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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 690835 20 pageshttpdxdoiorg1011552013690835

Review ArticleUnderstanding the Dengue Viruses and Progress towardsTheir Control

Rosmari Rodriguez-Roche1 and Ernest A Gould23

1 ldquoPedro Kourirdquo Tropical Medicine Institute WHOPAHO Collaborating Centre for the Study of Dengue and Its VectorAutopista Novia del Mediodıa Km 6 12 La Lisa PO Box 601 Marianao 13 11300 Havana Cuba

2UMR190 Emergence des Pathologies Virales Aix-Marseille Universite Institut de Recherche pour le DeveloppementEcole des Hautes Etudes en Sante Publique Unite des Virus Emergents Faculte de Medecine de Marseille 27 Boulevard Jean Moulin13005 Marseille cedex 05 France

3 CEHWallingford Maclean Building Benson Lane Crowmarsh Gifford Oxfordshire OX10 8BB UK

Correspondence should be addressed to Rosmari Rodriguez-Roche rosmariipksldcu

Received 13 March 2013 Accepted 8 May 2013

Academic Editor Vittorio Sambri

Copyright copy 2013 R Rodriguez-Roche and E A Gould This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Traditionally the four dengue virus serotypes have been associated with fever rash and the more severe forms haemorrhagic feverand shock syndrome As our knowledge as well as understanding of these viruses increases we now recognise not only that theyare causing increasing numbers of human infections but also that they may cause neurological and other clinical complicationswith sequelae or fatal consequences In this review we attempt to highlight some of these features in the context of dengue viruspathogenesis We also examine some of the efforts currently underway to control this ldquoscourgerdquo of the tropical and subtropicalworld

1 Introduction

Dengue fever is a mosquito-borne virus disease of humansIn terms of numbers of individuals infected it is by far themost devastating of all the recognised arthropod-transmittedvirus diseases It is estimated that more than 3 billion humanslive in dengue endemic regions of the world and currentlymore than 50 million infections occur annually with atleast 500000 individuals requiring hospitalisation [1] Ofthese tens of thousands have a high risk of developinghaemorrhagic disease potentially with fatal consequencesdepending to a large extent on the quality of the availablemedical services

The dengue viruses are positive stranded RNA viruses inthe genus Flavivirus family Flaviviridae [2] There are fourdistinct dengue virus (DENV) serotypes that share antigenicrelationships (DENV-1 DENV-2 DENV-3 and DENV-4)and although infection with one serotype confers lifelongprotection against that serotype it does not necessarilyprotect against a secondary infection with a heterologous

serotype Indeed nonprotective but cross-reactive antibodiesmay enhance disease severity [3] Currently there are noeffective vaccines or antiviral drugs against these virusesThisproblem is being addressed as a matter of urgency as failureto develop effective DENV control strategies will inevitablyresult in a further increase in the number of infected humansas predicted more than a decade ago [4]This problem is alsoexacerbated by the continuing dispersal of these viruses tonew geographic regions

This review therefore focuses on our current understand-ing of dengue virus pathology epidemiology pathogenesisevolution biogeography and disease control

2 Dengue FeverHaemorrhagicFeverShock Syndrome

21 Clinical Picture Inmost cases asymptomatic or relativelymild disease follows infection with dengue virus However totake into account the increasing number of clinical cases theWorld Health Organization (WHO) produced guidelines [5]

2 BioMed Research International

in which they identified the clinical pictures resulting frominfection with dengue virus The first known as denguefever (DF) is characterised by an abrupt onset of feveraccompanied by frontal headache and retroorbital painfollowed by a variety of possible clinical symptoms such asmyalgia arthralgia vomiting and weakness A generalisedmaculopapular rash appears one or two days after feverdefervescence Minor haemorrhagic manifestations such aspetechiae may be observed in some patients DF is generallyself-limiting and rarely fatal Most patients recover withoutcomplications around ten days after the onset of illness

The second clinical picture dengue haemorrhagic fever(DHF) is a more severe form of the disease and occurs inup to 5 of dengue cases It is initially characterized by thesame variety of clinical symptoms as are seen in DF Thecritical period in DHF starts at the moment of defervescencebut haemorrhagic manifestations may occur 24 hours earlierA positive tourniquet test indicates that the patient hasincreased capillary fragility Petechiae bleeding at venepunc-ture sites epistaxis gum bleeding and haematemesis mayalso be observed High fever haemorrhagic manifestationsthrombocytopenia (platelet count 100 000mm3 or less) andhaemoconcentration (gt20 difference) characterize DHFPlasma leakage is the most significant pathophysiologicalevent in determining the severity of the disease Signs ofcirculatory failure such as irritability cold clammy extrem-ities flushed face and restlessness may be observed Thiscrisis usually persists for 24ndash36 hours With appropriatesupportive medicine and carefully monitored intravenousisotonic crystalloid therapy to ensure adequate fluid replace-ment most patients recover However during this criticalperiod it is essential to look for characteristic warning signsof worse to come Patients progressing to shock (dengueshock syndromemdashDSS) show intense abdominal pain ortenderness persistent vomiting weak pulse and hypoten-sion If increased vascular permeability progresses to vascularcollapse the outcome is usually fatal as a result of irreversibleDSS In addition to DF DHF and DSS it is now recog-nised that other clinical manifestations can be associatedwith infection by dengue virus for example encephalitismyocarditis hepatitis cholecystitis myelitis and acute colitis[6ndash11]

Despite the rigour of the DFDHFDSS classification andits intrinsic worth in clinical case management in recentyears a growing number of clinicians and authors haveargued that the 1997WHO scheme for dengue clinical classi-fication should be reassessed [12ndash14] because it distinguishesstrictly between DF DHF and DSS whereas it is now recog-nised that the point of transition between DF and DHF is noteasily defined The requirements for the WHO definition ofDHF (fever haemorrhage thrombocytopenia and signs ofplasma leakage) are not always satisfied severe thrombocy-topenia may be observed in uncomplicated as well as severecases associated with ldquounusual manifestationsrdquo and thereforemay not be consistent with theDHFDSS classification [15] AWHOTDR-supported prospective clinical multicentre studyacross dengue-endemic regions was established to collectand coordinate specific criteria for classifying clinical cases

into levels of severity [16] The study confirmed that byusing a set of clinical andor laboratory parameters one seesa clear-cut difference between patients with severe denguefever and those with nonsevere dengue fever However forpractical reasons it was considered necessary to split thelarge group of patients with nonsevere dengue into twosubgroups patients with warning signs (abdominal pain ortenderness persistent vomiting clinical fluid accumulationmucosal bleeding lethargy restlessness liver enlargementgt2 cm and increase in hematocrit concurrent with rapiddecrease in platelet count) and those without these warningsigns On the other hand the criteria for severe dengue feverinclude extensive plasma leakage severe bleeding or severeorgan impairment

The third and most recent edition of the WHOTDRdengue guidelines for diagnosis treatment prevention andcontrol includes a new clinical classification [17] This publi-cation serves as an authoritative reference source for healthworkers and researchers These new guidelines provide arevised case classification which is intended to facilitateeffective triage and patient management and collection ofimproved comparative surveillance data [18] However dueto the recommendation that cases of dengue fever withwarning signs and also cases of severe dengue fever shouldbe admitted to hospital there is concern that this could resultin overadmission of patients to hospitals during epidemicsinevitably reducing the efficiency of patient triage andadversely affecting the quality of clinical case management[19 20] Furthermore there is additional concern that theWHOTDRclassificationmay impact significantly on denguepathogenesis research since it requires the identificationand study of distinct dengue syndromes Because the 2009WHO case definitions do not require laboratory tests for thediagnosis of severe dengue it is considered that retrospectiveidentification of patients with clinically significant vascularpermeability from data on hospital charts may be difficultif not impossible [21] The previous discussion highlightsthe difficulties of designing a totally acceptable classificationscheme for dengue pathogenesis

22 History of DF and DHF Clinically diagnosed DF waswidespread during the 18th and 19th centuries in Northand South America the Caribbean Basin Asia and Aus-tralia In the Americas this was largely due to the repeatedintroduction from Africa of Stegomyia (St) aegypti (formerlyAedes aegypti) [22ndash24] Moreover together with yellow fevervirus (YFV) DENV-infected humans and mosquitoes wereintroduced via the slave ships and other commercial vesselsthat crossed the Atlantic Ocean from Africa during the pastfive or more centuries [25ndash31]

It is also important to note that disease clinically com-patible with the more severe and often fatal syndromes DHFand DSS was sporadically reported from 1780 onwards [32]although it is not clear if the more severe cases were confinedto individuals of European descent

DF thus became endemic in Latin America and theCaribbean region periodically causing epidemics At thesame time YFV was also causing epidemics in South

BioMed Research International 3

America prompting the Pan American Health Organisation(PAHO) to introduce a mosquito-eradication programmewhich lasted from 1946 until the late 1970s Since both DENVand YFV are transmitted to humans via St aegypti this erad-ication campaign in South America also resulted in a lowerincidence of DF in South America Thus DF was confinedmainly to the Caribbean basin [33 34] Subsequently thegradual decline of mosquito control measures and increasingintroduction and dispersal of mosquitoes via transportationfor commercial andmilitary purposes led to the reemergenceof dengue as a major health problem during the mid andlater parts of the 20th CenturyThe incidence of dengue feverincreased dramatically in Southeast Asia duringWorldWar IIand continued to intensify with increased geographic spreadof the viruses and the principal mosquito vector St aegyptiIn addition to the major influence of increased shipping andair-traffic globally other major factors for the reemergence ofdengue fever include ecological and demographic changes inthe tropical zones [2 34ndash39]

During the 1980s and 1990s rapidly expanding popula-tions of St aegypti in Brazil resulted in successive epidemicsdue toDENV-1 DENV-2 andDENV-3 In Brazil these infec-tions presented mostly as DF with surprisingly few cases ofDHF This contrasts with Asia where the proportion of DHFcases was significantly higher during DF epidemics Thesedifferences have been partly attributed to the widespreadpresence of dengue virus resistance genes in Latin Americanswith African ancestry [33 40 41] The differences may alsobe partly explained by the high levels of antibody against theAmerican DENV-2 genotype and antigenically cross-reactiveDENV-1 both of which had been endemic in Latin Americafor many years

Today all four DENV serotypes circulate in Africa Southand Southeast Asia theWestern Pacific region theCaribbeanbasin and Central and South America [39 42ndash44] Frequentintroductions into the Southern states of North Americaare also regularly recorded although to date they have notresulted in epidemic outbreaks in the USA DF has thepotential to become reestablished as an endemic disease inthis country In fact sustained transmission of dengue hasoccurred in Florida during recent years Conditions existthat could facilitate sustained dengue transmission includingenvironmental factors competent mosquito vectors limitedvector and dengue surveillance increased domestic outdoordaytime activities in warmer months and low public aware-ness of the disease [45] Indeed dengue continues to spreadmore widely as demonstrated in 2010 by the first recordedcases of autochthonous dengue fever in southern France [46]and Croatia [47]

Many countries in the tropics and subtropical regionsshow cocirculation of at least two DENV serotypes [36] andincreasingly cocirculation of all 4 serotypes is being recordedin individual countries Taken together with the ecologicaland demographic changes this partly explains why thepattern of epidemics is gradually increasing from a frequencyof outbreaks every 3ndash5 years to approximately every 2 years[48] Additional explanations for this increased incidenceinclude the possibility that more highly pathogenic strainsof DENV are also emerging [44 49ndash51] Greater awareness

of this disease as the result of more extensive monitoringis also impacting on our understanding of and the apparentincreased periodicity of dengue virus epidemiology

Comparison of disease incidence in Asia and LatinAmerica reveals a distinct difference in the age distributionof DF and DHF In Asia hospitalizations principally involvechildren whereas in the Americas they tend to involve agreater proportion of adults [33] The reasons for this appar-ent difference have not been adequately defined However tocomplicate this issue a recent epidemic in the State of Riode Janeiro revealed that the incidence of DHF in childrenwas significantly higher than in previous epidemics in Brazil[52 53]

23 Risk Factors Associated with the Development of SevereDengue The principal vector associated with all 4 DENVserotypes is the African mosquito St aegypti an urban-dwelling anthropophilic mosquito However St albopicta theAsian ldquoTigerrdquo mosquito is also competent to reproduce andtransmitDENVbetween humans In contrast with St aegyptiSt albopicta is peridomestic with a preference for the ruralenvironment In some parts of Asia and Africa St albopictahas displaced St aegypti [54 55] A possible scenario of thischanging pattern ofmosquito distribution and the continuingdispersal of St albopicta is that the dengue viruses willdisperse even more widely gradually establishing in thewarmer regions of the temperate zones including Europe[46 47 56 57] the southern regions of North America [58]and more northern regions of Asia [59]

The pathogenetic basis for DHF has been a subject ofstudy for decades and whilst significant progress has beenmade in understanding the most important risk factorsinvolved the precise biochemical and immunological path-ways have not yet been defined [60ndash62] Amongst the severalpossibilities that have been identified there is compellingevidence that secondary infectionwith a heterologousDENVserotype or primary infection in infants born to dengue-immune mothers is an important individual risk factorfor DHFDSS [63ndash70] During secondary infection with adifferent serotype the presence of low levels of heterotypicneutralizing antibodies may reduce disease severity Alter-natively in the absence of such neutralizing antibodies het-erotypic cross-reactive antibodies may form complexes withthe virus and the Fc-receptors on these complexed antibodiesmay attach to mononuclear phagocytes thus enhancing theefficiency of infection and thereby increasing the number ofinfectedmononuclear phagocytes [71ndash74]This phenomenonis known as antibody dependent enhancement (ADE) [75]Humans infected with one serotype maintain a life-long pro-tective immunity to infection by the homologous virus butprotective immunity to infectionwith heterologous serotypesis relatively short-lived [76]The precisemechanism bywhichDENV replication is amplified in the infected cells remainsunclear One possibility is that there is a relationship betweenDENV-ADE infection suppression of nitric oxide during theinnate immune response and the corresponding cytokine-expression pattern in THP-1 cells [77] Recent evidencesuggests that viral susceptibility or resistance to nitric oxidemay be regulated by the viral NS5 protein [78]


It has also been argued that strain differences in virulencemay contribute to disease severity [51 79ndash84] However thefact that severe dengue disease is identified most consistentlyfollowing secondary dengue infections supports the view thatvirulence must be defined in a two-infection context [39]Host risk factors such as gender ethnicity the presence ofchronic disease (bronchial asthma diabetes mellitus andsickle cell anaemia) [85ndash87] and also the genetic character-istics of the individual are also likely predisposing factors forsevere illness Human leukocyte antigen (HLA) Fc120574R tumornecrosis factor- (TNF-) 120572 and dendritic cell-specific intra-cellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) among other genes have been associated with thepathogenesis of dengue [41 66 86 88ndash93] In addition T-cellmediated immune mechanisms involving skewed cytokineresponses resulting in plasma leakage are also risk factorsfor DHF [62] It has been proposed that an inappropriateimmune response to the secondary virus infection that inturn induces reactivation of cross-memory T cells specificfor the first rather than the secondary DENV infectionresults in delayed viral clearance [94 95] More recentlyit was suggested that the presence of an effective antiviralinflammatory response in the presence of adequate immuneregulation could be associated with protection during denguesecondary infection [96] However as highlighted in a recentreview on dengue pathogenesis it is worth noting that otherinfectious diseases and inflammatory disorders result inelevated cytokines without the attendant increased vascularpermeability seen in severe dengue [97] Indeed one of themajor challenges in denguemagnified due to absence of goodanimal models of disease is to dissect those elements of thehost immune response that are causally linked to capillarypermeability from those that simply reflect the normal hostimmune response to a pathogen [97]

Independent of this antibodies specific for the NS1viral protein may form immune complexes with the NS1protein in the circulation and on the surface of infected cellsleading to complement activation [98] An additional riskfactor for DHF is believed to be dependent on autoimmuneresponses against cross-reactive viral components For exam-ple antibodies specific for dengue virus NS1 protein mayinduce platelet lysis andor nitric oxide-mediated apoptosisof endothelial cells contributing to thrombocytopenia andvascular damage [99ndash104]

The serious complications observed during dengue infec-tion occur as plasma viremia is resolving This is thoughtto be immunologically mediated Tam et al performed arandomised trial to verify the effects of short-course oralcorticosteroid therapy in early dengue infection No associ-ation between treatment allocation and any of the predefinedclinical hematological or virological endpoints was found[105] Unexpectedly the steroid doses administered werenot immunosuppressive Based on these observations it wassuggested that rather than dengue-mediated vascular per-meability being T-cell mediated an alternative pathogeneticmechanism could involve the dengue soluble complementfixing antigen or the viral NS1 protein Indeed it was recentlyproposed that during the late stages of clinically apparentdengue infection secreted DENV NS1 protein may bind to

prothrombin and inhibit its activation which in turn couldcontribute to the prolongation of activated partial throm-boplastin time and haemorrhage in DHF patients [106]Also previous studies on the virological course of dengueinfections in monkeys have shown that the peak of cellularinfection occurs at the end of the viremic phase Accordinglyit was proposed that dengue vascular permeability syndromecould be the equivalent of a viral toxicosis induced bycirculating NS1 protein [107]

Whilst higher levels of viremia and circulating NS1protein have been associated with dengue disease severity[108] collective results arising from different epidemiologicalsettings are inconclusive showing variations depending onthe infecting serotype and patient immune status [109ndash112]Therefore the usefulness of these markers for the recognitionof patients with increased risk of progression towards themore severe forms of dengue is still limited

24 Underlying Basis for the Emergence of DHF in ParticularEpidemiological Settings Taking into account many of thefactors described earlier and based on observations madeduring the 1981 Cuban epidemic of DFDHF Kouri andcoworkers presented an integral hypothesis in which theassociation of different factors such as immunological statusgenetic background host condition viral strain and epi-demiological and ecological conditions determines whetheror not and to what extent DHF will be involved in anyparticular epidemic [113] Research conducted during thepast 20 years strongly supports this unifying view of thesituation [3 39 48 114]

With the exception of Chile Uruguay and Cuba thatexperience occasional epidemics resulting from introducedvirus DF is endemic in Latin America and the Caribbeanregion Cuba is a relatively small island with a well-integratedmedical health and research infrastructure When combinedwith the epidemiological history of DF in Cuba this situationhas provided a unique opportunity to investigate the specificrisk factors for severe illness in detail [48] Firstly it isimportant to realise that from the end of World War IIuntil 1977 dengue virus was not evident in Cuba This wassupported by a national seroepidemiological survey in 1975which identified only 26 of the population with DENVhemagglutination-inhibition (HI) antibodies Importantlymost of the positives were individuals older than 45 years[115 116] However in 1977 based on serological evidence[117] it was estimated that up to 445 of all Cubansbecame infected by an introduced Asian strain of DENV-1Nevertheless no cases of DHF were recorded These resultsdemonstrate that in the absence of heterologous immunityprimary DENV-1 infections did not result in cases of DHFand bearing in mind that subsequent DHF epidemics inCuba all involved secondary infections (see later) the resultsstrongly support the contention that secondary infections byheterologous serotypes are a very important risk factor forDHF as proposed previously [118]

During the past 28 years three DENV-epidemics involv-ing DHF have occurred in Cuba The first epidemic startedin 1981 [113] the second was seen in 1997 [65] and


the third occurred in 2001 [63] During each epidemicsecondary infection was demonstrated as themost importanthost risk factor for DHF Additionally specific sequentialvirus serotypes were associated with severe disease indepen-dent of the time-gap between the primary and secondaryinfection For example in Cuba two epidemics of DHF havebeen associated with primary infections due to an Asianstrain of DENV-1 and secondary infections due to DENV-2 (ie DENV-1DENV-2) [119 120] Significantly during the1997 Cuban epidemic it was demonstrated for the first timethat there was a higher risk of DHF in DENV-1DENV-2individuals when the average time gap between primary andsecondary infections was about 20 years as opposed to 4years a more commonly reported timespan [64] Moreovercomparison of attack rates and case fatality rates in the sameage groups revealed that during the 1997 epidemic the rate inpatients older than 15 years of age was 40 times higher thanduring the 1981 epidemic [121]

Subsequently another Cuban epidemic caused byDENV-3 and involving cases of DHF occurred in 2001ThusDHF occurred in DENV-3-infected Cubans 24 years afterprimary exposure to DENV-1 infection [122] InterestinglyCubans infected sequentially with DENV-1DENV-3 wereassociated with severe disease whilst those infectedsequentially with DENV-2DENV-3 were associated withmilder disease or asymptomatic infections [63 123] Addi-tionally the DENV-3 immune individuals infected duringthe 2001 Cuban epidemic revealed differences in the neu-tralization capability of their sera to different DENV-3 strainsbelonging to different genotypes [124] This observationmight be anticipated taking into account that differencesin neutralisation capability have been found using differentgenotypes of DENV-2 However it was highly significant tofind differences in neutralisation against strains belongingto the same genotype [125] Moreover the strains involvedin the Cuban 1981 1997 and 2001 epidemics [80 125 126]had previously been associated with severe epidemics andtherefore had the potential to produce DHF Neverthelessin all of these epidemics an extremely high number ofprimarily infected individuals were asymptomatic [64]

Using human volunteers Dr Albert Sabin was the firstscientist to demonstrate that heterotypic immunity can pre-vent disease induced by a different dengue virus serotype[127] Whilst DENV-1 immunity did not appear to preventDENV-2 infections partial immunity may have downregu-lated infections thus reducing severity tomild disease duringsecondary dengue infections It has been postulated thatif virological factors are involved in determining diseaseseverity they may reflect common antigenic determinantsshared between the first and second infecting viruses [39] Anexceptional illustration of this phenomenon is the neutraliza-tion of American genotype DENV-2 by human antibodies toDENV-1 [81] These results suggest that the apparent lowervirulence of American genotype DENV-2 results from aDENV-1 like surface epitope on the DENV-2 that permitspartial neutralization (and downregulation of disease) byDENV-1 antibodies [81] In contrast Asian genotypesDENV-2 are poorly neutralised by human antibodies to DENV-1 [128] Furthermore a significant increase in the mean

titre of homologous DENV-1 neutralizing antibodies and asignificant decrease in heterologous antibodies to DENV-2American genotype were reported in a long-term study inCuba [128]This finding may reflect time-dependent changesin severity of disease observed following secondary dengueinfection

On the other hand case fatality rates were observedto increase month by month during epidemics that werestudied in Cuba Taking into account that DENV-2 epidemicsoccurred in 1981 and 1997Guzman and coworkers proposed aneutralisation escape mutant hypothesis based on the associ-ation of severe disease with dengue secondary infection [129]Furthermore during the DENV-3 epidemic that occurred inHavana in 2001 the same sequential increase in case fatalityrates was observed [48 85 122]

Although specific viral factors alone probably do notdetermine the severity of dengue infection in individualcases the demonstrated increasing severity of infection withtime during a single epidemic strongly argues that significantchanges occur in the virus causing the epidemic Indeed hostfactors do not appear to explain this observation of increasingseverity with time because it is not logical to assume that themost susceptible individualswould all be infected towards theend of the epidemic

The 1997 Cuban epidemic was the most severe reportedin Cuba to date Nevertheless a search for evidence of theappearance of neutralisation-escape mutants proved nega-tive The structural gene sequences were highly conservedin viruses isolated at different times during the epidemic[126] However nucleotide substitutions were found in thenonstructural genes and in general they correlated withthe time of sampling showing a clear pattern of virusevolution during the epidemic [130] Therefore at least inthis study antibody-driven selection of escape mutants in thestructural genes was not the key selective force On the otherhand cytotoxic T-lymphocytes (CTLs) play a crucial rolein controlling infection in RNA viruses including dengueviruses [62 131] Variation in the epitopes recognized by CTLis common and frequently offers potential escape routes formutant virus Forthcoming studies will assess whether or notthe reported mutations in NS1 and NS5 proteins [130] arerepresented in antibody inducing or CTL epitopes

Regardless of which mechanism that is natural selectionor genetic drift is operating it is likely that a fitter virus couldbe selected during the period of high transmission in individ-uals that have experienced secondary infections However itis a very difficult task to study dengue epidemiology becauseit is not only endemic in most tropical countries but there arefour serotypes andmany different genotypes often cocirculat-ing Nevertheless Cuba represents a unique epidemiologicalsetting for this kind of research because epidemics caused byonly one serotype have occurred providing the opportunityfor carefully defined epidemiological studies

Mutations in the nonstructural genes of DENV-2 iso-lated during the Santiago de Cuba epidemic may corre-late with increased efficiency of virus replication Variationin nonstructural proteins has been also associated withincreasing severity in epidemiological settings correspond-ing to endemicepidemic transmission [132ndash136] However


the specific relevance of these types of mutation has not yetbeen investigated thoroughly This is in large part due to thelack of suitable animal models with which to study denguevirus ldquovirulencerdquo [51 137]

During the most recent and severe DENV-4 epidemicin Puerto Rico in 1998 viruses were distinguished by threeamino acid replacements in the NS2A protein (I14V V54Tand P101T) which were fixed more rapidly than wouldhave been expected by drift alone This study demonstratesthe significance of viral genetic turnover within a focalpopulation and the potential importance of adaptive evo-lution during viral epidemic expansion [132] In contrast aretrospective phylogenetic study of events on the SouthernPacific islands three decades ago where severe dengue wasdescribed in patients infected with the DENV-2 Americangenotype recorded attenuation of this virus following a seriesof outbreaks involving nonsynonymousmutations also in theNS2A gene [138]

Similarly study of the population structure of dengueviruses transmitted in Aragua Venezuela during the period2006-2007 under hyperendemic conditions also suggestedthat the nonstructural proteins could play an important rolein DENV evolution According to this particular epidemi-ological setting changes in NS1 NS2A and NS4B proteinswere either favourable or adverse in terms of viral fitnessTheauthors argued that specific mutations could be associatedwith severe disease but some could be associated with milddisease due to the appearance of naturally attenuated strains[139]

The flavivirus NS2A protein is a small hydrophobic mul-tifunctional membrane-associated protein involved in RNAreplication [140 141] host-antiviral interferon response [142ndash145] and assemblysecretion of virus particles [146ndash148] Inaddition the NS2A and the NS4B proteins may participatein the modulation of vector competence [149] Accordingto previous reports changes in NS1 and NS4 proteins couldbe involved in viral attenuation [150 151] On the contraryrecent studies have demonstrated that mutations in the NS4Bprotein may increase the efficiency of DENV replication Inaddition it has been suggested that mutations in this proteinmay also be involved in species tropism of DENV and mayevenmodulate the balance of efficient replication inmosquitoand mammalian cells [152] Moreover it has been shownthat a single amino acid in the nonstructural NS4B proteinnamely L52F confers virulence on DENV-2 in AG129 micethrough enhancement of viral RNA synthesis [153]

Whilst these results suggest a possible role for the NSgenes in determining viral fitness the importance of thestructural genes should not be overlooked The sequencescompared in the cited studies represent a consensus ofthose observed within each patient and may not necessarilyrepresent the dominant variant present in the original clinicalsample For example virus isolation usingmosquito cell lines[154] is known to perturb the distribution of variants inthe original clinical sample This is particularly importantgiven that studies of dengue virus populations sampled fromindividual humans or mosquitoes have revealed significantsequence variation [155] Consequently a greater focus onstudies of viral population variation during epidemics is

needed and the data should be obtained directly from clinicalsamples

The demonstration of long-term transmission of defec-tive dengue virus in humans andmosquitoes has added a newdimension to the study of dengue evolution The increasedfrequency of the ldquostop-codonrdquo strain was concomitant witha major reduction in DENV-1 prevalence in Myanmar Theauthors suggested that complementation between defectivevariants might provide a mechanism for the survival ofldquohyperparasitesrdquo and this process of viral complementationcould impact on pathogen transmission and virulence [156]

Obviously more comprehensive approaches includingsequencing of larger numbers of viral genomes obtaineddirectly fromdiverse clinical samples corresponding to longi-tudinal studies are needed to examine how the genetic struc-ture of dengue virus is influenced by heterotypic antibodies

Data obtained in two carefully planned clinical studiesof dengue in Nicaragua demonstrate that the complex inter-play between viral genetics and serotype-specific immunitydetermines the risk of severe dengue disease Indeed thesedata provide insights into viral evolution and the interactionbetween viral and immunological determinants of fitnessand virulence The abrupt increase in disease severity acrossseveral epidemic seasons of DENV-2 transmission coincidedwith clade replacement events Interestingly DENV-2 strainscorresponding toNI-1 clade caused severe disease specificallyin children who were immune to DENV-1 whereas DENV-3 immunity was associated with more severe disease amongNI-2B infections signifying that mutations altering the neu-tralization profile of someDENV strains can lead to increasedviral fitness [157]

Most dengue virus genomic studies have been directedat identifying the origin and genetic relatedness of theviruses causing epidemics Other studies have focussed onidentifying genetic markers associated with severe diseaseand comparing viruses isolated from DF and DHFDSScases within the same epidemiological setting Howevergenetic variations have not been consistently associated withdifferences in clinical outcome Conversely introduction ofnew genotypesserotypes with replacementdisplacement ofthe existing viruses or changes in viral populations duringan interepidemic period extinction events and sustainedtransmission of dengue virus due to repeated introductionshave all been related to changes in the severity pattern of localepidemics [134 158ndash161]

Recently in Vietnam the introduction of the Asian 1genotype of DENV-2 led to the complete replacement of theresident AsianAmerican genotype of DENV-2 The trans-mission fitness advantage of Asian 1 viruses was attributedto this virus attaining higher viremia levels in humans[162] However there are multiple factors implicated inthe transmission dynamic of DENVs that remain unclearEpidemiological data have suggested that fitness is alwayscontext dependent and that as the immunological landscapechanges viral lineages that evade cross-immunity will be at aselective advantage [163]

Clearly there is still wide scope for research on themolec-ular basis of dengue virus epidemiology and pathogenesisWe need to know whether or not a circulating dengue virus


that produces an asymptomatic infection in one host differsin sequence from the same virus that causes a fatal infectionin another host We also need to know if different tissues[164] within a single host house the same dominant denguevirus strain Similarly does the virus that circulates in asingle epidemic have the same sequence in individuals withdifferent histories of dengue infection

25 Intrahost Genetic Variation The population geneticsand evolutionary epidemiology of RNA viruses have beenreviewed [165] The authors describe migration or geneflow as a factor to consider during RNA virus evolutionAccordingly they advocate that migration must not only beunderstood at a macroscopic level (ie among hosts withina population among populations or between host species)but also within a single infected individual From the siteof inoculation viruses can be transported to several tissuesgenerating intrahost spatial variation This has been studiedin Hepatitis C virus family Flaviviridae [166] However theeffect of a nonhomogeneous population distribution on thespread fitness and variability of virus populations has notbeen studied extensively Nevertheless a positive correlationbetween migration rate and average fitness of the populationhas been observed [167] The outcome of acute Hepatitis Cinfection has been attributed to the evolution of viral quasis-pecies [168] Large-scale sequencing of complete viral RNAgenomes obtained directly from clinical samples is needed toinvestigate the role of variation in viral populations ondenguepathogenesis The purifying selection that phylogenies haverevealed [169] thus farmay bemisleading becausemost of thesequences analysed over the years were obtained from tissueculture viral isolates Additionally an intrinsic inadequacy ofutilising consensus sequences to make inferences concerningthe fitness of viral populations is that consensus sequencesonly reflect the majority nucleotide at any given position ofthe viral genome Consequently low frequency variants willremain undetected

Examination of the viral population structure inmosquitoes and patients has revealed that the sequences ofthe major variants are the same but the extent of sequencevariation seen with the mosquitoes is generally lower thanthat seen with the patients suggesting that the mosquitocontributes to the evolutionary conservation of denguevirus by maintaining a more homogenous viral populationand a dominant variant during transmission [170] Inaddition by studying the evolutionary relationships ofDENV-1 viruses that have circulated in French Polynesiaand the viral intrahost genetic diversity according toclinical presentation Descloux and coworkers suggestedfor the first time that clinical outcome may correlate withintrahost genetic diversity [171] On the other hand a recentstudy in Vietnam showed no relationship between theextent and pattern of DENV-1 genetic diversity and diseaseseverity immune status or level of viremia Interestinglydespite the high sequence conservation observed clearevidence for mixed infection with the presence of multiplephylogenetically distinct lineages present within the samehost was demonstrated [172]

Indeed most attempts to investigate intrahost geneticvariation in DENV characterised only a few viral genes ora limited number of full-length genomes A new study inNicaragua using a whole-genome amplification approachcoupled with deep sequencing to capture intrahost diversityacross the entire coding region ofDENV-2 showed significantgenetic diversity among genes [173] However the extensionof that diversity was less than expected suggesting strongpurifying selection across transmission events as have beenproposed previously [174ndash176]

Another point of view is that there is no reason toignore vector-driven selection [177] However the interactionbetween virus and vector has been less extensively exploredBy comparing the ability of DENV-1 isolates from Thailandspanning a 24-year period to infect and be transmittedby St aegypti Lambrechts et al found that a major cladereplacement event in the mid-1990s was associated with ahigher transmission potential of the isolates belonging tothe new clade Higher transmissibility was mainly due toa higher infectious titre of virus in the vectorrsquos haemocoelwhich is predicted to result in a higher probability oftransmissionThis finding supports the hypothesis thatmajorclade replacement events can be driven by natural selectionand emphasizes the potentially important role of vector-virusinteractions in DENV evolution [178]

Since the 1900s the extrinsic incubation period (EIP)that is the time taken for the viremic bloodmeal to beamplified in the mosquito and then transmitted to a newhost had been recognized as an important component ofDENV transmission dynamics The DENV EIP is generallyconsidered to be between 8 and 12 days [17] Nonethelessdifferent factors can induce variations in EIP For exampleconsiderable degrees of variation in EIP have been shownto vary depending on the specific DENV strain studiedMosquitoes feeding on humans infected with an unadaptedstrain of DENV-1 had shorter EIPs (14 days) thanmosquitoesfeeding on humans infectedwith strains at lowmouse passagelevels where the EIP was 22 days [127] In addition longEIPs have been observedwith dengue virus attenuated strains[179 180] Likewise highly controlled laboratory studieshave demonstrated the effect of distinct genotypes serotypesand mosquito population on the EIP [181 182] Taking intoaccount the advanced technologies available in molecularbiology new avenues for the study of virus-vector interac-tions should reveal newmechanisms involved in dengue virustransmission dynamics

3 Disease Control Strategies

Increasing human and associated mosquito population den-sities and mobility of humans and commercial goods arethe main factors that have determined the very successfulreemergence of DF and DHF during recent decades Incontrast with YFV which exploits the same mosquito species(St aegypti) to infect humans the dengue viruses haveevolved to become independent of the need for a reservoirsylvatic environment with which to sustain their epidemicityThus in the absence of effective control strategies we are


faced with the prospect of further increases in morbidityand mortality due to the dengue viruses Currently severaldifferent approaches (reviewed after) are being developed inthe future hope of alleviating this ldquoscourgerdquo of modern times

31 Vector Control History has shown that vector controlmeasures can be effective in reducing arthropod-borne virusdiseases [120 183ndash186] However many developing coun-tries do not have the necessary resources and infrastruc-ture for successful eradication measures to be implementedand sustainable in the manner that has been achieved inSingapore and Cuba [187] This situation is exacerbatedby the emergence of resistance to insecticides and theenvironmental issues arising from the use of potentiallytoxic chemicals [188] Today dissemination of insecticideresistance throughout vector populations is much faster thanthe rate of development of new insecticides In additionthe existence of cross-resistance based on the activation ofgeneral detoxifyingmechanisms in the vector can shorten theuseful lifespan of alternative insecticides or even prevent theirimplementation [189]

New approaches to vector eradication including the useof naturally occurring plant insecticideslarvicides [190] andvaccines that induce antibodies to impair vital functions inmosquitoes [191] are being considered but such approachesare unlikely to provide effective vector control measures inthe near future

Primary prevention of dengue is largely dependent onlarval and adult mosquito control St aegypti surveillancehas relied heavily upon larval indices However this hasbeen strongly criticised as they provide little informationto determine the risk of DENV transmission The studiesof Bisset Lazcano and colleagues used pupal surveillancefor the St aegypti control programme in Cuba and focusedon the most productive mosquito water containers [192]In urban areas St aegypti breed on water that collects inartificial containers such as plastic cups used tyres brokenbottles flowerpots and other water traps Elimination ofthese containers is the most effective way of reducing themosquito breeding grounds The use of insect repellentsmosquito traps and mosquito nets in the home can also bemoderately effective in reducing the number of bites due tomosquitoes

Novel alternative approaches have also been investigatedInVietnam trials were conducted inwhich children and localcommunities were encouraged to place a known mosquitopredator the crustacean Mesocyclops [193] in water tanksand discarded containers where St aegypti are known tothrive [194] The concept exploited the principle that thisprocedure might be more cost-effective and environmentallyfriendly than the use of pesticides Over a period of yearsand in defined rural provinces of Viet Nam a reductionin mosquitoes and dengue fever was observed [195 196]However such approaches are only likely to be successfulin regions of countries with the organisational infrastruc-ture and appropriate community attitudes A community

education strategy is utilised to promote participation indengue prevention in Cuba resulting in reduced mosquitovector infestation levels The main principle has been toincrease community participation in decision-making andstrengthening the competence of the medical teams andcommunity working groups [197] Whether or not a similarapproach could be successfully applied to major cities andurban areas in other countries remains to be seen

An alternative approach involves infecting St aegyptiwiththe bacteriumWolbachia [189] Early studies suggest that thisreduces the adult lifespan of the mosquito by 50 [198] Thisis important because the adult femalemosquito is the primaryvector of the virus Insects infected by Wolbachia transmitthem transovarially to the next generationThus by reducingthe lifespan of the mosquito virus-vector competence andvirus transmission efficiency a significant reduction of Staegypti should be observed Another important feature ofWolbachia is its ability to induce resistance to a variety ofpathogens including DENV in its insect hosts [199] Inthe transinfected St aegypti all the three different types ofWolbachia wAlbB wMelPop-CLA and wMel induce sig-nificant inhibition of DENV replication and disseminationresulting in either complete or partial block of virus trans-mission [200ndash202] Recent studies also show that Wolbachiainduces production of reactive-oxygen species which thenactivate the Toll-pathway to induce expression of antiviraleffectors [203] Interestingly it was recently demonstratedthat nativeWolbachia symbionts limit transmission of DENVin St albopictus by restricting the delivery of infectious viralparticles from the mosquito saliva when biting These resultsmight therefore explain the low vector competence of Stalbopictus for dengue and thus its relativelyweak contributionas an epidemic dengue vector [204]

Understanding how Wolbachia density is regulated bymosquito hosts and how the Wolbachia machinery controlsits replication will facilitate the current effort to eliminatedengue through Wolbachia-based population replacement[205]

The genetic structure of St aegypti populations and itsimplications for potential mosquito releases have been stud-ied in Queensland Australia [206] and Tri Nguyen villageVietnam [207 208] Populations of St aegypti artificiallyinfected with strains of Wolbachia pipientis thatinterfere with its vector competence are being backcrossedinto wild mosquito genetic backgrounds from northQueensland and assessed as potential candidates for release[209] In addition a pilot releasemdashhttpwwweliminatedenguecomprojectvietnemprogress mdashof infected mosquitoeshas been authorised to take place from April 2013 on TriNguyen village (611 households) on Hon Mieu Island incentral Vietnam Subject to satisfactory results larger scalestudies could be launched within five years [210]

Promisingly studies related with the effect of Wol-bachia on insecticide susceptibility in lines of St aegyptihave demonstrated that spreading Wolbachia infections areunlikely to affect the efficacy of traditional chemical methodsof controlling mosquito outbreaks [211]


32 Development of Vaccines against Dengue Viruses It isgenerally agreed that vaccination can provide an effectivemethod with which to control virus diseases In the case ofthe dengue viruses the four serotypes are sufficiently anti-genically different that it is considered necessary to producefour monovalent vaccines which will then be mixed to pro-duce a tetravalent immunological response This is a logicalapproach that has previously been employed successfullywith the three monovalent poliovirus vaccines However asdiscussed earlier the dengue viruses also present the problemof antibody mediated enhancement of disease severity [75118 212] Long-term protection is essential as severe denguehas been observed in individuals secondarily infected morethan 20 years after the primary infection [63 85] As it isvirtually impossible to test whether or not the tetravalentformula would overcome this potential problem an elementof uncertaintymight prevail following the introduction of thevaccines in Asia andor Latin America where dengue virusesare most prevalent In addition although the protectiverole of neutralizing antibodies is recognised correlates ofprotection need to be defined [213]

Several different approaches are being employed todevelop dengue virus vaccines The vaccine pipeline includeslive empirically attenuated vaccines newer live attenuatedvaccines developed using infectious clone technology geneticvaccines using virus and plasmid vectors and many recom-binant subunit vaccine candidates [214]

Potential vaccines already progressing through clinicaltrials include a live attenuated tetravalent vaccine producedvia serial subculture in primary dog kidney cells [215ndash217]Another approach involves the use of genetic modificationof dengue viruses to attenuate their virulence [218ndash222]Although vaccine candidates based on infectious virus haveshown the greatest progress amongst different dengue vaccineapproaches there are safety concerns associated with theiruse based on potential reactogenicity interference amongstthe viruses possible reversion to native virus and possibleincrease of virus infectivity andor virulence via antibodydependent enhancement [223]

An alternative approach is based on the use of the liveattenuated YFV 17D vaccine as a backbone for the productionof four chimaeric live attenuated viruses in which the prMandE genes of the 17D vaccine virus are replaced by the corre-sponding genes of the four dengue virus serotypes [224ndash226]Preclinical studies demonstrated that the tetravalent vaccineis genetically and phenotypically stable nonhepatotropic lessneurovirulent than the tried and tested YFV 17D vaccineand does not infect mosquitoes by the oral route Vaccinereactogenicity viremia induction and antibody responseshave been investigated in phase 1 trials in the USA thePhilippines andMexico Preclinical and clinical trials showedfavourable immunogenicity and short-term safety of thisvaccine [227] Relatively favourable results of phase 2 trialswere published recently A surprising lack of efficacy againstDENV2 was observed and the fact that DENV2 was theprevalent serotype during the study diminished the overallvaccine efficacy in this setting [228]

Several possible causes of this apparent failure have beenproposed including significant genetic differences between

the circulatingDENV-2 genotype and the strain incorporatedin the yellow fever chimaeric vaccine imbalanced viraemiasor immune responses due to interference NeverthelessSanofirsquos CYD dengue vaccine has been discussed as a poten-tial ldquo75 solutionrdquo referring to the vaccinersquos efficacy towardsthree of the four DENVs in the context of potential antibody-dependent enhancement The general view seems to be thatthis approach would be inappropriate [229] Probably themost relevant issue is related to the long-term safety of suchvaccines DENV-2 has been associated with severe diseasein several epidemiological settings In fact studies in Cubademonstrated that disease severity increased notably wheninfection with DENV-2 follows infection with DENV-1 atan interval of 20 years [64] probably due to a significantdecrease inmean titre of heterologous neutralizing antibodies[128] Thus there is justified concern that CYD vaccinatedindividuals could develop severe disease if infection withDENV-2 occurs after a relatively long interval of timeOn the other hand depending on the DENV-2 genotypethat might subsequently circulate it cannot be ruled outthat heterologous neutralisation might lead to a satisfactoryimmune outcome as occurred in Cuba during the 2001-2002 epidemic caused by DENV-3 where most DENV-2immune individuals (infected in 1981) developed asymp-tomatic DENV-3 infections whilst a high proportion of theDENV-1 immune cases suffered overt disease [63 230]In summary our limited understanding of the underlyingprocesses of the immunopathological response to primaryand successive infections with the four DENV largely deter-mines our inability to predict the clinical outcome [231 232]Nevertheless ongoing large-scale phase 3 studies in morethan 30000 volunteers in ten countries in Latin America andAsia should provide critical data with which to overcomeinitial problems identified with the CYD dengue vaccinecandidate

A similar approach is being developed based on theattenuated DENV-2 virus DENV-2 PDK-53 and three chi-maeric viruses containing the prM and E genes of DENV-1 DENV-3 and DENV-4 virus in the genetic backbone ofthe DENV-2 PDK-53 virus (termed DENVax) Based on thesafety immunogenicity and efficacy in preclinical studies inanimalmodels phase 1 clinical testing of tetravalent DENVaxhas been initiated [233ndash235] This candidate might haveadvantages as the DENV backbone could reduce the risk ofunanticipated effects due to the YFV NS proteins present inthe Sanofi vaccine candidate [228]

Potential vaccines not yet progressing through clinicaltrials include the development of subunit vaccines based ondomain III of the dengue virus envelope protein Recom-binant fusion proteins formed by domain III and P64kprotein from Neisseria meningitides expressed in E coliinduce functional and protective immunity in mice andnonhuman primate models inducing highly serotype specificimmune responses [236ndash238] Additionally the domainsof each serotype have been engineered in tandem in ayeast expression system [239] A recombinant adenovirussystem has been utilised to express the DENV NS1 proteins[240] The paediatric measles vaccine has been modified toexpress a fragment of the DENV-1M protein together with


domain III of the envelope protein [241] More recently theevaluation in mice of a novel domain III-capsid chimaericprotein expressed in E coli provides additional evidencefor a crucial role of cell-mediated immunity in protectionagainst dengue virus [242ndash245] Finally a novel single-doselipidated consensus dengue virus envelope protein domainIII (LcEDIII) subunit vaccine was shown to induce humoraland cellular immune responses in mice [246] This groupalso evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system termed PELC plusCpG oligodeoxynucleotides in potentiating the protectivecapacity of dengue-1 envelope protein domain III concludingthat it could be a promising adjuvant for recombinant proteincandidates [247]

Whilst subunit vaccines may have some advantages interms of type-specific neutralisation with a low potential forinducing ADE via cross-reactive antibodies and low reacto-genicity multiple doses are usually needed to ensure long-term immune protection However it has been suggested thatrecombinant domain III vaccine could function as a boosterif used in combination with live vaccine [223]

It is anticipated that within the next five years increasedunderstanding of the basis for dengue pathogenesis [248] andthe protective immune response to DENV will improve ourability to develop safer and more effective vaccines [223]

Critical issues in dengue vaccine development have beenreviewed [249] Of relevance is the potential impact ofvaccination on the evolution of naturally occurring DENVsVaccination could ultimately produce an environment whererelatively low transmission of natural DENV occurs This isespecially relevant if vaccination is focused on a selected por-tion of the population thereby increasing stochastic eventsthat will allow new DENV genotypes to emerge possiblywith greater virulence Furthermore dengue vaccinationmayproduce a background of low titres of enhancing antibodyto specific DENV serotypes resulting in the emergence ofspecific serotypes in a population Recent studies suggestthat strain diversity may limit the efficacy of monoclonalantibody therapy or tetravalent vaccines against DENV asneutralization potency generally correlates with a narrowedgenotype specificity [250] Consequently a better under-standing of dengue immunopathogenesis will assist not onlydevelopment of therapeutic interventions but also the under-standing of dengue vaccine efficacy or vaccine adverse events[97] Therefore laboratory surveillance of dengue needs tobe improved considerably to increase our knowledge of thecirculating viruses at the molecular level preferably beforethe introduction of a vaccine on a large-scale

33 Development of Antivirals againstDengueViruses Whilstno approved antiviral therapeutic agents are available totreat individuals presenting with symptoms of DFDHFseveral potential virus inhibitors are under consideration forfurther development The dengue viruses provide a varietyof potential targets for inhibitors of infectionreplication AsHepatitis C virus (HCV) is also a member of the Flaviviridaeantivirals under development to control disease due to HCVmay also prove to be effective against the dengue viruses

One of the major problems likely to be encountered is drugresistance Consequently the discovery and development ofat least two antivirals that attack different viral targets shouldbe aminimumgoal Anothermajor hurdle for dengue virusesis the lack of availability of a validated animal model thatfaithfully reflects DHF and DSS observed in patients

The NS5 viral RNA dependent RNA polymerase (RdRp)and the methyl transferase as well as the NS3 protease andhelicase are considered good targets for inhibitors of dengueinfection because they are all major components of thereplicative viral complex [251]The viral envelope (E) proteinis also a good target for antivirals The use of E protein-specific monoclonal antibodies has been shown to have somepotential in this context [252] The NS1 prM and capsidprotein of the dengue viruses have not been studied at thesame level of intensity and thus there are few if any potentialantivirals against these targets

One approach that appears quite successful both in vitroand in vivo has been the demonstration that specific antisensemorpholino oligomers can inhibit dengue virus replication[253 254] However major efforts are required to reducethe risk of toxicity and to provide safe and effective deliverysystems for these oligomers [252 255]

Other approaches are being utilised to identify fla-vivirus chemotherapeutic agents including screening knowninhibitors of other viruses rational design based on pro-tein crystal structures or secondary viral RNA structuresoptimization of known viral inhibitors use of humanizedantibodies use of immunoglobulins and nucleic acid-basedtherapy [256]

Polyoxotungstates and sulphated polysaccharides showsome potential as viral inhibitors They impair flavivirusadsorption and entry into host cells in vitro apparentlyby binding to the cell surface [257 258] Sulphated galac-tomannans protected mice from lethal YFV infection wheninoculated simultaneously with the virus [259]

The licensed drug Ribavirin has been used to treat anumber of RNA viral infections It functions as an RNAcap analogue and mutagen causing errors in syntheticpathways [260ndash262] However the in vitro and in vivoactivity of Ribavirin against YFV and DENV was poor [263ndash265] Prophylactic Ribavirin treatment of rhesus monkeysinfected with DENV had little effect on viremia [266] andin mice intraperitoneal administration of Ribavirin had noeffect on survival following intracerebral inoculation withDENVHowever treatment with Ribavirin-210158403101584051015840-triacetatea prodrug of Ribavirin resulted in a significantly increasedsurvival time and rate possibly due to its higher ability tocross the blood-brain barrier [267]

Nucleoside analogues characterized for chemotherapeu-tic use against HIV and Hepatitis B virus show inhibitoryactivity in cell culture against YFV DENV and West Nilevirus (WNV) [268] Rather than blocking RNA replicationsome analogues inhibit flaviviruses by inhibiting nucleo-side triphosphate synthesis in host cells For example 6-azauridine acetate pyrazofurin and 2 thio-azauridine inhibitorotidine monophosphate decarboxylase (OMPDC) In con-trast mycophenolic acid and Ribavirin-210158403101584051015840-triacetateinhibit inosine monophosphate dehydrogenase (IMPDH)


and block viral RNA synthesis [269 270] Carbamate pro-drugs have also been recommended as IMPDH inhibitorssince they show in vivo activity [271] Recently a uracil-based multifunctional compound was shown to have strongactivity against dengue virus It is likely that the mechanismof action of the antiviral activity of this compound is throughinhibition of the enzyme IMPDH [272]

An alternative strategy in the search for effective antiviralsthat potentially reduces the lead-time for their developmentis to identify drugs already licensed for use to control diseasesother than those caused by the target virus For examplethe aminoglycoside Geneticin (G418) was recently shownto have antiviral activity against bovine viral diarrhea virus(BVDV) Since BVDVDENV andYFV all belong to the virusfamily Flaviviridae it seems possible that a common stepin their life cycle might be affected by this aminoglycosideGeneticin prevented the cytopathic effect resulting fromDENV-2 infection of BHK cells in a dose-dependentmanner[273] However Geneticin had no detectable effect on YFV inBHK cells

Ivermectin a broadly used antihelmintic drug displaysspecific inhibitory unwinding activity against helicases fromseveral flaviviruses including YFV DENV and WNV withthe half maximal inhibitory concentration (IC

50) values in

the submicromolar range [274] Preliminary studies indicatehigher binding efficiency with YFV than with DENV Never-theless disappointingly Ivermectin did not protect hamstersagainst infection with YFV Structure-based optimizationmay result in analogues exerting potent activity againstflaviviruses both in vitro and in vivo

Doxorubicin is an antineoplastic antibiotic obtainedfrom Streptomyces peucetius This antibiotic exhibits in vitroantiviral activity against the YFV17D vaccine strain and theDENV-2 NGC strain Doxorubicin proved to be cytotoxicin uninfected host cells However a novel derivative ofdoxorubicin SA-17 showed excellent antiviral activity againstDENV andmarkedly reduced cytopathogenicity [275] Dose-dependent anti-DENVactivity was confirmed using a denguereporter virus Time-of-drug addition studies indicated thatSA-17 acts at an early stage of the replication cycle It does notinhibit the replication of the replicon and thus does not workat the level of the viral replication machinery Further studiesrevealed that SA-17 exerts it activity via a virucidal effect evenwhen using very high titres of the virus as the inoculum

A large number of small molecules derived by computermodelling of known enzyme domains were screened forinhibitory activity against DENV-2 virus Two of thesemolecules ARDP0006 and ARDP0009 inhibited DENV-2with high efficiency ARDP0009 had no apparent toxicityat the concentrations tested Selectivity indices calculatedfor ARDP0006 and ARDP0009 were comparable to thosecalculated for Ribavirinwhich has demonstrated inhibition ofthe DENV-2-O-methyltransferase NS5 [276] and HCV repli-cation when used in combination with interferon Antiviralactivity in vitro of 3101584051015840 di-O-trityluridine has also been iden-tified The compound inhibits DENV and YFV replicationby targeting the elongation process of the viral NS5 RNA-dependent RNA polymerase A nucleoside analogue (T-705)which is a substituted pyrazine compound that has been used

in clinical trials for the treatment of human influenza virusinfection is an analogue of T-1106 a knownHCV polymeraseinhibitor [277] T-705 significantly improved survival anddisease parameters in YFV-infected hamsters despite the lackof good in vitro antiviral activity These studies highlightthe possibility that nucleoside analogues could potentiallybe developed for flavivirus therapy although more potentcompounds with reduced toxic effects on the host cells willneed to be generated

Flaviviral inhibitory activity has also been observed withplant extracts Boesenbergia rotunda (L) Mansf Kulturpfl(BR) is a common spice belonging to a member of the gingerfamily (Zingiberaceae) Some of the BR compounds suchas flavanoids and chalcones have been shown to be phar-maceutically activeThe chalcone cardamonin isolated fromBR was recently reported to exhibit appreciable anti-HIV-1protease inhibition [278] Moreover inhibitory activity by sixcompounds isolated from BR has also been demonstrated onDENV-2 virus NS3 protease activity

In conclusion whilst mosquito control strategies havebeen shown to be successful in reducing the incidenceof dengue infections such methods are most effective inthose tropicalsubtropical countries that have well-developedhuman and environmental health infrastructures Clearlythere is a real need for more effort to understand thecomplex epidemiology and pathogenesis of the dengueviruses to expedite the development of suitable vaccinesandor antiviral therapies Although some vaccine candidatesappear promising as yet none has been licensed Due to thepresence of the four DENV serotypes these viruses presenta different situation from YFV tick-borne encephalitis virusand Japanese encephalitis virus The question of whether ornot deteriorating antibody levels will leave vaccinated peopleliable to the development of DHF via ADE will need to beaddressed Moreover the relationships between the presenceof neutralizing antibodies the level of protection affordedand the duration of protection by each of the four serotypeswill need to be critically assessed There is a pressing needfor global collective efforts to develop antiviral therapeuticswith which to combat dengue viruses The current trendof expanding our efforts on antiviral drug discovery isencouraging in this respect

Acknowledgments

The authors wish to thank Dr Maria G Guzman for help-ful advice and criticism in the preparation of this reviewProfessor Gould is supported under the EU 7th Framework-Health Programmes (Grant Agreement no 260644 - SILVERand Grant Agreement no 278433 - PREDEMICS) and DrRodriguez-Roche is supported by the Cuban Ministry ofPublic Health and under the EU 7th Framework-HealthProgramme (Grant Agreement no 282378 - DENFREE)

References

[1] F P Pinheiro and S J Corber ldquoGlobal situation of dengue anddengue haemorrhagic fever and its emergence in theAmericasrdquo


World Health Statistics Quarterly vol 50 no 3-4 pp 161ndash1691997

[2] D J Gubler ldquoThe global emergenceresurgence of arboviral dis-eases as public health problemsrdquo Archives of Medical Researchvol 33 no 4 pp 330ndash342 2002

[3] S B Halstead ldquoDenguerdquo The Lancet vol 370 no 9599 pp1644ndash1652 2007

[4] P M De A Zanotto E A Gould G F Gao P H Harvey andE C Holmes ldquoPopulation dynamics of flaviviruses revealed bymolecular phylogeniesrdquo Proceedings of the National Academy ofSciences of the United States of America vol 93 no 2 pp 548ndash553 1996

[5] WHO Dengue Haemorrhagic Fever Diagnosis Treatment Pre-vention and Control WHO Geneva Switzerland 2nd edition1997

[6] LM Ling AWilder-Smith and Y S Leo ldquoFulminant hepatitisin dengue haemorrhagic feverrdquo Journal of Clinical Virology vol38 no 3 pp 265ndash268 2007

[7] S Gulati and A Maheshwari ldquoAtypical manifestations ofdenguerdquo Tropical Medicine and International Health vol 12 no9 pp 1087ndash1095 2007

[8] R Kumar S Tripathi J J Tambe V Arora A Srivastava and VL Nag ldquoDengue encephalopathy in children in Northern Indiaclinical features and comparison with non denguerdquo Journal ofthe Neurological Sciences vol 269 no 1-2 pp 41ndash48 2008

[9] M Wasay R Channa M Jumani G Shabbir M Azeemuddinand A Zafar ldquoEncephalitis and myelitis associated with dengueviral infection Clinical and neuroimaging featuresrdquo ClinicalNeurology and Neurosurgery vol 110 no 6 pp 635ndash640 2008

[10] T Solomon N M Dung D W Vaughn et al ldquoNeurologicalmanifestations of dengue infectionrdquo The Lancet vol 355 no9209 pp 1053ndash1059 2000

[11] S B Park S Y Ryu K B Jin et al ldquoAcute colitis associated withdengue fever in a renal transplant recipientrdquo TransplantationProceedings vol 40 no 7 pp 2431ndash2432 2008

[12] A Balmaseda S N Hammond M A Perez et al ldquoShortreport assessment of the World Health Organization schemefor classification of dengue severity inNicaraguardquoTheAmericanJournal of Tropical Medicine and Hygiene vol 73 no 6 pp1059ndash1062 2005

[13] S Bandyopadhyay L C S Lum and A Kroeger ldquoClassifyingdengue a review of the difficulties in using the WHO caseclassification for dengue haemorrhagic feverrdquoTropicalMedicineand International Health vol 11 no 8 pp 1238ndash1255 2006

[14] J G Rigau-Perez ldquoSevere dengue the need for new casedefinitionsrdquo Lancet Infectious Diseases vol 6 no 5 pp 297ndash3022006

[15] J L Deen E Harris B Wills et al ldquoThe WHO dengueclassification and case definitions time for a reassessmentrdquoTheLancet vol 368 no 9530 pp 170ndash173 2006

[16] N Alexander A Balmaseda I C B Coelho et al ldquoMulticentreprospective study on dengue classification in four South-eastAsian and three Latin American countriesrdquo Tropical Medicineand International Health vol 16 no 8 pp 936ndash948 2011

[17] WHOTDR Dengue Guidelines for Diagnosis Treatment Pre-vention and Control WHO Press Geneva Switzerland 2009

[18] N A Akbar I Allende A Balmaseda et al ldquoRegardingldquoDenguemdashhow best to classify itrdquordquo Clinical Infectious Diseasesvol 54 no 12 pp 1820ndash1821 2012 author reply 1821-1822

[19] A Srikiatkhachorn A L Rothman R V Gibbons et alldquoDengue-how best to classify itrdquoClinical Infectious Diseases vol53 no 6 pp 563ndash567 2011

[20] S Kalayanarooj ldquoDengue classification current WHO vs thenewly suggested classification for better clinical applicationrdquoJournal of the Medical Association of Thailand vol 94 pp S74ndash84 2011

[21] S B Halstead ldquoDengue the syndromic basis to pathogenesisresearch Inutility of the 2009 WHO case definitionrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 88 no2 pp 212ndash215 2013

[22] J F Reinert R E Harbach and I J Kitching ldquoPhylogenyand classification of Ochlerotatus and allied taxa (DipteraCulicidae Aedini) based on morphological data from all lifestagesrdquo Zoological Journal of the Linnean Society vol 153 no1 pp 29ndash114 2008

[23] J F Reinert R E Harbach and I J Kitching ldquoPhylogenyand classification of Aedini (Diptera Culicidae) based onmorphological characters of all life stagesrdquo Zoological Journalof the Linnean Society vol 142 no 3 pp 289ndash368 2004

[24] J F Reinert R E Harbach and I J Kitching ldquoPhylogenyand classification of Finlaya and allied taxa (Diptera CulicidaeAedini) based on morphological data from all life stagesrdquoZoological Journal of the Linnean Society vol 148 no 1 pp 1ndash1012006

[25] E A Gould X de Lamballerie P M D A Zanotto and E CHolmes ldquoOrigins evolution and vectorplus 45 degree rulehostcoadaptations within the Genus Flavivirusrdquo Advances in VirusResearch vol 59 pp 277ndash314 2003

[26] G K Strode Yellow Fever McGraw-Hill New York NY USA1951

[27] W J Tabachnick ldquoEvolutionary genetics and arthropod-bornediseases The yellow fever mosquito Aedes aegyptirdquo AmericanJournal of Entomology vol 37 pp 14ndash24 1991

[28] L P Lounibos ldquoInvasions by insect vectors of human diseaserdquoAnnual Review of Entomology vol 47 pp 233ndash266 2002

[29] X De Lamballerie E Leroy R N Charrel K Ttsetsarkin SHiggs and E A Gould ldquoChikungunya virus adapts to tigermosquito via evolutionary convergence a sign of things tocomerdquo Virology Journal vol 5 article 33 2008

[30] A J Tatem R W Snow and S I Hay ldquoMapping the environ-mental coverage of the INDEPTH demographic surveillancesystem network in rural Africardquo Tropical Medicine and Interna-tional Health vol 11 no 8 pp 1318ndash1326 2006

[31] K J BloomTheMississippi Valleyrsquos Great Yellow Fever Epidemicof 1978 Louisiana State University Press Baton Rouge Lo USA1993

[32] B Rush ldquoAn account of the bilious remitting fever As itappeared in philadelphia in the summer and autumnof the year1780rdquoThe American Journal of Medicine vol 11 no 5 pp 546ndash550 1951

[33] S B Halstead ldquoDengue in the Americas and Southeast Asia dothey differrdquo Revista Panamericana de Salud Publica vol 20 no6 pp 407ndash415 2006

[34] D Gubler ldquoDengue and dengue hemorrhagic fever its historyand resurgence as a global public health problemrdquo in Dengueand Dengue Hemorrhagic Fever D Gubler and G Kuno Edspp 1ndash22 CAB International 1997

[35] D J Gubler ldquoThe emergence of denguedengue haemorrhagicfever as a global public health problemrdquo in Factors in theEmergence of Arbovirus Diseases J F Saluzzo and B DodetEds pp 83ndash92 Elsevier Paris France 1997

[36] D J Gubler ldquoEpidemic denguedengue hemorrhagic fever as apublic health social and economic problem in the 21st centuryrdquoTrends in Microbiology vol 10 no 2 pp 100ndash103 2002


[37] D J Gubler ldquoDenguedengue haemorrhagic fever history andcurrent statusrdquoNovartis Foundation Symposium vol 277 pp 3ndash16 2006 discussion 16ndash22 71ndash13 251ndash253

[38] S B Halstead ldquoDengue hemorrhagic fever two infections andantibody dependent enhancement a brief history and personalmemoirrdquo Revista Cubana de Medicina Tropical vol 54 no 3pp 171ndash179 2002

[39] S B Halstead ldquoDengue virus-mosquito interactionsrdquo AnnualReview of Entomology vol 53 pp 273ndash291 2008

[40] R E Blanton L K Silva V G Morato et al ldquoGenetic ancestryand income are associated with dengue hemorrhagic fever ina highly admixed populationrdquo European Journal of HumanGenetics vol 16 no 6 pp 762ndash765 2008

[41] B D L C Sierra G Kourı and M G Guzman ldquoRace a riskfactor for dengue hemorrhagic feverrdquo Archives of Virology vol152 no 3 pp 533ndash542 2007

[42] MGGuzman andGKouri ldquoDengue and dengue hemorrhagicfever in the Americas lessons and challengesrdquo Journal ofClinical Virology vol 27 no 1 pp 1ndash13 2003

[43] N Singh T Kiedrzynski C Lepers and E K Benyon ldquoDenguein the Pacificmdashan update of the current situationrdquoPacificHealthDialog vol 12 no 2 pp 111ndash119 2005

[44] A Wilder-Smith and D J Gubler ldquoGeographic expansion ofdengue the impact of international travelrdquo Medical Clinics ofNorth America vol 92 no 6 pp 1377ndash1390 2008

[45] C Franco N A Hynes N Bouri and D A HendersonldquoThe dengue threat to the United Statesrdquo Biosecurity andBioterrorism vol 8 no 3 pp 273ndash276 2010

[46] E A Gould P Gallian X De Lamballerie and R N CharrelldquoFirst cases of autochthonous dengue fever and chikungunyafever in France from bad dream to realityrdquo Clinical Microbi-ology and Infection vol 16 no 12 pp 1702ndash1704 2010

[47] I C Kurolt L Betica-Radic O Dakovic-Rode et al ldquoMolecularcharacterization of dengue virus 1 from autochthonous denguefever cases in Croatiardquo Clinical Microbiology and Infection vol19 no 3 pp 163ndash165 2013

[48] M G Guzman andG Kouri ldquoDengue haemorrhagic fever inte-gral hypothesis confirming observations 1987ndash2007rdquo Transac-tions of the Royal Society of Tropical Medicine and Hygiene vol102 no 6 pp 522ndash523 2008

[49] M G Guzman S Vazquez E Martinez et al ldquoDengue inNicaragua 1994 reintroduction of serotype 3 in the AmericasrdquoBoletin de la Oficina Sanitaria Panamericana vol 121 no 2 pp102ndash110 1996

[50] MG Guzman D RosarioMMuneM Alvarez R RodrıguezandGKourı ldquoGenetic relatedness of the dengue 3 virus isolatedin the outbreak of dengue hemorrhagic fever in Nicaragua1994rdquo Revista Cubana de Medicina Tropical vol 48 no 2 pp114ndash117 1996

[51] R Rico-Hesse ldquoMicroevolution and virulence of denguevirusesrdquo Advances in Virus Research vol 59 pp 315ndash341 2003

[52] M G Teixeira M C N Costa G Coelho and M L BarretoldquoRecent shift in age pattern of dengue hemorrhagic feverBrazilrdquoEmerging InfectiousDiseases vol 14 no 10 p 1663 2008

[53] L P Cavalcanti D Vilar R Souza-Santos and M G TeixeiraldquoChange in age pattern of persons with dengue NortheasternBrazilrdquo Emerging Infectious Diseases vol 17 no 1 pp 132ndash1342011

[54] M Q Benedict R S Levine W A Hawley and L P LounibosldquoSpread of the tiger global risk of invasion by the mosquitoAedes albopictusrdquo Vector-Borne and Zoonotic Diseases vol 7no 1 pp 76ndash85 2007

[55] R N Charrel X De Lamballerie and D Raoult ldquoChikungunyaoutbreaksmdashthe globalization of vectorborne diseasesrdquoTheNewEngland Journal of Medicine vol 356 no 8 pp 769ndash771 2007

[56] C Sousa M Clairouin G Seixas et al ldquoOngoing outbreak ofdengue type 1 in the Autonomous Region of Madeira Portugalpreliminary reportrdquo Eurosurveillance vol 17 no 49 article 32012

[57] C Caminade J M Medlock E Ducheyne et al ldquoSuitability ofEuropean climate for the Asian tigermosquitoAedes albopictusrecent trends and future scenariosrdquo Journal of the Royal SocietyInterface vol 9 no 75 pp 2708ndash2717 2012

[58] W F Wright and B S Pritt ldquoUpdate the diagnosis andmanagement of dengue virus infection in North AmericardquoDiagnostic Microbiology and Infectious Disease 2012

[59] F Wu Q Liu L Lu J Wang X Song and D Ren ldquoDistribu-tion of Aedes albopictus (Diptera Culicidae) in northwesternChinardquo Vector-Borne and Zoonotic Diseases vol 11 no 8 pp1181ndash1186 2011

[60] M G Guzman ldquoDeciphering dengue the Cuban experiencerdquoScience vol 309 no 5740 pp 1495ndash1497 2005

[61] G K Tan and S Alonso ldquoPathogenesis and prevention ofdengue virus infection state-of-the-artrdquo Current Opinion inInfectious Diseases vol 22 no 3 pp 302ndash308 2009

[62] A Mathew and A L Rothman ldquoUnderstanding the contri-bution of cellular immunity to dengue disease pathogenesisrdquoImmunological Reviews vol 225 no 1 pp 300ndash313 2008

[63] M Alvarez R Rodriguez-Roche L Bernardo et al ldquoDenguehemorrhagic fever caused by sequential dengue 1ndash3 virusinfections over a long time interval Havana epidemic 2001-2002rdquo The American Journal of Tropical Medicine and Hygienevol 75 no 6 pp 1113ndash1117 2006

[64] MGGuzmanGKouri L Valdes et al ldquoEpidemiologic studieson dengue in Santiago de Cuba 1997rdquo American Journal ofEpidemiology vol 152 no 9 pp 793ndash799 2000

[65] M G Guzman M Alvarez R Rodriguez et al ldquoFatal denguehemorrhagic fever in Cubardquo International Journal of InfectiousDiseases vol 3 no 3 pp 130ndash135 1997

[66] M G Guzman G P Kouri J Bravo M Soler S Vazquezand L Morier ldquoDengue hemorrhagic fever in Cuba 1981 aretrospective seroepidemiologic studyrdquoTheAmerican Journal ofTropical Medicine and Hygiene vol 42 no 2 pp 179ndash184 1990

[67] J R BravoMGGuzman andG P Kouri ldquoWhydengue haem-orrhagic fever in Cuba I Individual risk factors for denguehaemorrhagic feverdengue shock syndrome (DHFDSS)rdquoTransactions of the Royal Society of Tropical Medicine andHygiene vol 81 no 5 pp 816ndash820 1987

[68] L Valdes M G Guzman G Kourı et al ldquoThe epidemiologyof dengue and dengue hemorrhagic fever in Santiago de Cuba1997rdquo Revista Panamericana de Salud Publica vol 6 no 1 pp16ndash25 1999

[69] N Sangkawibha S Rojanasuphot and S Ahandrik ldquoRiskfactors in dengue shock syndrome a prospective epidemiologicstudy in Rayong Thailand I The 1980 outbreakrdquo AmericanJournal of Epidemiology vol 120 no 5 pp 653ndash669 1984

[70] S BHalstead SNimmannitya andMRMargiotta ldquoDengue dchikungunya virus infection in man inThailand 1962ndash1964 IIObservations on disease in outpatientsrdquo The American Journalof Tropical Medicine and Hygiene vol 18 no 6 pp 972ndash9831969

[71] S B Halstead ldquoThe pathogenesis of dengue Molecular epi-demiology in infectious diseaserdquo American Journal of Epidemi-ology vol 114 no 5 pp 632ndash648 1981


[72] D M Morens ldquoAntibody-dependent enhancement of infectionand the pathogenesis of viral diseaserdquo Clinical Infectious Dis-eases vol 19 no 3 pp 500ndash512 1994

[73] B J Mady D V Erbe I Kurane M W Fanger and F A EnnisldquoAntibody-dependent enhancement of dengue virus infectionmediated by bispecific antibodies against cell surface moleculesother than Fc120574 receptorsrdquo Journal of Immunology vol 147 no 9pp 3139ndash3144 1991

[74] I Kurane B J Mady and F A Ennis ldquoAntibody-dependentenhancement of dengue virus infectionrdquo Reviews in MedicalVirology vol 1 no 4 pp 211ndash221 1991

[75] S Halstead Antibody-Dependent Enhancement of Infection amechanism for Indirect Virus Entry into Cells Cellular Receptorsfor Animal Viruses Harbor Laboraty Press Cold SpringHarborNY USA 1994

[76] A B Sabin and RWalter Schlesinger ldquoProduction of immunityto denguewith virusmodified by propagation inmice1rdquo Sciencevol 101 no 2634 pp 640ndash642 1945

[77] T Chareonsirisuthigul S Kalayanarooj and S Ubol ldquoDenguevirus (DENV) antibody-dependent enhancement of infectionupregulates the production of anti-inflammatory cytokinesbut suppresses anti-DENV free radical and pro-inflammatorycytokine production in THP-1 cellsrdquo Journal of General Virol-ogy vol 88 no 2 pp 365ndash375 2007

[78] S Ubol T Chareonsirisuthigul J Kasisith and C KlungthongldquoClinical isolates of dengue virus with distinctive susceptibilityto nitric oxide radical induce differential gene responses inTHP-1 cellsrdquo Virology vol 376 no 2 pp 290ndash296 2008

[79] R Cologna P M Armstrong and R Rico-Hesse ldquoSelectionfor virulent dengue viruses occurs in humans and mosquitoesrdquoJournal of Virology vol 79 no 2 pp 853ndash859 2005

[80] M G Guzman V Deubel J L Pelegrino et al ldquoPartialnucleotide and amino acid sequences of the envelope and theenvelopenonstructural protein-1 gene junction of four dengue-2 virus strains isolated during the 1981 Cuban epidemicrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 52 no3 pp 241ndash246 1995

[81] T J Kochel D MWatts S B Halstead et al ldquoEffect of dengue-1 antibodies on American dengue-2 viral infection and denguehaemorrhagic feverrdquoThe Lancet vol 360 no 9329 pp 310ndash3122002

[82] D M Watts K R Porter P Putvatana et al ldquoFailure ofsecondary infection with American genotype dengue 2 to causedengue haemorrhagic feverrdquo The Lancet vol 354 no 9188 pp1431ndash1434 1999

[83] R Rico-Hesse L M Harrison R A Salas et al ldquoOrigins ofdengue type 2 viruses associated with increased pathogenicityin the Americasrdquo Virology vol 230 no 2 pp 244ndash251 1997

[84] K C Leitmeyer DWVaughnDMWatts et al ldquoDengue virusstructural differences that correlate with pathogenesisrdquo Journalof Virology vol 73 no 6 pp 4738ndash4747 1999

[85] D Gonzalez O E Castro G Kourı et al ldquoClassical denguehemorrhagic fever resulting from two dengue infections spaced20 years ormore apart Havana Dengue 3 epidemic 2001-2002rdquoInternational Journal of Infectious Diseases vol 9 no 5 pp 280ndash285 2005

[86] D Limonta D Gonzalez V Capo et al ldquoFatal severe dengueand cell death in sickle cell disease during the 2001-2002Havanadengue epidemicrdquo International Journal of Infectious Diseasesvol 13 no 2 pp e77ndashe78 2009

[87] M-S Lee K-P Hwang T-C Chen P-L Lu and T-P ChenldquoClinical characteristics of dengue and dengue hemorrhagic

fever in a medical center of southern Taiwan during the 2002epidemicrdquo Journal of Microbiology Immunology and Infectionvol 39 no 2 pp 121ndash129 2006

[88] B Sierra R Alegre A B Perez et al ldquoHLA-A -B -C and -DRB1 allele frequencies in Cuban individuals with antecedentsof dengue 2 disease advantages of the Cuban population forHLA studies of dengue virus infectionrdquo Human Immunologyvol 68 no 6 pp 531ndash540 2007

[89] C LaFleur J Granados G Vargas-Alarcon et al ldquoHLA-DRantigen frequencies in Mexican patients with dengue virusinfection HLA-DR4 as a possible genetic resistance factor fordengue hemorrhagic feverrdquoHuman Immunology vol 63 no 11pp 1039ndash1044 2002

[90] H A F Stephens R Klaythong M Sirikong et al ldquoHLA-A and-B allele associations with secondary dengue virus infectionscorrelate with disease severity and the infecting viral serotype inethnic Thaisrdquo Tissue Antigens vol 60 no 4 pp 309ndash318 2002

[91] M L Paradoa Perez Y Trujillo and P Basanta ldquoAssociation ofdengue hemorrhagic fever with the HLA systemrdquo Haematolo-gia vol 20 no 2 pp 83ndash87 1987

[92] U C Chaturvedi R Nagar and R Shrivastava ldquoDengue anddengue haemorrhagic fever implications of host geneticsrdquoFEMS Immunology and Medical Microbiology vol 47 no 2 pp155ndash166 2006

[93] G Garcıa B Sierra A B Perez et al ldquoAsymptomatic dengueinfection in a cuban population confirms the protective role ofthe RR variant of the Fc120574RIIa polymorphismrdquo The AmericanJournal of TropicalMedicine andHygiene vol 82 no 6 pp 1153ndash1156 2010

[94] J Mongkolsapaya T Duangchinda W Dejnirattisai et al ldquoTcell responses in dengue hemorrhagic fever are cross-reactiveT cells suboptimalrdquo Journal of Immunology vol 176 no 6 pp3821ndash3829 2006

[95] K Clyde J L Kyle and E Harris ldquoRecent advances in deci-phering viral and host determinants of dengue virus replicationand pathogenesisrdquo Journal of Virology vol 80 no 23 pp 11418ndash11431 2006

[96] B Sierra A B Perez K Vogt et al ldquoSecondary heterologousdengue infection risk disequilibrium between immune regula-tion and inflammationrdquo Cellular Immunology vol 262 no 2pp 134ndash140 2010

[97] J Whitehorn and C P Simmons ldquoThe pathogenesis of denguerdquoVaccine vol 29 no 42 pp 7221ndash7228 2011

[98] P AvirutnanN Punyadee S Noisakran et al ldquoVascular leakagein severe dengue virus infections a potential role for thenonstructural viral protein NS1 and complementrdquo Journal ofInfectious Diseases vol 193 no 8 pp 1078ndash1088 2006

[99] C-F Lin S-W Wan M-C Chen et al ldquoLiver injury causedby antibodies against dengue virus nonstructural protein 1 ina murine modelrdquo Laboratory Investigation vol 88 no 10 pp1079ndash1089 2008

[100] H-J Cheng C-F Lin H-Y Lei et al ldquoProteomic analysis ofendothelial cell autoantigens recognized by anti-dengue virusnonstructural protein 1 antibodiesrdquo Experimental Biology andMedicine vol 234 no 1 pp 63ndash73 2009

[101] C-F Lin S-W Wan H-J Cheng H-Y Lei and Y-S LinldquoAutoimmune pathogenesis in dengue virus infectionrdquo ViralImmunology vol 19 no 2 pp 127ndash132 2006

[102] M G Brown Y Y Huang J S Marshall C A King DW Hoskin and R Anderson ldquoDramatic caspase-dependentapoptosis in antibody-enhanced dengue virus infection of


human mast cellsrdquo Journal of Leukocyte Biology vol 85 no 1pp 71ndash80 2009

[103] M G Brown C A King C Sherren J S Marshall and RAnderson ldquoA dominant role for Fc120574RII in antibody-enhanceddengue virus infection of human mast cells and associatedCCL5 releaserdquo Journal of Leukocyte Biology vol 80 no 6 pp1242ndash1250 2006

[104] A K I Falconar ldquoThe dengue virus nonstructural-1 protein(NS1) generates antibodies to common epitopes on humanblood clotting integrinadhesin proteins and binds to humanendothelial cells potential implications in haemorrhagic feverpathogenesisrdquo Archives of Virology vol 142 no 5 pp 897ndash9161997

[105] D T Tam T V Ngoc N T Tien et al ldquoEffects of short-course oral corticosteroid therapy in early dengue infection inVietnamese patients a randomized placebo-controlled trialrdquoClinical Infectious Diseases vol 55 no 9 pp 1216ndash1224 2012

[106] S-W Lin Y-C Chuang Y-S Lin H-Y Lei H-S Liu andT-M Yeh ldquoDengue virus nonstructural protein NS1 binds toprothrombinthrombin and inhibits prothrombin activationrdquoJournal of Infection vol 64 no 3 pp 325ndash334 2012

[107] S B Halstead ldquoDengue vascular permeability syndrome whatno T cellsrdquo Clinical Infectious Diseases vol 56 no 6 pp 900ndash901 2013

[108] D H Libraty P R Young D Pickering et al ldquoHigh circulatinglevels of the dengue virus nonstructural protein NS1 earlyin dengue illness correlate with the development of denguehemorrhagic feverrdquo Journal of Infectious Diseases vol 186 no8 pp 1165ndash1168 2002

[109] H T L Duyen T V Ngoc D T Ha et al ldquoKinetics of plasmaviremia and soluble nonstructural protein 1 concentrations indengue differential effects according to serotype and immunestatusrdquo Journal of Infectious Diseases vol 203 no 9 pp 1292ndash1300 2011

[110] V Tricou N N Minh J Farrar H T Tran and C P SimmonsldquoKinetics of viremia and NS1 antigenemia are shaped byimmune status and virus serotype in adults with denguerdquo PLoSNeglected Tropical Diseases vol 5 no 9 Article ID e1309 2011

[111] S I de la Cruz-Hernandez H Flores-Aguilar S Gonzalez-Mateos et al ldquoDetermination of viremia and concentrationof circulating nonstructural protein 1 in patients infected withdengue virus in Mexicordquo The American Journal of TropicalMedicine and Hygiene vol 88 no 3 pp 446ndash454 2013

[112] A Fox L N M Hoa C P Simmons et al ldquoImmunological andviral determinants of dengue severity in hospitalized adults inHa Noi Viet Namrdquo PLoS Neglected Tropical Diseases vol 5 no3 article e967 2011

[113] G P KouriMGGuzman and J R Bravo ldquoWhydengue haem-orrhagic fever in Cuba 2 An integral analysisrdquo Transactions ofthe Royal Society of Tropical Medicine and Hygiene vol 81 no5 pp 821ndash823 1987

[114] J L Kyle and E Harris ldquoGlobal spread and persistence ofdenguerdquo Annual Review of Microbiology vol 62 pp 71ndash922008

[115] P Mas Lago ldquoDengue fever in Cuba in 1977 some laboratoryaspectsrdquo PAHO Scientific Publication vol 375 pp 40ndash43 1979

[116] P Mas Lago R Palomera and M Jacobo ldquoDengue someepidemiologic aspectsrdquo Revista Cubana de Medicina Tropicalvol 22 no 6 1983

[117] N Cantelar de Francisco A Fernandez L Albert Molina andE Perez Balbis ldquoSurvey of dengue in Cuba 1978-1979rdquo RevistaCubana de Medicina Tropical vol 33 no 1 pp 72ndash78 1981

[118] S B Halstead S Nimmannitya and S N Cohen ldquoObserva-tions related to pathogenesis of dengue hemorrhagic fever IVRelation of disease severity to antibody response and virusrecoveredrdquo Yale Journal of Biology and Medicine vol 42 no 5pp 311ndash328 1970

[119] M G Guzman G Kouri J Bravo M Soler and E MartınezldquoSequential infection as risk factor for dengue hemorrhagicfeverdengue shock syndrome (DHFDSS) during the 1981dengue hemorrhagic Cuban epidemicrdquo Memorias do InstitutoOswaldo Cruz vol 86 no 3 p 367 1991

[120] G Kourı M G Guzman L Valdes et al ldquoReemergence ofdengue inCuba a 1997 epidemic in Santiago deCubardquoEmergingInfectious Diseases vol 4 no 1 pp 89ndash92 1998

[121] M G Guzman G Kourı L Valdes J Bravo S Vazquezand S B Halstead ldquoEnhanced severity of secondary dengue-2 infections death rates in 1981 and 1997 Cuban outbreaksrdquoRevista Panamericana de Salud Publica vol 11 no 4 pp 223ndash227 2002

[122] O Pelaez M G Guzman G Kouri et al ldquoDengue 3 epidemicHavana 2001rdquo Emerging Infectious Diseases vol 10 no 4 pp719ndash722 2004

[123] M Alvarez A Pavon-Oro S Vazquez LMorier AM Alvarezand M G Guzman ldquoViral infection sequences related todengue fever in dengue 3 epidemics occurred in the City ofHavana 2001-2002rdquo Revista Cubana de Medicina Tropical vol60 no 1 pp 18ndash23 2008

[124] M Alvarez A Pavon-Oro R Rodriguez-Roche et al ldquoNeu-tralizing antibody response variation against dengue 3 strainsrdquoJournal of Medical Virology vol 80 no 10 pp 1783ndash1789 2008

[125] R Rodriguez-Roche M Alvarez E C Holmes et al ldquoDenguevirus type 3 Cuba 2000ndash2002rdquo Emerging Infectious Diseasesvol 11 no 5 pp 773ndash774 2005

[126] R Rodriguez-Roche M Alvarez T Gritsun et al ldquoDenguevirus type 2 in Cuba 1997 conservation of E gene sequencein isolates obtained at different times during the epidemicrdquoArchives of Virology vol 150 no 3 pp 415ndash425 2005

[127] A B Sabin ldquoResearch on dengue during World War IIrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 1 no1 pp 30ndash50 1952

[128] M G Guzman M Alvarez R Rodriguez-Roche et al ldquoNeu-tralizing antibodies after infection with dengue 1 virusrdquo Emerg-ing Infectious Diseases vol 13 no 2 pp 282ndash286 2007

[129] M G Guzman G Kourı and S B Halstead ldquoDo escapemutants explain rapid increases in dengue case-fatality rateswithin epidemicsrdquoTheLancet vol 355 no 9218 pp 1902ndash19032000

[130] R Rodriguez-Roche M Alvarez T Gritsun et al ldquoVirusevolution during a severe dengue epidemic in Cuba 1997rdquoVirology vol 334 no 2 pp 154ndash159 2005

[131] J Mongkolsapaya W Dejnirattisai X-N Xu et al ldquoOriginalantigenic sin and apoptosis in the pathogenesis of denguehemorrhagic feverrdquo Nature Medicine vol 9 no 7 pp 921ndash9272003

[132] S N Bennett E C Holmes M Chirivella et al ldquoSelection-driven evolution of emergent dengue virusrdquo Molecular Biologyand Evolution vol 20 no 10 pp 1650ndash1658 2003

[133] C Klungthong C Zhang M P Mammen Jr S Ubol and E CHolmes ldquoThemolecular epidemiology of dengue virus serotype4 in Bangkok Thailandrdquo Virology vol 329 no 1 pp 168ndash1792004


[134] H-L Chen S-R Lin H-F Liu C-C King S-C Hsieh andW-K Wang ldquoEvolution of dengue virus type 2 during twoconsecutive outbreaks with an increase in severity in South-ern Taiwan in 2001-2002rdquo The American Journal of TropicalMedicine and Hygiene vol 79 no 4 pp 495ndash504 2008

[135] Y Tang P Rodpradit P Chinnawirotpisan et al ldquoComparativeanalysis of full-length genomic sequences of 10 dengue serotype1 viruses associated with different genotypes epidemics anddisease severity isolated in Thailand over 22 yearsrdquoThe Ameri-can Journal of Tropical Medicine and Hygiene vol 83 no 5 pp1156ndash1165 2010

[136] R Zhao P Chinnawirotpisan C Klungthong C Zhang and RPutnak ldquoEvidence for inter- and intra-genotypic variations indengue serotype 4 viruses representing predominant and non-predominant genotypes co-circulating inThailand from 1977 to2001rdquo Virus Genes vol 41 no 1 pp 5ndash13 2010

[137] R Rico-Hesse ldquoDengue virus evolution and virulence modelsrdquoClinical Infectious Diseases vol 44 no 11 pp 1462ndash1466 2007

[138] A Steel D J Gubler and S N Bennett ldquoNatural attenuationof dengue virus type-2 after a series of island outbreaks aretrospective phylogenetic study of events in the South Pacificthree decades agordquo Virology vol 405 no 2 pp 505ndash512 2010

[139] R Rodriguez-Roche E Villegas S Cook et al ldquoPopulationstructure of the dengue viruses Aragua Venezuela 2006-2007Insights into dengue evolution under hyperendemic transmis-sionrdquo Infection Genetics and Evolution vol 12 no 2 pp 332ndash344 2012

[140] T J Chambers D W McCourt and C M Rice ldquoYellowfever virus proteins NS2A NS2B and NS4B identification andpartial N-terminal amino acid sequence analysisrdquoVirology vol169 no 1 pp 100ndash109 1989

[141] J M MacKenzie A A Khromykh M K Jones and EG Westaway ldquoSubcellular localization and some biochemicalproperties of the flavivirus Kunjin nonstructural proteins NS2Aand NS4Ardquo Virology vol 245 no 2 pp 203ndash215 1998

[142] J L Wen B C Hua J W Xiang H Huang and A AKhromykh ldquoAnalysis of adaptive mutations in Kunjin virusreplicon RNA reveals a novel role for the flavivirus nonstruc-tural protein NS2A in inhibition of beta interferon promoter-driven transcriptionrdquo Journal of Virology vol 78 no 22 pp12225ndash12235 2004

[143] W J Liu X JWangD C ClarkM Lobigs R AHall andAAKhromykh ldquoA single amino acid substitution in the West Nilevirus nonstructural protein NS2A disables its ability to inhibitalphabeta interferon induction and attenuates virus virulenceinmicerdquo Journal of Virology vol 80 no 5 pp 2396ndash2404 2006

[144] J L Munoz-Jordan G G Sanchez-Burgos M Laurent-Rolleand A Garcıa-Sastre ldquoInhibition of interferon signaling bydengue virusrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 2 pp 14333ndash143382003

[145] W J Liu X J Wang V V Mokhonov P-Y Shi R Randall andA A Khromykh ldquoInhibition of interferon signaling by the NewYork 99 strain and Kunjin subtype of West Nile virus involvesblockage of STAT1 and STAT2 activation by nonstructuralproteinsrdquo Journal of Virology vol 79 no 3 pp 1934ndash1942 2005

[146] B M Kummerer and CM Rice ldquoMutations in the yellow fevervirus nonstructural protein NS2A selectively block productionof infectious particlesrdquo Journal of Virology vol 76 no 10 pp4773ndash4784 2002

[147] W J Liu H B Chen and A A Khromykh ldquoMolecular andfunctional analyses of Kunjin virus infectious cDNA clones

demonstrate the essential roles for NS2A in virus assemblyand for a nonconservative residue in NS3 in RNA replicationrdquoJournal of Virology vol 77 no 14 pp 7804ndash7813 2003

[148] J Y LeungG P PijlmanNKondratieva JHyde JMMacken-zie and A A Khromykh ldquoRole of nonstructural protein NS2Ain flavivirus assemblyrdquo Journal of Virology vol 82 no 10 pp4731ndash4741 2008

[149] K L McElroy K A Tsetsarkin D L Vanlandingham and SHiggs ldquoManipulation of the yellow fever virus non-structuralgenes 2A and 4B and the 31015840 non-coding region to evaluategenetic determinants of viral dissemination from the AedesaegyptimidgutrdquoTheAmerican Journal of Tropical Medicine andHygiene vol 75 no 6 pp 1158ndash1164 2006

[150] K A Hanley L R Manlucu L E Gilmore et al ldquoA trade-off inreplication in mosquito versus mammalian systems conferredby a point mutation in the NS4B protein of dengue virus type4rdquo Virology vol 312 no 1 pp 222ndash232 2003

[151] E P Kelly B Puri W Sun and B Falgout ldquoIdentification ofmutations in a candidate dengue 4 vaccine strain 341750 PDK20and construction of a full-length cDNA clone of the PDK20vaccine candidaterdquo Vaccine vol 28 no 17 pp 3030ndash3037 2010

[152] S Tajima T Takasaki and I Kurane ldquoRestoration ofreplication-defective dengue type 1 virus bearing mutationsin the N-terminal cytoplasmic portion of NS4A by additionalmutations in NS4Brdquo Archives of Virology vol 156 no 1 pp63ndash69 2011

[153] D Grant G K Tan M Qing et al ldquoA single amino acid innonstructural protein NS4B Confers virulence to dengue virusin AG129 mice through enhancement of viral RNA synthesisrdquoJournal of Virology vol 85 no 15 pp 7775ndash7787 2011

[154] R R Roche M Alvarez M G Guzman L Morier andG Kouri ldquoComparison of rapid centrifugation assay withconventional tissue culture method for isolation of dengue 2virus in C636-HT cellsrdquo Journal of Clinical Microbiology vol38 no 9 pp 3508ndash3510 2000

[155] W-KWang S-R Lin C-M Lee C-C King and S-C ChangldquoDengue type 3 virus in plasma is a population of closely relatedgenomes quasispeciesrdquo Journal of Virology vol 76 no 9 pp4662ndash4665 2002

[156] J Aaskov K Buzacott H MThu K Lowry and E C HolmesldquoLong-term transmission of defective RNA viruses in humansand Aedes mosquitoesrdquo Science vol 311 no 5758 pp 236ndash2382006

[157] M OhAinle A Balmaseda A R Macalalad et al ldquoDynamicsof dengue disease severity determined by the interplay betweenviral genetics and serotype-specific immunityrdquo Science Transla-tional Medicine vol 3 no 114 Article ID 114ra128 2011

[158] J E Foster S N Bennett C V F Carrington H Vaughan andW O McMillan ldquoPhylogeography and molecular evolution ofdengue 2 in the Caribbean basin 1981ndash2000rdquo Virology vol 324no 1 pp 48ndash59 2004

[159] W B Messer U T Vitarana K Sivananthan et al ldquoEpidemi-ology of dengue in Sri lanka before and after the emergence ofepidemic dengue hemorrhagic feverrdquo The American Journal ofTropical Medicine andHygiene vol 66 no 6 pp 765ndash773 2002

[160] H M Thu K Lowry L Jiang T Hlaing E C Holmes and JAaskov ldquoLineage extinction and replacement in dengue type1 virus populations are due to stochastic events rather than tonatural selectionrdquo Virology vol 336 no 2 pp 163ndash172 2005

[161] L T D Salda M D C Parquet R R Matias F F Natividad NKobayashi and K Morita ldquoMolecular epidemiology of dengue


2 viruses in the Philippines genotype shift and local evolutionrdquoThe American Journal of Tropical Medicine and Hygiene vol 73no 4 pp 796ndash802 2005

[162] T T H Vu E C Holmes V Duong et al ldquoEmergence of theAsian 1 genotype of dengue virus serotype 2 in viet nam in vivofitness advantage and lineage replacement in South-East AsiardquoPLoS Neglected Tropical Diseases vol 4 no 7 p e757 2010

[163] E C Holmes ldquoRNA virus genomics a world of possibilitiesrdquoJournal of Clinical Investigation vol 119 no 9 pp 2488ndash24952009

[164] D Limonta V Capo G Torres A B Perez andM G GuzmanldquoApoptosis in tissues from fatal dengue shock syndromerdquoJournal of Clinical Virology vol 40 no 1 pp 50ndash54 2007

[165] A Moya E C Holmes and F Gonzalez-Candelas ldquoThepopulation genetics and evolutionary epidemiology of RNAvirusesrdquoNature ReviewsMicrobiology vol 2 no 4 pp 279ndash2882004

[166] S Navas J Martın J A Quiroga I Castillo and V CarrenoldquoGenetic diversity and tissue compartmentalization of thehepatitis C virus genome in blood mononuclear cells liver andserum from chronic hepatitis C patientsrdquo Journal of Virologyvol 72 no 2 pp 1640ndash1646 1998

[167] R Miralles A Moya and S F Elena ldquoEffect of populationpatchiness and migration rates on the adaptation and diver-gence of vesicular stomatitis virus quasispecies populationsrdquoJournal of General Virology vol 80 no 8 pp 2051ndash2059 1999

[168] P Farci A Shimoda A Coiana et al ldquoThe outcome of acutehepatitis C predicted by the evolution of the viral quasispeciesrdquoScience vol 288 no 5464 pp 339ndash344 2000

[169] E C Holmes ldquoPatterns of intra- and interhost nonsynonymousvariation reveal strong purifying selection in dengue virusrdquoJournal of Virology vol 77 no 20 pp 11296ndash11298 2003

[170] S-R Lin S-C Hsieh Y-Y Yueh et al ldquoStudy of sequence vari-ation of dengue type 3 virus in naturally infected mosquitoesand human hosts implications for transmission and evolutionrdquoJournal of Virology vol 78 no 22 pp 12717ndash12721 2004

[171] E Descloux V-M Cao-Lormeau C Roche and X De Lambal-lerie ldquoDengue 1 diversity andmicroevolution French Polynesia2001ndash2006 connection with epidemiology and clinicsrdquo PLoSNeglected Tropical Diseases vol 3 no 8 article e493 2009

[172] K T D Thai M R Henn M C Zody et al ldquoHigh-resolutionanalysis of intrahost genetic diversity in dengue virus serotype1 infection identifies mixed infectionsrdquo Journal of Virology vol86 no 2 pp 835ndash843 2012

[173] P Parameswaran P Charlebois Y Tellez et al ldquoGenome-widepatterns of intrahuman dengue virus diversity reveal associ-ations with viral phylogenetic clade and interhost diversityrdquoJournal of Virology vol 86 no 16 pp 8546ndash8558 2012

[174] R L Costa CMVoloch andCG Schrago ldquoComparative evo-lutionary epidemiology of dengue virus serotypesrdquo InfectionGenetics and Evolution vol 12 no 2 pp 309ndash314 2012

[175] J Lourenco and M Recker ldquoViral and epidemiological deter-minants of the invasion dynamics of Novel Dengue GenotypesrdquoPLoSNeglected Tropical Diseases vol 4 no 11 article e894 2010

[176] V Wittke T E Robb H M Thu et al ldquoExtinction and rapidemergence of strains of dengue 3 virus during an interepidemicperiodrdquo Virology vol 301 no 1 pp 148ndash156 2002

[177] C Chevillon and A-B Failloux ldquoQuestions on viral populationbiology to complete dengue puzzlerdquoTrends inMicrobiology vol11 no 9 pp 415ndash421 2003

[178] L Lambrechts T Fansiri A Pongsiri et al ldquoDengue-1virus clade replacement in Thailand associated with enhancedmosquito transmissionrdquo Journal of Virology vol 86 no 3 pp1853ndash1861 2012

[179] W H Bancroft R M Scott and W E Brandt ldquoDengue-2vaccine infection of Aedes aegypti mosquitoes by feeding onviremic recipientsrdquo The American Journal of Tropical Medicineand Hygiene vol 31 no 6 pp 1229ndash1231 1982

[180] B R Miller B J Beaty and T H G Aitken ldquoDengue-2vaccine oral infection transmission and lack of evidence forreversion in themosquitoAedes aegyptirdquoTheAmerican Journalof Tropical Medicine and Hygiene vol 31 no 6 pp 1232ndash12371982

[181] D J Gubler S Nalim and R Tan ldquoVariation in susceptibility tooral infection with dengue viruses among geographic strains ofAedes aegyptirdquo The American Journal of Tropical Medicine andHygiene vol 28 no 6 pp 1045ndash1052 1979

[182] P M Armstrong and R Rico-Hesse ldquoDifferential susceptibilityof Aedes aegypti to infection by the American and SoutheastAsian genotypes of dengue type 2 virusrdquo Vector Borne ZoonoticDis vol 1 no 2 pp 159ndash168 2001

[183] F L Soper ldquoAedes aegypti and yellow feverrdquoBulletin of theWorldHealth Organization vol 36 no 4 pp 521ndash527 1967

[184] D J Gubler ldquoAedes aegypti and Aedes aegypti-borne diseasecontrol in the 1990s top down or bottom up Charles FranklinCraig Lecturerdquo The American Journal of Tropical Medicine andHygiene vol 40 no 6 pp 571ndash578 1989

[185] E-E Ooi K-T Goh and D J Gubler ldquoDengue prevention and35 years of vector control in Singaporerdquo Emerging InfectiousDiseases vol 12 no 6 pp 887ndash893 2006

[186] MGGuzmanO Pelaez G Kouri et al ldquoFinal characterizationof and lessons learned from the dengue 3 epidemic in Cuba2001-2002rdquo Revista Panamericana de Salud Publica vol 19 no4 pp 282ndash289 2006

[187] M Bonet JM Spiegel AM Ibarra G Kouri A Pintre Lic andA Yassi ldquoAn integrated ecosystem approach for sustainable pre-vention and control of dengue in Central Havanardquo InternationalJournal of Occupational and Environmental Health vol 13 no2 pp 188ndash194 2007

[188] J A Bisset M M Rodrıguez Y Ricardo et al ldquoTemephosresistance and esterase activity in the mosquito Aedes aegypti inHavana Cuba increased dramatically between 2006 and 2008rdquoMedical and Veterinary Entomology vol 25 no 3 pp 233ndash2392011

[189] R Maciel-de-Freitas R Aguiar R V Bruno et al ldquoWhy dowe need alternative tools to control mosquito-borne diseases inLatin Americardquo Memorias do Instituto Oswaldo Cruz vol 107no 6 pp 828ndash829 2012

[190] R A Sa N D D L Santos C S B D Silva et al ldquoLarvicidalactivity of lectins from Myracrodruon urundeuva on Aedesaegyptirdquo Comparative Biochemistry and Physiology Part C vol149 no 3 pp 300ndash306 2009

[191] P F Billingsley B Foy and J L Rasgon ldquoMosquitocidalvaccines a neglected addition to malaria and dengue controlstrategiesrdquo Trends in Parasitology vol 24 no 9 pp 396ndash4002008

[192] J A Bisset Lazcano M C Marquetti A Garcia et al ldquoPupalsurveillance of Aedes aegypti as a tool for control of the vectorin a municipality with low density of La Habana City CubardquoRevista Biomedica vol 19 pp 92ndash103 2008

[193] T T Tuyet Hanh P S Hill B H Kay and M Q TranldquoDevelopment of a framework for evaluating the sustainability


of community-based dengue control projectsrdquo The AmericanJournal of Tropical Medicine and Hygiene vol 80 no 2 pp 312ndash318 2009

[194] S N Vu T Y Nguyen B H Kay G G Marten and J W ReidldquoEradication of Aedes aegypti from a village in Vietnam usingcopepods and community participationrdquoTheAmerican Journalof Tropical Medicine and Hygiene vol 59 no 4 pp 657ndash6601998

[195] V S Nam N T Yen T V Phong et al ldquoElimination ofdengue by community programs using Mesocyclops (cope-poda) against Aedes aegypti in central VietnamrdquoThe AmericanJournal of Tropical Medicine and Hygiene vol 72 no 1 pp 67ndash73 2005

[196] B Kay and V S Nam ldquoNew strategy against Aedes aegypti inVietnamrdquoThe Lancet vol 365 no 9459 pp 613ndash617 2005

[197] L Sanchez D Perez L Alfonso et al ldquoA community educationstrategy to promote participation in dengue prevention inCubardquo Revista Panamericana de Salud Publica vol 24 no 1 pp61ndash69 2008

[198] C J McMeniman R V Lane B N Cass et al ldquoStableintroduction of a life-shortening Wolbachia infection into themosquito Aedes aegyptirdquo Science vol 323 no 5910 pp 141ndash1442009

[199] P Lu G Bian X Pan and Z Xi ldquoWolbachia induces density-dependent inhibition to dengue virus in mosquito cellsrdquo PLOSNeglected Tropical Diseases vol 6 no 7 Article ID e1754 2012

[200] T Walker P H Johnson L A Moreira et al ldquoThe wMelWolbachia strain blocks dengue and invades caged Aedesaegypti populationsrdquo Nature vol 476 no 7361 pp 450ndash4552011

[201] G Bian Y Xu P Lu Y Xie and Z Xi ldquoThe endosymbiotic bac-terium Wolbachia induces resistance to dengue virus in Aedesaegyptirdquo PLoS Pathogens vol 6 no 4 Article ID e1000833 2010

[202] L A Moreira I Iturbe-Ormaetxe J A Jeffery et al ldquoAWolbachia Symbiont in Aedes aegypti Limits Infection withDengue Chikungunya and Plasmodiumrdquo Cell vol 139 no 7pp 1268ndash1278 2009

[203] X Pan G Zhou J Wu et al ldquoWolbachia induces reactiveoxygen species (ROS)-dependent activation of the Toll pathwayto control dengue virus in themosquitoAedes aegyptirdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 109 no 1 pp E23ndashE31 2012

[204] L Mousson K Zouache C Arias-Goeta V Raquin P Mavin-gui and A B Failloux ldquoThe native wolbachia symbiontslimit transmission of dengue virus in Aedes albopictusrdquo PLOSNeglected Tropical Diseases vol 6 no 12 Article ID e1989 2012

[205] P E Cook C J McMeniman and S L OrsquoNeill ldquoModifyinginsect population age structure to control vector-borne diseaserdquoAdvances in Experimental Medicine and Biology vol 627 pp126ndash140 2008

[206] N M Endersby A A Hoffmann V L White S A RitchieP H Johnson and A R Weeks ldquoChanges in the geneticstructure of Aedes aegypti (Diptera Culicidae) populations inQueensland Australia across two seasons implications forpotential mosquito releasesrdquo Journal of Medical Entomologyvol 48 no 5 pp 999ndash1007 2011

[207] N M Endersby A A Hoffmann V L White et al ldquoGeneticstructure of Aedes aegypti in Australia and Vietnam revealedby microsatellite and exon primed intron crossing markerssuggests feasibility of local control optionsrdquo Journal of MedicalEntomology vol 46 no 5 pp 1074ndash1083 2009

[208] J A L Jeffery N T Yen V S Nam et al ldquoCharacterizing theAedes aegypti population in a Vietnamese village in preparationfor a Wolbachia-based mosquito control strategy to eliminatedenguerdquo PLoS Neglected Tropical Diseases vol 3 no 11 ArticleID e0000552 2009

[209] H L Yeap P Mee T Walker et al ldquoDynamics of the ldquopopcornrdquoWolbachia infection in outbredAedes aegypti informs prospectsfor mosquito vector controlrdquo Genetics vol 187 no 2 pp 583ndash595 2011

[210] R Macey ldquoDengue fever breakthroughrdquo Sydney MorningHerald 2009 httpwwwsmhcomaunewsspecialssciencedengue-fever-breakthrough200901021230681699070html

[211] N M Endersby and A A Hoffmann ldquoEffect of Wolbachia oninsecticide susceptibility in lines of Aedes aegyptirdquo Bulletin ofEntomological Research pp 1ndash9 2012

[212] S B Halstead ldquoAntibody macrophages dengue virus infectionshock and hemorrhage a pathogenetic cascaderdquo Reviews ofInfectious Diseases vol 11 pp S830ndash839 1989

[213] J Hombach M Jane Cardosa A Sabchareon D W Vaughnand A D T Barrett ldquoScientific consultation on immunologicalcorrelates of protection induced by dengue vaccines Reportfrom a meeting held at the World Health Organization 17-18November 2005rdquo Vaccine vol 25 no 21 pp 4130ndash4139 2007

[214] S Swaminathan G Batra and N Khanna ldquoDengue vaccinesstate of the artrdquo Expert Opinion on Therapeutic Patents vol 20no 6 pp 819ndash835 2010

[215] R Edelman S S Wasserman S A Bodison et al ldquoPhase I trialof 16 formulations of a tetravalent live-attenuated dengue vac-cinerdquo The American Journal of Tropical Medicine and Hygienevol 69 no 6 pp 48ndash60 2003

[216] S Simasathien S J Thomas V Watanaveeradej et al ldquoSafetyand immunogenicity of a tetravalent live-attenuated denguevaccine in flavivirus naive childrenrdquo The American Journal ofTropical Medicine andHygiene vol 78 no 3 pp 426ndash433 2008

[217] W SunDCunningham S SWasserman et al ldquoPhase 2 clinicaltrial of three formulations of tetravalent live-attenuated denguevaccine in flavivirus-naıve adultsrdquo Human Vaccines vol 5 no1 pp 33ndash40 2009

[218] A P Durbin S S Whitehead J McArthur et al ldquorDEN412059030a live attenuated dengue virus type 4 vaccine candidate issafe immunogenic and highly infectious in healthy adultvolunteersrdquo Journal of Infectious Diseases vol 191 no 5 pp 710ndash718 2005

[219] A P Durbin J McArthur J A Marron et al ldquoThe live atten-uated dengue serotype 1 vaccine rDEN1Δ30 is safe and highlyimmunogenic in healthy adult volunteersrdquo Human Vaccinesvol 2 no 4 pp 167ndash173 2006

[220] A P Durbin J H McArthur J A Marron et al ldquorDEN24Δ30(ME) a live attenuated chimeric dengue serotype 2 vac-cine is safe and highly immunogenic in healthy dengue-naıveadultsrdquo Human Vaccines vol 2 no 6 pp 255ndash260 2006

[221] J HMcArthur A P Durbin J AMarron et al ldquoPhase I clinicalevaluation of rDEN4Δ30-200201 a live attenuated dengue 4vaccine candidate designed for decreased hepatotoxicityrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 79 no5 pp 678ndash684 2008

[222] J E Blaney Jr N S Sathe L Goddard et al ldquoDengue virustype 3 vaccine candidates generated by introduction of deletionsin the 31015840 untranslated region (31015840-UTR) or by exchange of theDENV-3 31015840-UTR with that of DENV-4rdquo Vaccine vol 26 no 6pp 817ndash828 2008


[223] M G Guzman L Hermida L Bernardo R Ramirez andG Guillen ldquoDomain III of the envelope protein as a denguevaccine targetrdquo Expert Review of Vaccines vol 9 no 2 pp 137ndash147 2010

[224] F Guirakhoo S Kitckener D Morrison et al ldquoLive attenuatedchimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vac-cine phase I clinical trial for safety and immunogenicitymdasheffectof yellow fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypesrdquoHuman Vaccinesvol 2 no 2 pp 60ndash67 2006

[225] B Guy N Nougarede S Begue et al ldquoCell-mediated immunityinduced by chimeric tetravalent dengue vaccine in naive orflavivirus-primed subjectsrdquo Vaccine vol 26 no 45 pp 5712ndash5721 2008

[226] B Guy F Guirakhoo V Barban S Higgs T P Monath andJ Lang ldquoPreclinical and clinical development of YFV 17D-based chimeric vaccines against dengueWestNile and Japaneseencephalitis virusesrdquo Vaccine vol 28 no 3 pp 632ndash649 2010

[227] B Guy M Saville and J Lang ldquoDevelopment of Sanofi Pasteurtetravalent dengue vaccinerdquo Human Vaccines vol 6 no 9 pp696ndash705 2010

[228] A Sabchareon D Wallace C Sirivichayakul et al ldquoProtectiveefficacy of the recombinant live-attenuated CYD tetravalentdengue vaccine in Thai schoolchildren a randomised con-trolled phase 2b trialrdquo The Lancet vol 380 no 9853 pp 1559ndash1567 2012

[229] S Swaminathan N Khanna B Herring and S MahalingamldquoDengue vaccine efficacy trial does interference cause failurerdquoThe Lancet Infectious Diseases vol 13 no 3 pp 191ndash192 2013

[230] M G Guzman A Alvarez S Vazquez et al ldquoEpidemiologicalstudies on dengue virus type 3 in Playa municipality HavanaCuba 2001-2002rdquo International Journal of Infectious Diseasesvol 16 no 3 pp e198ndashe203 2012

[231] S B Halstead ldquoDengue vaccine development a 75 solutionrdquoThe Lancet vol 380 no 9853 pp 1535ndash1536 2012

[232] W M P B Wahala and A M de Silva ldquoThe human antibodyresponse to dengue virus infectionrdquo Viruses vol 3 no 12 pp2374ndash2395 2011

[233] J E Osorio C Y-H Huang R M Kinney and D T Stinch-comb ldquoDevelopment of DENVax a chimeric dengue-2 PDK-53-based tetravalent vaccine for protection against denguefeverrdquo Vaccine vol 29 no 42 pp 7251ndash7260 2011

[234] J E Osorio J N Brewoo S J Silengo et al ldquoEfficacy of atetravalent chimeric dengue vaccine (DENVax) in cynomolgusmacaquesrdquo The American Journal of Tropical Medicine andHygiene vol 84 no 6 pp 978ndash987 2011

[235] J N Brewoo RM Kinney T D Powell et al ldquoImmunogenicityand efficacy of chimeric dengue vaccine (DENVax) formula-tions in interferon-deficient AG129 micerdquo Vaccine vol 30 no8 pp 1513ndash1520 2012

[236] L Hermida L Bernardo J Martın et al ldquoA recombinant fusionprotein containing the domain III of the dengue-2 envelopeprotein is immunogenic and protective in nonhuman primatesrdquoVaccine vol 24 no 16 pp 3165ndash3171 2006

[237] L Bernardo A Izquierdo M Alvarez et al ldquoImmunogenicityand protective efficacy of a recombinant fusion protein contain-ing the domain III of the dengue 1 envelope protein in non-human primatesrdquo Antiviral Research vol 80 no 2 pp 194ndash1992008

[238] A Izquierdo L Bernardo J Martin et al ldquoSerotype-specificityof recombinant fusion proteins containing domain III of denguevirusrdquo Virus Research vol 138 no 1-2 pp 135ndash138 2008

[239] B Etemad G Batra R Raut et al ldquoAn envelope domainIII-based chimeric antigen produced in Pichia pastoris elicitsneutralizing antibodies against all four dengue virus serotypesrdquoThe American Journal of Tropical Medicine and Hygiene vol 79no 3 pp 353ndash363 2008

[240] G Gao Q Wang Z Dai et al ldquoAdenovirus-based vaccinesgenerate cytotoxic T lymphocytes to epitopes of NS1 fromdengue virus that are present in all major serotypesrdquo HumanGene Therapy vol 19 no 9 pp 927ndash936 2008

[241] S Brandler M Lucas-Hourani A Moris et al ldquoPediatricmeasles vaccine expressing a dengue antigen induces durableserotype-specific neutralizing antibodies to dengue virusrdquo PLoSNeglected Tropical Diseases vol 1 no 3 article e96 2007

[242] I Valdes L Bernardo L Gil et al ldquoA novel fusion proteindomain III-capsid from dengue-2 in a highly aggregated forminduces a functional immune response and protection in micerdquoVirology vol 394 no 2 pp 249ndash258 2009

[243] E Marcos L Gil L Lazo et al ldquoPurified and highly aggre-gated chimeric protein DIIIC-2 induces a functional immuneresponse in mice against dengue 2 virusrdquo Archives of Virologyvol 158 no 1 pp 225ndash230 2013

[244] L Lazo L Gil C Lopez et al ldquoA vaccine formulation consistingof nucleocapsid-like particles from Dengue-2 and the fusionprotein P64k-domain III from Dengue-1 induces a protectiveimmune response against the homologous serotypes in micerdquoActa Tropica vol 124 no 2 pp 107ndash112 2012

[245] L Gil L Bernardo A Pavon et al ldquoRecombinant nucleocapsid-like particles fromdengue-2 induce functional serotype-specificcell-mediated immunity in micerdquo Journal of General Virologyvol 93 part 6 pp 1204ndash1214 2012

[246] C-Y Chiang S-J Liu J-P Tsai et al ldquoA novel single-dosedengue subunit vaccine induces memory immune responsesrdquoPLoS ONE vol 6 no 8 Article ID e23319 2011

[247] C Y Chiang M H Huang C H Hsieh et al ldquoDengue-1envelope protein domain III along with PELC and CpGoligodeoxynucleotides synergistically enhances immuneresponsesrdquo PLOS Neglected Tropical Diseases vol 6 no 5Article ID e1645 2012

[248] S B Halstead ldquoControversies in dengue pathogenesisrdquo Paedi-atrics and International Child Health vol 32 supplement 1 pp5ndash9 2012

[249] S J Thomas and T P Endy ldquoCritical issues in dengue vaccinedevelopmentrdquo Current Opinion in Infectious Diseases vol 24no 5 pp 442ndash450 2011

[250] J D Brien S K Austin S Sukupolvi-Petty et al ldquoGenotype-specific neutralization and protection by antibodies againstdengue virus type 3rdquo Journal of Virology vol 84 no 20 pp10630ndash10643 2010

[251] M Bollati K Alvarez R Assenberg et al ldquoStructure andfunctionality in flavivirus NS-proteins perspectives for drugdesignrdquo Antiviral Research vol 87 no 2 pp 125ndash148 2010

[252] E A Gould T Solomon and J S Mackenzie ldquoDoes antiviraltherapy have a role in the control of Japanese encephalitisrdquoAntiviral Research vol 78 no 1 pp 140ndash149 2008

[253] R M Kinney C Y-H Huang B C Rose et al ldquoInhibitionof dengue virus serotypes 1 to 4 in vero cell cultures withmorpholino oligomersrdquo Journal of Virology vol 79 no 8 pp5116ndash5128 2005

[254] D A Stein C Y-H Huang S Silengo et al ldquoTreatment ofAG129 mice with antisense morpholino oligomers increasessurvival time following challenge with dengue 2 virusrdquo Journalof Antimicrobial Chemotherapy vol 62 no 3 pp 555ndash565 2008


[255] P Kumar HWu J L McBride et al ldquoTransvascular delivery ofsmall interfering RNA to the central nervous systemrdquo Naturevol 448 no 7149 pp 39ndash43 2007

[256] D Ray and P-Y Shi ldquoRecent advances in flavivirus antiviraldrug discovery and vaccine developmentrdquo Recent Patents onAnti-Infective Drug Discovery vol 1 no 1 pp 45ndash55 2006

[257] S Shigeta S Mori E Kodama J Kodama K Takahashi andT Yamase ldquoBroad spectrum anti-RNA virus activities of tita-nium and vanadium substituted polyoxotungstatesrdquo AntiviralResearch vol 58 no 3 pp 265ndash271 2003

[258] L B Talarico C A Pujol R G M Zibetti et al ldquoThe antiviralactivity of sulfated polysaccharides against dengue virus isdependent on virus serotype and host cellrdquo Antiviral Researchvol 66 no 2-3 pp 103ndash110 2005

[259] L Ono W Wollinger I M Rocco T L M Coimbra P A JGorin and M-R Sierakowski ldquoIn vitro and in vivo antiviralproperties of sulfated galactomannans against yellow fever virus(BeH111 strain) and dengue 1 virus (Hawaii strain)rdquo AntiviralResearch vol 60 no 3 pp 201ndash208 2003

[260] P Leyssen A Van Lommel C Drosten H Schmitz E DeClercq and J Neyts ldquoA novel model for the study of the therapyof flavivirus infections using the Modoc virusrdquo Virology vol279 no 1 pp 27ndash37 2001

[261] P Leyssen J Balzarini E De Clercq and J Neyts ldquoThepredominant mechanism by which ribavirin exerts its antiviralactivity in vitro against flaviviruses and paramyxoviruses ismediated by inhibition of IMP dehydrogenaserdquo Journal ofVirology vol 79 no 3 pp 1943ndash1947 2005

[262] J D Morrey D F Smee R W Sidwell and C Tseng ldquoIdentifi-cation of active antiviral compounds against a New York isolateofWest Nile virusrdquoAntiviral Research vol 55 no 1 pp 107ndash1162002

[263] J W Huggins ldquoProspects for treatment of viral hemorrhagicfevers with ribavirin a broad-spectrum antiviral drugrdquo Reviewsof Infectious Diseases vol 11 pp S750ndash761 1989

[264] J W Huggins R K Robins and P G Canonico ldquoSynergisticantiviral effects of ribavirin and the C-nucleoside analogstiazofurin and selenazofurin against togaviruses bunyavirusesand arenavirusesrdquoAntimicrobial Agents and Chemotherapy vol26 no 4 pp 476ndash480 1984

[265] J Neyts A Meerbach P McKenna and E De Clercq ldquoUseof the yellow fever virus vaccine strain 17D for the study ofstrategies for the treatment of yellow fever virus infectionsrdquoAntiviral Research vol 30 no 2-3 pp 125ndash132 1996

[266] F J Malinoski S E Hasty M A Ussery and J M DalrympleldquoProphylactic ribavirin treatment of dengue type 1 infection inrhesus monkeysrdquo Antiviral Research vol 13 no 3 pp 139ndash1501990

[267] W C Koff R D Pratt J L Elm Jr C N Venkateshan and SB Halstead ldquoTreatment of intracranial dengue virus infectionsin mice with a lipophilic derivative of ribavirinrdquo AntimicrobialAgents and Chemotherapy vol 24 no 1 pp 134ndash136 1983

[268] P LaColla and J P Sommadossi ldquoMethods and compositionsfor treating flaviviruses and pestivirusesrdquo US6812219 2004

[269] M S Diamond M Zachariah and E Harris ldquoMycophenolicacid inhibits dengue virus infection by preventing replicationof viral RNArdquo Virology vol 304 no 2 pp 211ndash221 2002

[270] M D Sintchak M A Fleming O Futer et al ldquoStructureand mechanism of inosine monophosphate dehydrogenase incomplex with the immunosuppressant mycophenolic acidrdquoCell vol 85 no 6 pp 921ndash930 1996

[271] D P Stamos and R S Bethiel ldquoProdrugs of carbamateinhibitors of IMPDHrdquo US6825224 2004

[272] V Nair G Chi Q Shu J Julander and D F Smee ldquoAheterocyclic molecule with significant activity against denguevirusrdquo Bioorganic and Medicinal Chemistry Letters vol 19 no5 pp 1425ndash1427 2009

[273] X G Zhang P W Mason E J Dubovi et al ldquoAntiviral activityof geneticin against dengue virusrdquo Antiviral Research vol 83no 1 pp 21ndash27 2009

[274] E Mastrangelo M Pezzullo T De Burghgraeve et al ldquoIver-mectin is a potent inhibitor of flavivirus replication specificallytargeting NS3 helicase activity new prospects for an old drugrdquoJournal of Antimicrobial Chemotherapy vol 67 no 8 pp 1884ndash1894 2012

[275] S J F Kaptein T De Burghgraeve M Froeyen et al ldquoA derivateof the antibiotic doxorubicin is a selective inhibitor of dengueand yellow fever virus replication in vitrordquoAntimicrobial Agentsand Chemotherapy vol 54 no 12 pp 5269ndash5280 2010

[276] D Benarroch M-P Egloff L Mulard C Guerreiro J-LRomette and B Canard ldquoA structural basis for the inhibition ofthe NS5 dengue virus mRNA 21015840-O-methyltransferase domainby ribavirin 51015840-triphosphaterdquo Journal of Biological Chemistryvol 279 no 34 pp 35638ndash35643 2004

[277] J G Julander K Shafer D F Smee J D Morrey and YFuruta ldquoActivity of T-705 in a hamster model of yellow fevervirus infection in comparison with that of a chemically relatedcompound T-1106rdquo Antimicrobial Agents and Chemotherapyvol 53 no 1 pp 202ndash209 2009

[278] S Cheenpracha C Karalai C Ponglimanont S Subhadhi-rasakul and S Tewtrakul ldquoAnti-HIV-1 protease activity ofcompounds from Boesenbergia panduratardquo Bioorganic andMedicinal Chemistry vol 14 no 6 pp 1710ndash1714 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of

BioMed Research International


Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y


Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


in which they identified the clinical pictures resulting frominfection with dengue virus The first known as denguefever (DF) is characterised by an abrupt onset of feveraccompanied by frontal headache and retroorbital painfollowed by a variety of possible clinical symptoms such asmyalgia arthralgia vomiting and weakness A generalisedmaculopapular rash appears one or two days after feverdefervescence Minor haemorrhagic manifestations such aspetechiae may be observed in some patients DF is generallyself-limiting and rarely fatal Most patients recover withoutcomplications around ten days after the onset of illness

The second clinical picture dengue haemorrhagic fever(DHF) is a more severe form of the disease and occurs inup to 5 of dengue cases It is initially characterized by thesame variety of clinical symptoms as are seen in DF Thecritical period in DHF starts at the moment of defervescencebut haemorrhagic manifestations may occur 24 hours earlierA positive tourniquet test indicates that the patient hasincreased capillary fragility Petechiae bleeding at venepunc-ture sites epistaxis gum bleeding and haematemesis mayalso be observed High fever haemorrhagic manifestationsthrombocytopenia (platelet count 100 000mm3 or less) andhaemoconcentration (gt20 difference) characterize DHFPlasma leakage is the most significant pathophysiologicalevent in determining the severity of the disease Signs ofcirculatory failure such as irritability cold clammy extrem-ities flushed face and restlessness may be observed Thiscrisis usually persists for 24ndash36 hours With appropriatesupportive medicine and carefully monitored intravenousisotonic crystalloid therapy to ensure adequate fluid replace-ment most patients recover However during this criticalperiod it is essential to look for characteristic warning signsof worse to come Patients progressing to shock (dengueshock syndromemdashDSS) show intense abdominal pain ortenderness persistent vomiting weak pulse and hypoten-sion If increased vascular permeability progresses to vascularcollapse the outcome is usually fatal as a result of irreversibleDSS In addition to DF DHF and DSS it is now recog-nised that other clinical manifestations can be associatedwith infection by dengue virus for example encephalitismyocarditis hepatitis cholecystitis myelitis and acute colitis[6ndash11]

Despite the rigour of the DFDHFDSS classification andits intrinsic worth in clinical case management in recentyears a growing number of clinicians and authors haveargued that the 1997WHO scheme for dengue clinical classi-fication should be reassessed [12ndash14] because it distinguishesstrictly between DF DHF and DSS whereas it is now recog-nised that the point of transition between DF and DHF is noteasily defined The requirements for the WHO definition ofDHF (fever haemorrhage thrombocytopenia and signs ofplasma leakage) are not always satisfied severe thrombocy-topenia may be observed in uncomplicated as well as severecases associated with ldquounusual manifestationsrdquo and thereforemay not be consistent with theDHFDSS classification [15] AWHOTDR-supported prospective clinical multicentre studyacross dengue-endemic regions was established to collectand coordinate specific criteria for classifying clinical cases

into levels of severity [16] The study confirmed that byusing a set of clinical andor laboratory parameters one seesa clear-cut difference between patients with severe denguefever and those with nonsevere dengue fever However forpractical reasons it was considered necessary to split thelarge group of patients with nonsevere dengue into twosubgroups patients with warning signs (abdominal pain ortenderness persistent vomiting clinical fluid accumulationmucosal bleeding lethargy restlessness liver enlargementgt2 cm and increase in hematocrit concurrent with rapiddecrease in platelet count) and those without these warningsigns On the other hand the criteria for severe dengue feverinclude extensive plasma leakage severe bleeding or severeorgan impairment

The third and most recent edition of the WHOTDRdengue guidelines for diagnosis treatment prevention andcontrol includes a new clinical classification [17] This publi-cation serves as an authoritative reference source for healthworkers and researchers These new guidelines provide arevised case classification which is intended to facilitateeffective triage and patient management and collection ofimproved comparative surveillance data [18] However dueto the recommendation that cases of dengue fever withwarning signs and also cases of severe dengue fever shouldbe admitted to hospital there is concern that this could resultin overadmission of patients to hospitals during epidemicsinevitably reducing the efficiency of patient triage andadversely affecting the quality of clinical case management[19 20] Furthermore there is additional concern that theWHOTDRclassificationmay impact significantly on denguepathogenesis research since it requires the identificationand study of distinct dengue syndromes Because the 2009WHO case definitions do not require laboratory tests for thediagnosis of severe dengue it is considered that retrospectiveidentification of patients with clinically significant vascularpermeability from data on hospital charts may be difficultif not impossible [21] The previous discussion highlightsthe difficulties of designing a totally acceptable classificationscheme for dengue pathogenesis

22 History of DF and DHF Clinically diagnosed DF waswidespread during the 18th and 19th centuries in Northand South America the Caribbean Basin Asia and Aus-tralia In the Americas this was largely due to the repeatedintroduction from Africa of Stegomyia (St) aegypti (formerlyAedes aegypti) [22ndash24] Moreover together with yellow fevervirus (YFV) DENV-infected humans and mosquitoes wereintroduced via the slave ships and other commercial vesselsthat crossed the Atlantic Ocean from Africa during the pastfive or more centuries [25ndash31]

It is also important to note that disease clinically com-patible with the more severe and often fatal syndromes DHFand DSS was sporadically reported from 1780 onwards [32]although it is not clear if the more severe cases were confinedto individuals of European descent

DF thus became endemic in Latin America and theCaribbean region periodically causing epidemics At thesame time YFV was also causing epidemics in South
























































































50) values in







Acknowledgments


References













































































































































































































































































































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































































50) values in







Acknowledgments


References













































































































































































































































































































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































































50) values in







Acknowledgments


References













































































































































































































































































































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































































50) values in







Acknowledgments


References













































































































































































































































































































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































50) values in







Acknowledgments


References













































































































































































































































































































Volume 2014

Zoology





Volume 2014


















Advances in

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