Hantavirus

Stephanie Bagley Eugene Khandros Nicholas Bevins

History
Hantaviruses are rodent-borne viruses which may be transmitted to humans in aerosolized urine, feces, or saliva, and occasionally by bite. Hantaviruses often cause either hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS) Other hantaviruses are not known to be human pathogens.

Hantavirus Outbreak in the US

HPS was first described in the United States in May 1993 during the investigation of a cluster of cases of acute adult respiratory distress in the Four Corners region. HPS was found to be caused by a previously unknown hantavirus, Sin Nombre, detected in deer mice. Sin Nombre caused approximately 200 confirmed cases of HPS during the outbreak, that led to a 50% mortality rate.

Hantavirus in the US
Prior to the HPS outbreak, the only known hantaviruses were those that caused HFRS At least three other hantaviruses, New York Virus, Bayou Virus, and Black Creek Canal virus have since been confirmed to cause HPS in the U.S.

HPS in South America

The discovery of HPS in North America led to retrospective studies in South America. More than 140 cases of HPS confirmed in Argentina. The Andes hantavirus was discovered in long tailed pygmy rice rats in southern Argentina. Andes Virus is the only known hantavirus to be transmitted person-to-person.

History of HFRS
While HPS has only been identified since 1993, HFRS has a much longer and complex history. HFRS may have been recognized in China as early as 1000 years ago. HFRS described in 1913 Russian records

History of HFRS
Outbreaks of HFRS in the 1930s: Russia (1932) Japanese troops in Manchuria (1934) Sweden (1934)

Characterization of HFRS
1934 -Japanese outbreak led to studies by Japanese physicians 1940- Japanese physicians compiled a clinical and pathological description of what was then called epidemic hemorrhagic fever. First to implicate mice in disease transmission. Physicians injected filtrates of tissue from Apodemus agrarius carrying the hantavirus Hantaan into human subjects.

History of HFRS
1939-Russian studies also implicated mice in disease transmission. 1961- Moscow outbreak affecting 113 of 186 workers linked to rodent shipment

Characterization of HFRS
1967-Russian scientists Yankovski and Povalishina provide more insight into disease. Incubation period up to 6 weeks Cycles of virus activity parallel population cycles of field mice Transmission of disease occurred via inhalation of dust contaminated by rodent excretions.

Recent HFRS Cases

1951- outbreak in Korea during the war affected 3,200 soldiers and resulted in a 7-15% mortality rate. Korean hemorrhagic fever (KHF) drew widespread international attention and was reclassified as HFRS in 1983 by the WHO. 1983-Several past outbreaks including KHF, epidemic hemorrhagic fever , Russian outbreaks all reclassified as HFRS by the WHO

Family Bunyaviridae
5 genera, 250 species
Genus Bunyavirus Human disease LaCrosse encephalitis, others

Phlebovirus
Nairovirus Tospovirus

Rift Valley fever, sandfly fever

Crimean-Congo hemorrhagic fever Plant virus, no known human disease

Hantavirus

Hemorrhagic fever with renal syndrome

Hantavirus pulmonary syndrome

Hantavirus Genus
Hantavirus Similarities
RNA viruses Lipid membrane Tri-segmented genome

Hantavirus Differences
Hantavirus transmitted through aerosolized rodent urine, feces and saliva. Others genera transmitted through arthropod vectors.

Epidemiology and Rodent Hosts

Each strain of hantavirus has a specific rodent host Hantavirus species appear to have coevolved with host rodent species Rodents carrying hantavirus are asymptomatic

Transmission of Hantaviruses
Chronically infected rodent Horizontal transmission of infection by intraspecific aggressive behavior

Virus is present in aerosolized excreta, particularly urine

Virus also present in throat swab and feces

Secondary aerosols, mucous membrane contact, and skin breaches are also sources of infection
Courtesy of CDC

Rodent Hosts
Virus Strain
Hantaan Dobrav Seoul a Thailand Prospect Hill Puumala Sin Nombre New York

Rodent Host
Apodemus agrarius
Apodemus flavicollis Rattus norvegicus

Murinae

Bandicota indicus
Microtus pennsylvanicus Clethrionomys glareolus Peromyscus maniculatus Peromyscus leucopus

Arvicolinae

Oryzomys palustris Bayou Black Creek Canal Sigmodon hispidus

Sigmodontinae

New World Hantaviruses

Sin Nombre
Peromyscus maniculatus

New York
Peromyscus leucopus

Muleshoe
Sigmodon hispidus

Prospect Hill
Microtus pennsylvanicus

Bloodland Lake

Isla Vista
Microtus californicus

Microtus ochrogaster

El Moro Canyon
Reithrodontomys megalotis

Bayou Oryzomys palustris Black Creek Canal

Sigmodon hispidus

Calabazo
Zygodontomys brevicauda Sigmodon alstoni Oligoryzomys Rio Mamore fulvescens Oligoryzomys microtis

Rio Segundo
Reithrodontomys mexicanus

Choclo

Cao Delgadito Orn

Unknown Host Laguna Negra Calomys laucha

Juquitiba

Maciel

Necromys benefactus

Hu39694

Oligoryzomys longicaudatus

Unknown Host

Bermejo
Oligoryzomys chacoensis

Lechiguanas
Oligoryzomys flavescens

Andes
Oligoryzomys longicaudatus

Pergamino
Courtesy of CDC

Akodon azarae

Hantavirus Pulmonary Syndrome

Countries with reported cases of HPS (no of cases)
Canada (36)

United States (335)

Countries with no reported cases of HPS

Panama (31) Bolivia (20) Chile (273)

Brazil (168)

Paraguay (74)
Uruguay (23) Argentina (404)

Rodent Hosts in the United States

Deer mouse (Peromyscus maniculatus) Carrier of Sin Nombre strain, primary agent of HPS in the US. 250-300 cases since discovery.

Rodent Hosts in the United States

White-footed Mouse (Peromyscus leucopus) Carrier of New York strain.

Molecular Biology of Hantavirus

Physical Properties Structure Genetics Replication Cycle Pathogenesis

Molecular Biology of Hantavirus

Physical Properties Structure Genetics Replication Cycle Pathogenesis

Virion Properties
Spherical or oval-shaped. 80-120 nm diameter Unique grid-like surface pattern, with 7-8 nm projections Lipid bilayer envelope Granulofilamentous interior Survive 12 hours at 4C, high salt concentration and nonphysiological pH. Survives 1-3 days after drying. Exposure to lipid solvents and nonionic detergents destroys viral envelope

Molecular Biology of Hantavirus

Virion Properties Structure Genetics Replication Cycle Pathogenesis

Structure

Structural Proteins
Membrane glycoproteins (G1 and G2) Polymerase (L)

Nucleocapsid proteins (N)

Membrane Glycoproteins
G1: 64-67kDa G2: 54 kDa, highly conserved Integral membrane proteins G1-G2 heterodimers form 8 nm projections on virion surface Cysteine-rich Contain asparagine-linked sugar groups Important in cell entry and pathogenesis

Nucleocapsid Protein (N)

48 kDA Complexes with genomic vRNA in virus, as well as with cRNA after infection, but not with mRNA Necessary for virus replication and packaging

Polymerase (L)
247 kDA RNA-dependent RNA polymerase (RdRp) Complexed with ribonucleocapsids in virion Endonuclease activity to cleave host mRNA Transcriptase activity for making cRNA and mRNA from vRNA Helicase activity to unwind vRNA during transcription

Molecular Biology of Hantavirus

Physical Properties Structure Genetics Replication Cycle Pathogenesis

Genomic Organization
Tripartite negative sense genome Small (S) segment, 1.7-2.1kb, codes for N nucleocapsid protein Medium (M) segment, 3.6-3.7kb, codes for G1 and G2 glycoproteins Large (L) segment, 6.5 kb, codes for L polymerase protein

Coding Strategy

Panhandle Structure
Conserved repeated complementary sequences at 5 and 3 ends form panhandle structures

Transcription
Viral polymerase transcribes negative-strand vRNA to mRNA Polymerase acts as a endonuclease and cleaves host mRNAs 7-18 nt from the 5 cap The capped oligonucleotides act as primers required to initiate transcription After transcription is primed and the first repeat of the terminal sequence is transcribed, polymerase slips and realigns the nascent RNA, then continues transcription

Replication
Viral polymerase transcribes negativestrand vRNA to sense cRNA cRNA is used as template to make more negative-strand vRNA pppG is used to prime cRNA and vRNA synthesis Same prime and realign strategy

Prime and Realign

Transcription and Replication

Molecular Biology of Hantavirus

Physical Properties Structure Genetics Replication Cycle Pathogenesis

Attachment

Entry
Uncoating Transcription Replication Release

Assembly Translation

Attachment
Viral G1 and G2 glycoproteins interact with cell surface receptors Pathogenic hantaviruses bind 3 integrins Non-pathogenic hantaviruses bind 1 receptors

Entry and Uncoating

Virus particles bound to integrin receptors are taken in by receptor mediated endocytosis Newly formed vesicles are acidified Acidic environment changes conformation of G1 and G2 Viral and cell membrane fuse Genomic material and polymerase are released into cytoplasm

Entry

Primary Transcription
Transcription of negative sense vRNA to mRNA Viral polymerase (RdRp) transcribes nucleoprotein-coated vRNA Capped oligonucleotides from cells own mRNA are used to prime transcription Follows prime and realign model

Translation
L and S segment mRNA is translated on free ribosomes in cytoplasm M segment mRNA translated on ER-bound ribosomes G1 and G2 peptides produced from M mRNA are cleaved cotranslationally Separate signal sequences for G1 and G2 cause ER attachment and embed the peptides in ER membrane (Signal Hypothesis)

Translation

Genome Replication
vRNA is used as a template by viral polymerase to make sense strand cRNA cRNA is used as a template to make more negative strand vRNA More genetic material means more virions produced

Secondary Transcription
Extra vRNA synthesized during replication is used as template to make mRNA Since more template is present after vRNA is replicated, more mRNA can be transcribed, and more viral proteins can be made

Virion Assembly
Membrane-bound G1 and G2 peptides are transported to Golgi and carbohydrates are attached by N-linked glycosylation vRNA complexes with N nucleopcapsid protein, forms looped panhandle structure, and complexes with L polymerase

Virion Assembly

Virion Release Scenario 1

Nucleocapsid complexes bud into the Golgi membrane with G1 and G2 embedded Virion particle is formed inside the Golgi Virions are transported to cell membrane by vesicles and released by exocytosis, just like in secretion Viruses may prefer different cell surfaces for release

Virion Release Scenario 2

Sin Nombre and Black Creek Canal viruses G1 and G2 embedded into cell membrane through Golgi vesicles Virions bud from cell membrane, not through Golgi

Molecular Biology of Hantavirus

Physical Properties Structure Genetics Replication Cycle Pathogenesis

Hantavirus and Host Cells

Virus replication typically halts host macromolecule synthesis Hantavirus replication does not affect host cells natural functions Hantavirus release does not require host cell lysis Hantavirus is able to establish a persistent infection in rodent host cells

Integrins
Heterodimeric receptors composed of and subunits Present on endothelial cells, macrophages, and platelets cells affected by Hantavirus infection Normally involved in regulation of endothelial cell adhesion, platelet aggregation, Ca++ channel activation, and extracellular matrix interactions, including cell migration

3-Integrins
Required for infection by pathogenic Hantaviruses 1 integrins are used by non-pathogenic strains Attachment of G1/G2 proteins of viroid to integrin initiates endocytosis, but also activates the receptor Variation in virus G1/G2 protein may account for severity of disease

Hantavirus Infection Pathogenesis

Binding of Hantavirus glycoproteins to 3 integrin causes disruption of vascular integrity Capillaries become more permeable Arteriole vasoconstriction and vasodilation are disrupted Binding to platelet receptors affects clotting and platelet function

Immune Reaction
Immune system activated against Hantavirus epitopes Virus epitopes expressed on surface of host cells triggers cytotoxic T-cell attack on host tissues Symptoms are consistent with inflammatory response

Laboratory Diagnosis of Hantavirus

Hantavirus is difficult to culture, so morphological identification is difficult RT-PCR using primers for conserved genome regions allows confirmation of infection PCR product can be sequenced and compared to known viral sequence database for species identification

Clinical Presentation of Hantavirus Infection

Three different clinical manifestations of hantavirus infection caused by different viral strains
Hemorrhagic fever with renal syndrome (HFRS) Found in Europe and Asia Nephropathia Epidemica (NE) Found in Europe Hantavirus pulmonary syndrome (HPS) Found in north and south America

HFRS
A group of clinically similar diseases that occur throughout Europe and Asia Includes several diseases that formerly had other names, including Korean hemorrhagic fever, epidemic hemorrhagic fever and nephropathia epidemica ~15% fatality

Stages of Hemorrhagic Fever with Renal Syndrome (HFRS)

1)Incubation (4-40 days) 2)Febrile Phase (3-5 days) 3)Hypotensive Phase (hours to days) 4)Oliguric Phase (3-7 days) Recovery: 5)Diuretic Phase (2-21 days) 6)Convalescent Phase (2-3 months)

 3-5 days Characterized by fever, chills Headache, severe myalgia (muscle pain), nausea Blurred vision, photophobia, eye pain caused by movement Flushing of face, V-area of the neck and back Petechiae (small red spots on skin) Abdominal pain and back pain. Thirst, edema, hemoconcentration, postural hypotension

Febrile Phase

Hypotensive phase
Hours to days Blood pressure decrease, hypovolemia (decreased blood volume), shock Worsening of bleeding manifestations: petechiae, epistaxis (nosebleed), gastrointestinal and intracranial bleeding Levels of urea and creatinine in blood rise, proteinuria (excessive protein in urine) Leukocytosis, thrombocytopenia (decreased # of platelets)

Oliguric Phase
3-7 days Marked by decreased urine production due to renal (kidney) dysfunction Hypervolemia (high blood volume) leading to hypertension Blood electrolyte imbalance Continuation of hemorrhagic symptoms Severe complications: cardiac failure pulmonary edema (swelling of lungs), and cerebral bleeding

Diuretic Phase
Several days to several weeks Beginning of recovery 3-6 liters of urine/ day; return to normal renal activity Anorexia, fatigue due to dehydration

Convalescent Phase
2-3 months Progressive improvement in glomerular filtration, renal blood flow, and urine concentrating ability

Clinical Testing for HFRS

Thrombocytopenia (low platelet count) is a signifier Urine tests for albuminuria (abnormally high amounts of the plasma protein albumin in the urine) Urine tests for microhematuria (microscopic amounts of blood in the urine)

Problems Diagnosing HFRS

Early symptoms resemble influenza More serious symptoms of hypotensive phase have acute onset

Nephropathia Epidemica (NE)

Puumala hantavirus strain Common mild form of HFRS in Europe Similar sequence of symptoms as HFRS, but much milder Only 6% of serologically confirmed cases require hospitalization

HPS
1993 four corners outbreak Cases found in almost all of the Americas ~50% fatality

Stages of Hantavirus Pulmonary Syndrome (HPS)

1) 2) 3) 4) 5) Incubation (4-30 days) Febrile phase Cardiopulmonary phase Diuretic phase Convalescent phase

Febrile Phase
3-5 days Fever, myalgia, malaise Other symptoms: headache, dizziness, anorexia, nausea, vomiting, and diarrhea.

Cardiopulmonary Phase
4-24 hours Presentation and rapid progression of shock and pulmonary edema (4-24h non-productive cough and tachypnea (shortness of breath) Hypovolemia due to progressive leakage of high protein fluid from blood to lung interstitium and alveoli Hypotension and oliguria Thrombocytopenia (often present in febrile phase as well) Death within 24-48 hours due to hypoxia (lack of oxygen) and/or myocardial failure

Diuretic Phase
Several days to several weeks Beginning of recovery Rapid clearance of pulmonary edema Resolution of fever and shock Anorexia, fatigue due to dehydration

Convalescent Phase
Up to 2 months Results in chronic decreased small-airway volume and diminished alveolar diffusing capacity

Clinical Testing for HPS

Many lab tests and radiographs appear normal Serological tests more effective ELISA IgM capture assay, using either SNV, Laguna Negra, or Andes antigens are used in all countries that have previously detected cases Immunofluorescent test for the presence of antibodies Blood analysis also may find thrombocytopenia with platelet count less than 150,000 mm in 98% of cases

Problems Diagnosing HPS

Symptoms often confused with influenza Common signs of upper respiratory disease such as sore throat, sinusitis, and ear pain not usually present Abdominal pain often misinterpreted as appendicitis Many doctors outside endemic regions fail to recognize or have sufficient testing

Treatment of Hantavirus Infection

General care, alleviation of symptoms Ribavirin (HFRS) ECMO (HPS)

General Care
HFRS General treatment for renal failure Hydration Dialysis HPS General treatment for pulmonary pathology Administration of oxygen

Extra Corporeal Membrane Oxygenation

Removes blood from the body and artificially removes CO2 and adds O2 Costly Difficult

ECMO

Ribavirin
Administered intravenously Shown to be effective against HFRS causing strains Not shown to be effective against HPS causing strains

Terrorism

Terrorism
A policy intended to strike with terror those against whom it is adopted; the employment of methods of intimidation; the fact of terrorizing or condition of being terrorized.

One mans terrorist is another mans freedom fighter.

Considerations of Pathogens for Use in Bioterrorism

Health effects Epidemiology Cost effectiveness Psychological effects Economic impact

CDC classifications
Category A Highest priority organisms that can be easily disseminated or transmitted form person to person, results in high mortality rates and have the potential for major public health impact, might cause public panic and social disruption, and require special action for public health preparedness.

CDC Classifications
Category A Anthrax (bacillus anthracis) Botulism (clostridium botulinum) Plague (Yersinia pestis) Smallpox (variola major) Tularemia (Francisella tularensis) Viral hemorrhagic fevers

CDC Classifications
Category B Second highest priority organisms that are moderately easy to disseminate, results in moderate mortality rates, and require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance.

CDC Classifications
Category B Typhus fever Viral encephalitis Ricin toxin Food and water safety threats

CDC Classifications
Category C Third highest priority organisms that include emerging pathogens that could be engineered for mass dissemination in the future because of availability, ease of production and dissemination, and potential for high morbidity and mortality rates and major health impact.

CDC Classifications
Category C Nipah Virus Crimean-Congo Hemorrhagic Fever virus Yellow fever Multi-drug resistant TB Influenza Rabies

CDC Classifications
HFRS causing strains are Category A because of high infectivity and morbidity HPS causing strains are Category C because of low infectivity

Considerations of Pathogens for Use in Bioterrorism

Health effects Epidemiology Cost effectiveness Psychological effects Economic impact

Health Effects
High lethality No or ineffective treatment

Health Effects
Hemorrhagic Fever with Renal Syndrome Medium lethality Dramatic visual change in patients (psychological) Some success with antiviral treatments Hantavirus Pulmonary Syndrome High Lethality No effective treatment

Considerations of Pathogens for Use in Bioterrorism

Health effects Epidemiology Cost effectiveness Psychological effects Economic impact

Epidemiology
Medium incubation time in order to cause secondary infections Rodent vector Spread through aerosol HFRS causing strains known to transmit human to human Suspected human to human transmission of HPS causing strain in Argentina (Andes Virus)

Andes Virus
1996 outbreak in rural Argentina Spread to people whose only contact was a car ride Spread to several doctors caring for HPS patients Low rodent population in Argentina at the time (early spring)

Aerosolization
Virus only infectious for 1-3 days outside of a host because of a weak lipid envelope The number of particles needed to cause a human infection is not known

Considerations of Pathogens for Use in Bioterrorism

Health effects Epidemiology Cost effectiveness Psychological effects Economic impact

Cost Effectiveness
What resources would be required to successfully disseminate Hantavirus into a large population?

Cost Effectiveness
Preparation of virus using cell culture (difficult, need training and equipment) Aerosolization? Contaminated water? Creation of infected rodent population?

Considerations of Pathogens for Use in Bioterrorism

Health effects Epidemiology Cost effectiveness Psychological effects Economic impact

Psychological Effects
HFRS causes dramatic visual effects in patients HPS would be especially difficult to diagnose outside of its normal range Media coverage super flu hemorrhagic fever

Mouse Virus in City! Everyone will Die!

Nothing to Fear say Liberal Doctors

Considerations of Pathogens for Use in Bioterrorism

Health effects Epidemiology Cost effectiveness Psychological effects Economic impact

Economic Impact
What effect would an outbreak of hantavirus have on the economy? Treatment Disruption of work Hysteria Even a small outbreak could cause a large disruption

Why Hantavirus Would Be a Good Terrorist Weapon

There is no cure for HPS infection Fairly long incubation period between infection and onset of symptoms Difficult diagnosis of HPS High lethality of HPS

Why Hantavirus Would Not Be a Good Terrorist Weapon

Low infectivity of HPS Difficult production Not stable

Prevention
Vaccines Hygiene

Vaccines
E. Coli expressed truncated nucleocapsid as an immunogen Naked DNA Recombinant non-pathogenic virus Rodent brain-derived Cell culture derived Inactivated virus

Hygiene
Prevent aerosolization of virus from rodent excrement Dampen surfaces with detergent before cleaning General hygiene

Weapons of mass destruction, including evil chemistry and evil biology, are all matters of great concern, not only to the United States but also to the world community ~ John Ashcroft