Viral Genetics as a Basis of Dengue Pathogenesis

Suchita Chaudhry, Sathyamangalam Swaminathan and Navin Khanna

RGP Laboratory, International Centre for Genetic Engineering & Biotechnology, P.O. Box 10504,
Aruna Asaf Ali Marg, New Delhi 110067, India

Abstract
Dengue is the most widespread mosquito-borne human viral disease. The disease is now endemic in
more than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-East Asia and the
Western Pacific regions. Dengue viruses cause dengue infection, which ranges from mild febrile illness
(dengue fever, DF) to fatal haemorrhagic manifestation (dengue haemorrhagic fever, DHF) leading to
shock syndrome (dengue shock syndrome, DSS). In recent years, DHF epidemics have intensified and
become a major cause of morbidity. Understanding the pathogenesis of DHF/DSS has been hampered
by the lack of in vitro and in vivo models of the disease. Two theories, which are not mutually exclusive,
are frequently cited to explain the basis of DHF/DSS pathogenesis. One is based on antibody-dependent
enhancement (ADE) and the other on inherent dengue virus virulence. There is epidemiological and
laboratory evidence to support both hypotheses. However, neither has been definitively proven because
of the lack of an appropriate animal model. Nevertheless, the ADE hypothesis has gained wider
recognition and is in fact the basis for current dengue vaccine approaches. In recent years, evidence has
been increasingly accumulating to support a role for viral genetics in dengue pathogenesis. This review
focuses on the hypothesis that the recent increase in the incidence of DHF/DSS epidemics correlates
with the emergence of viral strains with higher virulence and epidemic potential.
Keywords: Dengue, pathogenesis, antibody-dependent enhancement; viral determinants; epidemic potential.

Introduction billion people at risk. The resurgence of dengue

Dengue is currently the most important
arthropod-borne viral disease caused by any of
the four closely related, yet antigenically distinct,
serotypes of dengue viruses (DENV-1, DENV-
2, DENV-3 and DENV-4) of the Flaviviridae
family.[1] These are transmitted to humans by
the Aedes aegypti mosquito. Dengue infections
may be either asymptomatic or may cause a
spectrum of illnesses ranging from mild fever
to severe, potentially fatal disease. Once a
sporadic illness, dengue is on the rise and is
currently endemic in several regions of the
world, particularly South-East Asia, the Western
Pacific, and the Americas, placing about 2.5
in the latter half of the 20th century, which
began in the Asian and Pacific regions, is
thought to be the result of ecological disruption
and demographic changes in the wake of World
War II. Dengue epidemics, first recorded in
the 1950s in the South-East Asian countries,
have been occurring in waves every 3 to 5
years, spreading to India, Sri Lanka, Maldives
and China by the 1980s, and have become
the leading cause of hospitalization and death
among children in Asia. In the Pacific islands,
small dengue outbreaks were recorded in the
mid- to late 1960s, followed in the first half of
the next decade by explosive epidemics in Fiji,
Tahiti, New Caledonia and Niue.[2-5] In the

navin@icgeb.res.in;
+91-11-26177357/Ext.272; Fax +91-11-26162316

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Viral Virulence and Dengue Pathogenesis
Americas, the successful mosquito eradication
programmes in effect during the 1940s and
1950s to the early 1970s kept dengue at bay.
The failure of this programme coincided with
the return of dengue to the Americas in the
late 1970s, with more than 30 countries
reporting dengue activity by 2002.[6] It is
currently estimated that there are 50-100
million cases of dengue fever (DF) per annum
worldwide, about 1% of which result in the
severe forms of the disease, dengue
haemorrhagic fever (DHF) and dengue shock
syndrome (DSS).[2-6]
Despite dengue having been around for
several decades now, there is as yet no
consensus on the mechanism of DHF/DSS
pathogenesis, mainly because of the lack of
reliable in vitro and in vivo model systems of
the disease. Available information suggests that
the pathogenesis underlying severe disease
could be the result of the interplay of multiple
factors such as the presence of cross-reactive,
non-neutralizing antibodies, viral virulence,
genetic predisposition, age, nutritional status
and underlying chronic disease conditions of
the human host.[7,8] Data supporting the role
of many of the host-related factors are weak.
Two hypotheses, based on the first two factors,
have been developed to explain dengue
pathogenesis. While neither one is conclusively
proven, epidemiological and laboratory
evidences are available to support both
hypotheses.[4] However, one of these, known
as the immune enhancement hypothesis,
based on the prior presence of heterotypic
antibodies during a secondary infection, has
gained wider acceptance and popularity and
provides the basis for the current live
attenuated four-in-one ‘tetravalent’ vaccine
approaches.[9] The purpose of this article is to
briefly present the salient features of the
immune enhancement hypothesis and then
focus on the increasing evidence that
emphasizes the significance of viral factors in
the pathogenesis of DHF/DSS.
122
Immune enhancement
The most commonly accepted theory that seeks
to explain the pathogenesis of DHF/DSS is
known as the secondary-infection or immune
enhancement hypothesis.[10,11] Several in-depth
reviews of the antibody-dependent
enhancement (ADE) hypothesis are
available.[8,12,13] Only the salient aspects of this
hypothesis are presented here. According to
this, patients experiencing a second infection
with a heterologous DENV serotype are at a
significantly higher risk of developing DHF and
DSS. Pre-existing heterologous dengue
antibody, which recognizes and binds, but does
not neutralize the infecting virus, is believed
to facilitate its internalization via
immunoglobulin Fc receptors on the cell
membrane of leukocytes, especially
macrophages. This ADE of virus uptake into
cells of the mononuclear cell lineage and
subsequent augmented replication of the virus
is believed to be responsible for the production
and secretion of vasoactive mediators leading
to increased vascular permeability,
hypovolemia, haemorrhage, and ultimately,
shock. In addition to cross-reacting antibodies,
this hypothesis has been expanded in recent
times to include a role for cross-reactive T cells
as well in DHF/DSS. [8,12,13] The strongest
epidemiological data supporting a pathological
role for the immune response in DHF has come
from the Cuban dengue epidemics. Guzman
et al. [14] obtained these data based on a
comparison of two epidemics, one caused by
DENV-1 and the other, nearly two decades
later, by DENV-2 in 1997, made possible by a
meticulous national dengue surveillance
programme. This study showed that all patients
with symptomatic dengue in the 1997 epidemic
were adults who had been born prior to the
earlier DENV-1 epidemic and the majority of
these (98%) experienced secondary DENV
infection. In contrast, almost all those who
seroconverted without illness (97%)
Dengue Bulletin – Volume 30, 2006

experienced a primary infection.[14,15] However,
evidence for the role of ADE in human disease
is by and large still circumstantial.[8,12,13]
Inherent viral virulence
The inadequacy of the ADE hypothesis to
explain the association of DHF/DSS in
confirmed cases of primary infection and the
rarity of severe dengue disease in epidemic
situations wherein the scope for secondary
infection is particularly high (see below) suggest
that dengue virus strains differing in pathogenic
potential over a wide range exist in nature.
The epidemiological and molecular evidence
supporting this notion and possible factors that
may contribute to genetic variation in dengue
virus are discussed below.
Epidemiological evidence
The concept that viral determinants may have
a role in the pathogenesis of severe disease
was first proposed by Rosen based on the
dengue epidemic which occurred in Niue Island
in the South Pacific region in 1972.[16] This
epidemic was characterized by a high incidence
of haemorrhagic manifestations and a number
of deaths, including that of children. A
retrospective epidemiological and serological
investigation showed that this epidemic was
exclusively due to DENV-2 and that there was
no evidence of any dengue activity during the
preceding 25 years.[17] This indicated strongly
that DHF/DSS in the 1972 Niue Island
epidemic did not arise out of sequential
infection with heterologous serotypes, but was
the result of primary dengue infection. Thus,
prior dengue infection is not a prerequisite to
the pathogenesis of DHF/DSS. This conclusion
is strengthened by the observations of Gubler
and coworkers in 1974 and 1975 in the Pacific
Island Kingdom of Tonga.[18] Contrary to what
has been proposed by the ADE hypothesis, this
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Viral Virulence and Dengue Pathogenesis
study found that a DENV-1 outbreak in the
Kingdom of Tonga in 1975, associated with
severe haemorrhagic manifestations, which
followed a DENV-2 epidemic the year before,
was largely the result of primary, and not
secondary, infection. As the severity of the
1975 outbreak did not correlate with prior
immune status, these authors proposed that
the DENV-1 strain responsible for the severe
outbreak in 1975 must be inherently more
virulent than the DENV-2 strain that caused
the dengue epidemic during the preceding
year.[18] However, DENV-2 does not always
cause mild infections. It has been associated
with explosive epidemics on other islands in
the South Pacific in previous years.[3] Two
different genetic types (genotypes) of DENV-2
have been recognized, based on phylogenetic
studies: the ‘South-East Asian’ and ‘American’
genotypes. The former has been associated
with DHF, and the latter, with mild clinical
manifestations of dengue.[19] Interestingly, the
American genotype DENV-2 failed to cause
DHF in a major epidemic in Peru in 1995 even
though the same population had experienced
a DENV-1 epidemic about five years earlier.[20]
This observation, which is clearly not consistent
with the prediction of the ADE hypothesis, has
been attributed to the capacity of pre-existing
anti-DENV-1 antibodies to attenuate the
severity of the secondary infection by the
American DENV-2 genotype.[21] However, given
the high secondary transmission rate (86%), one
may argue that the reason for the absence of
severe clinical manifestations in the Peruvian
epidemic is more a consequence of the lower
level of inherent virulence of the American
DENV-2 genotype, rather than its attenuation
by heterologous anti-DENV-1 antibodies.
Another line of epidemiological evidence
that supports the notion that the dengue disease
severity may be linked to the degree of
inherent virulence comes from the information
available from hyperendemic (co-circulation of
multiple DENV serotypes) areas. For example,
123
Viral Virulence and Dengue Pathogenesis
in the Americas where multiple DENV
serotypes co-exist, the occurrence of DHF was
not documented until the 1981 Cuban
epidemic. This coincided with the introduction
of a new genetic type of DENV-2.[19,22] Similarly,
DHF was relatively uncommon in Sri Lanka prior
to 1989, despite hyperendemicity. However,
after 1989, the incidence of DHF, which
increased dramatically, was frequently
associated with DENV-3.[23,24] Again, as noted
for DENV-2 above, DENV-3 also exists in mild
and virulent forms. This is quite vividly evident
from a comparison of the dengue epidemics
recorded in Central Java during the late
1970s.[25] Even though the predominant virus
isolated in both these epidemics was DENV-3,
one was associated with milder and the other
with more severe illness. Similarly, in regard to
DENV-1 in the South and Central Pacific islands,
some have experienced silent transmissions,
while others in contrast have had explosive
epidemics.[26] The existence of mild and virulent
strains within each serotype suggests that the
dengue virus changes in its epidemic potential
as it moves through populations.[3,4]
Molecular evidence
Genetic variation in dengue virus isolates had
been initially documented on the basis of cross-
neutralization assays [27] and RNAse T1
oligonucleotide fingerprinting.[28] However,
both these methods that recognize serotype-,
group- and family-specific epitopes fail to detect
phenotypically silent differences. In order to
delineate the evolutionary and epidemiological
relationships between many isolates of the
same virus, Rico-Hesse developed a primer
extension-based limited sequencing approach.
Based on a careful analysis of a 240-nucleotide
sequence spanning the E/NS1 junction of 40
isolates each of DENV-1 and DENV-2, she
identified five distinct genotypes for each of
these serotypes.[22] Rico-Hesse’s method was
modified by Lanciotti’s group into an RT-PCR-
124
based sequencing method, focusing on the
entire envelope region of the dengue virus
genome. Their studies, which confirmed the
existence of 5 distinct genotypes of DENV-2,[29]
also identified 4 distinct subtypes of DENV-
3[30] and two distinct subtypes of DENV-4.[31]
These researchers discerned a correlation
between disease severity and virus genotype,
based on the phylogenic data in conjunction
with epidemiology, in their DENV-3 study.
While DENV-3 subtype 4 had never been
associated with severe disease, DENV-3
subtypes 1 (in Indonesia and Thailand) and 3
(in Sri Lanka and India) have been isolated from
DHF patients. Furthermore, their phylogenetic
analysis suggests that these DHF strains
presumably were the result of genetic drift of
the endemic strains.[30] In regard to DENV-2,
specific mutations associated with changes in
virulence have been identified using infectious
clones,[32,33] chimeric constructs[34] and by direct
comparison of wild-type and attenuated
strains.[33,35]
The most convincing evidence
demonstrating a genetic basis to dengue
virulence comes from the work of Rico-Hesse’s
group. Their molecular epidemiological studies,
in conjunction with clinical data, have
demonstrated an association between the
introduction of a South-East Asian genotype of
DENV-2 and the appearance of DHF in the
Americas. Furthermore, the native DENV-2
American genotype, known to cause only the
milder disease, appears to have been displaced
by the virulent imported South-East Asian
genotype.[19] In a subsequent study, this group
determined the full nucleotide sequences of
DENV-2 strains directly from clinical samples
from DF and DHF patients in the Americas
and Thailand, respectively. From a comparison
of 11 different strains, they identified several
consistent structural differences between the
low (American genotype) and high (South-East
Asian genotype) virulence strains, summarized
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 Viral Virulence and Dengue Pathogenesis

in Table 1.[36] Based on their comparative
molecular analysis they have determined that
the primary determinants of virulence reside
in the E gene, the 5’ non-translated region (5’
NTR) and the 3’ NTR. Interestingly, all these
are of critical importance in the virus replication
cycle. For instance, they identified a mutation
corresponding to amino acid (aa) residue 390
on the E protein as a potential determinant of
virulence. This observation ties up with the data
showing that replacement of aa 390, which is
part of the E domain involved in host cell
surface receptor binding, [37] impacts the
neurovirulence of the virus in mice.[35] The
NTRs fold into secondary structures comprising
a number of stable stem-loops, that appear to
be highly conserved and are expected to play
an important role during viral infection in
coordinating gene expression and the onset of
RNA replication. For example, the 5’ NTR binds

Table 1: Structural changes in the DF strain with reference to the DHF strain#[36]

#
refers to DENV-2 serotype
*denotes nucleotide positions on the DENV-2 genome
**denotes aa residue position from the N-terminus of the respective mature protein
Abbreviations: A, Adenine; T, Thymine; G, Guanine; Asn, Asparagine; Asp, Aspartic acid; Glu, Glutamic acid;
Lys, Lysine; Val, Valine; Thr, Threonine; Ser, Serine; Leu, Leucine; Ile, Isoleucine; His, Histidine; Arg, Arginine;
Lys, Lysine; Gln, Glutamine; prM, pre-membrane protein; NS, non-structural protein.

Dengue Bulletin – Volume 30, 2006 125

Viral Virulence and Dengue Pathogenesis
to the ribosomes to initiate translation. More
recently, it has been shown to function as a
promoter for the transcription of the minus
sense genomic RNA which eventually
templates plus sense RNA synthesis during the
viral replicative cycle.[38] This entails a role for
the 3’ end of the minus RNA intermediate
(complement of 5’ NTR) in the initiation of
(+) RNA synthesis. Finally, our own studies
have implicated a role for the 5’ end of the
minus sense RNA (complement of the 3’ NTR)
in viral replication. [39] Essentially, all the
functions of the NTRs are mediated by their
specific interactions with viral and host proteins.
Table 2 provides a summary of the host factors
documented to interact with DENV and other
flaviviral NTRs. [39-48] Consistent with their
importance in various aspects of the viral life-
cycle, mutations that alter the predicted folding
of the NTRs can influence the degree of viral
virulence. A comparison of the predicted
secondary structures of the 5’ NTRs of the
American and South-East Asian DENV-2
genotypes, based on the data of Leitmeyer and
colleagues,[36] revealed minor, subtle changes
(Figure, panels A and B). In contrast, however,
a set of mutations identified by these
researchers in the 3’ NTR of DENV-2 American
genotype predicts a significantly different
secondary structure compared to the 3’ NTR
of the South-East Asian genotype of DENV-2
(Figure, panels C and D). Others have also
subsequently corroborated these 3’ NTR
secondary structural differences between the
low and high virulence genotypes of DENV-
2.[49] To address the question if the putative
viral determinants do indeed determine the
degree of virulence, Cologna and Rico-Hesse[50]
generated a panel of chimeric infectious clones
wherein they introduced the E390 substitution,
and the 5’ and 3’ NTRs of DENV-2 American
genotype into the background of the South-
East Asian genotype singly and in various
combinations. Their analysis showed that all
three American genotype-specific mutations
126
acted synergistically to result in a large
reduction in virus replication and output. This
study showed definitively that American
genotype structures decreased the replicative
ability of the South-East Asian genotype-derived
infectious clone. This may explain at least
partially the reason why the American genotype
has never been associated with DHF.[50] More
recent studies based on an analysis of the
replication efficiencies of several American and
South-East Asian genotypes in primary cell
cultures and in mosquitoes support the notion
that viruses of the latter genotype have greater
pathogenic potential by virtue of their capacity
to replicate to significantly higher levels in the
human hosts.[51]
Factors contributing to genetic
diversity
As discussed above, dengue viruses are prone
to change giving rise to strains with varying
degrees of virulence. Increasingly it is becoming
apparent that many instances of epidemic DHF
are associated with the circulation of viral strains
with increased virulence. What drives such
change? While a definitive answer is not
available, several factors have been attributed
to be potential contributors to the observed
genetic diversity of dengue viruses.[52] Firstly,
genetic diversity is apparently an inherent
property of dengue viruses as illustrated by the
existence of four serotypes. As RNA viruses,
dengue viruses rely on an error-prone RNA-
dependent RNA polymerase (RdRp)-mediated
replication mechanism, estimated to produce
one error per round of replication.[53] However,
it is regarded that the overall spontaneous
mutation rate may be subject to functional
constraints stemming from the need of the
dengue viruses to replicate in two disparate
hosts, mosquitoes and humans. Secondly, co-
infection of a single host cell by two viral strains
can facilitate recombination wherein the RdRp
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 Viral Virulence and Dengue Pathogenesis

Table 2: Host cellular factors known to interact with flaviviral NTRs

Abbreviations: PTB, Pyrimidine tract binding protein; HnRNP, Heterogeneous nuclear ribonucleoprotein; EF-1,
Elongation factor 1; eIF3: Eukaryotic initiation factor 3; PCBP, Poly C binding protein; PDI, Protein disulphide
isomerase; Mov34, Mouse brain protein. CHMP2A, Charged multivesicular body protein 2A; CapZ-b, Actin
capping protein b subunit; RuvBL-2, RuvB like protein 2; WNV, West Nile virus; DENV, Dengue virus; HCV,
Hepatitis C virus; JEV, Japanese encephalitis virus; UVCL, UV induced cross-linking; EMSA, Electrophoretic
mobility shift assay; IP, Immunoprecipitation assay; * unpublished data.

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Viral Virulence and Dengue Pathogenesis

Figure: Comparison of computer-predicted secondary structures of NTRs of

DENV-2 American (Am) and South-East Asian (As) strains

(A) Am 5’ NTR (B) As 5’ NTR

(C) Am 3’ NTR (D) As 3’ NTR

128 Dengue Bulletin – Volume 30, 2006


switches mid-replication from one template to
another. This possibility draws support from the
work of Worobey et al.[54] who performed a
diversity analysis of 71 published dengue virus
sequences and found several hybrid sequences
which were mosaics comprising gene regions
with conflicting evolutionary histories. Their
work indicates that dengue viruses manifest
widespread recombination within, but not
between, serotypes in natural populations. This
study supports the role of recombination as a
significant factor in shaping the genetic diversity
of dengue viruses. Thirdly, it has been proposed
that genetic variation can be introduced through
migration, or gene flow, given that the hosts
and vectors are often transported across large
distances enabling wide geographical
distribution of viral strains.[22] This, in turn, could
lead to greater mixing of viral strains and the
generation of diversity through
recombination.[52] Finally, the origin of such
diversity in dengue viruses may simply be the
result of enhanced opportunities for
transmission stemming from the increase in
size and density of the human host
population.[55] Thus, it appears that genetic
variation evident in dengue viruses may be the
result of the action of multiple factors at work.[52]
Conclusions
Dengue infection has re-emerged as a public
health problem of global proportions. The
pathogenesis of severe dengue disease is not
fully understood, as appropriate in vitro and in
vivo models are not available to investigate the
progression of mild DF to sever DHF/DSS. Out
of the several explanations proposed based on
epidemiological and experimental evidence, as
discussed at the beginning, one proposed
mechanism, which invokes disease
exacerbation through the ADE phenomenon
upon secondary infection with a heterotypic
DENV serotype, is most widely accepted.
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Viral Virulence and Dengue Pathogenesis
However, not all cases of DHF can be
accounted for by the ADE hypothesis.
Molecular evidence accumulated in recent
years is increasingly pointing to the involvement
of viral factors in DHF/DSS pathogenesis.
Preliminary proof-of-concept experiments have
shown that viral determinants modulate the
degree of virulence, and therefore, disease
severity. Again, not all cases of DHF can be
explained by viral virulence alone. That immune
enhancement and virulence are not the only
factors in determining the severity of dengue
pathogenesis is dramatically illustrated by the
situation reported in Haiti recently.[56] Despite
hyperendemicity and the presence of a virulent
DENV-2 genotype, the two prerequisites
postulated by the immune enhancement and
viral virulence hypotheses respectively, DHF
has not been detected in the Haitians. This
interesting observation points to the existence
of a putative dengue resistance gene in the
Haitian population. In fact, several other host-
related have also been implicated in the
disease pathogenesis. Thus, it is likely that DHF
pathogenesis may have a multifactorial basis.
The recent description of murine models which
manifests the hallmarks of dengue disease, such
as fever, rashes and decreased platelet
counts [57] and increased vascular
permeability,[58] may prove useful in delineating
the role of all putative factors and pave the
way towards an integrated understanding of
DHF pathogenesis.
Acknowledgements
This work was supported by internal funds from
the International Centre for Genetic Engineering
and Biotechnology (ICGEB) and a grant from
the Department of Biotechnology, Government
of India. SC is a recipient of a senior research
fellowship from the Council of Scientific and
Industrial Research, Government of India.
129
Viral Virulence and Dengue Pathogenesis
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