West Nile Virus: An Emerging and Reemerging Infectious Disease

Jyoti S. Kumar*; Paban Kumar Dash1; Priyanka Singh Tomar;1; Divyanshi Karothia1; Manmohan

Parida2

1Defence Resarch and Development Establishment, Jhansi Raod, Gwalior-474 002, Madhya Pradesh,

INDIA.2Defence Food Research Laboratory, Siddhartha Nagar, Mysore (Karnataka) India - 570 011

*Correspondence to: Jyoti S Kumar, Defence Resarch and Development Establishment , Jhansi Raod, Gwal-

ior-474 002, Madhya Pradesh, INDIA.

Email: jyotishukla2@yahoo.co.in, Phone: 0751-2390292

Chapter 3

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Abstract

WestNileVirus(WNV),aflavivirusoftheFlaviviridaefamily,ismaintainedinnatureinanenzootictransmissioncyclebetweenbirdsandornithophilicmosquitovectors,althoughthevirusrarelyinfectsotherver-tebrates.WNVcausesdiseaseinhorsesandhumans,whichdevelopsfe-brileillness,meningitis,encephalitisandflaccidparalysis.Untilrecently,itsmedicalandveterinaryhealthconcernwasrelativelylow;however,thenumber,andseverityofoutbreakswithneurologicalconsequencesinhu-mansandhorseshave increased inEuropeancountriesand theMediter-raneanbasin.SinceitsintroductionintheAmerica,thevirusspreadacrossthecontinentwithworrisomeconsequencesinbirdmortalityandaconsid-erablenumberofoutbreaksamonghumansandhorses,whichhavedevel-opedinthelargestepidemicsofneuroinvasiveWNVdiseaseeverdocu-mented.Evengreatadvanceshavebeenobtained latelyregardingWNVinfection,andalthoughefficientequinevaccinesareavailable,nospecifictreatmentsorvaccinesavailableforhumanuse.ThisreviewupdatesthemostrecentinvestigationsofWNVparticularlypathogenesis,transmissiondynamics, host range, clinical symptoms, epidemiology, diagnosis, con-trol,andprevention,andhighlightssomeapproachesthatcertainlyrequirefurtherresearch.

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Vector-BorneDiseases&TreatmentKum

arJS

1. Introduction

WestNilevirus(WNV)isaneuroinvasivehumanpathogenthatisthecausativeagentofWNVinhumanandhorses[1,2].In2000,theWNVspreadoutin12statesandtheColumbiaDistrictofU.S.WNVinfectmanyavianandmosquitospeciesthroughoutNorthAmerica[3,4].Morethan2.5millionpeopleaffectedbyWestNilefeverandencephalitisbetween1999to2010.TheoutbreaksofWestNilevirusoccurredinU.S,Israel,Egypt,India,France,andSouthAfrica.WestNilevirusbelongstothefamilyFlaviviridae. ThefamilyFlaviviridae comprises3genera:theflaviviruses,whichcontainWNV,dengueVirus(DENV),andyellowfevervirus(YFV);thehepaciviruses,whichcontainhepatitisBandCviruses;andthepestiviruses,whichcausesencephalitisinhoofedmammals.WithintheFlavivirus genus,whichcontainsmorethan70viruses,virusescanbefurtherclassifiedintotick-borneandmosquito-borneviruses.Themosquito-bornevirusesclassifiedintotheencephaliticclade,ortheJEserocomplex,whichcontainWNVandJapaneseEncephalitisVirus(JEV),andthenonencephaliticorhemorrhagicfever clade,which containDENVandYFV, and there are10 serologic/genetic complexes[5,6,7].Theglobaldistributionofthemosquito-borneflavivirusesdependsonthehabitatofthemosquito vector.Culex mosquitoes transmitting encephaliticflavivirusesmostly in theNorthernHemisphere.

2. Structure of the Virus

WNV isanenvelopedvirushavinga single-stranded,positive- senseRNAgenome.Thegenomecomprisesofasingleopenreadingframeofabout11kblength.Boththe5’and3’noncodingregionsofthegenomesynthesizestem-loopstructuresthathelpinreplication,transcription,translation,andpackaging[8,9,10].TheWestNileRNAdirectlytranslatedintoasinglepolypeptideandthencleavedbyhostandviralproteases.Followedbytheformationofthreestructural(capsid,envelope,andpremembrane)andsevennonstructural(NS1,NS2A,NS2B,NS3,NS4A,NS4B,andNS5)proteins.StructuralproteinsareessentialforentryofvirusintohostcellandalsoaidinfusionencapsidationoftheviralRNAduringassembly[Figure 1].

Figure 1:SchematicofgenomicOrganizationofWestNileVirus.Theauthoracknowledgethesupportfrom;http://cme.cwru.edu/brochures/2003/WESTNILE

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The nonstructural proteins play important role and perform several functions. NS1proteinhas“cellular”aswellassecretedformandhashighlyimmunogenicbutdonotplayimportantroleduringencapsulationandpackaging,butitplayssignificantroleinreplicationofviralgenome[11,12,13,14].NS3proteinsuggestedtoplayimportantrole incleavingofviralpolyproteinswiththeaidoftheviralproteasesandencodesenzymeactivities.TheNS5proteinisessentialforviralreplicationbecauseithasviralpolymeraseactivityandencodesamethyltransferase.Othernonstructuralproteins,includingNS2A,NS2B,NS4A,andNS4B,havebeenshowntopreventseveralcomponentsoftheinnateimmunesystemagainstviralinfection[15,16,17].WestNilevirionisanicosahedralparticle.Thecapsidproteinassociatingwith the viral genome to form the nucleocapsid and lipid bilayer is present outside of thenucleocapsid.Ahighratioofcapsidproteinlocalizestothenucleus,whileviralassemblyalwaysoccursinthecytoplasm.Buddingistakesplaceintheendoplasmicreticulum(ER)[18,19,20].Althoughinsidethenucleusfunctionsofcapsidarenotfullyknown,recentresearchessuggestsaroleingeneregulation.Duringvirusassembly,theenvelopeproteinenclosedfirmlyinthelipidbilayerofthevirusanddisplaytothevirionsurface[21,22,23,24].Theenvelopeproteinisnecessaryforbindingtothereceptoronthecellsurfaceforviralentry.TheprMproteinisalsoknowntoenclosedfirmlyinthelipidbilayer.Atthetimeofinfection,theviruspopulationcomprisesbothmatureandimmaturevirionparticles[25].

3. Life Cycle

WNVentersintothehostcellthroughreceptor-mediatedendocytosisprocess.SeveralmoleculeshavebeenservesasreceptorsforWestNilevirus,likeDC-SIGN,mannosereceptor,and severalglycosaminoglycans etc.Endosomeof thevirusmaturesduring internalizationfromthecellsurface,withthepHdecreasesdownfromneutraltoslightlyacidicintheearlyendosomeandhavingmoreacidicatthetimeofmaturationintothelateendosome.Insidethelateendosome,theenvelopeproteinchangestheirconformationandthenvirallipidmembranefusewiththeendocyticmembraneandviralRNAgenomereleasesintothecellcytosol.Aftercapsiddisassociation,theviralgenomeisreplicatedandassembled.Theviralpolyproteinistranslatedandprocessedonintracellularmembranesofcellorganellesandexpressionofthe10viralproteinsoccurs.Theviralgenome is replicatedwith thehelpofviral andcellularproteins[26].

Immatureflavivirusparticlesalsoplaysignificantroleduringinfection.ThesimmatureflavivirusparticlesformduringinefficientcleavageoftheprMproteinatthetimeofmaturationand budding. Immature flavivirus particleswere traditionally thought to be noninfectious,severalreportshaveshownthat immatureWNVparticlesmaybepotentiallyimmunogenicandinfectious in vitro and in vivowhenlinkedbyantibodiesagainst theEorprMprotein.Theseantibody-linkedimmaturevirusparticlespenetratetheimmunecellsviatheFcreceptoroftheantibodyresultinginfectionoccurs[27][Figure 2].

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4. Host Reservoirs

WNVissurviveinnatureinacyclebetweenmosquitoesandanimalhosts.Somespecieslikebirdsshowsymptomsofdisease,andmaydie,whileothersbecomeonlyinfecteddonotshowsymptomsandactascarriers[28,29,30].Althoughhousesparrowsandcrowsareplayminorroleintransmission.TheAmericanrobinplayimportantroleintransmissionofWNVintheUnitedStates[31,32].Humansareconsideredas“dead-end”hostsforWNV,asthelowlevelofviremiainmammalsisusuallynotsufficienttobetransmittedtomosquitoes,therebyendingthetransmissioncycle.TheabilityofmammalstoservesashostsAedes mosquitoes,feedprimarilyonhumans,becomeprimarytransmissionvectorsforWNV[33,34][Figure 3].

Figure 2:TheWestNileviruslifecycle.Duringviralentry,theEproteininteractswithoneormorecellsurfacereceptor(s).ItisnotcompletelyclearwhichcellularreceptorsareinvolvedinWNVbinding,howeverDC-SIGN,alphaVbeta3integrinandlaminin-bindingproteinhavebeenreportedaspotentialreceptors.Afterbindingtothecell,thevirusistakenupviaclathrin-mediatedendocytosisandintheacidifiedendosometheEproteinundergoesconformationalchangesresultinginfusionbetweentheviralandcellularmembranes.Afterthefusioneventthepositive-strandedRNAgenomeisreleasedintothecytoplasmofthecell.TheviralRNAistranslatedintoasinglepolyprotein,whichisproteolyticallyprocessed to yield three structural proteins (the envelope proteinE; themembrane precursor protein prM; and thecapsidproteinC)andsevenNon-Structural(NS)proteins(NS1,NS2a,NS2b,NS3,NS4a,NS4b,andNS5).WhereasthecleavagesatthejunctionsC-prM,prM-E,E-NS1,NS4A-NS4B,andlikelyalsoNS1-NS2A,areperformedbythehostsignalpeptidaselocatedwithinthelumenoftheER,theremainingpeptidebondsarecleavedbythevirusencodedNS3protease.FlavivirusesreplicationrequirestheviralproteinNS5,whichisanRNA-dependentRNApolymerase.An"antisense"negativestrandRNAisproducedbythisenzyme,whichthenservesasatemplateforthesynthesisofmanynewcopiesoftheinfectiouspositivestrandRNAgenome.TheauthoracknowledgeCDCtoobtainthisimage.

Figure 3:TransmissioncycleofWestNileVirusTheauthoralsothankfultoCDCtogetaboveimage

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5.Vector Response to Infection

There have been many recent discoveries purposed at clarifying the transcriptomicandproteomicresponse toflavivirus infection in themosquitovector.WNVdemonstrateaconsistentinfectioninmosquitocellsin vitro andinlivemosquitoes,thereisgrowingevidencethatthemosquitodoesshowsomeimmuneresponsesagainstvirusinfection.Reportsaboutthe insect immune system come from experimentswithDrosophila melanogaster, thoughrecentexaminationofthemosquitoimmuneresponsesisstartingtoexposerelatedproteinsandpathways[35,36,37].Themosquitoantiviralresponseisincludetwopathways:theinnateimmunepathwayandtheRNAinterference(RNAi)pathway.Theinnateimmuneresponseisincludedofthreesignalingpathways:Toll,JAK-STAT,andIMD.TheTollandIMDpathwaysbothendsinNFkB-mediatedexpressionofantimicrobialpeptides(AMPs)andIMDsignalinghasbeenshowntopreventRNAvirusinfectioninDrosophila [38,39,40].NotmuchisknownabouttheroleofmosquitoAMPsinantiviralimmunity.Theirexpressionisinducedbyviralinfection.BothTollsignalingandtheJAK-STATpathwayshavebeenshowsignificantroleintheinhibitionofDENVinfectioninAedes aegypti [52,53]andmayalsobesignificantatthetimeofinfectionofCulex withWNV.TheRNAipathwayinmosquitoesisbothactivatedbyviraldsRNAandhasbeenshowntobecrucialforpreventingalphavirusinfectioninAedes andAnopheles [41,42,43].TheRNAipathway is inducedat the timeofWNVinfection inCulex pipiens [44].InfectionwithDENVwasalsofoundtoactivelycontrolmosquitoimmuneresponses in vitro [45].Evidence for a transcriptomic approachofflavivirus infectionwasfoundatthetimeofacomprehensivestudyofAedes aegypti infectedwithWNV,DENV,andYFV[46].

Genesplaysignificantroleintranscriptionandionbinding.Thereisupregulationanddownregulationofgenestakesplacehereandgenescodingforproteasesandcuticleproteinsweretobeupregulatedatthetimeofinfectionwithallthreeviruses.Serineproteasesplaysignificantroleduringviralpropagationandblooddigestion,althoughtherehavebeenseveralstudiesrelatedtheireffectonflaviviralinfectioninthemosquito[47,48].AnotherreportofflaviviralinfectioninDrosophilarecognizedseveralinsecthostfactorsrelevantatthetimeofdenguevirusinfectioninthemosquito.InregardingtoWNVinfection,arecenttranscriptomicanalysisofCulex quinquefasciatusexposedthatseveralgenestakespartinmetabolismandtransportareupregulatedat the timeof infection[49,50].Thevirus infectavarietyofcelltypesandorgansinthemosquitovector,andotherhostfactorswhichplaysignificantroleinWNVinfectionofthemosquitothathaveyettobereported.

WestNile(WNV)virusismosquito-transmittedflavivirusthatcausesignificantmorbidityandmortalityworldwide.Disease severity and pathogenesis ofWNV infection in humansdependonmanyfactors,includingpre-existingimmunity,strainvirulence,hostgeneticsandvirus–hostinteractions[51,52,53].Amongtheflavivirus-hostinteractions,viralevasionoftype

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Iinterferon(IFN)-mediatedinnateimmunityhasacriticalroleinmodulatingpathogenesis.DENVandWNVhaveevolvedeffectivemechanismstoevadeimmunesurveillancepathwaysthatleadtoIFNinductionandtoblocksignalingdownstreamoftheIFN-α/βreceptor.MolecularmechanismsofDENVandWNVantagonizethetypeIIFNresponseinhumancells[54,55].

6. Molecular Classification

Classifications ofWNVwere depends on cross-neutralization reactions and showedthatWNVisafamilymemberoftheJapaneseencephalitisvirusserocomplex.RecentstudiesonmolecularphylogenysuggestthisantigenicclassificationandexposethepresenceofuptoeightdifferentgeneticlineagesofWNV.Lineage1isfurtherdividedintothreeclades.Clade1aconsistsofAfrican,EuropeanandAmericanisolates;clade1bgroupstheAustralianKunjinVirus(KUNV),whichhasbeenrevealasubtypeofWNVandclade1cclustersisolatesfromIndia.Clade1arevealclosegeneticrelationshipsbetweengloballydifferentareaswhicharesuggestedtobetheresultofWNVtransmissionvia migratorybirdsWNVinsideclade1acanbefurtherclassifiedintovariousclusters.TheonlyoneendemicgenotypehasbeenidentifiedinIndia(1c)andoneinAustralia(1b),suggeststhatWNVwasspreadintotheseareasonlyonce,aswellasitoccurredintheAmericancontinent,whereWNVwasendemicin1999intheEastCostoftheUS[56].

ThefirstNorthAmericanWNVisolatewasshowrelevancytoastrainisolatedfromadeadgooseinIsrael(lineage1)NorthAmericanWNVwasoriginatedfromthisepidemicintheyear1998outbreak.However,recentstudiessuggestthatthe1998IsraelepidemicwasnotthedirectlyrelevantofNorthAmericanepidemics,butratherthatbothepidemicsderivedfromthesame(unknown)areas.Lineage2initiallyconsistsofWNVstrainsonlyidentifiedinAfricaandMadagascar,which have been speculated to be less neuroinvasive than those consistsin lineage1.Recentoutbreaksoccurs inEurope (Austria,HungaryandGreece)havebeenrelatedtolineage2strains.OtherlineagesofWNVofunidentifiedhumanpathogenicityduetolineage3(Rabensburgisolate97-103),isolatedfromCulex pipiens mosquitoesintheCzechRepublicin1997andLineage4(LEIVKrnd88-190),isolatedfromDermacentor marginatus ticksin1998inRussia.IthasbeensuggestedthatWNVIndianisolatesthatwereclassifiedaslinage1cformsanewclustertermedlineage5.AnewlineageofWNV(strainHU2925/06)that includes evolutionary branchwith lineage 4 has been recently reported in Spain [57][Figure 4].

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7. Molecular Epidemiology

TheWNVoutbreakoccurredinNorthAmericainthelatesummerof1999inNewYorkCityduetothisoutbreakofmosquitoborneencephalitisdeathoccurredofhumans,birds,andhorses[58].WNVtransportthroughouttheUnitedStatesandintoCanada,Mexico,andtheCaribbean[Figure 5].From1999to2016,46,086caseswerereportedtotheCDC,with2,017(4%)deaths,and21,574reportedcasesofneuroinvasivedisease,with1,888(9%)deaths[Figure6].AsinmostcasesthevirusisspreadbytheCulex mosquitovector.WNVtransmissionmayoccurthroughbloodtransfusion,organtransplantation,andlaboratory-acquiredinfectionhasalsobeenreported.ThefirstreportedAcuteEncephalitisSyndrome(AES)outbreakinKerala,India,occurred inKuttanadregionbetweenJanuaryandFebruary1996,causing105casesand31deaths.InIndia,presenceofWestNileantibodiesinhumanswasfirstreportedfromBombay(nowMumbai)byBankerin1952.SmithburnetalconfirmedthereportbydetectingtheWNVneutralizingantibodies.Duringapostsero-epidemiologicalstudy,detectedWNVneutralizingantibodiesamonghumansatSouthArcotdistrictofTamilNadu.WNVhasbeenisolated fromsporadiccasesofencephalitisandmosquitoes.WorkpostulatedahypothesisofazoogeographicalinterfaceofJapaneseencephalitisandWestNilevirus.Thehypothesis

Figure 4: Phylogenetic tree ofWestNile virus strains based on a 282nt fragment of the Envelope gene.The treewasconstructedwiththeprogramMEGA(MolecularEvolutionaryGeneticAnalysis)byneighbor-joining.Bootstrapconfidencelevel(1,000replicates)andaconfidenceprobabilityvaluebasedonthestandarderrortestwerecalculatedbyMEGA.WNVstrainsarenamedaccordingtothefollowingrules:asetofletterscorrespondingtotheplacewherethestrainwasisolated(Fr,France;Mo,Morocco;It,Italy;Ro,Romania;Ken,Kenya;Tu,Tunisia;Hu,Hungary;NY,NewYork;Is,Israel;Tx,Texas;Eg,Egypt;Chin,China;Ind,India;Rus,Russia;Rab,Rabensburg;CAR,CentralAfricanRepublic;SA,SouthAfrica,Ug,Uganda),2numbersfortheisolationyear(ex:00=2000,96=1996),andGenBankaccessionnumber.Sequencesobtainedfromthe2horsesamplesinHungary2008arehighlighted(rectangle).JEV,acloseflavivirus,wasusedtorootthephylogenetictree.Theauthorthankstoresearchgateforthisimage.

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proposedtheinterminglingdistributionofJEVandWNVatthesouthIndianpeninsularregion[59].

8. Clinical Features

Infectionsinhumansaremainlysubclinical,butreportedinfectiondemonstrationmayrangefromfeverandCNSaffectedinencephalitiscausesdeath.Encephalitisoccursonlyinasmallsubsetofpatients;concatenationtochronicneurologicalconditionmayinduceacuteparalysisafterencephalitis.Individualswithneurologicalinvolvementthatmayresultindeathandmayhavehigh riskofmortalityafter acute illness. Inabout75%of infectionspeoplehavefewornosymptoms.About20%ofpeopledevelopafever,headache,vomiting,orarash.Inlessthan1%ofpeople,encephalitisormeningitisoccurs,withassociatedneckstiffness,confusion,orseizures.Recoverymaytakeweekstomonths.Theriskofdeathamongthoseinwhomthenervoussystemisaffectedisabout10%.Amongpeopleover70yearsofage,thecase-severityraterangesfrom15%to29%[60-62].Highermortalityisalsoseenininfectedinfantsandinimmunocompromisedpatients.Riskfactorsforencephalitisarecardiovasculardiseaseorchronicrenaldisease,hepatitisCvirusinfection,andimmunosuppression.Insomecasesconvalescentpatientsmayhavepersistentorsevereinfectionidentifiedthroughmolecular

Figure 5:WorldwideoutbreaksofWestNilevirus.TheauthorsalsothankfullyacknowledgetheILRIClippingsforthisimage.

Figure 6:WestNilevirusneuroinvasivediseaseincidencereportedtoCDCbyyear,1999-2016.TheauthoracknowledgetheCDCtogetabovementionedinformation

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basedassayslikePCRoftheurine,whichsuggestedongoingviralreplicationinkidneytissues[63,64].

9. Pathogenesis

WNVisaneuroinvasivediseaseinvolveinprogressionofmeningitisorencephalitisto poliomyelitis- like conditionwith acuteflaccid paralysis that can also cause respiratorytroubles. It isestimated that10%casesare fatal [65,66].Olderage individualsareathighrisk of infection but neuroinvasive diseases have been also reported in young people andchildren.HypertensionanddiabetesareconsideredaspotentialriskfactorforsevereWND,Immunocompromisedpeopleeasilyinfectedbythisdisease.Afullyfunctionalimmune(innateandadaptive)response(humoralandcellular)hasbeenshowntobenecessarytocompetewithWNVinfectioninanimalmodels.Itiswellfamiliarthatoverall,humoralimmuneresponsehascapableofcontrolviralloadwhileT-cellmediatedresponseisnecessaryforclearanceofthevirusfromtheCentralNervousSystem(CNS).WNVisabletoinfectneuronsoftheCNS,brain,stemandspinalcord[67,68].

ThepathogenesisofWNVinfectionissimilartothatofotherFlaviviruses.Afterprimaryinoculation,WNVisreplicateinskinLangerhansdendriticcellsbeforeittravelstothelymphnodesandbloodstreamfromwhereittransferstothespleenandkidneysand,finallyreachestotheCNSresultingininflammationofthemedulla,brainstemandspinalcord.TheViralentrymechanism inCNS is yet to be fully elucidated.Themainmechanisms include:via leukocytes,directentryacrossthebrainbarrier[69,70].

Asseveralviruses,WNVhasdevelopeddifferentmechanismstoblocktheactionofimmunesystemlikeitinhibitInterferongamma(IFN)and,thus,toescapethehostantiviralactivityofIFN-stimulatinggenes.DifferentreportssuggestthatnonstructuralproteinsNS1,NS2A,NS4BandNS5playimportantroletocontrolIFNα/βsignalingbyseveralpathways[71,72].

10. Laboratory Diagnostics

Routine laboratorydiagnosisofWNV infection isprimarilybasedon serodiagnosis,followedbyvirusisolationandidentification.Serologically,WNVinfectioncanbeinferredbyimmunoglobulinM(IgM)andimmunoglobulinG(IgG)captureELISA.Recentlyseveralinvestigators have reported PCR-based detection systems for the rapid diagnosis ofWNVinfection inclinical specimens thatarenegative forvirus isolation, suggesting thatnucleicacid-basedassaysholdgreatpromiseforthediagnosisofWNVinfection.Inaddition,otherPCR-basedmethods,likeReversetranscriptionloop-mediatedisothermalgeneamplification(RT-LAMP)assay,havebeendevelopedforthediagnosisofWNVRNA[73].

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10.1. Immunological diagnosis

The detection ofWNV infection ismainly based on clinical criteria and testing forantibodyresponses.TheincubationperiodforWNVinfectionisabout2to14days.Theanti-WNV IgMmainly fromCerebrospinalFluid (CSF), is used for detection.Cross-reactivitywith related flaviviruses (Japanese encephalitis virus, St. Louis encephalitis virus, YFV,and DENV), if suspected, can be accessed through plaque neutralization assays (PRNT).ReplicationofWNVhasbeenreportedinhumanmonocytesin vitro andwithhigherefficiencyinpolymorphonuclearleukocytes;thiscouldcausetransmissionviabloodtransfusion[74,75].ThusmanyrapiddiagnosticshavebeendevelopedforblooddonortestingusingNucleicAcidTesting(NAT),anamplification-basedtranscriptiontechnique,whichdetectsWNV-infectedindividualsbeforetheybecomesymptomatic[76,77].RNAmostlybecameundetectableafter13.2days,althoughitwasrarelyfoundtopersistfor40days.IgMandIgAantibodiesdecreasessignificantly,whiletheIgGlevelleftelevatedfor1yearafterdetectionofviremia.AntibodytoWNVNS5persistsin vivo,andthusNS5antibodycannotbeusedtodifferentiaterecentfromoldWNVinfection[78,79].

10.2. Molecular diagnosis

SeveralmethodsforidentificationofviralRNAhavebeenusedforWNVsurveillance.GenerallyReverseTranscriptionPolymeraseChainReaction(RT-PCR)technique,quantitativereal-timeRT-PCRandIsothermalgeneamplificationtechniqueslikeLAMPthatisone-step,singletube,costeffectivereal-timereversetranscriptionloop-mediatedisothermalamplification(RT-LAMP)assaywasdevelopedforidentifyingtheWestNile(WN)virus[80].TheRT-LAMPassayisanoveltechniqueofnucleicacidamplificationthatishighlyspecific,sensitive,rapidandcosteffectiveunderisothermalconditionswithasetofsixspeciallydesignedprimersthatrecognizeeightdistinctregionsofthetarget.Thecompleteprocessisverysimpleandrapid,andamplificationcanbeoccurinlessthan1hbyincubatingallofthereagentsinasingletubewithreversetranscriptaseandBst DNApolymeraseat63°C.Bst DNApolymerasehasstranddisplacingactivity.Thereareseveralformatsforendpointdetectionofgeneamplificationlikeagarosegelelectrophoresis,real-timemonitoringinaninexpensiveturbidimeter,inpractice,generally thevisual inspection foramplification isperformed throughobservationofcolorchangefollowingadditionofSYBRgreenIdye,(afluorescencedsintercalatingdye)WhenthesensitivityoftheRT-LAMPassaywascomparedtothatofconventionalRT-PCR,itwasfoundthattheRT-LAMPassaydemonstrated10-foldhighersensitivitycomparedtoRT-PCR.Alltheseassayshavebeenextensivelyusedinmosquitopools,animalandhumansamples[81,82].

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11. Therapeutics and Vaccines

Advance therapeutic alternatives againstWNVaremainly supportive; there are noFDA-approvedvaccinesortreatmentsavailable.RNAinterference(RNAi)isamajorpathwayofantiviraldefense inplantsand insects.Thepathway involves theprocessingofDouble-StrandedRNA(dsRNA)intoshort21–22bpeffectorRNAmoleculesbytheRNAseIIIdomain-containingDicer2enzyme.ThefidelityofDicercleavageanddownstreamstrandselectionintheRNAiprocessismaintainedbyDicer-associatedproteins(TRBPandPACTinmammaliancells)ThesedsRNAserveastargetingmoietieswhenloadedintoaRISCcomplextoselectivelydownregulatemRNAtargets.Argonauteproteins(e.g.Ago2)playamajorroleintheknockdownofgeneexpressionbytheRISCcomplex,mediatingselectiveendonucleolyticcleavageoftargetRNAs.ThesuccessfulavoidanceordownregulationoftheRNAimachineryisvitalforarbovirusestoproductivelyinfecttheirarthropodvectors.Thereareseveralreportstoidentifyindividual susceptibilitymarkers, recombinant antibodies, peptides,RNA interference, andsmallmoleculeswiththeabilitytodirectlyorindirectlyneutralizeWNV.Tillnoweffectivetherapyisstilllacking[83].

Arecentapproach tosearchfornewantiviralagentcandidates is theassessmentoflong-useddrugs commonly administeredby clinicians to treat humandisorders, as part ofdrugrepositioning(findingofnewapplicationstolicenseddrugs).Amongsomeofthedrugsalreadytestedasantiviralsarelithium,statins,orvalproicacid[84,85].Sincethereisevidencesupporting thatWNVinfectionsharescommonpointswithParkinson’sdisease, studywasundertakenwhetherdrugsusedforthetreatmentofParkinson’sdiseasecouldprovidenoveltoolsforantiviralintervention.Inthisway,assessedtheeffectoffourantiparkinsoniandrugs(l-dopa, Selegiline, Isatin, andAmantadine) inWNVmultiplication in cultured cells fromdifferentorigin.l-dopa,Isatin,andAmantadinetreatmentssignificantlyreducedtheproductionofinfectiousvirusinallcelltypestested,butonlyAmantadinereducedviralRNAlevels.ThisresultsindicatesthatAmantadine,aspossibletherapeuticcandidatesforthedevelopmentofantiviralstrategiesagainstWNVinfection.

TherearerecentlyfourUSDA-licensedvaccinesavailableforequinesinwhichtwoare attenuated wholeWNV one is a non-replicating live canary pox recombinant vectorvaccine,andanotherisanattenuatedflaviviruschimericvaccine.Passiveimmunizationhasbeenusedinasomecases;Sometimesitalsocausesallergicreactions.AcasestudyoftwoWNVencephalitis patients treatedwith alpha interferon, the standardof care for infectionwiththerelatedflavivirushepatitisCvirus,showedsignificantmeliorationandanimprovedrecoverycourse.Severalstrategiesarebeingusedfortheadvancementofavaccineinhumansthatmaysignificantforuse.Reportsincludeliveinactivatedvaccines,recombinantsubunitvaccines,vectorizedvaccines,DNAvaccineswithconstructsthatexpresstheWNVEprotein,liverecombinantvaccines,andaninactivatedstrainbasedonnonglycosylatedEandmutant

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NS1protein[86].AneutralizingWNV-specificmonoclonalantibodyE16(MGAWN1),whichentersintheCNSinanimalmodels,generatesneutralizingantibodiesinphaseItrials.VeryeffectiveresultswereseenwithachimericvaccinebasedontheWNVprMandEproteinsinsertedintotheyellowfever17Dvaccinemoiety(ChimeriVax-WN02).Itwasshowntobeeffective,safeandimmunogenicinphaseIIclinicaltrials,withhighseroconversionrates,yetitisnolongeravailable[87,88].

12. Preventive measures and control

PreventionandcontrolofWNDrequire strategy that includesvaccination,mosquitocontrolandclinicalmanagement.PreventivetoolsarenecessaryforinhibitingWNVinfectionsotherthandrugsandvaccinescanalsoaidtoarmedthetransportoftheinfectionbyavoidingmosquitobites[89].Thesesimplemeasurescanbesummarizedunderthetitle‘fightthebite!’andconsistsoftheuseofinsectrepellents,theremovalofstandingwaterwheremosquitoeslayeggs,thereducestheoutdooractivitiescooccurencewiththemaximumactivityofmosquitoes,reportingdeadbirdstolocalauthoritiesandhelpingmosquitocontrolprograms[90].

13. Future Directions

WNVhasnowpersistedandcausesendemicsinNorthAmerica.TransmissionofthemosquitovectorsharboringWNVtoincludeAedes albopictus,amammal-bitingmosquito.ItisbelievedthattheincreaseinourknowledgeofWNVwiththemosquitovectorwillleadtonewboulevardfortherapeuticsandpreventivemeasures.Mosquitoresponsesatthelevelsofproteinandgeneexpression[91].ManystrategiestonoveltargetstoconcentrateoureffortstopreventorblockWNVinfection.Forexample,asingle-chainhumanmonoclonalantibodydeveloped throughphagedisplay against the envelope protein reveled both protection andtherapeuticefficacywhendetectinthemurinemodel.CurrentadvancementinnanoparticletechnologyhavealsobeenusedinvaccinationstudiesofmurineWNVinfectionandshowsignificant efficacy of TLR9-targeted biodegradable nanoparticles, which produce a largenumberofcirculatingeffectorTcellsandantigen-specificlymphocytes.Potencytorelevantviralsusceptibilitymechanisms,processinghostantagonismofchemokineresponsesashasbeennotedininfectionwiththerelatedflavivirushepatitisCvirus.mayshowinfectiousmechanismsusedbyWNV.Therateofvectordiscovery,virus,andhostmoleculesofpathogenesisprovidecriticalinsightsforthecontrolsandtherapeuticsforWNV[92].

14. Conclusion

TherecentamanationandtransportofWNVinAmericaandincreaseinnumberandseverityofoutbreaks inEuropeshownoneof themajorzoonotic treats inyears.AlthoughourinformationaboutWNVinfectionhasincreasedinrecentyears,somepanoramaofWNVactivitystillessentialtobefurtheraddressed:thewaysbywhichWNVcolonizesnewhabitats

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andtherolethatclimatic(temperature,humidity,etc.)factorsplay;thedifferencesinWNVdiseasedemonstrationbetweentheUSandotherpartsoftheworld,mainlyCentralandSouthAmerica; a better knowledge aboutWNV immunity, pathogenicity, and the factorswhichcausesvirulence;thelong-termmanifestationsofWNVinfectionandtheresultsofpersistentinfections; the progression of national and international surveillance programs tomonitorWNVoutbreaksandtotakeappropriatestrategytocontrolit.Thesearchformoreefficient,rapid,andspecificandsensitivediagnosticassaysthatcanbeeasilyadoptedinallovertheworld;and thesearch forcheaphumanvaccines forhigh risk targetedpopulationsandfornewantiviraltargetsfortherapeuticusage.AdvancementonourcurrentknowledgeonWNVinfectionwillgreatlyhelptofightnotonlyfuturetransmissionofWNVtohabitatsaroundtheworld,butalsoofotherFlaviviruses.

15. Acknowledgment

The authors thankDr.D.K.Dubey,Director, DefenceResearch andDevelopmentEstablishment (DRDE), Ministry of Defence, Govt. of India for his support, constantinspirationandprovidingthenecessaryfacilitiesforthisstudy.ThismanuscriptisassignedDRDEaccessionno.DRDE/VIRO/009/2018.

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