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2584DIAMONDETAL.plesFIG.postinfectionwere5.collectedPassiveadministrationfrommicethatofwereserumnaïto MTmice.Serumsam-administeredwithto MTWNV.miceAfter1dayheatinactivation,veorimmune0.5mloforserumat4dayswasexperimentsofWNV.withDatapriortoandafterinfectionwith102PFU verepresentmiceperresultscondition.

fromatleastthreeindependenttionbeganperipherallyand,inasubsetofanimals,dissemi-natedtotheCNSbyahematogenousroute.CongenicmicethatlackedBcellsandantibodyweremoreviremic,andthiscorrespondedwithhigh-gradedisseminationintheCNSandadverseoutcome.

Infectionmodelinwild-typeC57BL/6mice.Wild-typemicereplicatedviruslocallyindraininglymphnodesanddevelopedapeaklevelofviremiawithin2daysofinfection.Byday4,viralinfectionwasdetectedinthespleenandmultiplesitesintheCNS,apatternmostconsistentwithhematogenousspread.Overall,high-gradeviraldisseminationtothebrainwaslimitedtoasubsetofwild-typeanimals:thesemiceweremorbidlyillwhereaswild-typemicethathadlowerviralburdensintheCNSexhibitedmilderclinicalsymptoms.Sinceallwild-typemiceshowedevidenceofperipheralinfection,theextentofCNSdisseminationmaybedeterminedbythekineticsofpro-ductionofanti-WNVantibodyduringtheearlyphaseofin-fection.Interestingly,clearanceofviralinfectionintheserumandspleenlaterintheinfectioncoursedidnotcorrelatewithCNSdisseminationoroutcome;wild-typemoribundmicewithevidenceofdisseminatedCNSinfectionnonethelessretainedtheabilitytoclearinfectiousvirusfromtheseperipheralcom-partments.

WNVreplicationwasobservedatseveralextraneuralsites,includinglymphnodes,spleen,andkidney(datanotshown).Ourdataareconsistentwiththoseofpriorstudies(26,50)thatdocumentedWNVreplicationatseveralextraneuralsites,in-cludingspleen,kidney,andmuscle.Atpresent,theextraneuralcellulartargetofWNVreplicationremainsuncertain;his-topathologicandinsituhybridizationstudiestode nepermis-sivecelltypesinthespleenareunderway.Basedoncellcultureinfectionstudies,cellsofmyeloidorigin(9,10,17,18),includingtissuemacrophagesanddendriticcells,maybetar-getsforWNVinfection.

Therewaslittledifferenceinoverallsurvivaloraveragesurvivaltimesoverabroadrangeofinoculatingdoses.This

J.VIROL.

strikinglackofdose-dependentmortalitywasnotseeninpre-viousstudieswithWNVinwhichC3H/HeNoroutbredmicewereinoculatedviatheintraperitonealroute(15,16,49).Itwas,however,observedafterinfectionwiththerelatedMurrayValleyencephalitis avivirus(30).

Infectioninimmunode cientmice.RAG1and MTmiceweremorevulnerabletolethalWNVinfection.Animalsde-velopedarapid-onsetparalysis,andhighlevelsofvirusandviralRNAweredetectedperipherallyandintheCNS.Simi-larly,SCIDmicedevelopedhigh-gradeviremiaandCNSin-fectionandsuccumbedtoinfectionafterinoculationwithacandidateWNVvaccinestrain(22).Bcellsandantibodyhavebeenproposedtoprotectagainstencephalitiscausedbyother aviviruses(8,25,32,38)andnon- aviviruses(6,19,20,33,37,43,44,48).Insomeoftheseinfectionmodels(e.g.,Sindbis,murinehepatitis,andlymphocyticchoriomeningitisviruses),antibodyandBcellscontributetotheeradicationofinfectionbyclearingvirusfromthebrain(19,29)orpreventingviralrecrudescence(6,43).OurdatasuggestthatantibodyandBcellsdirectlylimitthedisseminationofWNVintheCNSearlyduringthecourseofinfection. MTmicehadan 500-foldincreaseinserumviralloadatday4afterinfection;thisledtoamarkedlyincreasedviralburdeninneuronsintheCNSatday6andprovokedarapidlyfatalencephalitis.

Antibodyprotection.Speci cantibodiesagainstWNVwereinitiallydetected4daysafterinfectioninwild-typeanimals,whichwasthesametimewhenhigh-gradeviremiawas rstdetectedin MTmice.Anisotype-speci cELISAcon rmedthatthesewereexclusivelyIgM.Nonetheless,inducedIgMagainstWNV,derivedfromwild-typemice4daysafterinfec-tion,prolongedbutdidnotguaranteesurvivalof MTmice.AlthoughthesesameIgMantibodiesweredemonstratedtohaveneutralizingcapacity,theycouldnoteradicateinfectionin mineMTwhethermice.Additionalhigherdosesstudiesofanti-WNVmustbeIgMperformedcanconfertodeter-com-pleteprotectionin MTmiceandwhetherequivalentamountsofanti-WNVIgMfromday4serumcanprotectwild-typemice.Higherdosesofanti-WNVIgMmaynevereliminateWNVinfectionin MTmicebecauseIgMcannottriggerthematureIgGresponsethatisnecessaryforeradication.Indeed,onlyimmuneserumthatcontainedbothanti-WNVIgMandIgGpreventedmorbidityandmortalityin MTmice.Speci cIgMmayhaveadualroleearlyduringviralinfection:tolimitdisseminationbytemporarilycontainingviremiaandtotriggeranadaptiveIgGresponsethateliminatesviralinfection(35).Recentexperimentswithcomplement-de cientmice(M.Di-amond,E.Mehlhop,andM.Engle,unpublishedobservations)suggestthatanti-WNVIgMmayinduceamaturehumoralresponsebyactivatingcomplementandfacilitatingT-cell-de-pendentand-independentantibodyproduction(36).

Incontrasttoinducedantibody,“natural”antibody(primar-ilyIgMgeneratedfromCD5 B-1cells[3,11,34])obtainedfromnaïvemicedidnotattenuateWNVinfection.Ourresultscontrastwithdataobtainedwithvesicularstomatitisvirus(34),wherepassiveadministrationofnaturalantibodyto MTmiceimprovedsurvivalafterinfection.Severalvariablesmayin u-encetheef cacyofnaturalantibodiesinpreventinginfection,includingtheviralinoculum,thekineticsofviralreplication,thesiteofvirusinoculationandantibodyadministration,andtheabsoluteamountofnaturalantibodytransferred.

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