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ActaOceanolo~eaSinica2O08,Vo1.27,No.2,P.126132

    ;http://www.oceanpress.con.cn

    ;E-mail:hyxbe@263.net

    ;Invivoneutralizationassaysbymonoclonalantibodiesagainstwhitespot ;syndromevirusincrayfish(Cambarusproclarkii)

    ;WANGYinan,ZHANWenbin,XINGJing,JIANGYousheng

    ;1.LaboratoryofPathologyandImmunologyofAquaticAnimals,LaboratoryofMarieuhureofMinistryofEducation,OceanUni

    ;versityofChina,Qingdno266003,China

    ;Received5June2007;accepted19December2007

    ;Abs![ract

    ;Theneutralizingactivitiesofeightmonoclonalantibodies(MAbs)againstwhitespotsyndromevirus(WSSV)(2D2,2B2,1D2,

    ;1D5,1C2,4A1,6A4and6B4)wereanalyzedbyinvivoexperiments.GillsfromWSSV-infectedshrimpwerehomogenizedand

    ;tenfoldseriallydilutedbyPBS,andthenincubatedwithMAbs(hybridomaculturesupematant),respectively.Themixtureof

    ;WSSVandMAbswereinjectedintocrayfish(Procambarusclarkii).Afterchallenge,thedeathratesoferayfishwerecountedto

    ;determinetheneutralizingactivitiesofMAbs.Atthesametime,themixtureofmyelomaculturesupematantandWSSVorPBS

    ;wasservedaspositiveornegativecontrol,respectively.Theresultsshowedthat,ateachvirusdilution,themeantimetodeathof

    ;thecrayfishinjectedwithMAbtreatedviruswassignificantlylongerthanthatinthepositivecontrol,thoughtheyallshowed100%

    ;mortalitywithin25d,andmeanwhile,fewcrayfishdiedinthenegativecontro1.AmongtheeightMAbs,2D2,2B2,1D2and

    ;1D5,especiallytheformertwo,delayedthemortalitysignificantly,and1C2,4A1and6A4delayedthemortalityaswellbutnot

    ;soefficiently,whileMAb6B4wasefficientonlywhenthevirusconcentrationincreased.TheresultsindicatedthattheantiWSSV

    ;MAbscanneutralizeWSSVindiflferentvirusdiutions.

    ;Keywords:crayfish,WSSV,monoclonalantibodies,invivoneutralization ;1Introduction

    ;Whitespotsyndromevirus(WSSV),emerging

    ;duringtheearly1990s,hasquicklyspreadtothe

    ;shrimpfarmingareasthroughouttheworldandbe

    ;cameoneofthemajorpathogensinculturedpenaeid

    ;shrimp(Wueta1.,2005;Corbeleta1.,2001;

    ;Ljghtnereta1.,1998).Thevirusexistsinawide ;rangeofhostsincludingallculturedshrimpandother ;aquaticorganismssuchascrabsandcrayiish,and ;hascausedlargeeconomiclosses(Rajendraneta1., ;1999;Wangeta1.,1998).Therefore,extensive ;studieshavebeencarriedout,especiallyinthemot- ;phologyandgenomestructureofWSSV,whichclas- ;siftedWSSVasasolerepresentativeofanewvirus

    ) ;group(familyNimaviridae;genus

    ;(Vlaketa1.,2005;Cheneta1.,2002).However, ;Foundationitem:TheNationalBasicResearchProgramofChinaundercontractNo.

    ;2006CB101806andtheNational”863”ProjectofChinaundereor1.

    ;tractNo.2006AAl0o3l2.

    ;Correspondingauthor.Email:wbzhan@OUC.edu.en

    ;

    ;WANGYinaneta1.ActaOceanologicaSinica2008,Vo1.27,No.2,P.126132

    ;themechanismofvirusentryintotheshrimpandthe ;spreadofthevirusinthecrustaceanbodyarenot ;knownyetthoughtheenvelopeproteinswereas

    ;sumedtoberelatedtothevirusinfection(IJieta1., ;2006:vanHuheneta1.,2001).

    ;Neutralizationexperimentsareconductedrou

    ;tinelytostudythefunctionofviralenvelopeproteins ;andidentifyviralproteinepitopesinvolvedinthevi

    ;rusinfectionprocess,whichmightleadtopreventive ;approachestocontrollingvirusdiseases(Burtonet ;2000;Schofieldeta1.,2000).Polyclonalan ;tibodiesraisedinrabbitsagainsttheindividual ;WSSVenvelopeproteinhasbeenlargelyusedin ;WSSVneutralizationassays,andsomeofthem ;showedneutralizingabilityagainstthevirus(Huang ;eta1.,2005:Wueta1.,2005;vanHuheneta1., ;2001).However,aphenomenonwasfoundthatthe ;n0rmalrabbitserumcouldalsoinactivateWSSV,so ;theneutralizingabilityshowedbyantiserumshould ;bejudgedcarefully(Robalinoeta1.2006).On ;the0therhand.specificmonoclonalantibodies ;(MAbs)againstWSSVhavebeenmainlyappliedin ;thedetectionofvirus,andtherearefewreportson ;theneutralizingevaluationofMAbs(Liueta1., ;2002:Pouloseta1.,2001).

    ;Traditionally,variousneutralizationassayswere ;developedfordifferentviruses.Formanyvertebrate ;viruses,neutralizationexperimentsweresuccessfully

    ;carriedoutinvivoaswellasinvitrousingthecell ;line.Intermsofshrimpvirus,developmentofin ;vitroexperimentswasrestrictedbytheabsenceofa ;continHOHSshrimpcellline,andthathasbeenacrit

    ;icallimitingfactorforthevirusinfectionstudy ;(Wangeta1.,2000;TongandMiao,1996;Luede

    ;manandLightner,1992).

    ;Inthispaper,weexaminedtheneutralizationof ;WSSVbyMAbsagainstthevirus,andtheinvivo ;approachusingcrash(Procambarusclarkii)was ;performedinneutralizationtests.

    ;2Materialsandmethods

    ;Experimentalanimals

    ;127

    ;Crayfish(Procambarusclarkii)usedintheex

    ;perimentswere8.10cminsizeandculturedat ;about25~Cinaquaria,eachcontaining15,andhalf ;ofthewaterwasrefreshedeveryday.Inordertoen

    ;surethecrayfishwereWSSVfreebeforethevira1 ;challenge,randomsamplesofgilltissuefromthe ;crayfishwerepickedupanddetectedbyimmunoflu

    ;orescenceassays(IFAs),employingamixtureof ;MAbsagainstWSSVastheprimaryantibody(Zhan ;eta1.,1999)andtwostepPCR(polymerasechain

    ;reaction)withnestedprimersets(Loeta1., ;1996).

    ;2.2Virusdilutions

    ;AWSSVstockwasproducedfromthegillsof ;naturallvinfectedPenaeuschinensisthathadbeen ;storedat80~C.Thegilltissuewashomogenizedin ;PBS(0.01mol/dm.pH7.4)at10%(ratioof ;masstovolume)at4cI=,thenthehomogenatewas ;centrifugedat3000r/minfor20rainat4cCand ;passedthrougha450nmmembranefilter,finally, ;thefihratecontainingtheviruswaslabeledasvirus ;stockandstoredat80?.

    ;Virusstockwastenfolddilutedwithsterilized

    ;PBSandaninvivovirustitrationwasperformed ;fvanHuheneta1.,2001).Afterthetest,thedilu

    ;tions0f1:10.1:10and1:10virussolution,re

    ;suhingin100%mortalityofcrayfish,wereselected ;touseintheneutralizingassay,respectively. ;2.3MonoclonalantibodiesagainstWSSV ;EightMAbsagainstWSSVproducedpreviously

    ;byDr.Zhanwereusedintheexperiments,among ;which.1C2(IgM),lD2(IgM),1D5(IgG1), ;2D2(IgG1),6B4(IgM),4A1(IgM)and2B2

    ;

    132 ;128WANGYinaneta1.ActaOceanologicaSinica2008,Vo1.27,No.2,P.126

    ;(IgM)werespecificforenvelope,and6A4 ;(IgG2b)fornucleocapsid(Zhaneta1.,1999). ;2.4Invivoneutralizationassay

    ;Twentyfivemicrolitresdilutionsof1:10, ;1:10and1:102virussolutionweremixedwithan ;equalvolumeofeachMAb,respectively,andincu

    ;batedfor2hatroomtemperature,thenthemixture ;wasinjectedintothecrayfishattheabdomen,onthe ;otherhand,mixtureofPBSplusmyelomaculturesu

    ;pernatantandmixtureofvirusplusmyelomaculture ;superuatantwereusedasnegativecontrolandposi

    ;tivecontrol(Table1).Theneutralizingassayatthe ;dihtionsof1:104.1:10and1:10wasdeftnedre. ;spectivelyasGroups1,2and3.

    ;Table1.Constitutionofinjectionsolution ;Notes:Thefourthcolumnshowsthenumberofcrayfishusedin ;eachtest.

    ;Afteri.jection,thecrayfishwereobservedfora ;periodof25dandthemortalitywasmonitoreddaily, ;meanwhilethegillsofdeadcrayfishweretestedby ;IFAs.Differenceinthetimetodeathafterchallenge ;betweeneachgroupwasexaminedusingoneway ;analysisofvariance(ANOVA),andsubsequent ;Duncan’spairwisecomparisonswiththestatistical

    ;significancebeingsetatthe95%or99%confidence ;interval(P<0.05orP<0.01).A1ldataanalyses ;wereperformedusingtheStatisticalPackageforSo

    ;cialSciences(SPSS)13.0software(SPSSInc., ;Chicago,IL).

    ;3Resuits

    ;ThemortalityofallgroupsinjectedwithWSSV, ;treatedwithMAbormyelomaculturesuperuatant, ;reached100%within25dandthecrayfishwere ;confirmedtobeinfectedwithWSSV(datanot ;shown).Bycontrast,fewcrayfishdiedinthenega- ;tivecontrolgroupinjectedwithPBSandmyeloma ;culturesupematant.

    ;InGroup1,crayfishofpositivecontrolbeganto ;dieonthe2nddayandshowed100%mortalityby

    ;the16thday,whilecrayfishinMAb2D2treatedtest ;diedfromthe8thdayandthemortalityreached ;100%bythe25thday,moreover,treatmentth ;MAb2B2resultedindeathoccurringonthe7thday ;andthemortalityreached100%bythe24thday. ;thenwithMAb1D2and1D5,thecrayfishdiedon ;the6thdayandthemortalityreached100%bythe ;23rdday,themeantimetodeathoftheseMAbtrea.

    ;tedtestswasallsignificantlylongerthanthatofposi

    ;tivecontro1.ComparedwiththesefourMAbs,the ;meantimetodeathfortheremainingfourMAbswas ;significantlyshorter,thoughitwassignificantlylon

    ;gerthanthatofthepositivecontroltoo.Insummary, ;themeantimetodeathoftheMAb.treatedtestswas ;allsignificantlylongerthanthatofthepositivecon

    ;trol,andthevalueofMAb2D2treatedtestwasthe ;longestandsignificantlylongerthanthatoftheother ;MAbtreatments(P<0.O1)(seeFig.1). ;ResultsofGroup2weresimilartoGroup1, ;briefly,crayfishofpositivecontrolbegantodieon ;the2nddayandthemortalityreached100%bythe ;15thday,andforMAb2D2,thedeathperiodCOY- ;

    ;WANGYinaneta1.ActaOceanologicaSinica2008,Vo1.27,No.2,P.126132

    ;eredthe5thdaytothe24thday.forMAb2B2it ;coveredthe4thdaytothe23rdday,thenforMAb ;1D2and6B4itcoveredthe4thdaytothe22ndday. ;SimilartoGroup1.themeantimetodeathforall ;MAbtreatmentswassignificantlylongerthanthatof ;positivecontrol(P<0.05),andthevalueofthere

    ;129

    ;mainingfourMAbswassignificantlyshorterthanthat ;fourgivenabove.Furthermore,themeantimeto ;deathwithMAb2D2and2B2treatmentwassimilar ;toeachotherbutsignificantlylongerthanthatofthe ;otherMAbtreatments(P<0.01)(Fig.2). ;23456789

    ;rest

    ;IWSSV13[usMAb2B2

    ;2WSSVl:)IusMAb2D2

    ;3WSSVl3lusMAbID2

    ;4WSSVI)1usMAbII)5

    ;5WSSVl:)lusMAbIC2

    ;6WSSVI)lusMAb4AI

;7WSSVI)lusMAb6A4

    ;8WSSVplusMAb6B4

    ;9WSSVplusmyeloma

    ;culturesupernataut

    ;Fig.1.MeantimetodeathininvivoneutralizationtestsofGroup1(WSSVinoculumat1:10d

    ilution).

    ;Barsindicatethestandarderror.Barswithdifferentlettersaresignificantlydifferent(P<

    0.05).

    ;23456789

    ;rest

    ;1wssvplusMAb2B2

    ;2WSSVplusMAb21)2

    ;3WSSVplusMAI)II)2

    ;4WSSVplusMAbII)5

    ;5WSSVplusMAbIC2

    ;6WSSVplusMAb4AI

    ;7WSSVplusMAb6A4

    ;8WSSVplusMAb6B4

    ;9WSSVplusmyeh)ma

    ;culturesupernataut

    ;Fig.2.MeantimetodeathininvivoneutralizationtestsofGroup2(WSSVinoculumat1:10d

    ilution)

    ;Barsindicatethestandarderror.Barswithdifferentlettersaresignificantlydifferent(P<

    O.05).

    ;InGroup3,thedeathperiodofpositivecontrol ;coveredthe2nddaytothe16thday,whileforMAb ;2132and2132itcoveredthe4thdaytothe23rdaay, ;andforMAb6B4itcoveredthe5thdaytothe23rd ;clay.Similartotheprevioustwogroups,themeantime ;todeathofalltheMAbtreatmentswassignificantlylon- ;gerthanthatofpositivecontrol(P<0.O1).Mean

    ;while.themeantimetodeathforMAb6B4,2D2and ;2B2,withsomeoverlap,wassignificantlylongerthan ;thatofotherMAbtreatments.andMAb6B4hadthe ;longestvalue(P<O.05)(seeFig.3).

    ;WiththethreedilutionsofWSSVinoculum.all ;MAbsdelayedthemortalityofWSSVchallenged ;crayfishtosomeextent.andtheeffectofallMAbs ;except6B4becamelowerwhenthevirusconcentra

    ;tionincreased.Furthermore.accordingtothemean ;08642086420

    ;舌日0_=日苫

    ;8642086420

    ;?0_=日苫

;

    ;130WANGYinaneta1.ActaOceanologicaSinica2008,Vo1.27,No.2,P.126132

    ;timetodeath,MAb2B2and2D2wereexcellentat ;virusdilutionsof1:10and1:10.andMAb6B4 ;exceededa11theothersatthedilutionof1:102. ;Clearly,neutralizingactivitieswouldvarynotonly ;withdifferentMAbsbutalsowiththeWSSVconcen- ;tration.

    ;23456789

    ;Test

    ;1WSSVplusMAb2B2

    ;2WSSVplusMAb2D2

    ;3WSSVplusMAb1D2

    ;4WSSVplusMAb1D5

    ;5WSSVplusMAb1C2

    ;6WSSVplusMAb4A1

    ;7WSS~plusMhb6A4

    ;8WSSVplusMAb6B4

    ;9WSSVplusmyeloma

    ;culturesupernatant

    ;Fig.3.MeantimetodeathininvivoneutralizationtestsofGroup3(WSSVinoculumat1:10d

    ilution)

    ;Barsindicatethestandarderror.Barswithdifferentlettersaresignificantlydifferent(P<

    0.05).

    ;4Discussion

    ;Crayfishhavebeendevelopedasamodelfor ;proliferatingWSSVinvivo(Huangeta1.,1999) ;becausethelimitedavailabilityofcultivatedpenaeid ;shrimp.Herewealsofoundthemtobeagoodmodel ;f0rinvivoneutralizationexperiments.becauseitwas ;easiertoculture,moreavailable,andcheaperthan ;penaeidshrimp.Fromtheinvivoneutralizationtests ;performedincrayfish,wefoundthatenvelopespeciG ;icMAbofWSSVcoulddelaymortalityinWSSV ;challengedcrayfish.Thatsupportedthegeneral ;contentionthatantienvelopeantibodiesmayneutral- ;izeavirusorshowantiviralactivity(Burtoneta1., ;2000).Furthermore,experimentsalsodemonstrated ;thatthenucleocapsidspecificMAb6A4hadneutral

    ;izingactivityaswellindicatingthattheepitoperec

    ;ognizedby6A4mightbeinvolvedinthevirusinfec- ;tion.ItmaybeimportantforWSSVinvasion,repli

    ;cationorproliferation,andthatwouldbestudiedin ;moredetail.

    ;Throughtheneutralizationtestsofpolyclonal ;antibodies,severalenvelopeproteinsofWSSVinclu

    ;dingVP28,VP76,VP68,VP281,VP466,VP36B ;andVP31havebeenassumedtobeinvolvedinthe ;virusinfection(Lieta1.,2006;Huangetal ;2005;Wueta1.,2005;vanHulteneta1.,2001). ;Inthepresentstudy,MAbs2B2and2D2gavethe ;bestneutralizingactivityandtheirepitopesappeared ;tobeinvolvedintheWSSVinfectionprocess.How- ;ever,wefailedtoidentifythetargetproteinsby ;Western.blot(datanotshown).Thereasonmaybe ;thattheepitopesofthetwoMAbsareeonformational- ;lydeterminantrespectivelyandtheycouldnotreact ;withthedenaturedWSSVafterSDSPAGE.Fur-

    ;therresearchisrequiredtoidentifytheproteinsrec- ;ognizedbythesetwoMAbs.

    ;Themechanismsofviralneutralizationinclude ;aggregation,inhibitionoftargetcellattachmentand ;antibody——induceddefectsinvirusfunctionthatOC?? ;curafterattachment,includinginhibitionoffusion, ;endoeytosis,virusuncoatingandtranscription ;(YuanandParrish,2000;ZollaPazner,1996;

    ;Christenseneta1.,1995).Furthermore,bindingof ;antibodycaninduceeonformationalchangesinviral ;proteinsandthesemaybeessentialfortheneural- ;izationprocess(EdwardsandDimmock,2000). ;Sinceeachvirushasitsownuniqueinfectionsys- ;tem,themechanismsforneutralizationwillvaryfrom ;642O8642O

    ;吾日00?巨【1c0

    ;

    ;WANGYinaneta1.ActaOceanologicaSinica2008,Vo1.27,No.2,P.126132

    ;typetotype.Itisacomplexandmuhifactorial ;processgovernedbyfactorssuchasvirusstrain, ;epitopes,hostcelltypesandtheisotype,affinityand ;concentrationofantibodies(EdwardsandDimmock. ;2000).Thus,thefindingthatourMAbcouldnot ;neutralizeWSSVcompletelymaynotbesurprising. ;Onecuriousresultwasthefactthatneutralizing ;activitiesofmostoftheMAbsbecamelowerasthe ;concentrationofWSSVincreased.exceptforMAb ;6B4thatbecamemoreeffectiveatahigherconcen

    ;tration(WSSVinoculumat1:10dilution).The ;reasonforthisisunknownyetbutcouldbeasubiect

;offurtherstudy.

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