圆环病毒cap蛋白功能

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VeterinaryImmunologyandImmunopathology

journalhomepage:www.elsevier.com/locate/vetimm

Researchpaper

Porcinecircovirustype2(PCV2)CapandRepproteinsareinvolvedinthedevelopmentofcell-mediatedimmunityuponPCV2infection

MariaForta,MarinaSibilaa,MiquelNofraríasa,EvaPérez-Martína,AlexOlveraa,EnricMateua,b,JoaquimSegalésa,b,∗

ab

CentredeRecercaenSanitatAnimal(CReSA),UAB-IRTA,CampusdelaUniversitatAutònomadeBarcelona,08193Bellaterra,Barcelona,SpainDepartamentdeSanitatid’AnatomiaAnimals,FacultatdeVeterinària,UniversitatAutònomadeBarcelona,08193Bellaterra,Barcelona,Spain

articleinfoabstract

Theaimofthepresentstudywastoinvestigatetheroleofthecapside(Cap)andreplicase(Rep)proteinsofPorcinecircovirustype2(PCV2)aswellasthewholePCV2(bothPCV2aandPCV2bgenotypes)intheinductionofcell-mediatedimmunityuponinfection.At6weeksofage,sixpigswereintranasallyinoculatedwiththeStoon1010(Stoon)isolate(PCV2a)andsevenwiththeSp-7-10-54-13(Sp)isolate(PCV2b).NoneofthepigsdevelopedclinicaldiseasebuttheSpgrouphadsignificantlyhigherproportionofpigswithPCV2-associatedlesionsandPCV2loadintissuescomparedtotheStoongroup.Inbothgroups,developmentofIFN-␥secretingcells(SC)inresponsetothewholePCV2andCapproteinwasdetectedbymeansofanELISPOTfromday7post-inoculation(PI)totheendofthestudy(21daysPI).SignificantresponsesagainstRepproteinwereonlydetectedinSp-inoculatedpigs.NodifferencesinELISPOTresultswereseenwheneitherPCV2aorPCV2bwasusedinvitrotorecallperipheralbloodmononuclearcells(PBMC)inanygroup.StimulationofPBMCwiththewholevirusbutnotwithCaporRepproteininducedIL-10-SCinallpigsregardlessoftheirPCV2infectionstatus,indicatinganinnateoriginofthisresponse.TheresultsfromthisstudydemonstratethatPCV2-infectedpigsdevelopedcell-mediatedimmunitytoCapandRepproteinsandthat,inthecourseofasub-clinicalinfection,developmentandstrengthofsuchresponsesarepossiblyrelatedtothelevelsofPCV2replication.

©2010ElsevierB.V.Allrightsreserved.

Articlehistory:

Received12December2009

Receivedinrevisedform27April2010Accepted26May2010

Keywords:

Cell-mediatedimmunity

Porcinecircovirustype2(PCV2)CapproteinRepprotein

1.Introduction

Porcinecircovirustype2(PCV2)isacircularsingle-strandedDNAvirusthatcausesthepostweaningmultisys-temicwastingsyndrome(PMWS),amultifactorialdiseaseaffectingweanedpigletsandcharacterizedbygrowthretardation,lossofweightanddeath(Segalésetal.,2005).Atpresent,threegenotypesofPCV2havebeenreported:a,bandc(Carmanetal.,2006;Dupontetal.,2008;Gagnonetal.,2007;Olveraetal.,2007;Segalésetal.,2008),ofwhichPCV2bhasbeensuggestedaspotentiallymorevir-

∗Correspondingauthor.Tel.:+34935814561,fax:+34935814490.E-mailaddress:joaquim.segales@cresa.uab.cat(J.Segalés).0165-2427/$–seefrontmatter©2010ElsevierB.V.Allrightsreserved.doi:10.1016/j.vetimm.2010.05.013

ulentthanPCV2a(Gagnonetal.,2007;Grau-Romaetal.,2008).

PCV2genomeiscomposedof1767–1768nucleotidescodifyingfor11putativeopenreadingframes(ORF)(Hameletal.,1998).Sofar,proteinexpressionhasbeenonlydescribedforthreeofthoseORFs.ORF1encodesforthereplicase(Rep)anditssplicingvariantRep󰀅,bothconsid-eredessentialproteinsforviralreplication(Cheung,2003).ORF2encodesforthecapsid(Cap)protein,theonlystruc-turalproteinofPCV2(Nawagitguletal.,2000).TheproductencodedbyORF3isanon-structuralproteinthathasbeenrecentlyassociatedwithviralreplicationandpathogen-esisinvivo(Karuppannanetal.,2009;Liuetal.,2006).Atpresent,Capisconsideredtobethemostimmuno-genicproteinofPCV2(Blanchardetal.,2003;Pogranichnyy

M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234227

etal.,2000)andatleastthreeconformationalneutraliz-ingepitopeswithinthisproteinhavebeenidentifiedsofar(Lekcharoensuketal.,2004;Shangetal.,2009).TheroleofRepproteinintheimmuneresponsesagainstPCV2hasnotbeenextensivelystudiedyet.Underexperimen-talconditionsitwasdemonstratedthatcaesarean-derivedcolostrum-deprived(CD-CD)pigletsinoculatedwithPCV2developedantibodiesagainstthreedifferentviralproteinswithmolecularmassesof28(Cap),28.5(Rep󰀅)and35(Rep)kDa(Pogranichnyyetal.,2000).Inaddition,aserologi-calsurveyperformedonPMWS-affectedfarmsrevealedthepresenceofCapandRepantibodiesinbothPCV2sub-clinicallyinfectedandPMWS-affectedpigs(Pérez-Martínetal.,2008).Inbothstudies,Cap-specificantibodiesappearedearlierandreachedhighertitresthananti-Repantibodies.ThefactthatallcommercialPCV2vaccinesarebasedoneitherinactivatedvirusorCapprotein(Opriessnigetal.,2007)suggeststhattheprotectiveimmunityagainstPCV2involvesthedevelopmentofCapantibodies.

Uptonow,moststudiesontheimmuneresponsestakingplaceduringPCV2infectionoraftervaccinationhavefocusedmainlyonthedevelopmentofantibodies(Blanchardetal.,2003;Bolinetal.,2001;Fenauxetal.,2004a;Fortetal.,2008;Ladekjaer-Mikkelsenetal.,2002;Meertsetal.,2006;Opriessnigetal.,2008;Pogranichnyyetal.,2000;Roviraetal.,2002).However,bothhumoralandcellularcompartmentsoftheimmunityareappar-entlyinvolvedintheimmuneresponsesdevelopeduponPCV2infectionandvaccination(Fortetal.,2009a;Fortetal.,2009b;Meertsetal.,2005).Besides,highlevelsofviralreplicationleadingtoPMWShavebeenassociatedwithimpairedneutralizingandT-cellimmuneresponses(Krakowkaetal.,2002;Meertsetal.,2006;Nielsenetal.,2003).

TheaimofthepresentstudywastogaininsightintotheimmunologicalresponsesdevelopedbypigsinfectedwithaPCV2aoraPCV2bisolate,withemphasisoncell-mediatedimmunityandtheroleofRepandCapproteinsonitsdevelopment.2.Materialsandmethods2.1.Virusesandviralproteins

Stoon-1010(PCV2agenotype)andSp-10-7-54-13(PCV2bgenotype)isolateswereusedasinocula.Forinoc-ulationofpigs,viruseswerepropagatedinPCV-freePK15cellsandharvestedafterlysisandcentrifugationofcellcul-tures.Forinvitrostudies,onlycellculturesupernatantswerecollected.UninfectedPK-15supernatantswereusedasmock-stimulus.Toevaluatewhetherthedifferenceinthepassagenumberatwhichthestrainswereused(pas-sage30thand14thforStoon-1010andSp-10-7-54-13,respectively)couldaccountfordifferencesinaminoacidcomposition,acomparativeanalysisofthegenomeamongthechallengevirusesandtheirparentalviralseedswasperformed.SinceaveryearlypassageofStoon-1010wasnotavailable,thefirstsequenceofthisstrainsubmittedtotheGenBankdatabase(accessionnumberAF055392)wasconsideredasitsparentalsequence.Thewholegenomeofthevirusesusedasinocula,Sp-10-7-54-13andStoon-1010,

aswellastheoriginalSp-10-7-54-13strain(PCRproductobtainedfromthepigserum)weresequencedasprevi-ouslydescribed(Fenauxetal.,2000;Fenauxetal.,2002).ThecorrespondingsequencesweredepositedintheGen-BankdatabaseundertheaccessionnumbersGU049342,GU049340andGU049341,respectively.

BaculovirusPCV2viruslikeparticles(VLPs)(Cap)andbaculoviruspreparationswithoutVLPs(Bac)werekindlyprovidedbyBoehringerIngelheimVetmedicaInc.(St.Joseph,MO,USA).TheRepproteinandthemockbac-ulovirusextract(Ni)wereexpressedinbaculovirusandproducedinTrichoplusianilarvae(Pérez-Martínetal.,2008).BothCapandRepproteinswerebasedonPCV2astrains.

2.2.Experimentaldesignandsampling

SeventeenconventionalPCV2PCRnegativepigletsinserum,nasalcavityandfaeceswereobtainedfromaPCV2seropositivefarmandincludedintheexperiment.AnimalswerefreefromPorcinereproductiveandrespi-ratorysyndromevirus(PRRSV),Porcineparvovirus(PPV),Aujeszky’sdiseasevirus(ADV),Brachyspirahyodysenteriae,andMycoplasmahyopneumonia.PigswerecheckedforthepresenceofPCV2antibodiesbyanimmunoperoxidasemonolayerassay(IPMA)(Fortetal.,2007),andrandomlydistributedintothreegroups.PCV2IPMAtitersrangedfrom4.3to6.3log2inallgroupswithnosignificantdif-ferencesamonggroups.At6weeksofage,pigswereintranasallyinoculatedwithatotalof5×106.5TCID50/pigofeitherStoon-1010(Stoongroup;n=6)orSp-10-7-54-13(Spgroup,n=7)isolates.FourpigswerekeptascontrolsandweregivenanequalvolumeofPBS(PBSgroup)bythesamerouteofadministration.

Bloodsamplesweretakeninduplicate(siliconizedandheparinizedtubes)atchallenge,7,14and21(necropsy)dayspost-inoculation(PI).Serumwasstoredat−80◦Cuntilfurtheruse.Peripheralbloodmononuclearcells(PBMC)wereimmediatelyisolatedfromheparinizedbloodbydensitygradientcentrifugationusingHistopaque1.077(Sigma–Aldrich).

Theanimalcareactivitiesandthestudyproceduresper-formedinthepresentworkwereauthorisedbytheEthicalandAnimalWelfareCommitteeoftheUniversitatAutònomadeBarcelona.

2.3.Clinicalandpathologicalstudies

Rectaltemperaturesandbodyweightweremeasuredevery2daysandweekly,respectively.Theexperimentwasterminatedatday21PIwhenpigswereeuthanizedbyanoverdoseofsodiumpentobarbital.Animalswerenecrop-siedandsamplesofmesenteric,superficialinguinalandmediastinallymphnodes,tonsilandlungswerecollectedandfixedbyimmersionin10%bufferedformalin.Tissuesweresubsequentlyembeddedinparaffin,andtwocon-secutivesectionswerecutat4␮mthick.Oneslidewasstainedwithhaematoxylin-eosinandthesecondonewassubjectedtoaninsituhibridization(ISH)techniquetodetectPCV2nucleicacid(Roselletal.,1999).Threeparameterswereevaluatedperlymphoidtissue:lymphocytedeple-

228M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234

tion,granulomatousinflammationandpresenceofPCV2genomebyISH.LungtissuewasevaluatedforpresenceofinflammationandPCV2nucleicacidaswell.Allparameterswerescoredfrom0(nolesion/negativetoISH)to3(severelesion/highamountofPCV2genome)(Roselletal.,1999).2.4.PCV2viremia

PCV2DNAquantificationwasperformedonserumsam-plesbymeansofaquantitativePCR(Q-PCR)performedaspreviouslydescribed(Olveraetal.,2004).Resultswereexpressedaslog10PCV2genomecopies/mlofserum.2.5.PCV2-specificIPMAantibodies

LevelsofPCV2-specificantibodiesweremeasuredinserumcollectedatallsamplingdaysbymeansofanIPMA(Fortetal.,2007).

2.6.PCV2Cap-specificlymphoproliferation

Forallexperiments,PBMCwereculturedinRPMI10medium(Gibco)supplementedwith10%foetalcalfserum(FCS),1%glutamine,1%non-essentialaminoacids,1%sodiumpyruvate,1%peniciline–streptamicin,0.5%gen-tamicinand5×10−5M2-mercaptoethanol.ViabilityofPBMCwasassessedwithTrypanbluestaining.

ProliferativeresponsesofPBMCinducedbyPCV2Capproteinweremeasuredatdays0,7and21PIusingacommercialcellproliferationkit(CellproliferationBio-trackELISASystem,AmershamBiosciences).Briefly,PBMCwereculturedat37◦Cand5%CO2(1×105cells/well)inthepresenceofCapprotein(0.6␮g/ml).Phytohaemag-glutinin(PHA)(10␮g/ml)andBacwereusedaspositiveandnegativecontrols,respectively.After48hofincuba-tion,5-bromo-2󰀅-deoxyuridine(BrdU)wasaddedtoeachwell(finalconcentration10␮M)andplateswereincu-batedinthedarkfor16additionalhoursat37◦Cin5%CO2.Plateswerethencentrifuged,driedandfixedusingabsoluteethanol.Finally,theBrdUincorporatedduringtheDNAsynthesisoftheproliferatingcellswasquantifiedbyanELISAperformedfollowingthemanufacturerrecom-mendations.LymphoproliferativeresponsesspecificofCapwerecalculatedastheopticaldensity(OD)obtainedusingCapasstimulusminustheODobtainedusingBac.Foreachanimalandday,theresultwasgivenastheaverageoftworeplicates.2.7.IFN-󰀂ELISPOT

FrequenciesofspecificIFN-␥secretingcells(IFN-␥-SC)inPBMCweredetectedatdays0,7,14and21PIbyanELISPOTperformedasdescribedpreviously(Fortetal.,2009b).WholePCV2(PCV2-StoonandPCV2-Sp)(multi-plicityofinfectionof0.05)andCapandRep(0.6␮g/ml)proteinswereusedasstimuli.Asnegativecontrols,super-natantsfrommock-infectedcellcultures,BacandNiwereused.

Foreachindividualandstimulus,theELISPOTcountwasreportedastheaveragenumberofspotsofreplicatesminus

theaveragenumberofspotsinthecorrespondingnega-tivecontrolwells(namely,mock-infectedsupernatantsforPCV2,BacforCapproteinandNiforRepprotein).Foreachstimulus,acut-offvaluewascalculatedastheaverageoftheELISPOTcountsofPBS-inoculatedpigsplusthreestan-darddeviations.ResultsofrespondingpigswereexpressedasthenumberofIFN-␥-SCcellsper5×105PBMC.2.8.IL-10ELISPOT

IL-10secretingcells(IL-10-SC)weremeasuredbyELISPOTatthesametimesandusingthesamestim-ulimentionedabovefortheIFN-␥ELISPOT.Anti-IL-10(clone945A4C437B1)andbiotinylatedanti-IL-10(clone945A1A926C2)antibodiesfromBiosource(Spain)wereusedascapture(10␮g/ml)anddetection(2␮g/ml)anti-bodies,respectively.SincePCV2isknowntoinducehighlevelsofIL-10ininvitroculturedPBMC(Kekarainenetal.,2008b),apreliminaryassaywasperformedtosetuptheoptimalcellconcentrationthatallowedaproperreadingofthetest(datanotshown);thisvaluewasestablishedat2.5×104PBMC/well.Apartfromthat,thetechniquewasperformedfollowingthesameprotocolmentionedabovefortheIFN-␥ELISPOT.TheELISPOTcountforeachstimuluswasreportedastheaveragenumberofspotsofreplicatesminustheaveragenumberinthecorrespondingnegativecontrolwells.Resultswereexpressedasthenumberofrespondingcellsper105PBMC.2.9.Statistics

StatisticalanalysesweredoneusingSPSSv.15.TheShapiro–Wilktestwasusedtoevaluatethenormalityofthedistributionoftheexaminedvariables.ANOVAandtheTukey’sfollow-uptestwereusedforcomparisonofmeansofrectaltemperature,weightgain,viremia,Cap-lymphoproliferationandIFN-␥andIL-10ELISPOTs.Dataonserology(IPMA)wasanalysedbytheKruskall–Wallisnon-parametrictest.TheChi-squaretestwasappliedtocomparetheproportionofpositiveandnegativeresultsinviremia,lesionsandISH,andIFN-␥ELISPOT.AlinearregressionmodelwasbuilttocorrelateIFN-␥-andIL-10-SCdetectedbetweenPCV2-Sp-andPCV2-Stoon-stimulatedPBMC.Thesignificancelevel(˛)forallanalyseswassetat0.05.3.Results

3.1.SequencingofPCV2

Attheaminoacidlevel,thegenomesequencesfromtheSp-10-7-54-13usedasinoculumanditsparentalstrainobtainedfromthepigwereidentical.Incontrast,theStoon-1010genomedifferedfromtheparentalstraininsixnucleotidesintheORF2gene,leadingtotwoaminoacidchanges(V30L,I200A)andastopcodonintheaminoacidposition232,resultinginaORF2of2aminoacidshorter.NochangesweredetectedintheORF1geneoftheStoon-1010isolate.

M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234

229

Table1

IndividualresultsofviralloadinserumdetectedbyQ-PCR(log10PCV2DNAcopies/ml)fromday0(Challenge)today21post-inoculation(PI).Group

Pig

Dayspost-infection0

71421170.00.00.00.0PBS

590.00.00.00.0600.00.00.00.00.00.00.0

0.0

190.04.8a

a

390.00.04.10.0Stoon

410.00.04.25.9420.04.74.94.9680.00.00.04.2690.00.00.00.0220.04.84.84.0270.05.34.54.4290.05.05.20.0Sp

450.00.04.10.0520.05.76.05.8880.04.15.55.020

0.0

0.0

4.9

4.4

a

Deadpiglet.

3.2.Clinicalandpathologicalresults

NoneofthepigsshowedwastingorotherclinicalsignscompatiblewithPMWSduringthestudyperiodandnosig-nificantdifferencesweredetectedinthebodyweightgainbetweenPCV2-inoculatedgroupsandthecontrolone.TheStoongrouphadhigherrectaltemperatureat4,7,9and11daysPIcomparedtocontrolgroup(P<0.05).MeanrectaltemperatureoftheSpgroupwashigherthancontrolgroupat9,11and14daysPI(P<0.05)(datanotshown).Nodif-ferencesinrectaltemperaturewereobservedbetweentheStoonandtheSpgroup.

OnepigfromtheStoongroupdevelopedcolibacillardiarrhoeawithinthefirstweekPIandwaseuthanizedonday10PI.HistopathologicalstudiesrevealednoevidencesofPCV2-associatedlesionsorPCV2genomeintissuesofthispig.TenoutofthetwelvePCV2-inoculatedpigsthatfinishedthestudyhadPMWS-likelesionsand/orpresenceofPCV2DNAinthestudiedtissues.PCV2-associatedmicro-scopiclesions,characterizedbylowtomoderatedepletionoffollicles,granulomatousinflammationinlymphoidtis-suesandlunginflammation(interstitialpneumonia),weredetectedin2/5oftheStoon-inoculatedandin7/7oftheSp-inoculatedpigs(P<0.05).PCV2DNAwasrevealedintis-suesof1/5and6/7pigsinStoonandSpgroups,respectively(P=0.07).3.3.PCV2viremia

AllPCV2-inoculatedpigsbutonefromtheStoongroupbecameviremicduringthestudy(Table1).Nosignifi-cantdifferencesweredetectedintheproportionofPCV2viremicpigs,norinthemeanviralloadofpositivepigsbetweenStoonandSpgroups.3.4.PCV2antibodies

Atthebeginningofthestudy,pigletshadIPMAtitresrangingfrom4.3to6.3log2(meantiterof5.6±1.0log2)

Fig.1.Serologicalprofileobtainedbyimmunoperoxidasemonolayerassay(IPMA)ineachoftheexperimentalgroupsfromdayofchallengetotheendofthestudy.Differentletters(a–c)indicatesignificantdifferencesamonggroups(P<0.05).

andnosignificantdifferenceswereobservedamonggroups.SeroconversionagainstPCV2occurredinallPCV2-inoculatedpigsbetweendays7and14PI.Atday14,IPMAtitresofStoon-andSp-inoculatedpigs(8.3±1.7and9.8±2.0,respectively)werehigherthanthoseofcontrolpigs(3.2±2.2)(P<0.05),butnodifferencesweredetectedbetweenchallengedgroups(P>0.05).Nevertheless,bytheendofthestudy,thelevelsofPCV2antibodiesinSp-inoculatedpigswerehigherthanthoseinStoon-inoculatedanimals(11.3log2vs.8.8log2,P<0.05).TheevolutionofPCV2IPMAtitresisshowninFig.1.3.5.Cap-specificlymphoproliferation

WhenPBMCwerestimulatedwithPHA,nodifferencesintheproliferativeresponsesweredetectedamonggroups,neitheramongsamplingdays(meanOD:2.3±1.0).Incon-trast,usingtheCapprotein,ODsobtainedfromPBMCofSp-inoculatedpigsweresignificantlyhigherthanthosefromcontrolsfromday7PIonwards(P<0.05).FortheStoon-inoculatedanimals,althoughtheODsobservedinresponsetoCapstimulationincreasedwithtime(0.4±0.2at21PIvs.0.1±0.1atchallenge,P<0.05),valuesdidnotdiffersignificantlyfromthoseofcontrolpigs.ProliferativeresponsesofSp-inoculatedpigswerehigherthanthoseofStoon-inoculatedonesbothatday14PI(0.7±0.5vs.0.2±0.3,P=0.06)and21PI(0.7±0.2vs.0.4±0.2,P=0.05).LymphoproliferationinresponsetothePCV2CapproteinissummarizedinFig.2.3.6.IFN-󰀂ELISPOT

DevelopmentofIFN-␥-SCinresponsetothediffer-entstimuli(PCV2-Stoon,PCV2-Sp,andCapandRepproteins)foreachexperimentalgroupisdisplayedinFig.3.Thecut-offvalueforeachstimulus(meanofcon-trolgroup±3×standarddeviation)was9,6and2forPCV2(bothisolates),CapandRepproteins,respectively.Althoughatlowfrequency,thereweretwoStoon-andtwoSp-inoculatedpigsthatshowedanELISPOTresultabovethecut-offatchallenge.Sincethebackgroundofthetech-nique(unspecificresponsetothestimulus)waseliminatedbysubtractingtheresponseobservedinthecontrolpigs,itwasassumedthatPBMCofthosepigsweresomehowactivatedatthetimetheywereculturedtoperformthe

230M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234

Fig.2.LymphoproliferativeresponsestotheCapproteinofPCV2detectedatdayofchallenge,and7and21dayspost-inoculation(PI)ineachexperimentalgroup.

ELISPOTand,therefore,showedanoccasionalnon-specificresponsetothestimulus.

Inresponsetothewholevirus,nosignificantdifferencesweredetectedwheneitherPCV2-StoonorPCV2-Spwasusedasrecallantigen,andthefrequenciesinducedbybothstimuliwerecorrelated(r=0.94,P<0.01).Thus,onday7PI,whenPBMCwerestimulatedwithPCV2-Stoon(PCV2a),IFN-␥-SCweredetectedin2/6Stoon-inoculatedpigsandin7/7Sp-inoculatedones(P<0.05).Similarly,usingthePCV2-Sp(PCV2b)asstimulus,1/6Stoon-and6/7Sp-inoculatedpigsresponded(P<0.05).Ondays14and21PI,4/5and3/5Stoon-inoculatedpigsrespondedtothePCV2-StoonandPCV2-Sp,respectively,withlowIFN-␥-SCfrequenciesandnosignificantdifferencesbetweenbothstimuli.IntheSp-inoculatedgroup,allpigs(6/6)respondedwithhighfrequenciestobothPCV2-StoonandPCV2-Spisolates.

WhenCapproteinwasusedasstimulus,boththenum-berofrespondingpigsaswellastheIFN-␥-SCfrequenciesofpositivepigswererelativelyhighercomparedtotheresultsobtainedwiththewholevirusstimulation.Thus,onday7PI,7/7Sp-inoculatedpigsrespondedanddisplayedsignificantlyhigherfrequenciesthanthe4/6respondingStoon-inoculatedpigs(98±70vs.15±4,P<0.05).Fromthenonwards,allPCV2-inoculatedpigsrespondedtotheCapprotein.Interestingly,pigsinoculatedwiththeSp-10-7-54-13isolateshowedsignificantlyhighercountsofIFN-␥-SCthanthosethatreceivedStoon1010,bothondays14PI(187±39vs.59±40;P<0.05)and21PI(203±90vs.41±31,P<0.05).

IntheStoongroup,RepinducedIFN-␥-SCcouldbedetectedonlyinonepigandatverylowfrequencies(3and5IFN-␥-SCondays7and14PI,respectively).Incontrast,allpigsoftheSpgroupbutonerespondedtotheReppro-tein.Inthislattergroup,thehighestresponsewasdetectedonday14PI,when6/7pigswerepositivewithfrequenciesrangingfrom25to194Rep-specificIFN-␥-SC.Bytheendofthestudy,Rep-IFN-␥-SCfrequenciesslightlydecreasedinallSp-inoculatedpigsbutone(meanRep-specificIFN-␥-SC:78±65).3.7.IL-10ELISPOT

StimulationofPBMCwiththewholevirus(eitherPCV2-StoonorPCV2-Sp)inducedIL-10responsesinboth

inoculatedandnon-inoculatedpigs.Theseresponsesweredetectedatalldays,withnodifferencesbetweengroupsatanytime(Fig.4).IL-10-SCinducedbybothisolateswerecorrelated(r=0.55,r=0.83,r=0.73andr=0.atdays0,7,14and21PI;P<0.01).AlthoughfrequenciesinducedbyPCV2-Stoonwereslightlyhigher,theydidnotdiffersta-tisticallyfromthoseinducedbyPCV2-Sp.ContrarilytotheresultsobtainedintheELISPOTforthedetectionofIFN-␥-SC,stimulationwitheitherCaporRepproteindidnotresultinanysignificantinductionofIL-10-SCthroughoutthewholeexperiment.

4.Discussion

Thepresentstudyaimedtogaininsightintothecell-mediatedimmunitydevelopeduponPCV2infectionandtheinvolvementofdifferentviralproteinsinitsdevel-opment.TorepresentgeneticvariationwithinPCV2,onePCV2a(Stoon-1010)andonePCV2b(Sp-10-7-54-13)iso-lateswereusedforchallengeaswellasforinvitroassays.Sinceviralstockswereproducedatrelativelyhighpas-sages(30thand14thforStoon-1010andSp-10-7-54-13isolates,respectively),sequencingofthePCV2genomewasperformedtoelucidatepotentialdifferencesintheaminoacidcompositionbetweenthevirusesusedasinoculaandtheircorrespondingparentalseeds.Thus,forSp-10-7-54-13isolate,nochangewasdetectedafter14passagesincellculture.Conversely,theORF2ofStoon-1010usedasinoculumandthefirstsequenceofthisstrainsubmittedtotheGenBankdatabasedifferedintwoaminoacids(V30L,I200A)andthetruncationofthetwolastaminoacidsduetoastopcodon.Whetherthesechangesmightaccountfordifferencesinvirulenceand/orantigenicitycannotberuledout,sinceithasbeendemonstratedthatminimalchanges(P110AandR191S)inthecapsidofaPCV2isolateafter120passagesincellcultureenhancedPCV2replicationinvitrobutattenuatedthevirusinvivo(Fenauxetal.,2004b).Inaddition,I200AandthestopcodonarenexttoregionsexperimentallydefinedasepitopesbyLekcharoensuketal.(2004)andMahéetal.(2000).Nevertheless,toeluci-datetheeffectonviralattenuationoftheabove-mentionedchanges,aninvivostudycomparingtheStoon-1010usedasinoculumandtheoriginalstrainshouldbeperformed.

M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234231

Fig.3.ProportionofpigswithpositiveresultsbytheIFN-␥ELISPOTandmeanfrequenciesofIFN-␥-SCofpositiveanimalsinStoonandSpgroupsatchallenge,7,14an21dayspost-inoculation(PI)inresponsetoPCV2isolates(Stoon-1010andSp-10-7-54-13),andCapandRepproteins.Differentlettersindicatesignificantdifferences(P<0.05)betweengroupsintheproportionofpositivepigs(A,B)andthemeanfrequenciesofIFN-␥-SCofpositives(aandb).*PBMCfrom1/7Sp-inoculatedpigswerenotavailableonday21.

232M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234

Fig.4.FrequenciesofIL-10-SCdetectedinStoonandSpgroupsatchallenge,7,14an21post-inoculation(PI)inresponsetoPCV2isolatesStoon-1010andSp-10-7-54-13.

AlthoughPMWSwasnotreproduced,challengewithbothStoon-1010andSp-10-7-54-13isolatesresultedinthedevelopmentofasub-clinicalPCV2infection.Com-parisonamongchallengegroupsrevealedsubstantialdifferencesintheprogressionofPCV2infection.Thus,theSpgrouphadhigherproportionofindividualswithPCV2-associatedlesionsandpresenceofPCV2DNAintissuescomparedtotheStoongroup.PreviousstudiesfoundthatreducedviremiaandPCV2-associatedlesionsofinoculatedpigswererelatedtothepresenceofhighlevelsofPCV2MDAatchallenge(Fortetal.,2009b;McKeownetal.,2005;Ostanelloetal.,2005).Inthepresentwork,sinceallpigswerePCV2PCRnegativebeforechallengeandcontrolpigsremainednegativeuntiltheendofthestudy,thePCV2anti-bodiespresentatchallengewereassumedtobematernallyderived.However,thoseremainingMDAwerenotappar-entlyatthebaseoftheabove-mentionedvirologicalandpathologicaldifferencesbetweenStoon-andSp-inoculatedpigs,sinceallofthemhadIPMAtitresbelow6.3log2atthestartoftheexperimentandnosignificantdifferencesinantibodylevelsweredetectedamonggroups.Onepotentialexplanationmightbetheexistenceofdifferencesinviru-lencebetweenthetwoisolates,whichwouldbecoherentwiththepostulatedhigherpathogenicityofPCV2bstrains(Gagnonetal.,2007;Grau-Romaetal.,2008).However,sinceonlyoneisolatefromeachgenotypewascompared,whethertheobserveddifferencesarerelatedtothegeno-typeortotheparticularstrainsusedinthepresentstudycannotbedetermined.

TheadaptiveimmunitydevelopedbypigstoPCV2infectiondependedontheisolateusedforchallenge.Althoughtheonsetofhumoralresponseoccurredinbothgroupsbetween7and14daysPI,Sp-inoculatedpigsshowedsignificantlyhigherantibodytitrescomparedtoStoon-inoculatedonesattheendofthestudy.Inaddi-tion,remarkabledifferenceswereobservedinregardstocell-mediatedimmunity.Thus,intheSpgroup,lym-phoproliferativeresponsestotheCapweredetectedasearlyasday7PI,withincreasinglevelsuntiltheendofthestudy.Incontrast,valuesforStoon-inoculatedpigsweremuchlowerandsimilartothoseofcontrolpigs.IntheIFN-␥ELISPOT,theSpgrouphadsignificantlyhigherproportionofrespondingpigsaswellasfrequenciesofIFN-␥-SCinresponderscomparedtotheStoongroup.ThedistinctimmunologicalprofilesobservedbetweenSp-andStoon-inoculatedpigscouldbeattributedtotheobserveddifferencesinreplicationbetweenbothisolatesbutalsotodifferencesinantigenicity.However,thefactthatasimilarprofileofIFN-␥-SCwasdevelopedwheneitherPCV2isolatewasusedasrecallantigenissuggestiveoftheexistenceofconservedT-cellimmunodominantepitopesbetweenbothstrains.ThishypothesiswouldbesupportedbythehighestresponsesobservedinpigsinoculatedwithSp-10-7-54-13(PCV2bgenotype),takingintoaccountthatthestimuliusedinthelymphoproliferative(Cap)andELISPOT(CapandRep)assayswerebasedonPCV2astrains.Thesameappliesfortheserologicalresults,sinceIPMAwasper-formedonPCV2a-infectedPK-15cells.Itcanbespeculated,therefore,thatthehigherlevelofreplicationexhibitedinvivobySp-10-7-54-13isatthebaseofthestrongerimmunologicalresponsedevelopedbySp-inoculatedpigs.IntheSpgroup,theonsetofhumoralandcellularresponseswasfollowedbyadecreaseorevenresolutionofviremia.Thus,thehighestpercentageofviremicpigsaswellasthepeakofviralloadofQ-PCRpositivepigswasobservedonday14PI,coincidentallywiththehighestfrequenciesofRep-IFN-␥-SC.Onday21PI,2/7pigsoftheSpgroupbecameQ-PCRnegativetoPCV2inserum,andtherest(5/7)showedadecreaseonviralload.TheseresultssuggestacorrelationbetweenthedevelopmentandstrengthofbothhumoralandcellularimmunityandthelevelsofPCV2replicationinPCV2-infectedpigs.However,suchassoci-ationwasnotasclearinpigsfromtheStoongroup.Asdiscussedabove,theimmuneresponsedevelopedbypigsfromthatgroupwasnotasstrongasintheSpgroup,andonlyonepigshowedadecreaseonviralloadattheendofthestudy.OnepossibleexplanationcouldbethatthestrainStoon-1010hasaslowergrowthpatterncom-paredtotheSpisolate,thusresultinginadelayedpeakofviremiaand,inturns,adelayedimmuneresponseofStoon-

M.Fortetal./VeterinaryImmunologyandImmunopathology137(2010)226–234233

inoculatedpigs.Toexclude/confirmthatpossibility,pigsinoculatedwithStoon-1010shouldbefollowed-upforalongerperiodoftimetoassesstheirserologicalandviro-logicalprofiles.Interestingly,differencesintheappearanceandintensityofcell-mediatedresponsestoCapandRepproteinswereobserved.Thus,allPCV2-inoculatedpigs,eventhoseshowinglowviremialoads,developedIFN-␥-SCinresponsetotheCap,indicatingthatthisproteinisagoodT-cellimmunogen.TakingintoaccountthatPCV2isabletoinfectandpersistinmonocyte-macrophagelin-eagecellsforlongperiodsoftimewithnoapparentactivereplication(Gilpinetal.,2003;Vincentetal.,2003),theinductionofcell-mediatedimmunityagainstCapmightbeofcrucialimportancetoavoidviralpersistenceinthosecells.ContrarytotheresultsobtainedwithCap,onlypigswithdetectableviralloadandpresenceofPCV2-associatedlesionsintissuesdevelopedsignificantIFN-␥responseswhenRepwasusedasrecallantigen.Thisfindingsug-geststhatcertainlevelofvirusreplicationisrequiredtotriggerdetectableresponsestothisprotein.SincetheRepishighlyexpressedincellssupportingPCV2replica-tion,cellularresponsestothisproteinmightbeimportanttoconstrainPCV2replicationandpreventtheprogres-sionofPCV2infectiontowardsPMWS.Inarecentfieldstudy,itwasshownthatPMWS-affectedpigscouldbedif-ferentiatedfromhealthyandwastednon-PMWS-affectedanimalsbyhavingimpairedanti-Capandanti-Repanti-bodyresponses,andinthecaseofanti-Repantibodies,lowertitreswerealreadydetectedpriortotheappearanceofdisease(Pérez-Martínetal.,2008).

Asreportedpreviously(Fortetal.,2009b),stimulationofPBMCwitheitherthewholePCV2ortheCapproteinyieldeddifferentresultsattheIFN-␥ELISPOT.Onepossi-bleexplanationisthattheabilityofPCV2toreplicateoninvitroculturedPBMC(Yuetal.,2007)hadanimpactontheresponseobservedwhencultureswerestimulatedwiththewholevirus.Inaddition,Kekarainenetal.(2008a)demon-stratedthatthewholevirusanditsDNAbutnottheCapproteinrepresstheproductionofIFN-␥inPMBCsuponrecallantigen,factthatmightalsoexplainthehighfre-quenciesofIFN-␥-SCobservedwhentheCapproteinwasusedasstimulus.Alltheseobservationsshouldbeconsid-eredandusedadvantageouslyforrefiningthepresentandotherassaysaimedtomeasuretheimmuneresponsestoPCV2.

IncontrasttotheresultsobtainedintheIFN-␥ELISPOT,frequenciesofIL-10-SCwerenotassociatedwiththechal-lengegroup,neitherrelatedtothesamplingday,furtherindicatingthatIL-10wasreleasedmainlyasaresultofanaturalimmuneresponsetothevirus.Inaddition,induc-tionofIL-10-SCdependedonwhetherthewholePCV2ortheviralproteinswereusedasstimuli.NeitherCapnorRepproteininducedIL-10-SCwhenaddedtoPBMCcultures.Incontrast,veryhighfrequencieswereobservedwhenthewholeviruswasused.TheseresultsareinagreementwithapreviousstudyinwhichPCV2butnotPCV2-derivedVLPsinducedIL-10inPBMC(Kekarainenetal.,2008a).Presentdataindicatethat,asreportedfortheCap,theRepisnotdirectlyinvolvedinthePCV2-inducedup-regulationofIL-10.ComparisonamongstimulirevealednosignificantdifferencesonthefrequenciesofIL-10-SCinducedbyeither

theStoon-1010ortheSp-10-7-54-13isolates.SincethosetwostrainsbelongtogenotypesPCV2aandPCV2b,respec-tively,thisfindingsuggeststhatthePCV2IL-10inducingcapabilityisnotapparentlyrelatedtogenotype.FurtherstudiesincludingalargersetofPCV2aandPCV2bisolateswouldclarifywhetherdifferentialinteractionswiththeimmunesystemdoexistamongPCV2strains.

Insummary,thepresentstudyindicatesthatcell-mediatedimmunitydevelopeduponPCV2infectioninvolvesbothCapandRepproteins.Furtherstudiesusingamodelofclinicaldiseasearerequiredtoelucidatetherel-evanceofCapandRepTcellresponsesintheprotectiveimmunityagainstPMWS.Acknowledgements

ThisworkhasbeenfundedbytheEuropeanPCV2AwardofBoehringerIngelheimAnimalHealth,Germany.TheauthorsacknowledgeDr.JoseM.EscribanofromtheInstitutoNacionaldeInvestigaciónyTecnologíaAgrariayAlimentaria(INIA),Dr.FernandoRodríguezfromtheCen-tredeRecercaenSanitatAnimal(CReSA)andthecompanyAlternativeGeneExpression(Algenex)forprovidingtheRepproteinexpressedinbaculovirusanditsproductioninTrichoplusianiinsectlarva.TheauthorsthankMercedesMora,AnnaLlorens,MónicaPérez,RaquelMaesoandNúriaPujolfortheirtechnicalassistance.PhDstudiesofMs.Fortarefundedbyapre-doctoralFIgrantoftheGovernmentofCatalunya(Spain).References

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