想

throughoutthecourseofinfection.Incontrast,afterday4,infectiousvirus( 104PFU/g)andviralRNA( 2 104copiesofWNVRNAper gof18SrRNA)weredetectedintheliversofall MTmice.Atlatertimepoints,thelevelsofinfectiousWNVinliversof MTmiceexceededthosefoundintheserum(Fig.3A).

RoleofBcellsandimmunoglobulin.Althoughthevirologic,histologic,andclinicalanalysesshowedthat MTmicehadincreasedCNSviralburdensandmortalityratesrelativetowild-typemice,themechanismforthisremainedunclear.Wespeculatedthatspeci cantibodyagainstWNVdirectlypre-venteddisseminationofWNVintheCNS.Toevaluatethishypothesis,weassessedthekineticsofneutralizing-antibodyformationbyaviralplaquereductionassay(Fig.4A).Asex-pected,noneutralizingantibodiesweredetectedin MTmiceatanypointduringthecourseofinfection.Incontrast,lowlevels(inhibitorytiterof1/10to1/20)ofneutralizingantibod-iesweredetectedinwild-typemiceatday4afterinfection.Afterday4,inhibitorytitersincreased.Finally,atthelevelofsensitivityofourplaquereductionassay,neutralizingantibod-ieswerenotdetectedinserumfromnaïveanimalsorfromwild-typeanimalswithin2daysoftheinitialinfection.

Todirectlyaddresstheprotectivenatureofantibody,inde-pendentofBcells, MTmicewerepassivelyadministeredseracollectedfromwild-typemicethatwerenaïveorimmunetoWNVorthatwereexposedtoWNVfor4days,theearliesttimewhenneutralizingantibodiesweredetected. MTmicewereinoculatedwith0.5mlofheat-inactivatedserum1daypriortoandafterinfectionwith102PFUofWNV(Fig.5).Althoughsimilarquantitiesofnaïveserumprotected MTmiceagainstinfectionwithotherviruses(34),ithadnosignif-icanteffectonmortalityoraveragesurvivaltime(P 0.7).When MTmiceweregivenimmuneserumobtainedfromwild-typemice,theywerecompletelyprotectedagainstinfec-tionwith102PFUofWNV(P 0.0001).Treatmentof MTmicewithserumfromwild-typemicethatwereat4dayspostinfectionledtoanintermediatephenotype;althoughtherewasasigni cantincreaseinaveragesurvivaltime(14versus10days;P 0.0001),allanimalsultimatelysuccumbedtoinfec-tion.TodeterminetheroleofIgMorIgGinmediatingpro-tection,anisotype-speci cELISAagainstsolid-phaseWNVantigenwasperformed.Speci cIgMwasdetectedasearlyasday4afterinfection,whereasspeci canti-WNVIgGwasnotdetecteduntil8daysafterinfection(Fig.4B).ChemicalandimmunologicdepletionofIgMcon rmedthisresult.Treat-mentwith0.05M -mercaptoethanol(whichdestroysIgMbutnotIgG[42])orpreclearingwithanti-IgMagarosecompletelyabolishedtheneutralizingactivityofserumobtainedatday4afterinfectionbutnotatday10or28(Fig.4Canddatanotshown).Thus,day10andimmuneseracontainedprimarilyIgG-speci cantibodiesagainstWNVbutday4serumcon-tainedexclusivelyIgM-speci cantibodiesagainstWNV.

DISCUSSION

AlthoughpriorWNVinfectionmodelsinrodentsdocu-mentedtheneurotropiccharacterofthevirus(15,16,49,50),themechanismofviralspreadandtheroleoftheadaptiveimmunesysteminlimitingdisseminationwerenotdetermined.Inthispaper,bycomparinginfectioninwild-typeandcongenic

B-cell-andantibody-de cientmice,weestablishedthekineticsofthespreadofWNVinfectionfromlymphoidtissuetoserumtotheCNSanddeterminedhowthedevelopmentofantibodyimpedesthisprocess.Inwild-typeC57BL/6mice,WNV

infec-

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