomol 32:605–617
Mutebi JP, Gianella A, Travassos da Rosa AP, Tesh RB, Barrett AD, Higgs S (2004) Infectivity
of yellow fever virus for Bolivian Aedes aegypti mosquitoes. Emerg Infect Dis 10:1657–1660
Nasar F, Palacios G, Gorchakov R, Guzman H, Travassos da Rosa AP, Savji N, Popov VL, Sherman
MB, Lipkin WI, Tesh RB, Weaver SC (2012) Eilat virus, a unique Alphavirus with host range
restricted to insects by RNA replication. Proc Nat Acad Sci 109:14622–14627
Navarro JC, Giambalvo D, Hernandez R, Auguste AJ, Tesh RB, Weaver SC, Montanez H, Liria J,
Lima A, Travassos da Rosa JF, da Silva SP, Vasconcelos JM, Oliveira R, Vianez JL Jr, Nunes
MR (2016) Isolation of Madre de Dios Virus (Orthobunyavirus: Bunyaviridae), an Oropouche
virus species reassortant, from a monkey in Venezuela. Am J Trop Med Hyg 95:328–338
Noble GA, Tesh RB (1974) Monoecocestus diplomys sp. N. (Cestoda: Anoplocephalidae) from the
rat Diplomys darling. J Parasitol 60:605–607
Nunes Neto JP, de Souza WM, Acari GO, Romeiro MF, Fumagalli MJ, Viera CL, Medeiros DB,
de Lima JA, de Lima CPS, Cardos JF, Rodrigues SG, Figueiredo LTM, da Silva SP, Tesh RB,
Nunes MR, Vasconcelos PF (2017) Characterization of the Bujaru, Frijoles and Tapara anti-
genic complexes into the Sandfly Fever group and two unclassified phleboviruses from Brazil.
J Gen Virol 98:585–594
Nunes MR, Travassos da Rosa AP, Weaver SC, Tesh RB, Vasconcelos PF (2005) Molecular epide-
miology of group C viruses (Bunyaviridae, Orthobunyavirus) isolated in the Americas. J Virol
79:10561–10570
Nunes MRT, Faria NR, de Vasconcelos JM, Golding N, Kraemer MU, de Oliveira LF, Azevedo
Rdo S, da Silva DE, da Silva EV, da Silva SP, Carvalho VL, Coelho GE, Cruz AC, Rodrigues
SG, Vianez JL Jr, Nunes BT, Cardoso JF, Tesh RB, Hay SI, Pybus OG, Vasconcelos PE (2015a)
Emergence and potential for spread of Chikungunya virus in Brazil. BMC Med 13:102. https://
doi.org/10.1186/s12916-015-0348-x
Nunes MR, Silva SP, Carvalho VL, Vasconcelos JMV, da Silva DE, Oliveira LF, Nunes Neto JP,
Rodrigues SG, Azevedo RS, Monteiro HA, Cardoso JF, Guzman H, Tesh RB, Vasconcelos
PF, Vianez-Junior JL, Martins LC (2015b) Emergence of new insect-restricted viruses in
Amazon Region. Genome Announcements 3(2):e00131–e00115. https://doi.org/10.1128/
genomeA.00131-15
O’Neill SL, Pettigrew M, Sinkins S, Braig HR, Andreadis TG, Tesh RB (1997) In vitro cultivation
of Wolbachia pipientis in an Aedes albopictus cell line. Insect Mol Biol 6:33–39
Ohlund P, Lunden H, Bloomstrom AL (2019) Insect-specific virus evolution and potential effects
on vector competence. Virus Genes. https://doi.org/10.1007/s11262-018-01629-9
Palacios G, Savji N, Hui J, Travassos da Rosa A, Popov V, Briese T, Tesh RB, Lipkin WI (2010)
Genomic and phylogenetic characterization of Merino Walk virus, a novel arenavirus isolated
in South Africa. J Gen Virol 91:1315–1324
Palacios G, Tesh RB, Travassos da Rosa A, Savji N, Sze W, Jain K, Serge R, Guzman H,
Guevara C, Nunes MRT, Nunes-Neto JP, Kochel T, Hutchison S, Vasconcelos PFC, Lipkin
WI (2011a) Characterization of the Candiru antigenic complex (Bunyaviridae: Phlebovirus),
a highly diverse and reassorting group of viruses affecting humans in tropical America. J Virol
85:3811–3820
Palacios G, Travassos da Rosa AP, Savji N, Sze W, Wick I, Guzman H, Hutchison S, Tesh RB,
Lipkin WI (2011b) Aguacate virus, a new antigenic complex of the genus Phlebovirus (family
Bunyaviridae). J Gen Virol 92:1445–1453
Palacios G, Forrester NL, Savji N, Travassos da Rosa AP, Guzman H, Detoy K, Popov VL, Walker
PJ, Lipkin WI, Vasilakis N, Tesh RB (2013a) Characterization of Farmington virus, a novel
virus from birds that is distantly related to members of the family Rhabdoviridae. Virol J
10:219. https://doi.org/10.1186/1743-422X-10-219
Palacios G, Savji N, Travassos da Rosa A, Desai A, Sanchez-Seco MP, Guzman H, Lipkin WI, Tesh
RB (2013b) Characterization of the Salehabad virus species complex of the genus Phlebovirus
(Bunyaviridae). J Gen Virol 94:837–842
Palacios G, Savji N, Travassos da Rosa A, Guzman H, Yu X, Desai A, Rosen GE, Hutchison S,
Lipkin WI, Tesh RB (2013c) Characterization of the Uukuniemi virus group (Phlebovirus:
Bunyaviridae): evidence for seven distinct species. J Virol 87:3187–3195
532 R. B. Tesh
Palacios G, Tesh RB, Savji N, Travassos da Rosa APA, Guzman H, Bussetti AV, Desai A, Ladner
J, Sanchez-Seco M, Lipkin WI (2014) Characterization of the Sandfly fever Naples species
complex and description of a new Karimabad species complex (genus Phlebovirus, family
Bunyaviridae). J Gen Virol 95:292–300
Palacios G, Wiley M, Travassos da Rosa APA, Guzman H, Quiroz E, Savji N, Carrera JP, Bussetti
AV, Ladner J, Lipkin WI, Tesh RB (2015) Characterization of the Punta Toro species complex
(genus Phlebovirus, family Bunyaviridae). J Gen Virol 96:2079–2085
Petrischeva PA, Alymov AJ (1938) On transovarial transmission of virus of pappataci fever by
sandflies. Arch Biol Sci 53:138–144. (in Russian)
Pettersson JHO, Shi M, Eden JS, Holmes EC, Hesson JC (2019) Meta-transcriptomic comparison
of the RNA viromes of the mosquito vectors Culex pipiens and Culex torrentium in northern
Europe. Viruses 11:1033. https://doi.org/10.3390/v11111033
Popov VL, Tesh RB, Weaver SC, Vasilakis N (2019) Electron microscopy in discovery of novel
and emerging viruses from the collection of the World Reference Center for Emerging Viruses
and Arboviruses (WRCEVA). Viruses 11(5):E477. https://doi.org/10.3390/v11050477
Powers AM, Mercer DR, Watts DM, Guzman H, Fulhorst CF, Popov VL, Tesh RB (1999)
Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a
rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru. Am J Trop Med Hyg
61:92–98
Powers AM, Aguilar PV, Chandler LJ, Brault AC, Meakins TA, Watts D, Vasconcelos PE, Travassos
da Rosa AP, Weaver SC, Tesh RB (2006) Genetic relationships among Mayaro and Una viruses
suggest distinct patterns of transmission. Am J Trop Med Hyg 75:461–469
Presti R, Zhao G, Beatty WL, Mihindukulasuriya KA, Travassos APA, Popov VL, Tesh RB, Virgin
HW, Wang D (2009) Quaranfil, Johnston Atoll and Lake Chad viruses are novel members of
the family Orthomyxoviridae. J Virol 83:11599–11606
Ratterree MS, Gutierrez RA, Travassos da Rosa APA, Dille JB, Beasley DW, Bohm RP, Desai SM,
Didier PJ, Bikenmeyer LG, Dawson GJ, Leary TP, Schochetmann G, Phillippi-Falkenstein
AJ, ADT B, Tesh RB (2004) Experimental infection of rhesus macaques with West Nile virus:
level and duration of viremia and kinetics of the antibody response. J Infect Dis 189:669–676
Ribeiro JMC, Vachereau A, Modi GB, Tesh RB (1989a) A novel vasodilatory peptide from the
salivary glands of the sand fly Lutzomyia longipalpis. Science 243:212–214
Ribeiro JMC, Modi GB, Tesh RB (1989b) Salivary apyrase activity of some Old World phleboto-
mine sand flies. Insect Biochem 19:409–412
Rogers MB, Cui L, Fitch A, Popov V, Travassos da Rosa APA, Vasilakis N, Tesh RB, Ghedin E
(2014) Whole genome analysis of Sierra Nevada virus, a novel mononegavirus in the family
Nyamiviridae. Am J Trop Med Hyg 91:159–164
Rogers MB, Gulino KM, Tesh RB, Cui L, Fitch A, Unnasch TR, Popov VL, Travassos de Rosa
A, Guzman H, Carrera J-P, Vasilakis N, Ghedin E (2017) Characterization of five novel or
unclassified orthobunyaviruses (Bunyaviridae) from Africa and the Americas. J Gen Virol
98:2258–2266. https://doi.org/10.1099/jgv.0000899
Rosen L, Tesh RB, Lien JC, Cross JH (1978) Transovarial transmission of Japanese encephalitis
virus by mosquitoes. Science 199:909–911
Rosen L, Shroyer DA, Tesh RB, Frier JE, Lien JC (1983) Transovarial transmission of dengue
viruses by mosquitoes: Aedes albopictus and Aedes aegypti. Am J Trop Med Hyg 32:1108–1119
Russell KL, Montiel Gonzalez MA, Watts DM, Lagos-Figueroa RC, Chauca G, Ore M, Gonzalez
JE, Moron C, Tesh RB, Vinetz JM (2003) An outbreak of leptospirosis among Peruvian mili-
tary recruits. Am J Trop Med Hyg 69:53–57
Sadeghi M, Popov V, Guzman H, Phan TG, Vasilakis N, Tesh R, Delwart E (2017) Genomes of
viral isolates derived from different mosquitos species. Virus Res 242:49–57
Sadeghi M, Altar E, Deng X, Barker CM, Fang Y, Coffey LL, Delward E (2018) Virome of >12
thousand Culex mosquitoes from throughout California. Virology 523:74–88
Saidi S, Tesh RB, Javadian E, Nadim A (1976) The prevalence of human infection with West Nile
virus in Iran. Iranian J Pub Hlth 5:8–13
Saidi S, Tesh RB, Javadian E, Sahabi Z, Nadim A (1977) Studies on the epidemiology of sandfly
fever in Iran. II. The prevalence of human and animal infection with five phlebotomus fever
virus serotypes in Isfahan Province. Am J Trop Med Hyg 26:288–293
Samuelson J, Lerner E, Tesh RB, Titus R (1991) A mouse model of Leishmania braziliensis brazil-
iensis infection produced by co-injection with sand fly saliva. J Exp Med 173:49–54
Sbrana E, Xiao SY, Guzman H, Travassos da Rosa AP, Tesh RB (2004) Efficacy of post-exposure
treatment of yellow fever with ribavirin in a hamster model of the disease. Am J Trop Med Hyg
71:306–312
Sbrana E, Tonry JH, Xiao SY, Travassos da Rosa AP, Higgs S, Tesh RB (2005) Oral transmission
of West Nile virus. Am J Trop Med Hyg 72:325–329
Sbrana E, Xiao SY, Popov VL, Newman PC, Tesh RB (2006a) Experimental yellow fever virus
infection in the golden hamster (Mesocricetus auratus). III.Clinical laboratory values. Am J
Sbrana E, Mateo RI, Xiao SY, Popov VL, Newman PC, Tesh RB (2006b) Clinical laboratory,
virologic and pathologic changes in hamsters experimentally infected with Pirital virus
(Arenaviridae): a rodent model of Lassa fever. Am J Trop Med Hyg 74:1096–1102
Sbrana E, Xiao SY, Newman PC, Tesh RB (2007a) Comparative pathology of North American and
Central African strains of monkeypox virus in a ground squirrel model of the disease. Am J
Sbrana E, Jordan R, Hruby D, Mateo RI, Xiao SY, Siirin M, Newman PC, Travassos da Rosa AP,
Tesh RB (2007b) Efficacy of the antipoxvirus compound Siga ST-246 for the treatment of
severe orthopoxvirus infection. Am J Trop Med Hyg 76:768–773
Shelokov A, Peralta PH (1967) Vesicular stomatitis virus, Indiana type: an arbovirus infection of
sandflies and humans. Am J Epidemiol 86:149–157
Shi M, Lin XP, Vasilakis N, Tian JH, Li CX, Chen LJ, Eastwood G, Diao XN, Chen MH, Chen
X, Qin XC, Widen S, Wood T, Tesh RB, Xu J, Holmes E, Zhang YZ (2015) Divergent viruses
discovered in arthropods and vertebrates revise the evolutionary history of the Flaviviridae and
related viruses. J Virol 90:659–669
Siirin MT, Duan T, Lei H, Guzman H, Travassos da Rosa APA, Watts DM, Xiao S-Y, Tesh RB
(2007) Chronic St. Louis encephalitis virus infection in the golden hamster (Mesocricetus
auratus). Am J Trop Med Hyg 76:299–306
Silva SP, Dilcher M, Weber F, Hufert FT, Weidmann M, Cardoso JF, Carvalho VL, Chiang JO,
Martins LC, Lima CP, Da Silva DE, Vianez-Júnior JL, Popov VL, Travassos da Rosa AP, Tesh
RB, Vasconcelos PF, Nunes MR (2014) Genetic and biological characterization of selected
Changuinola viruses (Reoviridae, Orbivirus) from Brazil. J Gen Virol 95(pt10):2251–2259
Solomon T, Fisher AF, Beasley DWC, Mandava P, Granwehr BP, Langsjoen H, Travassos da Rosa
AP, Barrett A, Tesh RB (2003) Natural and nosocomial infection in a patient with West Nile
encephalitis and extrapyramidal movement disorders. Clin Infect Dis 36:140–145
Swetnam D, Widen SG, Wood TG, Symonds DA, Reyna M, Wilkerson L, Debboun M, Mead DG,
Beaty BJ, Guzman H, Tesh RB, Barrett A (2018) Terrestrial birds migration and West Nile
Virus circulation, United States. Emerg Infect Dis 24:2184–2194
Tesh RB (1970a) Observations on the natural history of Diplomys darlingi. J Mammal 51:197–199
Tesh RB (1970b) Notes on the reproduction, growth and development of echimyid rodents in
Panama. J Mammal 51:199–202
Tesh RB (1978) The prevalence of encephalomyocarditis virus neutralizing antibodies among vari-
ous human populations. Am J Trop Med Hyg 27:144–149
Tesh RB (1979) A method for the isolation of dengue viruses, using mosquito cell cultures. Am J
Tesh RB (1988) The genus Phlebovirus and its vectors. Ann Rev Entomol 33:169–181
Tesh RB (1995) Control of zoonotic visceral leishmaniasis; Is it time to change strategies? Am J
Trop med Hyg 52:287–292
Tesh RB, Cameron R (1970) Laboratory rearing of the climbing rat, Tylomys nudicaudatus. Lab
Animal Care 20:93–96
534 R. B. Tesh
Tesh RB, Cornet M (1981) The location of San Angelo virus in developing ovaries of transovari-
ally infected Aedes albopictus mosquitoes as revealed by fluorescent antibody technique. Am
Tesh RB, Johnson KM (1975) Vesicular stomatitis. In: Hubbert WT, WF MC, Schurrenburger
PR (eds) Diseases transmitted from animals to man, 6th edn. Charles C. Thomas, Springfield,
pp 897–910
Tesh RB, Modi GB (1983a) Growth and transovarial transmission of Chandipura virus
(Rhabdoviridae: Vesiculovirus) in Phlebotomus papatasi. Am J Trop Med Hyg 32:621–623
Tesh RB, Modi GB (1983b) Development of a continuous cell line from the sand fly, Lutzomyia
longipalpis (Diptera: Psychodidae), and its susceptibility to infection with arboviruses. J Med
Entomol 20:199–202
Tesh RB, Modi G (1984a) Studies on the biology of phleboviruses in sand flies (Diptera;
Psychodidae). 1. Experimental infection of the vector. Am J Trop Med Hyg 33:1007–1016
Tesh RB, Modi GB (1984b) A simple method for experimental infection of phlebotomine sand
flies with Leishmania. Am J Trop Med Hyg 33:41–46
Tesh RB, Modi GB (1987) Maintenance of Toscana virus in Phlebotomus perniciosus by vertical
transmission. Am J Trop Med Hyg 36:189–193
Tesh R, Schneidau JD (1966) Experimental infection of bats (Tadarida brasiliensis) with
Histoplasma capsulatum. Am J Trop Med Hyg 15:544–550
Tesh RB, Schneidau JD (1967) Naturally occurring histoplasmosis among bat colonies in the
southeastern United States. Am J Epidemiol 86:545–551
Tesh RB, Shroyer DA (1980) The mechanism of arbovirus transovarial transmission in mosqui-
toes: San Angelo virus in Aedes albopictus. Am J Trop Med Hyg 29:1394–1404
Tesh RB, Wallace GD (1978) Observations on the natural history of encephalomyocarditis virus.
Am J Trop med Hyg 27:133–143
Tesh RB, Shacklette MH, Diercks FH, Hirschl D (1964) Histoplasmosis in children. Pediatrics
33:894–903
Tesh RB, Peralta PH, Johnson KM (1969) Ecological studies of vesicular stomatitis virus.
I. Prevalence of infection among animals and humans living in an endemic area of VSV activ-
ity. Am J Epidemiol 90:255–261
Tesh RB, Peralta PH, Johnson KM (1970) Ecological studies of vesicular stomatitis virus.
II. Experimental infection of Panamanian wild animals. Am J Epidemiol 91:216–224
Tesh RB, Chaniotis BN, Aronson M, Johnson KM (1971a) Natural host preferences of Panamanian
phlebotomine sand flies as determined by precipitin test. Am J Trop Med Hyg 20:150–156
Tesh RB, Chaniotis BN, Johnson KM (1971b) Vesicular stomatitis virus, Indiana serotype:
Multiplication in and transmission by experimentally infected phlebotomine sand flies
(Lutzomyia trapidoi). Am J Epidemiol 93:491–495
Tesh RB, Chaniotis BN, Carrera B, Johnson KM (1972a) Further studies of the natural host prefer-
ences of Panamanian phlebotomine sand flies. Am J Epidemiol 95:88–93
Tesh RB, Chaniotis BN, Johnson KM (1972b) Vesicular stomatitis virus (Indiana serotype):
Transovarial transmission by phlebotomine sand flies. Science 175:1477–1479
Tesh RB, Chaniotis BN, Peralta PH, Johnson KM (1974) Ecology of viruses isolated from
Panamanian phlebotomine sandflies. Am J Trop Med Hyg 23:258–269
Tesh RB, Peralta PH, Shope RE, Chaniotis BN, Johnson KM (1975a) Antigenic relationships
among phlebotomus fever group arboviruses and their implications for the epidemiology of
sandfly fever. Am J Trop Med Hyg 24:135–144
Tesh RB, Gajdusek DC, Garruto RM, Cross JH, Rosen L (1975b) The distribution and prevalence
of group A arbovirus neutralizing antibodies among human populations in Southeast Asia and
the Pacific Islands. Am J Trop Med Hyg 24:664–675
Tesh RB, Saidi S, Gaidamovich SY, Rodhain F, Vesenjak-Hirjan J (1976a) Serologic studies on the
epidemiology of phlebotomus fever in the Old World. Bull WHO 54:663–674
Tesh RB, Saidi S, Javadian E, Nadim A, Sahabi Z (1976b) The distribution and prevalence of
human infection with Phlebotomus fever group viruses in Iran. Iranian J Pub Hlth 5:1–7
Tesh RB, Gubler DJ, Rosen L (1976c) Variation among geographic strains of Aedes albopictus in
susceptibility to infection with chikungunya virus. Am J Trop Med Hyg 25:326–335
Tesh RB, Saidi S, Javadian E, Loh P, Nadim A (1977a) Isfahan virus, a new Vesiculovirus infecting
humans, gerbils and sand flies in Iran. Am J Trop Med Hyg 26:299–306
Tesh RB, Saidi S, Javadian E, Nadim A (1977b) Studies on the epidemiology of sandfly fever in
Iran. I. Virus isolates obtained from Phlebotomus. Am J Trop Med Hyg 26:282–287
Tesh RB, McLean RG, Shroyer DA, Calisher CH, Rosen L (1981) Ross River virus infection (epi-
demic polyarthritis) in American Samoa. Trans R Soc Trop Med Hyg 75:426–431
Tesh R, Boshell J, Young DG, Morales AA, Corredor AA, Modi GB, Ferro de Carrasquilla C, de
Rodriguez C, Gaitan MO (1986a) Biology of Arboledas virus, a new phlebotomus fever sero-
group virus (Bunyaviridae: Phlebovirus) isolated from sand flies in Colombia. Am J Trop Med
Hyg 35:1310–1316
Tesh RB, Peleg J, Samina I, Magalit J, Bodkin DK, Shope RE, Knudson DL (1986b) Biological
and antigenic characterization of Netivot virus, an unusual new Orbivirus recovered from mos-
quitoes in Israel. Am J Trop Med Hyg 35:418–428
Tesh RB, Boshell J, Modi G, Morales A, Young D, Corredor A, Ferro C, de Rodriguez C, Walters
LL, Gaitan M (1987) Natural infection of humans, animals and phlebotomine sand flies with the
Alagoas serotype of vesicular stomatitis virus in Colombia. Am J Trop Med Hyg 36:653–661
Tesh RB, Boshell J, Young DG, Morales A, Ferro de Carrasquilla C, Corredor A, Modi GB,
Travassos da Rosa AP, RG ML, Rodriguez C, Gaitan MO (1989) Characterization of five
new phleboviruses recently isolated from sand flies in tropical America. Am J Trop Med Hyg
40:529–533
Tesh RB, Lubroth J, Guzman H (1992) Simulation of arbovirus over wintering: survival of Toscana
virus (Bunyaviridae: Phlebovirus) in its natural sand fly vector, Phlebotomus perniciosus. Am
Tesh RB, Wilson ML, Salas R, de Manzione NMC, Tovar D, Ksiazek TG, Peters CJ (1993) Field
studies on the epidemiology of Venezuelan hemorrhagic fever. Implication of the cotton rat
Sigmodon alstoni as the probable rodent reservoir. Am J Trop Med Hyg 49:227–235
Tesh RB, Jahrling PB, Salas RA, Shope RE (1994) Description of Guanarito virus (Arenaviridae:
Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever. Am J Trop Med Hyg
50:452–459
Tesh RB, Watts DM, Russell K, Damodaran C, Calampa C, Cabezas C, Ramirez G, Vasquez B,
Hayes CG, Rossi CA, Powers AM, Hice CL, Chandler LJ, Cropp BC, Karabatsos N, Roehrig
JT, Gubler DJ (1999) Mayaro virus disease: an emerging mosquito-borne zoonosis in tropical
South America. Clin Infect Dis 28:67–73
Tesh RB, Guzman H, Travassos da Rosa APA, Vasconcelos PFC, Dias LB, Bunnell JE, Zhang H,
Xiao S-Y (2001) Experimental yellow fever virus infection in the golden hamster (Mesocricetus
auratus). 1. Virologic, biochemical and immunologic studies. J Infect Dis 183:1431–1436
Tesh RB, Travassos da Rosa AP, Guzman H, Araujo TP, Xiao S-Y (2002a) Immunization with het-
erologous flaviviruses protects against fatal West Nile encephalitis. Emerg Infect Dis 8:245–251
Tesh RB, Arroyo J, Travassos da Rosa APA, Guzman H, Xiao S-Y, Monath TP (2002b) Efficacy
of a killed virus vaccine, live attenuated chimeric virus, and passive immunization serum for
prevention of West Nile virus encephalitis in a hamster model. Emerg Infect Dis 8:1392–1397
Tesh RB, Watts DM, Sbrana E, Siirin M, Popov VL, Xiao SY (2004) Experimental infection of
ground squirrels (Spermophilus tridecemlineatus) with monkeypox virus. Emerg Infect Dis
10:1563–1567
Tesh RB, Siirin M, Guzman H, Travassos da Rosa AP, Wu X, Duan T, Lei H, Nunes MR, Xiao
SY (2005) Persistent West Nile virus infection in the golden hamster (Mesocricetus auratus):
studies on its mechanism and possible implications for other flavivirus infections. J Infect Dis
192:287–295
Thangamani S, Huang J, Hart CE, Guzman H, Tesh RB (2016) Vertical transmission of Zika virus
in Aedes aegypti mosquitoes. Am J Trop Med Hyg 95:1169–1173
536 R. B. Tesh
Tonry JH, Xiao SY, Siirin M, Chen H, Travassos da Rosa APA, Tesh RB (2005) Persistent shed-
ding of West Nile virus in urine of experimentally infected hamsters. Am J Trop Med Hyg
72:320–324
Travassos da Rosa APA (2016) The history of arbovirology at Instituto Evandro Chagas, Belem,
Para, Brazil, from 1954 to 1998. Revista Pan-Amaz Saude 7:61–70
Travassos da Rosa AP, Tesh RB, Pinheiro FP, Travassos da Rosa JF, Peralta PH, Knudson DL
(1984a) Characterization of the Changuinola virus serogroup (Reoviridae: Orbivirus).
Intervirology 21:38–49
Travassos da Rosa AP, Tesh RB, Travassos da Rosa JF, Main AJ (1984b) Carajas and Maraba
viruses, two new vesiculoviruses isolated from phlebotomine sand flies in Brazil. Am J Trop
Med Hyg 33:999–1005
Travassos da Rosa A, Turrell MJ, Watts DM, Powers AM, PFC V, Jones J, Dohm D, Shope RE,
Degallier N, Popov VL, Russell KL, Weaver SC, Guzman H, Brault AC, Lemon AP, Tesh RB
(2001) Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes
in the Amazon Basin. Am J Trop Med Hyg 64:93–97
Utrera A, Duno G, Ellis BA, Salas RA, de Manzione N, Fulhorst CF, Tesh RB, Mills JN (2000)
Small mammals in agricultural areas of the western llanos of Venezuela: community structure,
habitat associations, and relative densities. J Mammol 81:536–548
Vasconcelos PFC, Bryant JE, Travassos da Rosa APA, Tesh RB, Rodrigues SG, ADT B (2004)
Genetic divergence and dispersal of yellow fever virus, Brazil. Emerg Infect Dis 10:1578–1584
Vasilakis N, Widen S, Travassos da Rosa APA, Wood TG, Walker PJ, Holmes EG, Tesh RB (2013a)
Malpais Spring virus is a new species in the genus Vesiculovirus. Virol J 10:69. https://doi.org/1
0.1186/1743-422X-10-69
Vasilakis N, Widen S, Mayer SV, Seymour R, Wood TG, Popov V, Guzman H, Travassos da Rosa
APA, Ghedin E, Holmes EC, Walker PJ, Tesh RB (2013b) Niakha virus: A novel member of the
family Rhabdoviridae isolated from phlebotomine sandflies in Senegal. Virology 444:80–89
Vasilakis N, Castro-Llanos F, Widen SG, Aguilar PV, Guzman H, Guevara C, Fernandez R,
Auguste AJ, Wood TG, Popov V, Mundal K, Ghedin E, Kochel TJ, Holmes EC, Walker PJ,
Tesh RB (2014a) Arboretum and Puerto Almendras viruses; two novel rhabdoviruses isolated
from mosquitoes in Peru. J Gen Virol 95:787–792
Vasilakis N, Guzman H, Firth C, Forrester NL, Widen SG, Wood SG, Rossi SL, Ghedin E, Popov
V, Blasdell KR, Walker PJ, Tesh RB (2014b) Mesoniviruses are insect-specific viruses with
extensive geographic distribution and host range. Virol J 11:97. https://doi.org/10.1186/
1743-422X-11-97
Vasilakis N, Tesh RB, Popov VL, Widen SG, Wood TG, Forrester NL, Gonzalez JP, Saluzzo JF,
Alkhovsky S, Lam SK, Mackenzie JS, Walker PJ (2019a) Exploiting the legacy of the arbovi-
rus hunter. Viruses 11(5):E471. https://doi.org/10.3390/v11050471
Vasilakis N, Tesh RB, Widen SG, Mirchandani D, Walker RJ (2019b) Genomic characterization of
Cuiaba and Charleville viruses: arboviruses (family Rhabdoviridae, genus Sripuvirus) infect-
ing reptiles and amphibians. Virus Genes 55:85–94
Walker PJ, Widen SG, Firth C, Blasdell KR, Wood TG, Travassos da Rosa APA, Guzman H, Tesh
RB, Vasilakis N (2015) Genomic characterization of Yogue, Kasokero, Issyk-Kul, Keterah,
Gossas and Thiafora viruses: nairoviruses naturally infecting bats, schrews and ticks. Am J
Walker PJ, Tesh RB, Guzman H, Popov VL, Travassos da Rosa APA, Reyna M, Nunes MRT, de
Souza WM, Contreras-Gutierrez MA, Patroca S, Vela J, Salvato V, Bueno R, Widen AG, Wood
TG, Vasilakis N (2019) Characterization of three novel viruses from families Nyamiviridae,
Orthomyxoviridae, and Peribunyaviridae, isolated from dead birds collected during West
Nile virus surveillance in Harris County, Texas. Viruses 11(10):937. https://doi.org/10.3390/
v11100927
Walters LL, Chaplin GL, Modi GB, Tesh RB (1989a) Ultrastructural biology of Leishmania
(Viannia) panamensis (Leishmania braziliensis panamensis) in the sand fly, Lutzomyia gomezi
(Diptera: Psychodidae): a natural host-parasite association. Am J Trop Med Hyg 40:19–39
Walters LL, Modi GB, Chaplin GL, Tesh RB (1989b) Ultrastructural development of Leishmania
chagasi in its vector, Lutzomyia longipalpis (Diptera: Psychodidae). Am J Trop Med Hyg
41:295–317
Walters LL, Irons KP, Modi GB, Tesh RB (1992) Refractory barriers in the sand fly Phlebotomus
papatasi (Diptera: Psychodidae) to infection with Leishmania panamensis. Am J Trop Med
Hyg 46:211–228
Walters LL, Irons KP, Chaplin G, Tesh RB (1993a) Life cycle of Leishmania major (Kinetoplastida:
Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae).
Walters LL, Irons KP, Guzman H, Tesh RB (1993b) Formation and composition of the peritrophic
membrane in the sand fly, Phlebotomus perniciosus (Diptera: Psychodidae). J Med Entomol
30:179–198
Walters LL, Irons KP, Guzman H, Tesh RB (1995) Peritrophic envelope of Lutzomyia spinicrassa
(Diptera: Psychodidae). J Med Entomol 32:711–725
Warburg A, Tesh RB, McMahon-Pratt D (1989) Studies on the attachment of Leishmania flagella
to sand fly midgut epithelium. J Protozool 36:613–617
Watts DM, Tesh RB, Siirin M, Travassos da Rosa A, Newman PC, Clements DE, Ogata S, Coller
BA, Weeks-Levy C, Lieberman MM (2007) Efficacy and durability of a recombinant sub-
unit West Nile vaccine candidate in protecting hamsters from West Nile encephalitis. Vaccine
25:2913–2918
Weaver SC, Salas RA, de Manzione N, Fulhorst CF, Duno G, Utrera A, Mills JN, Ellis BA, Ksiazek
TG, Tesh RB (2000) Guanarito virus (Arenaviridae) isolates from endemic and outlying locali-
ties in Venezuela: sequences comparisons among and within strains isolated from Venezuelan
hemorrhagic fever patients and rodents. Virology 266:189–195
Weaver SC, Salas RA, de Manzione N, Fulhorst CF, Travassos da Rosa APA, Duno G, Utrera A,
Mills JN, Ksiazek TG, Tovar D, Guzman H, Kang W, Tesh RB (2001) Extreme genetic diver-
sity of Pirital virus (Arenaviridae) isolates from western Venezuela. Virology 285:110–118
Whittingham HE (1924) The etiology of phlebotomus fever. J State Med 32:461–469
Widman DG, Ishikawa T, Giavedoni LD, Hodara VL, de la Garza M, Montalbo JA, Travassos
da Rosa AP, Tesh RB, Patterson JL, Carrion R Jr, Bourne N, Mason P (2010) Evaluation of
RepliVAX WN, a single-cycle flavivirus vaccine, in a non-human primate model of West Nile
virus infection. Am J Trop Med Hyg 82:1160–1167
Wu X, Lu L, Guzman H, Tesh RB, Xiao SY (2008) Persistent infection and associated nucleotide
changes of West Nile virus serially passaged in hamsters. J Gen Virol 89:3073–3079
Xiao SY, Zhang H, Guzman H, Tesh RB (2001a) Experimental yellow fever virus infection in the
golden hamster (Mesocricetus auratus). II. Pathology. J Infect Dis 183:1437–1444
Xiao SY, Guzman H, Zhang H, Travassos da Rosa AP, Tesh RB (2001b) West Nile virus infection
in the golden hamster (Mesocricetus auratus): a model of West Nile encephalitis. Emerg Infect
Dis 7:714–721
Xiao SY, Zhang H, Yang Y, Tesh RB (2001c) Pirital virus (Arenaviridae) infection in the Syrian
golden hamster, Mesocricetus auratus: a new animal model for arenaviral hemorrhagic fever.
Xiao SY, Guzman H, Travassos da Rosa APA, Zhu HB, Tesh RB (2003) Alteration of clinical
outcome and histopathology of yellow fever virus infection in a hamster model by previous
infection with heterologous flaviviruses. Am J Trop Med Hyg 68:695–703
Xiao SY, Sbrana E, Watts DM, Siirin M, Travassos da Rosa AP, Tesh RB (2005) Experimental
infection of prairie dogs with monkeypox virus. Emerg Infect Dis 11:539–545
Young DG, Duncan MA (1994) Guide to the identification and geographic distribution of
Lutzomyia Sand Flies in Mexico, the West Indies, Central and South America (Diptera:
Psychodidae), Memoirs of the Amer Entomological Inst No. 54. Associated Publishers
American Entomological Institute, Gainesville
538 R. B. Tesh
Young DG, Morales A, Kreutzer RD, Alexander JB, Corredor A, Tesh RB (1987) Isolations of
Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) from cryopreserved Colombian
sand flies (Diptera: Psychodidae). J Med Entomol 24:587–589. Erratum in: J Med Entomol
24(6):703, 1987
Zhao G, Droit L, Tesh RB, Popov V, Virgin HW, Wang D (2011) The Genome of Yoka poxvirus.
J Virol 85:10230–10238
Ziegler SA, Lu L, da Rosa AP, Xiao S-Y, Tesh RB (2008) An animal model for studying the patho-
genesis of chikungunya virus infection. Am J Trop Med Hyg 79:133–139
Zirkel F, Roth H, Kurth A, Drosten C, Ziebuhr J, Junglen S (2013) Identification and characteriza-
tion of genetically divergent members of the newly established family Mesoniviridae. J Virol
87:6346–6358
Index
A C
Aedes aegypti, 65, 73, 79, 89, 130, 145, 146, Cardiopulmonary syndrome by Araraquara
164, 166, 167, 169, 172, 179–181, hantavirus, 503
238, 239, 242, 245–248, 250, 268, Career, vii, 4, 23–25, 33, 35, 36, 39, 41, 45,
273–276, 279, 373, 377, 393, 413, 51, 57, 61, 62, 71, 72, 77, 78, 85,
414, 425–461, 469, 473, 493–495, 94–103, 105, 107–109, 135, 138,
500, 507, 508 148, 155, 159–162, 164, 181, 183,
Africa, vii, 35, 72, 74, 116, 181, 188, 189, 184, 206, 223, 228, 229, 231, 255,
191–195, 197–200, 211, 221–223, 257, 282, 295, 360, 378, 383, 384,
249, 253, 331, 361, 362, 365, 367, 390, 391, 394, 395, 402, 403,
372, 374, 378, 382, 391, 406, 415, 415–417, 419, 426, 465,
418, 486, 493, 516, 521 500, 513–523
Anopheles, 222, 439, 468 Career path, 3, 4, 24–36, 97, 108, 206, 225, 360
Antibody-dependent enhancement of dengue Centers for Disease Control (CDC), v, vii, 11,
virus infection, 255, 259–260, 263 17, 31, 60, 81, 82, 85, 175, 179,
Araraquara hantavirus (ARQV), 502–503 189, 197, 235, 268, 273, 285, 295,
Arbovirology, vi, vii, ix, x, 3–109, 127, 130, 296, 316, 332, 344, 346, 347,
149, 151, 159, 160, 164, 180, 188, 350–352, 359–361, 363, 367, 369,
205–225, 228, 229, 238, 254, 257, 370, 372, 374, 378, 383, 386, 397,
295, 331, 332, 360, 365, 381–420, 399–401, 404, 406–408, 411, 415,
428, 432, 458, 460, 465, 486, 490, 417–420, 426, 428, 429, 432, 447,
493–508, 513–523 473, 489, 519
Arbovirus, 4, 115, 180, 188, 208, 228, 329, Chikungunya, ix, x, 35, 53, 55, 57, 65, 66, 70,
359, 382, 428, 465, 485, 493, 514 79, 81, 84, 127, 130, 134, 136, 151,
Arbovirus diagnosis, 43, 76 152, 177, 182, 192, 195, 196,
Arbovirus Information Exchange, 11, 387, 392 227–298, 331, 362, 392, 396, 469,
Armed Forces Research Institute of Medical 486, 507, 516, 523
Sciences, 138 Cohort, 57, 73, 105, 120, 240, 246–247, 251,
282, 290, 296, 342–344, 382, 429,
434–436, 441, 442, 459, 460
B
Culex pipiens, 35, 172, 469, 470, 472
Bwamba, 192, 195, 205–225
Culex tarsalis, 30, 427, 467–472, 475, 476
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 539
Springer Nature Switzerland AG 2023
Field, https://doi.org/10.1007/978-3-031-21999-3
540 Index
D F
Defective interfering particles, 488, 489 Field trials, 177, 178, 285, 394
Dengue, 22, 118, 164, 191, 228, 362, 382, Flaviviruses, 38, 41, 53, 56, 63, 67, 70, 82, 88,
426, 469, 486, 500, 516 93, 118, 119, 148, 195, 196, 250,
Dengue hemorrhagic fever (DHF), 54, 55, 260, 262, 263, 265, 291, 361, 366,
165, 166, 179, 182, 228, 237, 238, 389, 393, 404, 476, 478, 488, 490,
240, 241, 244, 245, 247, 248, 491, 497, 500–502, 504, 516
251–256, 259, 260, 263, 266–271, 406th Medical General Laboratory
274, 277, 279, 281–283, 290, (MGL), 228–232
291, 383, 388, 395–397, 399,
406, 519
Dengue outbreaks, 130, 133, 135, 151, 254, G
271, 281, 429, 500 Gender equality, 3–109
Dengue shock syndrome (DSS), 165, 166, Genetic resistance, 41, 234, 291
171, 179, 182, 238, 240–248,
251–256, 260, 266, 267, 271, 277,
281–283, 290, 291, 388, 396, 399 H
Dengue vaccines, 130, 143, 256, 265, Haddow, A.J., 206–208, 210, 211, 213–216,
282–284, 291–298 219, 220, 391, 405
Diagnostics, 4, 22, 23, 35, 43, 50, 51, 55, Hantaan virus (HTNV), 14, 317–322, 326,
56, 60, 72, 73, 76, 93, 120, 122, 329–331, 338, 405
125, 127, 134, 146, 150–153, Hantaviridae, 316, 350
155, 162, 181, 191, 197, 232, Hantavirus pulmonary syndrome
236, 242, 321–323, 325, 328, (HPS), 316, 329, 339, 342–344,
329, 344, 349, 363, 367, 351, 419
455–458, 467, 500, 514 Hemagglutination-inhibition (HI) antibodies,
Diapause, 469, 470 233, 245, 246, 255, 256
Hemorrhagic fever, 12, 13, 15, 41, 49,
187–200, 237, 242, 254, 281, 318,
E 321, 365, 372, 378, 383, 384, 388,
East African Virus Research Institute 396–397, 405–412, 417, 418, 519
(EAVRI), 206–209, 212, 224, Hemorrhagic fever with renal syndrome
361, 387 (HFRS), 49, 237, 315–320,
Ebola, 17, 19, 36, 72, 189–190, 197–200, 352, 322–331, 337, 342–344
406, 407, 418, 419 Histoplasma capsulatum, 514
Emerging infectious diseases, 81, 91, 279, History of Medicine, 364
297, 350, 406, 517, 520 History of Structural Virology, 491
Entomology, 51, 58, 65, 68, 80, 82, 89, Host selection, 472, 473
160–162, 165, 188, 190, 274, 365,
402, 404, 431–440, 444, 448, 452,
455, 460, 465–478, 515 I
Epidemic hemorrhagic fever (EHF), 237, 315, Inapparent infection, 240, 369, 456
316, 318, 319, 321, 322 Institute of Research for Development (IRD),
Epidemics, 13, 115, 164, 189, 233, 315, 360, ix, 188, 190, 194, 200
382, 427, 467, 485, 495, 522
Epidemiology, ix, x, 4, 24, 43, 55, 59, 68, 71,
73, 74, 77, 85, 94, 123, 150, 155, J
161, 162, 165, 190, 193–194, 234, Japanese encephalitis, 51, 118, 125,
238, 240, 247, 253–256, 266, 272, 134, 144, 147–149, 153,
296, 315–352, 360, 361, 389, 392, 227–298, 389
411, 414, 427, 459, 466, 467, 490,
497, 498, 500, 504, 513–517,
519, 523 K
Epizootics, 94, 193, 196, 361, 390, 515 Korean hemorrhagic fever (KHF), 232,
Evidence-based intervention, 57 316–322, 344, 383, 396, 405
Index 541
L R
Lassa fever, 200, 322, 367–370, 372, 383, 407, Ribas, E., 494, 495
408, 412, 418 Rocio encephalitis, 498
Lutz, A., 495, 496 Rockefeller Foundation (RF), ix, 5, 6, 8, 9,
116, 192, 206, 238–240, 242, 245,
252–254, 256, 268, 272–274, 284,
M 285, 288, 291–294, 328, 331–333,
Manaus, 502, 505 359, 362, 382, 384–387, 390–393,
Mayaro Virus, 403, 449 403, 406, 407, 416, 428, 486, 514,
Medical history, 85, 147, 287, 359, 365 518, 519
Mentor, 4, 24–27, 32–34, 42, 43, 104, 107, Rockefeller Foundation Virus Lab (RFVL), 6,
160, 161, 183, 215, 257, 362, 402, 9, 362, 518
416, 426, 428, 429, 514 Rodent-borne viruses, 188, 341, 351, 519
Mosquito colonization, 468 Ruwenzori, 209–211
Mosquitoes, 5, 115, 161, 191, 206,
232, 325, 360, 388, 427, 466, 488,
493, 522 S
Sampling and laboratory methods,
360, 472–475
N Seoul virus (SEOV), 316, 326, 329–331, 333,
Nephropathia epidemica (NE), 318, 334, 336–349, 351
324–325, 344 Sindbis virus, 38, 49, 88, 89, 115, 128, 192,
Neutralizing antibodies, 173, 233, 244, 252, 195, 391, 403, 487–489
260, 263, 265–267, 271, 281, 291, Southeast Asia, 164, 188, 267, 383, 389,
319, 320, 366, 478, 515 396, 406
St. Louis encephalitis virus (SLEV), 27, 30,
86, 94, 265, 467, 469, 471,
O 475–477, 497, 500, 521, 522
Oropouche virus (OROV), 505 Students, 4, 22, 24–27, 30, 35, 38, 41, 43, 62,
Overt, 233, 235, 246, 260, 267, 328, 490 63, 75, 78, 85, 94, 96–98, 103–108,
116, 118, 138–140, 142, 153, 155,
159, 160, 162, 205, 221, 252, 258,
P 260, 262, 269, 295, 323, 334, 337,
Pasteur, ix, 9, 14, 42, 43, 64, 65, 67, 68, 74, 363, 394, 403, 413, 429, 442, 445,
134, 144, 188–196, 264, 282, 283, 466, 486–489, 500–503, 517,
291–293, 295, 297, 352, 361, 364, 521, 523
367, 372, 374, 378, 387, 519 Subclinical, 233, 240
Pathogenesis, ix, 40, 54–56, 62, 67, 71, 74, 85,
88, 93, 118, 129, 131, 135, 234,
238, 252, 254, 260, 270, 283, 295, T
298, 397, 398, 514, 521 Thailand, vii, 66, 130, 141, 151, 152, 216,
Pediatric dengue vaccine initiative, 291–298 220, 228, 235, 236, 238–240, 242,
Pet rats, 347–349 246–248, 251–253, 255, 256, 258,
Physiopathology of Hantavirus 263, 265, 267, 271, 272, 282, 288,
Cardiopulmonary Syndrome, 503 296, 330, 338, 396, 415, 419,
Prospective cohort studies, 246, 247, 431, 466
251, 434 Togaviruses, 38, 262, 490
Public health, 8, 141, 159, 228, 330, 360, 385,
426, 467, 494, 514
U
Uganda, 81, 82, 87, 198, 206, 208, 210–217,
Q 219, 220, 222–224, 240, 261, 377,
Quality assurance, 127, 147, 151, 442 391, 405
542 Index
University of Hawaii, 161, 164, 238, 257, 260, W

264, 265, 267, 292 Walter Reed Army Institute of Research
Urban infectious diseases, 446 (WRAIR), 229, 230, 234–236, 242,
Urban rats, 332, 333, 337, 339–340, 344–347 244, 253, 260, 264, 265, 285, 297,
386, 390
West Africa, 17, 187–200, 290, 359–378, 407
V Western equine encephalitis virus (WEEV),
Vaccines, 4, 125, 181, 189, 228, 325, 360, 30, 274, 467, 471, 475, 476
382, 486 West Nile virus (WNV), 24, 25, 30, 31, 35, 41,
Vector competence, 30, 82, 88, 165, 471, 475, 62, 66, 82, 84, 85, 88, 191, 275,
476, 517, 523 383, 391, 396, 402, 405, 466, 467,
Vector biology, 89, 274, 360, 517 469, 472, 475–478, 504, 521, 522
Vector borne diseases, 31, 64, 66, 79, 81, 89, Work-life balance, 25
120, 147, 180, 181, 188, 430
Vector control, 73, 128, 273–275, 293, 388,
404, 431, 432, 434–436, 438, 439, Y
448, 450, 455, 459, 460, 477, 522 Yellow fever, 5, 125, 181, 188, 207, 232, 329,
Viral diseases, 17, 22, 55, 188, 197, 361, 367, 360, 382, 431, 486, 493, 521
383–388, 398, 399, 406 Yellow Fever Research Institute (YFRI),
Viral pathogenesis, 56, 234 206, 212
Virology, vi, vii, 10, 14, 16, 22, 23, 25, 30, 38,
40, 41, 48, 50, 52, 54, 55, 58, 60,
71–73, 82, 94, 128, 134–137, 140, Z
143, 145, 149, 188–191, 229, 232, Zika, vi, ix, x, 53–55, 57, 63, 65, 66, 70, 79,
234, 239, 244, 245, 268, 270, 277, 81, 82, 84, 85, 88, 94, 127, 151,
315, 328, 359, 360, 362, 384, 387, 177, 191, 192, 195, 206, 208, 212,
388, 390, 396, 409, 414, 416, 214, 259, 263, 297, 391, 405, 415,
485–491, 496, 498, 500, 502 454, 455, 469, 486, 491, 506,
Virus Research Center in Ribeirão 507, 523
Preto, 493–508 Zoonotic diseases, x, 94, 332, 414

History of Arbovirology: Memories From The Field: Nikos Vasilakis Laura D. Kramer Editors

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History of Arbovirology: Memories From The Field: Nikos Vasilakis Laura D. Kramer Editors

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Nikos Vasilakis

Laura D. Kramer Editors

ISBN 978-3-031-21998-6 ISBN 978-3-031-21999-3 (eBook)

Placitas, NM, USA Karl M. Johnson

As of today, despite all the technological advances in understanding arbovirus

Galveston, TX, USA Nikos Vasilakis

Frederick AM (2012) The foundations of virology: discoverers and discoveries, inventors

Part I Personal Reflections

Twenty-Seven Years of Field Studies on Dengue and Aedes aegypti

Nikos Vasilakis Professor Nikos Vasilakis is the vice

Department of Pathology, Center for Vector-borne and

Laura D. Kramer Professor Emeritus Laura Kramer

Former (retired) Director Arbovirus Laboratory,

John Aaskov Queensland University of Technology, Brisbane, QLD, Australia

Sergey V. Alkhovsky D.I. Ivanovsky Institute of Virology, Ministry of Health of

Desiree A. LaBeaud Stanford University, Stanford, CA, USA

Abstract Women have made significant contributions to arbovirology, which

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 3

It is now almost 60 years since I (LDK) became a doctoral student, enthusiastically

2 The Pioneers: Profiles of Selected Female Scientists (Who

Laura D. Kramer and Martine Jozan

Fig. 4 Sonja Buckley and

Fig. 5 Delphine Clark.

Fig. 6 Dr. Robert Shope

Because of the proliferation of arbovirus research, a conference was convened at

visceral (hemorrhagic) tick-borne viruses, e.g., Crimean-Congo hemorrhagic fever

According to Telford Work, Helen Leshchinskaya had seen more hemorrhagic

Fig. 12 Helena Leschinskaya. (Kindly provided by Martine Jozan)

Fig. 13 Helena Leschinskaya in field. (Kindly provided by Martine Jozan)

Fig. 17 Patricia Webb. (Photo courtesy of Fred Murphy 1976)

Weapons Working Committee of the South African Non-proliferation Council, as

Fig. 21 Margaretha Isaacson in field. (Photo in public domain)

• Recommended reading. The Memoirs of Margaretha Isaacson. Annals of the

Fig. 22 Pedro Galindo, Enid Rodaniche, Harold Trapido

Fig. 23 Gladys Sather

3 My Career Path: Coming Full Circle from Southam

Fig. 33 My mentors – (1)

Fig. 34 (Mentor 2, PhD)

Fig. 35 (Mentor 3,

Fig. 36 (Mentor 4, career)

Fig. 38 (Mentor 5, ProMED) Jack Payne Woodall (1935–2016)

considered, Wadsworth Center is a unique environment with a large number of

arbovirology as the third female in its history by the American Committee on

Laura D. Kramer and Martine Jozan

Fig. 43 Diane Griffin

Diane Griffin, PhD (Fig. 43)

Fig. 44 Margot Brinton

Margo Brinton, PhD (Fig. 44)

now, in the arbovirus laboratory (Claude Hannoun, Director) I continued working

Fig. 47 Carol Blair

Fig. 50 Patricia Nuttall in front of tick painting

Fig. 51 Polly Roy

understood virus that has contributed to fundamental discoveries in molecular and

Fig. 55 Mary Jane

• Recommended reading. (Schrage 2019) Q&A: Husker alumna talks small-town

Fig. 56 Elizabeth Ann McGraw

Andrea Gamarnik, PhD (Figs. 57 and 58)

Fig. 57 Andrea Gamarnik

Fig. 58 Andrea Gamarnik

Fig. 59 Ana Fernandez

Fig. 61 Rebecca Rico-Hesse netting birds

antibodies, for efficacy in treating the clinical signs of DF or DHF in humanized

Fig. 62 Eva Harris

Placitas, NM, USA Karl M. Johnson

Galveston, TX, USA Nikos Vasilakis

Part I Personal Reflections

Twenty-Seven Years of Field Studies on Dengue and Aedes aegypti

2 The Pioneers: Profiles of Selected Female Scientists (Who

3 My Career Path: Coming Full Circle from Southam

5 Challenges in Pursuing a Professional Career

5.3 Academic Faculty Positions