PERSPECTIVES

DENV molecular pathogenesis that have

V E C TO R - B O R N E D I S E A S E S — O P I N I O N
been particularly difficult to understand
using the current experimental methodolo-
Barriers to preclinical investigations gies. We also discuss the paradox that fur-
ther substantial advances in the field might
of anti-dengue immunity and dengue be possible only through controlled animal
experimentation, which has inherent limita-
pathogenesis tions because animal models do not pre-
cisely replicate human disease. We propose
that this contradiction can be reconciled
Ashley L. St. John, Soman N. Abraham and Duane J. Gubler with the choice of appropriate animal mod-
els to answer specific experimental questions
Abstract | Dengue virus (DENV) is a human pathogen that causes severe and and with cautious interpretation of results,
potentially fatal disease in millions of individuals each year. Immune-mediated keeping in mind the multifaceted complexity
pathology is thought to underlie many of the complications of DENV infection in of the interactions between DENV and the
humans, but the notable limitations of the available animal models have impeded human host.
our knowledge of the interactions between DENV and the immune system. In this Dengue virus infection in humans
Opinion article, we discuss some of the controversies in the field of dengue DENV is a member of the family Flaviviridae
research relating to the interaction between DENV and the mammalian host. We (TABLE 1), which contains several other
highlight key barriers hindering our understanding of the molecular pathogenesis notable human pathogens. There are four
of DENV and offer suggestions for the most effective ways in which the role of the antigenically distinct DENV serotypes
(DENV1–DENV4), which have a distribu-
immune system in the protection from, and pathology of, DENV infection can be
tion throughout tropical regions of the world
addressed experimentally. similar to that of their principal mosquito
vector, Aedes aegypti 2. The key pathophysio­
Dengue fever has emerged as a major global analyse the mechanisms underlying DENV- logical host response to DENV infection
public-health problem over the past four induced pathologies. Early key studies that manifests in the form of increased vascular
decades. Closely tied to unprecedented elucidated the course of infection, the inci- permeability, plasma leakage, microvascular
human population growth, urbanization, dence of pathological complications and the bleeding and reduced functioning of the
globalization and the lack of effective vector characteristic symptoms were carried out in coagulation cascade. Current estimates sug-
control, both the causative viruses and the experimentally infected human subjects4,5. gest that between 1% and 70% of individuals
mosquito vectors that transmit them have Today, there are limitations on human who experience infection have mild haem-
spread globally in the tropics, resulting in experimentation because of ethical and reg- orrhagic manifestations such as petechiae,
an increased frequency and magnitude of ulatory concerns, and because of the expense purpura, ecchymoses and epistaxis (nose-
epidemics and the emergence of the severe of undertaking large studies in genetically bleed)6,7 (FIG. 1). Severe dengue, known as
form of disease, dengue haemorrhagic fever diverse populations. Furthermore, human DHF and dengue shock syndrome (DSS),
(DHF)1,2. In 2013, an estimated 3.6 billion studies using patient populations (as occurs in <1% of infections2 and can be life-
people, more than half of the world’s popula- opposed to experimental infections) may threatening. During DHF–DSS, bleeding
tion, live in areas that are at risk for dengue show a correlation, but do not show une- involves multiple organs, frequently includ-
virus (DENV) infection3. There are neither quivocal causation, as these studies lack the ing the gastrointestinal tract, and fluid may
vaccines nor specific antiviral therapies ability to measure the responses of patients pool within body cavities (FIG. 1); lesions in
available for this disease. in comparison to exact controls. For dengue, the blood vessels or signs of necrosis have not
The research that has defined our additional variables could include unidenti- been observed, even in fatal cases8. DSS, the
understanding of the pathogenesis and fied factors that might influence the host’s most severe form of DHF, is characterized by
immunology of DENV infection can be susceptibility to disease in addition to influ- a rapid, weak pulse and sudden drop in blood
predominantly segregated into three types encing the disease course. Although animal pressure, which is the result of collapse of
of studies: those examining patients with models have supplemented our knowledge, the vascular system owing to hypovolaemia
dengue, to describe the clinical presentation; unfortunately a single animal model has not caused by vascular leakage.
in vitro studies to define the viral life cycle been able to faithfully reproduce all aspects It is not fully understood why most indi-
or investigate direct interactions between of human DENV infection. viduals resolve DENV infections quickly
DENV and cultured cells; and controlled In this Opinion article, we highlight the and without complications, whereas others
hypothesis-driven animal experiments to key aspects of anti-dengue immunity and experience a potentially fatal vascular leak

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© 2013 Macmillan Publishers Limited. All rights reserved


Table 1 | Important flavivirus human pathogens
Flavivirus species Prominent pathologies during human infection
Dengue virus (DENV1–DENV4) Fever–arthralgia–rash syndrome, plasma leakage, haemorrhage
and severe disease associated with organ failure
All four serotypes can cause a spectrum of illness ranging from
asymptomatic to severe and fatal disease, as determined by
a complex interaction of the virus with the innate immune
response of the individual host; the strain of virus, the
previous infection history and age of the host, and host
genetics all influence disease severity.
Yellow fever virus Fever, nausea, abdominal pain, jaundice and haemorrhage
West Nile virus Fever, myalgia, encephalitis and long-term neurological defects
Japanese encephalitis virus Fever, encephalitis and long-term neurological defects
Tick-borne encephalitis virus Meningitis, encephalitis, mild fever and long-term neurological
defects
syndrome or even severe frank haemorrhag- human skin explants15, a detailed analysis of
ing. Risk factors for severe disease include the DC and monocyte subsets that sustain
the presence of heterologous antibodies infection within the skin has not been car-
from a previous infection9, certain strains ried out. Most efforts to define the human
or subtypes of infecting virus10,11, and the cell types infected by DENV have sought
age and genetic background of the human to identify viral replication at a much later
host 2,12–14. stage of infection: in the blood during peak
viraemia. In monkeys and mice, antigen
Key questions for dengue virus immunity has been detected in cells that are morpho­
Dengue remains a disease with many logically consistent with mononuclear
unanswered questions. There are multiple phagocytes19,20 (which are thought to be one
levels of DENV–host interaction from the of the prime targets of DENV infection),
time virus particles are injected by mos­ but this is not well documented in human
quitoes, to the establishment of systemic infection. Phenotypic differences potentially
infection, to its resolution. Each stage exist among the target cells for infection in
requires specialized considerations for the skin (early infection), in the lymphoid
effective experimental design to investigate organs (during viral dissemination) and in
the mechanisms underlying disease. various tissues when systemic infection has
been established.
The course of host–virus interactions
Initial target cells for infection. Little is Clearance of dengue virus from the injec-
understood regarding the early events fol- tion site. Early in DENV infection, immune
lowing DENV inoculation into the skin, cells are targets of infection, but also pro-
including immune responses at the site of mote DENV clearance from the skin. Much
inoculation and the impact of the infecting emphasis is placed on the role of DENV-
viral strain or subtype, both of which could specific neutralizing antibodies in viral clear-
potentially determine the host’s success in ance21. However, during primary infection,
viral clearance. The role of the infecting viral substantial levels of neutralizing antibodies
strain is discussed below. Subcutaneously should not be present until the later stages
injected virus can potentially encounter of disease, as the adaptive immune response
several cells of the immune system, includ- requires time to develop. This suggests that
ing tissue-resident dendritic cells (DCs), in the early days of infection additional,
macrophages and mast cells15,16 (FIG. 2). The antibody-independent mechanisms of viral
cell types that are first infected in the skin clearance from the injection site are required.
have not been extensively studied, but it It is possible that, as occurs in West Nile
is thought that macrophages and DCs are virus infection, γδ T cells play a part in viral
initial targets, and receptors have been iden- clearance from the skin22. In mice, natural
tified for DENV uptake by both cell types: killer (NK) cells and NK T cells (recruitment
the mannose receptor on macrophages and of which is promoted by activated skin-
DC‑SIGN (DC-specific ICAM‑3‑grabbing resident mast cells) have been implicated in
non-integrin 1) on DCs17,18 (FIG. 2). Aside viral clearance at the site of skin injection
from the demonstration that Langerhans and in draining lymph nodes16. Stromal and
cells are permissive to DENV infection in endothelial cells can also produce cytokines,
NATURE REVIEWS | MICROBIOLOGY VOLUME 11 | JUNE 2013 | 421
© 2013 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES
particularly when these cells are activated
by pro-inflammatory factors derived from
neighbouring immune cells23. Although
most immune cells release pro-inflammatory
mediators when they are infected with
DENV, some, such as mast cells16, can
directly detect and respond to the virus with-
out being infected. This emphasizes the fact
that there are likely to be additional mecha-
nisms of DENV structural recognition by the
immune system beyond antibody detection.
For cells that are infected, pro-inflammatory
responses to DENV replication are initiated
by intracellular pathogen recognition recep-
tors, including vesicle-associated Toll-like
receptor 3 (TLR3) and the cytosolic sensors
retinoic acid-inducible gene I protein (RIG‑I)
and melanoma differentiation-associated
protein 5 (MDA5; also known as IFIH1)24,25.
These pathways lead to the production
of pro-inflammatory cytokines, includ-
ing tumour necrosis factor (TNF), type‑I
interferons (IFNs), IFNγ and interleukin‑6
(IL‑6)24,25.
Systemic dengue virus infection. As DENV
disseminates in a host, it is detected first in
draining lymph nodes and then in remote
lymph nodes19. In humans and non-human
primates, this results in viraemia (which
can be detectable 24–48 hours before the
onset of clinical symptoms4), and DENV
can be isolated from the blood during the
acute phase of disease. At this stage, sub-
stantial changes occur to the cellularity of
the haematopoietic system, as would be
expected during systemic viral infection;
these changes include leukopenia, neutro-
penia, thrombocytopenia and, occasionally,
eosinophilia26 (FIG. 2). DENV infection is also
characterized by altered bone marrow cellu-
larity, but the mechanisms or consequences
of these changes are unknown26.
Vascular pathology, including microvas-
cular permeability of endothelial cells, occurs
as viraemia declines27. Although infected
endothelial cells have been observed in human
infection28, it is uncertain how frequently this
happens, because of the paucity of autopsies
on human patients with dengue. Human
endothelial cells can be infected in cell culture,
and infection of liver sinusoidal cells occurs
in immunocompromised animals29. Although
controversial, it has been suggested that
endothelial cells are not commonly infected
during human infection with DENV30,31 and
that the breakdown in the endothelial cell
barrier is consistent with other mechanisms
of vascular permeability26. Owing to both
the lack of evidence that endothelial cells are
directly targeted by DENV and the massive
PERSPECTIVES

not yet explained. Unfortunately, the com-

Headache, fever plexity of the interactions between DENV
and the host cannot be adequately modelled
Bleeding gums,
nose and eyes by in vitro manipulation of human cells and
Vascular symptoms:
Hypovolaemia
biological products outside the organs and
Low blood pressure systems that govern their responses to infec-
Vascular symptoms:
Leukopenia Shock tion. Not only are the specific target cells of
Thrombocytopenia DENV not clearly known, but also in vitro
Neutropenia Hepatic injury analysis does not capture the responses
Late eosinophilia Fluid pooling in
Reduced coagulation of cells that do not become infected but
body cavities
Gall bladder might nonetheless respond to DENV and
thickening to the inflammatory products in their
Skin symptoms: Haemorrhaging
Rash microenviron­ment in vivo. As a further
within organs
Bruising complication, interactions between DENV
Petechiae and host cells can change over the course of
Purpura Vomiting infection, which begins as a localized cuta-
Intestinal bleeding
neous infection with innate immune activa-
tion and develops into a systemic infection
characterized by viral replication in target
organs (including the lymphoid system) and
high viral titres in the blood19. The most
Infrequent
complications: severe pathologies associated with DENV
Joint pain Encephalitis infection frequently manifest in patients
Acute pancreatitis as viraemia resolves and fever begins to
Renal failure
Myocarditis subside27, but it is unclear at what stage of
Altered haematopoiesis
Splenic rupture infection these pathological processes are
Pulmonary haemorrhage initiated.

The role of immunological memory

Primary DENV infection results in long-
lasting immunity to the infecting serotype
and, potentially, partial immunity to subse-
Figure 1 | Dengue virus pathogenesis in humans.  Systemic infection with dengue virus (DENV)
Nature Reviews | Microbiology quent infection with other serotypes. Many
affects multiple organ systems. This diagram depicts the clinical symptoms and pathogenesis of
dengue in humans, across the spectrum of mild to severe disease. Clinical manifestations associated individuals in dengue-endemic countries
with dengue fever are listed in blue boxes, those associated with dengue haemorrhagic fever are listed have experienced DENV infection previ-
in purple boxes, and more rare complications of DENV infection are listed in the green box. ously. Re‑infection with the same serotype
as that which caused the primary infection
has not been documented, demonstrating
pro-inflammatory response that is detectable versus DHF–DSS in humans. Some evidence that immunity to a homologous DENV
in the serum of patients with dengue32, it has has been obtained for there being both lower strain can be highly protective and lifelong 5.
been postulated that immune factors act as expression of TNF receptors on granulo- Pre-existing neutralizing antibodies must
intermediaries in the response of endothe- cytes during DHF–DSS35 and an association have a major role in preventing subsequent
lial cells to DENV. In particular, cytokine between DHF–DSS and certain TNF poly- infection with the same DENV serotype.
storm (elevated levels of many cytokines in morphisms12. In one study, serum TNF Secondary infection with a heterologous
the serum) has been identified as a poten- was elevated during both DHF–DSS and serotype can cause a broad spectrum of
tial underlying mechanism of vascular dengue fever compared with levels in healthy illnesses, ranging from asymptomatic infec-
pathology21. controls, but a significant difference in TNF tion to severe haemorrhagic disease. For
During DENV infection, copious amounts levels between DHF–DSS and dengue fever example, some epidemiological studies have
of certain pro-inflammatory and vaso­active groups was not detected36. Thus, it is possible shown that patients with a secondary DENV
cytokines, including TNF and vascular that TNF has a role in DENV infection, but infection and babies born to mothers who
endothelial growth factor A (VEGFA), are whether it is (or can be, in some instances) have previously been infected with DENV
produced32. However, although these fac- a significant cause of DHF–DSS in humans are more likely to develop severe disease37,38.
tors are frequently elevated in the serum is still unclear. Multiple pro-inflammatory One theory for why this occurs was first
of patients with dengue, some studies have factors might act in concert to promote described in reference to other flaviviruses
raised questions about their direct role in vasculopathy. (Murray Valley encephalitis virus, West Nile
dengue pathology. For example, in spite of virus and Japanese encephalitis viruses)39
the well-established role of TNF in promot- Drawbacks of in vitro analysis. Ultimately, and is termed antibody-dependent enhance-
ing vascular leakage in other diseases and the mechanisms of many of the immune ment of infection (ADE). ADE involves
experimental contexts33,34, there has been no changes that occur in response to DENV the binding of immune complexes of non-
clear correlation shown between levels of infection, and the roles of these immune neutralizing antibody and infectious virus
TNF and the manifestation of dengue fever changes in protection versus pathology, are to the Fc receptors of immune cells29,40,41

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© 2013 Macmillan Publishers Limited. All rights reserved


and has been shown to enhance DENV
infectivity in vitro in human cells42. Studies
of DENV in vitro and in primates infused
with enhancing antibody have shown that
ADE can enhance infection of monocytes
by promoting viral uptake and intracellular
replication40,43. However, it should be noted
that this enhancement occurs in only a small
fraction of secondary infections and with
only certain strains of virus2,44.
Another immunological concept that
was first identified in the context of another
virus — in this case, influenza virus45 — and
later shown to be relevant to DENV infec-
tion46,47 is original antigenic sin. This theory
describes how amplification of pathogen-
specific lymphocytes during secondary
heterologous challenge can constitute a
misallocation of resources for the immune
system if those lymphocytes are not as effec-
tive in promoting clearance of the second-
ary challenge strain as they were for the
primary strain. It has been reported that
T cell responses in children experiencing a
secondary DENV infection have a low strain
specificity, resulting in T cell anergy and
apoptosis, and possibly higher viral titres
owing to a lack of optimal viral clearance
by T cells46. In other experimental models
resulting in original antigenic sin, T cell
responses are usually highly specific for the
primary challenge strain, and this might also
be the case for DENV48.
ADE and original antigenic sin have both
been offered as explanations for the asso-
ciation of secondary DENV infection with
an increased incidence of DHF–DSS21,46,
as both theories provide an understanding
of how immunological memory could be
detrimental to the host when a secondary
challenge is highly similar to, but antigeni-
cally distinct from, the primary infecting
virus. Each theory provides a potential
mechanism for achieving high viral titres
in vivo. Although viraemia levels have been
associated with disease severity 49,50, people
with secondary infections frequently do
not have higher levels of viraemia or severe
disease. Also, contrary to the widely held
view, ADE (as a specific mechanism to
explain secondary infection as a risk factor
for DHF–DSS) has not been demonstrated
to occur in vivo in humans, although it can
be achieved with human products in an
ex vivo experimental setting 51. DHF–DSS
is often diagnosed in patients with primary
infection, and capillary leakage has been
reported to more frequently accompany pri-
mary infection than secondary infection in
certain studies52. Secondary infection is thus
not a requirement for DHF–DSS and should
NATURE REVIEWS | MICROBIOLOGY VOLUME 11 | JUNE 2013 | 423
PERSPECTIVES
Epidermis
Mannose
DENV DC-SIGN
receptor
Viral RNA
Infected DC
Cellular
Infected cytotoxicity
Activated macrophage
mast cell
T cell
Dermis
NK cell
Lymphatic
vessel Trafficking
to lymph node Cellular
recruitment
Blood
vessel
Figure 2 | Host responses to cutaneous dengue virus injection.  Most infections by dengue virus
Nature Reviews | Microbiology
(DENV) occur after subcutaneous injection of the virus into the skin. Released viral particles may infect
nearby cells (thought to be predominantly monocytes or dendritic cells (DCs)) or activate resident
immune cells such as mast cells. A local inflammatory response to DENV in the skin prompts the recruit-
ment of leukocytes from the vasculature, including natural killer (NK) cells and T cells, which promote
the killing of virus-infected cells at the site of injection. DENV is thought to then travel to draining
lymph nodes via lymphatic vessels to establish systemic infection. These localized inflammatory
responses occur many days before there are any signs of severe infection.
not be a requirement for experimental whether unidentified factors released along
design to understand dengue pathogenesis. a similar time course could be more con-
Understanding the differential processes that sequential to pathology. Recent data show
lead to dengue pathogenesis during primary that mast cell proteases such as chymase
versus secondary infection is an important are released early during acute infection,
aim for the field. promoting DENV-induced vascular leakage
in mice, and serum levels of chymase have
The role of immune pathology been correlated with the severity of disease
Some of the emphasis on the role of anti- in human patients20. This is one example
body in dengue pathogenesis comes from of an immunomodulatory product that is
the experimental demonstration that anti- released along with cytokines early during
body alone or post-immune serum is suf- acute DENV infection. Another group of
ficient to enhance the pathology of DENV vasoactive lipid products, the leukotrienes,
infection in animal models29,53. Similar also seem to enhance DENV-induced vascu-
studies demonstrating causation are still lar leakage in the immunocompetent mouse
needed for host responses that, on the basis model20. Blockade of TNF can reduce vascu-
of human studies, are associated with vascu- lar pathology in immunocompromised ani-
lar leak syndrome, such as cytokine storm. mals during terminal disease54, but its role
Factors that might be correlated with dengue in the development of DHF–DSS in humans
severity include TNF and VEGF, but animal remains unclear 36. Cytokine storm can also
studies have not yet clearly established the occur as a result of infections of entirely
role of these host factors in disease progres- different aetiology without achieving the
sion, the cellular source of these factors, or highly specific symptoms that are attributed
© 2013 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES
to DENV infection, suggesting that more
information is required if we are to fully
understand the mechanisms underlying
DENV-induced vascular pathology.
Virus-intrinsic or host-associated risk factors
Virus-intrinsic factors are likely to influ-
ence the ability of certain strains to replicate
robustly or induce severe pathology. For
example, mutations in the DENV polymerase
non-structural protein 3 (NS3) and envelope
protein enhance viral fitness and promote
neurovirulence, potentially by altering viral
binding to cell surface receptors or increasing
the efficiency of NS3 (REF. 55). There are many
examples of interactions between DENV
proteins and immune products (for example,
NS1 binds to complement component
C4 (REF. 56), and NS5 binds to signalling inter-
mediary STAT2 (signal transducer and activa-
tor or transcription 2) to antagonize the IFN
pathway 57). Phylogenetic analyses have shown
that the emergence of a new DENV variant
can correlate with an increased likelihood
of DHF–DSS in humans11. Certain changes
within DENV structural proteins could alter
antibody binding to viral epitopes58, but
some regions that have shown variation do
not correspond to surface epitopes of mature
DENV virions11. Evolution of non-structural
genes has also been associated with increased
epidemic potential59 and reduced disease
severity 10, supporting the existence of a
DENV-intrinsic component to virulence
that requires further evaluation.
Conversely, some host genetic factors
are either associated with or protective
against the development of DHF–DSS.
In addition to TNF12, certain isoforms of
major histocompatibility complex (MHC)
class I molecules, MHC class II molecules,
immuno­globulin G Fc region receptor II
(FcγRII) molecules, cytotoxic T lymphocyte
protein 4 (CTLA4) and transforming growth
factor β1 (TGFβ1) have all been linked to
DHF–DSS13,60–63, whereas in some cases,
particular isoforms seem to be protective61.
Certain polymorphisms in TAP1 and TAP2,
genes associated with antigen presentation
and peptide loading on MHC, also have posi-
tive or negative correlations with DHF–DSS64.
These studies have identified target host
factors that might influence dengue patho-
genesis; however, as many of these factors
have been identified in an individual popu-
lation or outbreak, they require corrobora-
tion in independent studies and subsequent
experimental validation.
Virus–host interactions are not the
same for all infected patients or strains of
virus. This fact is underscored by the broad
424 | JUNE 2013 | VOLUME 11 www.nature.com/reviews/micro
spectrum of clinical outcomes for patients
infected with DENV, from asymptomatic
infection to classical dengue fever and, occa-
sionally, lethal complications of haemor-
rhage and/or shock. These complex factors
and seemingly contradictory observations
will probably be reconciled only with con-
trolled experimental investigation, which
would require preclinical studies using ani-
mal models and a range of unadapted viral
strains.
Animal models of dengue virus infection
Many animal models have been used in
DENV research, including multiple strains
of immunocompetent and immunocom-
promised mice, New World and Old World
primates, and even humanized animals65.
In the pursuit of an ideal model, however,
self-imposed boundaries have emerged
with regards to acceptable in vivo experi-
mentation using the tools that are currently
available. Non-human primates have been
underused, in part because results have been
highly variable, depending on the primate
species and infecting viral strain used. In
some studies, primates have shown mild
symptoms of DENV infection or vascular
leakage66–68. Primates have also been used to
test the safety, immunogenicity and protec-
tive capacity of vaccine candidates before
these candidates are tested in human clinical
trials69. This stage of preclinical testing in
primates is virtually inevitable for dengue
vaccine candidates and itself warrants fur-
ther study of the pathophysiological changes
caused by DENV in the primate model.
Immunocompetent animals (including
primates, but especially wild-type mice)
are considered a poor choice for DENV
research, even in studies addressing immune
function directly, owing to their relative
resistance to infection compared with
humans70,71. It should be noted, however, that
each model has distinct benefits and limita-
tions. In addition, many of the accepted
limitations of certain models (such as the
expectation that immunocompetent mice
would not support infections with replicat-
ing viruses) are not adequately represented
in the literature with data, but rather, are
discussed anecdotally 70,71. This makes assess-
ing other potential causes of model ‘failure’,
such as the choice of viral strain, difficult.
Some of these concepts regarding the resist-
ance of certain species to infection date
back to the earliest in vivo studies carried
out using DENV72, and scarce supporting
data have been published in the intervening
time. As new techniques have now become
available, more sensitive means of measuring
© 2013 Macmillan Publishers Limited. All rights reserved
viral and immune outcomes might also be
useful. Examples that are currently being
applied in some studies include quantita-
tive real-time reverse transcription PCR
(qRT-PCR), which can be used as a sensi-
tive substitute for plaque assays (although
this has the caveat that qRT-PCR does not
accurately measure infectious DENV),
and flow cytometry, which can be used in
place of immunohistochemistry to quantify
rare infected populations. Therefore, it is
worthwhile revisiting lesser-used models to
determine whether additional information
about dengue pathogenesis and the host
immune response can be garnered using
modern techniques and a broader range of
low-passage viral strains.
In response to the difficulty of assess-
ing DENV virology in immunocompe-
tent animals, and in an effort to achieve
uniform severity of infection and clearly
interpretable results, most recent animal
studies have gravitated towards the use of
immunocompromised models (such as the
AG129 IFN-deficient mouse model) and
‘antibody-enhanced’ infection (often using
monoclonal antibodies to DENV, along with
mouse-adapted DENV). These systems have
clear advantages for answering certain ques-
tions. For example, investigations with the
aim of identifying antiviral drugs might be
most definitive in immunocompromised
mice, in which almost unrestricted DENV
replication can occur and there is uniform
disease severity 73. However, we must be
careful not to overextend the conclusions
from such experiments. In many aspects,
particularly relating to host immunity and
potentially also to immune-mediated path­
ology, there might be differences between
the mouse immune response and the
response that develops during human infec-
tion. Moreover, we do not know whether the
underlying mechanisms of key pathological
events such as vascular leakage would be
the same in mice as during human disease
or whether the same cell types that become
infected in humans are infected in immuno-
compromised animals. The absence of key
inflammatory pathways could allow other-
wise resistant mouse cells to be permissive
for DENV replication. In our opinion, the
animal models that have lent themselves to
studying the basic virology of DENV, such
as immunocompromised mice, might not be
the most appropriate for studying immunity
or immune pathogenesis. We do not advo-
cate the adoption of any one existing model
as the gold standard because they each have
their pros, cons and caveats. Rather, as out-
lined below, researchers should select the

most appropriate existing model, with these
considerations in mind, to address a unique
experimental question.
Investigations that aim to understand
the development of effective anti-dengue
immunity and to use that understanding as
a basis for rational vaccine design require an
intact immune system to fully encompass
the immune response. Immunocompetent
mice are thought to be relatively resistant
to DENV compared with humans, but this
fact also affords us the opportunity to study
effective anti-dengue immunity in a host
that can respond to, and clear, the infection.
Using selective knockout mice could provide
a mechanistic understanding of how unique
cell types or inflammatory factors contrib-
ute to immunity and pathogenesis. There
is likely to be a complex interplay between
the processes of anti-dengue immunity and
DENV-induced pathology, as supported by
the association of secondary infection with
variable disease severity during dengue out-
breaks27,74–76. Studying immune pathology
mechanistically has proved difficult because
mice with functional immune systems rarely
fully recapitulate the severity of human
DHF. However, we challenge the premise
that full recapitulation of the disease time
course or pathological phenotype is a neces-
sity. Changes that occur in vivo in response
to DENV infection need not be equally
detrimental to the survival of animals and
humans to reveal information regarding the
progression of disease, and such information
could be crucial for identifying a therapeu-
tic or prophylactic strategy. Using animal
models that have fallen out of favour for
dengue research, such as wild-type immuno-
competent mice and non-human primates,
to study those physiological changes will
require careful experimental design, a real-
istic assessment of the likelihood that con-
clusions can be extended to human disease,
the use of selected unpassaged viral strains
and subtypes, and the use of more sensitive
measures of pathology than the extreme
outcomes of haemorrhaging, prolonged
viraemia or death. Some potential surrogates
for end-stage pathology include more sensi-
tive methods to objectively measure vascular
permeability or immune cell signalling, sta-
tistically significant changes in the number
of haematopoietic cells (for example, loss of
platelets or proliferation of lymphocytes),
the levels of immune factors upstream of
vascular leakage (such as heightened levels
of cytokines or other vasoactive factors),
and alternate time points of assessment to
those thought to be analogous to the human
course of infection. Thus, individual animal
NATURE REVIEWS | MICROBIOLOGY VOLUME 11 | JUNE 2013 | 425
models for dengue might have limitations,
but should not be dismissed as inherently
flawed. It should be noted that severe dengue
does not manifest uniformly in humans, so
lack of severity in a model should not pre-
vent the study of dengue in varied models.
Conversely, as the limitations of animal
models for dengue are well known, the
results of these studies should be used to
inform our collective knowledge of DENV
virology, pathogenesis and immunity in
order to introduce new theories and concepts
to the field, but not to limit the potential of
similar investigations in humans. For exam-
ple, there seem to be differences in the ability
of humans and mice to develop neutralizing
antibodies to certain epitopes of DENV
proteins77. For immunocompromised mice,
drugs might reduce dengue pathology but
never allow long-term survival of animals
that cannot clear the infection. Thus, limita-
tions in the degree to which we can extend
conclusions from animal studies should not
inhibit our pursuit of understanding this
pathogen in a range of animal models as well
as in reasoned human trials.
Conclusions
The field of dengue research has made great
progress over the past 50 years, particularly
in terms of defining dengue clinically, deter-
mining how to identify and treat dengue
with supportive care, and understanding the
epidemiology and basic biology of DENV,
including viral structure and replication.
Animal models have been developed for
antiviral drug testing, and many groups
have designed promising vaccine candidates.
Importantly, many of the remaining ques-
tions about DENV as a pathogen centre on
how the immune system, both innate and
acquired, perceives and responds to DENV
infection, how genetic variation in both the
host and the virus influence this perception,
and where the fine line lies for protection
versus pathology. These questions cannot
be definitively answered in the context of
an immunocompromised system alone,
but must be directly addressed with bold
experimental design of preclinical studies,
including in immunocompetent animals,
to fully define anti-dengue immunity. We
believe that meeting this challenge will be
a prerequisite to identifying the most effec-
tive targeted therapies and vaccine strategies
against dengue.
Ashley L. St. John, Soman N. Abraham and Duane
J. Gubler are at the Program in Emerging Infectious
Diseases, Graduate Medical School, Duke-National
University of Singapore, 8 College Road,
169857, Singapore.
© 2013 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES
Ashley L. St. John and Soman N. Abraham
are also at the Department of Pathology,
Duke University Medical Center.
Soman N. Abraham is also at the Department of
Immunology and the Department of Molecular Genetics
and Microbiology, Duke University Medical Center,
Durham, North Carolina 27710, USA.
Correspondence to D.J.G. 
e‑mail: duane.gubler@duke-nus.edu.sg
doi:10.1038/nrmicro3030
Published online 8 May 2013
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Acknowledgements
The authors thank A. P. S. Rathore for critical reading of the
manuscript. This work was supported by the National Medical
Research Council of Singapore (grant NIG/1053/2011) and by
the Duke-National University of Singapore Signature Research
Program, funded by the Ministry of Health, Singapore.
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
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