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Dengue Bulletin Volume 30, 2006 121

[email protected]; +91-11-26177357/Ext.272; Fax +91-11-26162316

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 inmore than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-East Asia and theWestern 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 toshock syndrome (dengue shock syndrome, DSS). In recent years, DHF epidemics have intensified andbecome a major cause of morbidity. Understanding the pathogenesis of DHF/DSS has been hamperedby the lack ofin vitro and in vivo models of the disease. Two theories, which are not mutually exclusive,are frequently citedto explain the basis of DHF/DSS pathogenesis.One is based on antibody-dependentenhancement (ADE) and the other on inherent dengue virus virulence. There is epidemiological andlaboratory evidence to support both hypotheses. However, neither has been definitively proven becauseof the lack of an appropriate animal model. Nevertheless, the ADE hypothesis has gained widerrecognition and is in fact the basis for current dengue vaccine approaches. In recent years, evidence hasbeen increasingly accumulating to support a role for viral genetics in dengue pathogenesis. This reviewfocuses on the hypothesis that the recent increase in the incidence of DHF/DSS epidemics correlateswith the emergence of viral strains with higher virulence and epidemic potential.

Keywords: Dengue, pathogenesis, antibody-dependent enhancement; viral determinants; epidemic potential.

Introduction

Dengue is currently the most importantarthropod-borne viral disease caused by any ofthe four closely related, yet antigenically distinct,serotypes of dengue viruses (DENV-1, DENV-

2, DENV-3 and DENV-4) of the Flaviviridaefamily.[1] These are transmitted to humans bytheAedes aegypti mosquito. Dengue infectionsmay be either asymptomatic or may cause aspectrum of illnesses ranging from mild feverto severe, potentially fatal disease. Once asporadic illness, dengue is on the rise and iscurrently endemic in several regions of theworld, particularly South-East Asia, the WesternPacific, and the Americas, placing about 2.5

billion people at risk. The resurgence of denguein the latter half of the 20th century, whichbegan in the Asian and Pacific regions, isthought to be the result of ecological disruptionand demographic changes in the wake of WorldWar II. Dengue epidemics, first recorded inthe 1950s in the South-East Asian countries,

have been occurring in waves every 3 to 5years, spreading to India, Sri Lanka, Maldivesand China by the 1980s, and have becomethe leading cause of hospitalization and deathamong children in Asia. In the Pacific islands,small dengue outbreaks were recorded in themid- to late 1960s, followed in the first half ofthe next decade by explosive epidemics in Fiji,Tahiti, New Caledonia and Niue.[2-5] In the


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122 Dengue Bulletin Volume 30, 2006

Viral Virulence and Dengue Pathogenesis

Americas, the successful mosquito eradicationprogrammes in effect during the 1940s and

1950s to the early 1970s kept dengue at bay.The failure of this programme coincided withthe return of dengue to the Americas in thelate 1970s, with more than 30 countriesreporting dengue activity by 2002.[6] It iscurrently estimated that there are 50-100million cases of dengue fever (DF) per annumworldwide, about 1% of which result in thesevere forms of the disease, denguehaemorrhagic fever (DHF) and dengue shocksyndrome (DSS).[2-6]

Despite dengue having been around forseveral decades now, there is as yet noconsensus on the mechanism of DHF/DSSpathogenesis, mainly because of the lack ofreliable in vitro and in vivo model systems ofthe disease. Available information suggests thatthe pathogenesis underlying severe diseasecould be the result of the interplay of multiplefactors such as the presence of cross-reactive,

non-neutralizing antibodies, viral virulence,genetic predisposition, age, nutritional statusand underlying chronic disease conditions ofthe human host.[7,8] Data supporting the roleof many of the host-related factors are weak.Two hypotheses, based on the first two factors,have been developed to explain denguepathogenesis. While neither one is conclusivelyproven, epidemiological and laboratoryevidences are available to support bothhypotheses.[4] However, one of these, knownas the immune enhancement hypothesis,based on the prior presence of heterotypicantibodies during a secondary infection, hasgained wider acceptance and popularity andprovides the basis for the current liveattenuated four-in-one tetravalent vaccineapproaches.[9] The purpose of this article is tobriefly present the salient features of theimmune enhancement hypothesis and thenfocus on the increasing evidence that

emphasizes the significance of viral factors inthe pathogenesis of DHF/DSS.

Immune enhancement

The most commonly accepted theory that seeksto explain the pathogenesis of DHF/DSS isknown as the secondary-infectionor immuneenhancement hypothesis.[10,11] Several in-depthreviews of the antibody-dependentenhancement (ADE) hypothesis areavailable.[8,12,13] Only the salient aspects of thishypothesis are presented here. According tothis, patients experiencing a second infectionwith a heterologousDENV serotype are at a

significantly higher risk of developing

DHF andDSS. Pre-existing heterologous dengueantibody, which recognizes and binds, but doesnot neutralize the infecting virus, is believedto facilitate its internalization viaimmunoglobulin Fc receptorson the cellmembrane of leukocytes, especiallymacrophages. This ADE of virus uptake intocells of the mononuclear cell lineage andsubsequent augmented replication of the virusis believed to be responsible for the productionand secretion of vasoactive mediators leadingto increased vascular permeability,hypovolemia, haemorrhage, and ultimately,shock. In addition to cross-reacting antibodies,this hypothesis has been expanded in recenttimes to include a role for cross-reactive T cellsas well in DHF/DSS.[8,12,13] The strongestepidemiological data supporting a pathologicalrole for the immune response in DHF has comefrom the Cuban dengue epidemics. Guzman

et al.[14]

obtained these data based on acomparison of two epidemics, one caused byDENV-1 and the other, nearly two decadeslater, by DENV-2 in 1997, made possible by ameticulous national dengue surveillanceprogramme. This study showed that all patientswith symptomatic dengue in the 1997 epidemicwere adults who had been born prior to theearlier DENV-1 epidemic and the majority ofthese (98%) experienced secondary DENVinfection. In contrast, almost all those whoseroconverted without illness (97%)


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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 toexplain the association of DHF/DSS inconfirmed cases of primary infection and therarity of severe dengue disease in epidemicsituations wherein the scope for secondary

infection is particularly high (see below) suggestthat dengue virus strains differing in pathogenicpotential over a wide range exist in nature.The epidemiological and molecular evidencesupporting this notion and possible factors thatmay contribute to genetic variation in denguevirus are discussed below.

Epidemiological evidence

The concept that viral determinants may havea role in the pathogenesis of severe diseasewas first proposed by Rosen based on thedengue epidemic which occurred in Niue Islandin the South Pacific region in 1972. [16] Thisepidemic was characterized by a high incidenceof haemorrhagic manifestations and a numberof deaths, including that of children. Aretrospective epidemiological and serologicalinvestigation showed that this epidemic wasexclusively due to DENV-2 and that there wasno evidence of any dengue activity during thepreceding 25 years.[17] This indicated stronglythat DHF/DSS in the 1972 Niue Islandepidemic did not arise out of sequentialinfection with heterologous serotypes, but wasthe result of primary dengue infection. Thus,prior dengue infection is not a prerequisite tothe pathogenesis of DHF/DSS. This conclusionis strengthened by the observations of Gublerand coworkers in 1974 and 1975 in the Pacific

Island Kingdom of Tonga.[18] Contrary to whathas been proposed by the ADE hypothesis, this

study found that a DENV-1 outbreak in theKingdom of Tonga in 1975, associated with

severe haemorrhagic manifestations, whichfollowed a DENV-2 epidemic the year before,was largely the result of primary, and notsecondary, infection. As the severity of the1975 outbreak did not correlate with priorimmune status, these authors proposed thatthe DENV-1 strain responsible for the severeoutbreak in 1975 must be inherently morevirulent than the DENV-2 strain that causedthe dengue epidemic during the precedingyear.[18] However, DENV-2 does not alwayscause mild infections. It has been associatedwith explosive epidemics on other islands inthe South Pacific in previous years.[3] Twodifferent genetic types (genotypes) of DENV-2have been recognized, based on phylogeneticstudies: the South-East Asian and Americangenotypes. The former has been associatedwith DHF, and the latter, with mild clinicalmanifestations of dengue.[19] Interestingly, the

American genotype DENV-2 failed to cause

DHF in a major epidemic in Peru in 1995 eventhough the same population had experienceda DENV-1 epidemic about five years earlier.[20]

This observation, which is clearly not consistentwith the prediction of the ADE hypothesis, hasbeen attributed to the capacity of pre-existinganti-DENV-1 antibodies to attenuate theseverity of the secondary infection by the

American DENV-2 genotype. [21] However, giventhe high secondary transmission rate (86%), one

may argue that the reason for the absence ofsevere clinical manifestations in the Peruvianepidemic is more a consequence of the lowerlevel of inherent virulence of the AmericanDENV-2 genotype, rather than its attenuationby heterologous anti-DENV-1 antibodies.

Another line of epidemiological evidencethat supports the notion that the dengue diseaseseverity may be linked to the degree ofinherent virulence comes from the information

available from hyperendemic (co-circulation ofmultiple DENV serotypes) areas. For example,


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in the Americas where multiple DENVserotypes co-exist, the occurrence of DHF was

not documented until the 1981 Cubanepidemic. This coincided with the introductionof a new genetic type of DENV-2.[19,22] Similarly,DHF was relatively uncommon in Sri Lanka priorto 1989, despite hyperendemicity. However,after 1989, the incidence of DHF, whichincreased dramatically, was frequentlyassociated with DENV-3.[23,24] Again, as notedfor DENV-2 above, DENV-3 also exists in mildand virulent forms. This is quite vividly evidentfrom a comparison of the dengue epidemicsrecorded in Central Java during the late1970s.[25] Even though the predominant virusisolated in both these epidemics was DENV-3,one was associated with milder and the otherwith more severe illness. Similarly, in regard toDENV-1 in the South and Central Pacific islands,some have experienced silent transmissions,while others in contrast have had explosiveepidemics.[26] The existence of mild and virulentstrains within each serotype suggests that the

dengue virus changes in its epidemic potentialas it moves through populations.[3,4]

Molecular evidence

Genetic variation in dengue virus isolates hadbeen initially documented on the basis of cross-neutralization assays[27] and RNAse T1oligonucleotide fingerprinting.[28] However,both these methods that recognize serotype-,group- and family-specific epitopes fail to detectphenotypically silent differences. In order todelineate the evolutionary and epidemiologicalrelationships between many isolates of thesame virus, Rico-Hesse developed a primerextension-based limited sequencing approach.Based on a careful analysis of a 240-nucleotidesequence spanning the E/NS1 junction of 40isolates each of DENV-1 and DENV-2, sheidentified five distinct genotypes for each of

these serotypes.[22]

Rico-Hesses method wasmodified by Lanciottis group into an RT-PCR-

based sequencing method, focusing on theentire envelope region of the dengue virus

genome. Their studies, which confirmed theexistence 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 correlationbetween disease severity and virus genotype,based on the phylogenic data in conjunctionwith epidemiology, in their DENV-3 study.While DENV-3 subtype 4 had never beenassociated with severe disease, DENV-3subtypes 1 (in Indonesia and Thailand) and 3(in Sri Lanka and India) have been isolated fromDHF patients. Furthermore, their phylogeneticanalysis suggests that these DHF strainspresumably were the result of genetic drift ofthe endemic strains.[30] In regard to DENV-2,specific mutations associated with changes invirulence have been identified using infectiousclones,[32,33] chimeric constructs[34] and by directcomparison of wild-type and attenuatedstrains.[33,35]

The most convincing evidencedemonstrating a genetic basis to denguevirulence comes from the work of Rico-Hessesgroup. Their molecular epidemiological studies,in conjunction with clinical data, havedemonstrated an association between theintroduction of a South-East Asian genotype ofDENV-2 and the appearance of DHF in the

Americas. Furthermore, the native DENV-2American genotype, known to cause only themilder disease, appears to have been displacedby the virulent imported South-East Asiangenotype.[19] In a subsequent study, this groupdetermined the full nucleotide sequences ofDENV-2 strains directly from clinical samplesfrom DF and DHF patients in the Americasand Thailand, respectively. From a comparisonof 11 different strains, they identified severalconsistent structural differences between thelow (American genotype) and high (South-East

Asian genotype) virulence strains, summarized


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showing that replacement of aa 390, which ispart of the E domain involved in host cell

surface receptor binding,[37] impacts theneurovirulence of the virus in mice.[35] TheNTRs fold into secondary structures comprisinga number of stable stem-loops, that appear tobe highly conserved and are expected to playan important role during viral infection incoordinating gene expression and the onset ofRNA replication. For example, the 5 NTR binds

in Table 1.[36] Based on their comparativemolecular analysis they have determined that

the primary determinants of virulence residein the E gene, the 5 non-translated region (5NTR) and the 3 NTR. Interestingly, all theseare of critical importance in the virus replicationcycle. For instance, they identified a mutationcorresponding to amino acid (aa) residue 390on the E protein as a potential determinant ofvirulence. This observation ties up with the data

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.


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to the ribosomes to initiate translation. Morerecently, it has been shown to function as a

promoter for the transcription of the minussense genomic RNA which eventuallytemplates plus sense RNA synthesis during theviral replicative cycle.[38] This entails a role forthe 3 end of the minus RNA intermediate(complement of 5 NTR) in the initiation of(+) RNA synthesis. Finally, our own studieshave implicated a role for the 5 end of theminus sense RNA (complement of the 3 NTR)in viral replication. [39] Essentially, all the

functions of the NTRs are mediated by theirspecific interactions with viral and host proteins.Table 2 provides a summary of the host factorsdocumented to interact with DENV and otherflaviviral NTRs.[39-48] Consistent with theirimportance in various aspects of the viral life-cycle, mutations that alter the predicted foldingof the NTRs can influence the degree of viralvirulence. A comparison of the predictedsecondary structures of the 5 NTRs of the

American and South-East Asian DENV-2

genotypes, based on the data of Leitmeyer andcolleagues,[36] revealed minor, subtle changes(Figure, panels A and B). In contrast, however,a set of mutations identified by theseresearchers in the 3 NTR of DENV-2 Americangenotype predicts a significantly differentsecondary structure compared to the 3 NTRof the South-East Asian genotype of DENV-2(Figure, panels C and D). Others have alsosubsequently corroborated these 3 NTR

secondary structural differences between thelow and high virulence genotypes of DENV-2.[49] To address the question if the putativeviral determinants do indeed determine thedegree of virulence, Cologna and Rico-Hesse[50]

generated a panel of chimeric infectious cloneswherein they introduced the E390 substitution,and the 5 and 3 NTRs of DENV-2 Americangenotype into the background of the South-East Asian genotype singly and in variouscombinations. Their analysis showed that allthree American genotype-specific mutations

acted synergistically to result in a largereduction in virus replication and output. This

study showed definitively that Americangenotype structures decreased the replicativeability of the South-East Asian genotype-derivedinfectious clone. This may explain at leastpartially the reason why the American genotypehas never been associated with DHF.[50] Morerecent studies based on an analysis of thereplication efficiencies of several American andSouth-East Asian genotypes in primary cellcultures and in mosquitoes support the notion

that viruses of the latter genotype have greaterpathogenic potential by virtue of their capacityto replicate to significantly higher levels in thehuman hosts.[51]

Factors contributing to geneticdiversity

As discussed above, dengue viruses are proneto change giving rise to strains with varying

degrees of virulence. Increasingly it is becomingapparent that many instances of epidemic DHFare associated with the circulation of viral strainswith increased virulence. What drives suchchange? While a definitive answer is notavailable, several factors have been attributedto be potential contributors to the observedgenetic diversity of dengue viruses.[52] Firstly,genetic diversity is apparently an inherentproperty 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)-mediatedreplication mechanism, estimated to produceone error per round of replication.[53] However,it is regarded that the overall spontaneousmutation rate may be subject to functionalconstraints stemming from the need of thedengue viruses to replicate in two disparatehosts, 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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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 disulphideisomerase; 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, Electrophoreticmobility shift assay; IP, Immunoprecipitation assay; * unpublished data.


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


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switches mid-replication from one template toanother. This possibility draws support from the

work of Worobey et al.[54] who performed adiversity analysis of 71 published dengue virussequences and found several hybrid sequenceswhich were mosaics comprising gene regionswith conflicting evolutionary histories. Theirwork indicates that dengue viruses manifestwidespread recombination within, but notbetween, serotypes in natural populations. Thisstudy supports the role of recombination as asignificant factor in shaping the genetic diversity

of dengue viruses. Thirdly, it has been proposedthat genetic variation can be introduced throughmigration, or gene flow, given that the hostsand vectors are often transported across largedistances enabling wide geographicaldistribution of viral strains.[22] This, in turn, couldlead to greater mixing of viral strains and thegeneration of diversity throughrecombination.[52] Finally, the origin of suchdiversity in dengue viruses may simply be theresult of enhanced opportunities for

transmission stemming from the increase insize and density of the human hostpopulation.[55] Thus, it appears that geneticvariation evident in dengue viruses may be theresult of the action of multiple factors at work.[52]

Conclusions

Dengue infection has re-emerged as a public

health problem of global proportions. Thepathogenesis of severe dengue disease is notfully understood, as appropriate in vitro and invivo models are not available to investigate theprogression of mild DF to sever DHF/DSS. Outof the several explanations proposed based onepidemiological and experimental evidence, asdiscussed at the beginning, one proposedmechanism, which invokes diseaseexacerbation through the ADE phenomenonupon secondary infection with a heterotypicDENV serotype, is most widely accepted.

However, not all cases of DHF can beaccounted for by the ADE hypothesis.

Molecular evidence accumulated in recentyears is increasingly pointing to the involvementof viral factors in DHF/DSS pathogenesis.Preliminary proof-of-concept experiments haveshown that viral determinants modulate thedegree of virulence, and therefore, diseaseseverity. Again, not all cases of DHF can beexplained by viral virulence alone. That immuneenhancement and virulence are not the onlyfactors in determining the severity of dengue

pathogenesis is dramatically illustrated by thesituation reported in Haiti recently.[56] Despitehyperendemicity and the presence of a virulentDENV-2 genotype, the two prerequisitespostulated by the immune enhancement andviral virulence hypotheses respectively, DHFhas not been detected in the Haitians. Thisinteresting observation points to the existenceof a putative dengue resistance gene in theHaitian population. In fact, several other host-related have also been implicated in the

disease pathogenesis. Thus, it is likely that DHFpathogenesis may have a multifactorial basis.The recent description of murine models whichmanifests the hallmarks of dengue disease, suchas fever, rashes and decreased plateletcounts[57] and increased vascularpermeability,[58] may prove useful in delineatingthe role of all putative factors and pave theway towards an integrated understanding ofDHF pathogenesis.

Acknowledgements

This work was supported by internal funds fromthe International Centre for Genetic Engineeringand Biotechnology (ICGEB) and a grant fromthe Department of Biotechnology, Governmentof India. SC is a recipient of a senior researchfellowship from the Council of Scientific andIndustrial Research, Government of India.


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