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RESEARCHOpenAccessIdentificationofanewcelllinepermissivetoporcinereproductiveandrespiratorysyndromevirusinfectionandreplicationwhichisphenotypicallydistinctfromMARC-145celllineChantaleProvost1,JianJunJia1,NedzadMusic1,CynthiaLévesque1,Marie-veLebel1,JérmeREdelCastillo1,2,MarioJacques1andCarlAGagnon1*AbstractBackground:Airbornetransmittedpathogens,suchasporcinereproductiveandrespiratorysyndromevirus(PRRSV),needtointeractwithhostcellsoftherespiratorytractinordertobeabletoenteranddisseminateinthehostorganism.
Pulmonaryalveolarmacrophages(PAM)andMA104derivedmonkeykidneyMARC-145cellsareknowntobepermissivetoPRRSVinfectionandreplicationandarethemoststudiedcellsintheliterature.
Morerecently,newcelllinesdevelopedtostudyPRRSVhavebeengeneticallymodifiedtomakethempermissivetothevirus.
TheSJPLcelllineoriginwasinitiallyreportedtobeepithelialcellsoftherespiratorytractofswine.
Thus,thegoalofthisstudywastodetermineifSJPLcellscouldsupportPRRSVinfectionandreplicationinvitro.
Results:TheSJPLcellgrowthwassignificantlyslowerthanMARC-145cellgrowth.
TheSJPLcellswerefoundtoexpresstheCD151proteinbutnottheCD163andneitherthesialoadhesinPRRSVreceptors.
Duringthecourseofthepresentstudy,theSJPLcellshavebeenreportedtobeofmonkeyorigin.
Nevertheless,SJPLcellswerefoundtobepermissivetoPRRSVinfectionandreplicationevenifthedevelopmentofthecytopathiceffectwasdelayedcomparedtoPRRSV-infectedMARC-145cells.
FollowingPRRSVreplication,theamountofinfectiousviralparticlesproducedinSJPLandMARC-145infectedcellswassimilar.
TheSJPLcellsallowedthereplicationofseveralPRRSVNorthAmericanstrainsandwerealmostefficientasMARC-145cellsforvirusisolation.
Interestingly,PRRSVis8to16timesmoresensitivetoIFNαantiviraleffectinSJPLcellincomparisontothatinMARC-145cells.
PRRSVinducedanincreaseinIFNβmRNAandnoupregulationofIFNαmRNAinbothinfectedcelltypes.
Inaddition,PRRSVinducedanupregulationofIFNγandTNF-αmRNAsonlyininfectedMARC-145cells.
Conclusions:Inconclusion,theSJPLcellsarepermissivetoPRRSV.
Inaddition,theyarephenotypicallydifferentfromMARC-145cellsandareanadditionaltoolthatcouldbeusedtostudyPRRSVpathogenesismechanismsinvitro.
Keywords:Porcinereproductiveandrespiratorysyndromevirus,PRRSV,SJPLcells,Virusreplication,Cellpermissivity,Type1IFN,IFNγ,TNF-α,Cytokines*Correspondence:carl.
a.
gagnon@umontreal.
ca1Groupederecherchesurlesmaladiesinfectieusesduporc(GREMIP),Centrederechercheeninfectiologieporcine(CRIP),FacultédemédecinevétérinaireUniversitédeMontréal,3200rueSicotte,Saint-Hyacinthe,J2S7C6,Québec,CanadaFulllistofauthorinformationisavailableattheendofthearticle2012Provostetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Provostetal.
VirologyJournal2012,9:267http://www.
virologyj.
com/content/9/1/267BackgroundPorcinereproductiveandrespiratorysyndrome(PRRS)ispresentworldwideandiseconomicallyspeaking,oneofthemostimportantinfectiousdiseasesinswinepro-duction[1].
PRRSdiseasewasfirstdescribedintheUni-tedStatesin1987[2,3]andafewyearslaterintheNetherlands[4].
Thediseasehasmanyclinicalmanifes-tationsbutthetwomostprevalentareseverereproduct-ivefailureinsowsandgilts(characterizedbylate-termabortions,anincreasednumberofstillborns,mummifiedandweak-bornpigs)[2,5]andrespiratoryproblemsinpigsofallagesassociatedwithanon-specificlympho-mononuclearinterstitialpneumonitis[2,5,6].
Theetiologicalagent,porcinereproductiveandre-spiratorysyndromevirus(PRRSV)wasidentifiedin1991byinvestigatorsintheNetherlandsandshortlyafterintheUSA[4,7,8].
PRRSVisanenveloped,single-strandedpositivesenseRNAvirus,approximately50–65nmindiameterclassifiedintheorderNidovirales,familyArteriviridae,genusArterivirusalongwithequinearter-itisvirus(EAV),lactatedehydrogenase-elevatingvirusofmice(LDV),andsimianhemorrhagicfevervirus(SHFV)[7,9].
PRRSVgenomeisapproximately15kbinlength.
TheviralRNAgenomeiscappedatthe5'endandpoly-adenylatedatthe3'endandencodesatleasttenopenreadingframes(ORFs)[10-12],eachofwhichisexpressedviathegenerationofa3'-coterminalnestedsetofsubgenomic(sg)mRNAs[13].
Thevirusisgenet-ically,antigenically,andpathogenicallyheterogeneous[14,15].
PRRSVisolatesarecurrentlydividedintotwodistinctgenotypes,theEuropeangenotype(EU)ortypeIrepresentedbytheLelystadvirus(LV)andtheNorthAmericangenotype(NA)ortypeIIrepresentedbytheATCCVR-2332strain[16].
PRRSVisknowntohaveaveryrestrictedcelltrop-ismbothinvivoandinvitro.
Invivo,thevirusinfectsmainlywell-differentiatedcellsofthemonocyte-macrophagelineage,inparticularporcinealveolarmacrophages(PAMs),theprimarytargetcellsofvirusandinterstitialmacrophagesinothertissuessuchasheart,thymus,spleenandPeyer'spatches,hepaticsinusoids,renalmedullaryinterstitium,andadrenalgland[17-20].
Inadditiontomacrophages,PRRSVRNAandnucleocapsidprotein(N)werefoundintes-ticulargermcells,endothelialcellsintheheart,inter-digitatingcellsinthethymus,dendriticcellsinthespleenandPeyer'spatches[19,21].
Inexperimentallyinfectedgnotobioticpigs,PRRSVantigenswerefoundinbronchiolarepithelialcells,arteriolarendothelialcells,monocytesaswellasinterstitial,alveolar,andintravascularmacrophagesusinganimmunogold-silverimmunohistochemicalstaining[22].
PRRSVRNAsandantigenswerealsofoundinbronchiolarepithelialcells[23],epithelium-likecellsofalveolarducts[24],andpneumocytes[23,25]inthenaturallyinfectedpigswhereastheywerenotfoundinthesetypesofcellsintheexperi-mentallyinfectedpigs[26].
Tissuessuchaslung,lymphoidtissues,Peyer'spatches,andkidneywerealsotheprefer-abletargetorgansofPRRSVinfection[27,28].
Invitro,PRRSVwasfirstisolatedonprimaryculturesofPAMs[4]andsofar,thesecellsaswellasfreshlyiso-latedbloodmonocytesormonocyticderiveddendriticcells[29-31],remaintheonlynon-geneticallymodifiedporcinecellsthatcanbeusedforviralpropagationsincetheycanbeinfectedbythevirusandallowitsreplica-tion.
Ontheotherhand,usingprimarycelllinespresentsomedisadvantagesaslownumberofcellsharvested,heterogeneityofthepopulation,andmoreimportantlyshortlifespanofcells.
Thus,usinginvitrocelllinespresentsomebenefitscomparedtoprimarycelllines.
Therearetwonon-porcinepermissiveimmortalizedcelllinesthatpermitthecompletereplicationcycleofPRRSV,theMARC-145andCL2621cells(subclonesofMA104monkeykidneycellline)[7,32,33]whichareroutinelyusedforinvitropropagationofPRRSVandforlargescaleproductionofPRRSVvaccinestrains.
Morerecently,newcelllineshavebeengeneticallymodifiedtobecomepermissivetoPRRSV,asimmortalizedPAMcellsexpressingtheCD163protein[34],immortalizedporcinemonomyeloidcellsexpressingthehumantel-omerasereversetranscriptase[35],PK-15cellsexpres-singthesialoadhesinprotein[36],andporcine,felineandbabyhamsterkidneycellsexpressingtheCD163protein[37].
Thus,allnewreportedcelllineshavebeengeneticallymodifiedtobepermissivetoPRRSV,leavingroomforthediscoveryofnon-geneticallymodifiedPRRSVpermissivecelllines.
PRRSVcanbeairbornetransmittedthroughlongdis-tance[38].
Airbornetransmittedpathogensneedtointeractwithhostcellsoftherespiratorytractsuchasepithelialcellsandalveolarmacrophagesinordertobeabletoenteranddisseminateinthehostorganism.
IfPRRSVisairbornetransmittedandPRRSVantigensandviralRNAcanbedetectedinepithelialcellsofthere-spiratorytractofinfectedpigs,thenitcanbespeculatedthat,inadditiontothealveolarmacrophages,epithelialcellsofrespiratorytractcouldbepermissivetoPRRSVreplicationinvitro.
Nonetheless,noimmortalizedepi-thelialcelloftherespiratorytractofswinehadbeenreportedsofartobepermissivetoPRRSVinfectionandreplicationinvitroandattemptstofindsuchcellshavepreviouslyfailed[4,39,40].
Thus,St-Judeporcinelungcells(SJPL)cells,whichwereatfirstreportedtobeanimmortalizedepithelialcellslineoftherespiratorytractofswineandwerepre-viouslydescribedtobesuitableforinfluenzavirusrepli-cation[41],weretestedfortheirPRRSVpermissivity.
Noteworthy,duringthecourseofthisstudy,theSJPLProvostetal.
VirologyJournal2012,9:267Page2of14http://www.
virologyj.
com/content/9/1/267celllinewasfoundtobeofmonkeyoriginbasedonkaryotypeandgeneticanalyses[42].
Nevertheless,theresultsofthepresentstudyshowthatSJPLcellsare:1)permissivetoPRRSVreplicationand2)phenotypicallydifferentfromMARC-145cells.
ResultsSJPLcellssusceptibilitytoPRRSVInordertoevaluatethesusceptibilityofepithelialcellsoftherespiratorytractofswineinregardstoPRRSV,twoepithelialcelllines,theNPTrandSJPLcells,wereinoculatedwithPRRSVIAF-Klopstrainat1multiplicityofinfection(MOI).
Asreportedpreviously,theNPTrcellswerenotpermissivetoPRRSV(datanotshown)[40].
However,theSJPLcellsinfectedbyPRRSVdevel-opedaverylightcytopathiceffect(CPE)at72hrspost-infection(pi)comparedtomockinfectedcellsasillustratedinFigure1,whichsuggestedthereplicationofPRRSV.
TheamountofCPEobservedinSJPLinfectedcellsincreasedovertimebutithasalwaysbeensignifi-cantlylowercomparedtoPRRSV-infectedMARC-145cells(datanotshownandFigure1).
ThedegreeofCPEat120hrspiinPRRSV-infectedSJPLcellswassimilartotheamountofCPEobservedat72hrspiinPRRSV-infectedMARC-145cells(datanotshown).
Interestingly,theSJPLcellsgrowthandcelldimensionwerehigher(doublingtime:32.
57±2.
58hrs,surface:4684.
41±2188.
94μm2,respectively)comparedtoMARC-145cells(doublingtime:21.
67±3.
30hrs,surface:3568.
96±1128.
47μm2,respectively)(Additionalfile1:FigureS1).
ToconfirmthePRRSVproteinsexpressioninSJPLinfectedcells,animmunofluorescentassay(IFA)wasperformed.
ThePRRSVNproteinwasdetectedinPRRSV-infectedSJPLcells(Figure1)whichindicatesthatPRRSVwasabletoexpressatleasttheNviralprotein.
MostoftheIFApositivecellshavepositivesig-nallocalizedinthecytoplasm(Figure1)suchaswhathasbeenreportedpreviouslyforPRRSV-infectedMARC-145cells[43].
IdentificationofPRRSVreceptorsinSJPLcellsThreecellularmoleculeshavebeenidentifiedtoplayacriticalroleinrenderingnon-permissivecellssusceptibletoPRRSVinfection:theCD163,CD151andsialoadhesin(Sn)[44-47].
Thus,thepresenceofthesemoleculesinSJPLcellswasdeterminedbyanimmunofluorescentassay(Figure2).
AsillustratedinFigure2A,theSJPLcellsexpresstheCD151suchasMARC-145cells(Figure2I)andtoalowerextentsuchasthePAMcells(Figure2E).
TheexpressionofCD163andSnproteinswasdetectedonlyinPAMcells(Figure2Fand2G).
InfectiousviralparticlesproductioninPRRSV-infectedSJPLcellsToestablishifSJPLcellsallowfullPRRSVreplicationcycleandinfectiousparticlesproductionafterbeingincontactwithinfectiousvirions,theamountofinfectiousPRRSVparticlesproducedbySJPLcellswasevaluatedduringfiveconsecutivepassages.
AsillustratedinFigure3,theamountofinfectiousvirusyieldfromtheinoculum(103.
3TCID50/106cells)comparedtothefirstpassageinSJPLcells(106.
6TCID50/106cells)increasedaround2000timeswhichindicatesclearlythatSJPLpermitstheproductionofinfectiousviralparticles.
Theamountofvirusyieldwasmaintainedduringsubse-quentpassageswhichfurtherindicatesthatinfectiousPRRSVparticlesareproduced(Figure3).
However,theoverallproductionofinfectiousparticlesinSJPLcellscomparedtoMARC-145cellsdoesnotseemtobesig-nificantlydifferent(P>0.
05).
Figure1ImmunofluorescencedetectionoftheNviralproteininPRRSV-infectedSJPLcells.
TheIFAwasdoneat72hrspiasdescribedinthematerialsandmethodssection.
Mockinfectedcellsareillustratedascontrolintheupperpanels.
Cellsinfectedat1MOIwithPRRSVIAF-Klopreferencestrainareillustratedinlowerpanels.
Cellswerevisualizedwithvisiblelight(phasecontrast)andUV(IFA).
ArrowsindicatetheSJPLcellmonolayerdisruptioninducedbyPRRSVinfection.
Provostetal.
VirologyJournal2012,9:267Page3of14http://www.
virologyj.
com/content/9/1/267InordertodeterminetheefficiencyofPRRSVproduc-tioninSJPLcellscomparedtoMARC-145cells,aPRRSVreplicationkineticexperimentusingtypeIandtypeIIPRRSVreferencestrains(LVandIAF-Klop,re-spectively)hasbeenconducted(Figure4).
Threesignifi-cantdualinteractionsoffixed-effectvariableswererecorded:cell*matrix(P=0.
0224),cell*time(P=0.
0006),andmatrix*time(P500TCID50ofPRRSV/gramwas100%and88%forMARC-145andSJPLcells,respectively(Table1).
TofurthercharacterizethePRRSVstrainsthatwereisolated,theORF5geneoffivecasesthatwerebothvirusisolationpositivewithMARC-145andSJPLcellsweresequenced.
SequenceanalysesrevealedthatallPRRSVstrainsaretypeIIiso-lates(datanotshown).
Thenucleotide(nt)identitiesbetweenthetissuesandthefourthcellpassageinbothcelllinesofeachcaseswere100%identicalindicatingthatthesamePRRSVstrains,thatwereidentifiedini-tiallyinthetissues,wereisolated.
Moreover,atthefourthcellpassage,theORF5sequencesofvirusesisolatedfromeachporcinetissuehomogenateinSJPLandMARC-145cellswere100%identicalwhichsug-geststhatSJPLcellsallowtheisolationofthesamestrainsasthoseisolatedwithMARC-145cells.
Se-quenceanalysesalsorevealedgeneticvariabilitybe-tweenstrainsthatwereisolatedfromeachporcinetissuehomogenatewithSJPLcells(86.
4%to93.
2%ntidentities)andcomparedtothePRRSVreferencestrainIAF-Klop(88.
3%to91.
0%ntidentities).
PRRSV-inducedapoptosisinSJPLcellsAsillustratedinFigure5,theprocaspases3/7activationbytheIAF-KlopPRRSVreferencestrainandseveralapoptoticinducerswasmorepronouncedinSJPLcellscomparedtoMARC-145cells(2.
7to4.
4timeshigher).
Inaddition,activationofprocaspases3/7inPRRSV-infectedMARC-145andSJPLcellswas3.
5to6.
2timeshigher(P6.
25,50,0.
78,3.
13,<6.
25aExpressinU/μL.
Note:theexperimentwasdoneinduplicate.
Provostetal.
VirologyJournal2012,9:267Page6of14http://www.
virologyj.
com/content/9/1/267virusproducedinPRRSV-infectedSJPLcells(Figure4)wassimilartoPRRSV-infectedMARC-145indicatingthatthosedifferencesdonotseemtoaffectthevirusproduction.
Inaddition,itissuggestingthatSJPLcouldreplacetheMARC-145cells(andrelatedcellsthatderiv-atefromMA104cells)inalargescalePRRSVliveorkilledvaccineproduction.
Interestingly,evenifSJPLcellsseemtobeslightlylesssensitiveforPRRSVisolationcomparedtoMARC-145cells(Table1),theSJPLcellswereabletoallowthereplicationofseveralPRRSVtypeIIORF5genomicvariantsandthetypeIrefe-rencestrainLV(Figure4andTable1)indicatingthatatleastthesecellsarepermissivetoawidespectrumofPRRSVisolates.
TheIFNαantiviraleffectagainstPRRSV-infectedMARC-145cellshasbeenpreviouslyreported[55].
Con-sequently,differentamountofIFNαwereaddedinthecellculturemediatoevaluateitsantiviraleffectinregardstobothPRRSV-infectedcelllines.
ItwasfoundthatSJPLcellsaremoreresponsivetotheIFNαantiviraleffectthanMARC-145cells(Table2).
Thelevelofcyto-kinemRNAexpressionsmeasuredbyqRT-PCRwasdif-ferentbetweenSJPLandMARC-145cells.
Aspreviouslydemonstratedinotherstudies,PRRSVinfectioninMARC-145orPAMcellsinducedanincreaseinIFNβ,IFNγandTNF-αmRNAexpressions[49-53]andsimilarresultswereobtainedinthisstudy.
Ontheotherhand,inSJPLcells,PRRSVinfectiononlyinducedIFNβmRNAexpressionatmuchloweramountcomparedtoMARC-145infectedcells,indicatingthatPRRSVmightescapeIFNtypeIandothercytokinesresponses[31,56,57].
SeveralpublishedreportsshowedthatPRRSVcontainedanabilitytosuppresstheIFNβactivityatthetranscriptionlevel[49,58,59].
Allthosestudieslookedei-theratthepromoterinduction(Luciferaseassay)oratthemRNAlevelbyqRT-PCRat0hruntil48hrspost-infection(pi).
Inthepresentstudy,IFNβmRNAwasquantifiedatadifferenttimepicomparedtopreviousstudies,i.
e.
at72hrspi,whichcouldexplainthediffer-encethathasbeenobserved.
InGeniniandcollaborators(2008),astronginductionofIFNβmRNAwasobservedinPRRSV-infectedPAMcellswithavariationintimepi,i.
e.
:,noinductionat0hr,inductionat3hrspi,noinduc-tionat6hrspiandverystronginductionat9and12hrspi,illustratingthatthereisavariationintimeofIFNβmRNAlevelthatdoesnotseemtobeconstantandproportional[53].
Furthermore,Leeandcollabora-tors(2004),havereportedthatdifferentstrainsofPRRSVareabletoinducedistinctiveinterferonpheno-typesinvitroindicatingthattheinductionofcytokinesFigure6RelativeexpressionofIFNα(A),IFNβ(B),IFNγ(C)andTNF-α(D)mRNAofSJPLandMARC-145cellsinducedbyPRRSV.
MARC-145andSJPLcellswereinfectedat0.
5MOIwithPRRSVIAF-Klopstrainortransfectedwithpoly(I:C)asapositivecontrolortreatedwithLPSasanIFNγinducer.
mRNArelativeexpressionofIFNα(A),IFNβ(B),IFNγ(C)andTNF-α(D)wasmeasureat72hrsbyqRT-PCRinPRRSVinfectedorpoly(I:C)treatedcells.
Valuesarepresentedas±standarddeviation(SD).
When2setsofdatawithinacelltypearelabeledwithsuperscriptsofdifferentlettersorwhenonlyonesetislabeledwithasuperscript,itindicatesthatthese2setsofdataarestatisticallydifferent(P<0.
05).
When2setsofdatafromdifferentcelltypesarelabeledwithasterisk,itindicatesthatthese2setsofdataarestatisticallydifferent(*P<0.
05,**P<0.
01,***P<0.
001).
Provostetal.
VirologyJournal2012,9:267Page7of14http://www.
virologyj.
com/content/9/1/267mRNAcanvarysubstantiallybetweenPRRSVstrains[60].
Inthepresentstudy,adifferentPRRSVstrain(IAF-Klop)wasusedcomparedtopreviousreportswhichmightexplainthedifferencesincytokinemRNApheno-typethatwasobserved.
Thus,thoseresultsdemonstratetheimportanceofusingmorethanoneinvitromodeltostudyPRRSVreplicationcycleandpathogenesis.
ManystudieshavedemonstratedthatPRRSVinducesapoptosisbothinvitroandinvivo[21,61-64]andseveraltechniqueshavebeenusedtodemonstratethatphenomenon,suchasprocaspase3activationinPRRSVIAF-KlopinfectedMARC-145cells[65].
TheCPEvisua-lizedbylightmicroscopyinPRRSV-infectedSJPLcellswasverymildandwasdelayedovertimecomparedtoPRRSV-infectedMARC-145cells(Figure1,datanotshown,respectively).
TheincreaseinTNF-αmRNAobtainedwithqRT-PCRininfectedMARC-145cellsandtheabsenceofitsupregulationininfectedSJPLcells(Figure6)couldsupportthedifferenceobservedinCPE.
Consequently,amountofcaspase3inMARC-145cellsinfectedbyPRRSVisexpectedtobehighercomparedtoSJPLcellsinfectedbyPRRSV.
Surprisingly,theoppositesituationwasobservedindicatingthatSJPLcellsaremoresuitedforprocaspases3/7activationthanMARC-145cells(Figure5).
ThislatestresultdemonstratesclearlythatSJPLcellsarephenotypicallycompletelydif-ferentfromMARC-145cellsandthatthelevelofpro-caspases3/7activationandTNF-αmRNAexpressioninducedbyPRRSVmaynotberelatedtothelevelofCPEthatcouldbeobservedbylightmicroscopy.
Infact,othercelldeathmechanismshavebeenreportedtooccurincellsinfectedbyPRRSVsuchasnecrosiswhichcouldexplainthisdifferenceobservedbetweenthecelllines[30,66].
ConclusionsInconclusion,SJPLcellsarephenotypicallydifferentfromMARC-145cellsandtheyresponddifferentlytoPRRSVinfection(Figures1,5,and6,Tables2,Additionalfile1:FigureS1).
SJPLcellshavealsobeenshowntorepresentaconvenientinvitromodelforthestudyofporcinebacterialpathogens[54].
Thus,studyingthePRRSV-SJPLinterac-tionsshouldgiveusnewinsightinregardstotheviralpathogenesisofPRRSV.
Inaddition,SJPLcellscouldserveasanewinvitromodeltostudyviral-bacterialinteractionsduringmixedinfections.
MethodsCellsandvirusesMARC-145cells,whichareasubcloneoftheAfricangreenmonkeykidneyMA104cellsthatishighlyper-missivetoPRRSV[33],weremaintainedasdescribedpreviously[59].
TheSt.
Judeporcinelung(SJPL)epithe-lialcelllinewasprovidedkindlybyDrR.
G.
Webster(St.
JudeChildren'sHospital,Memphis,TN,USA)[41].
Duringthisstudy,karyotypingandgenomesequenceanalysesoftheSJPLcellsrevealedthattheirspeciesori-ginwasnotporcinebutwasrathermonkey[42].
Thenewbornpigtracheaepithelialcellline(NPTr)waspro-videdkindlybyDr.
M.
Ferrari(InstitutoZooprofilatticoSperimental,Brescia,Italy)[40].
TheSJPLandNPTrcelllineswereculturedinDulbecco'smodifiedEagle'smedium(DMEM)(InvitrogenCorporation,GibcoBRL,Burlington,ON,Canada)supplementedwith10%fetalbovineserum(FBS)(WisentInc,St-Bruno,QC,Canada),1mMsodiumpyruvate,2mML-glutamine,1μMMEMnonessentialaminoacids,10U/mLofpenicillin,10μg/mLofstreptomycinand250g/Lantibiotic-antimycoticsolution(InvitrogenCorporation,GibcoBRL)asdescribedpreviously[40,41].
Pulmonaryalveolarmacrophages(PAMs)wereusedasapositivecontrolsforthedetectionofPRRSVreceptors.
PAMswereharvestedfromlungsof2to14weeksoldpigs.
Pigsweresacrificedfollowingethicprotocol12-Rech-1640approvedbytheInstitutionalethiccommitteefollowingtheguidelinesoftheCanadianCouncilonAnimalCare(CCAC).
Briefly,aninstillationofthelungswithPBScontaining10units/mLpenicillin,10μg/mLstreptomycinand100mg/Lgentamicin(InvitrogenCor-poration,GibcoBRL)wasrealized.
Then,PBSwascol-lectedandPAMsremovedfollowinglowspeedcentrifugation.
CellswerewashedwithmediumDMEMcomplementedwith2mML-glutamine,0,1mMHEPES,1μMNon-essentialaminoacids(InvitrogenCorpor-ation,GibcoBRL),250g/LAmphotericinB(WisentInc),10units/mLpenicillin,10μg/mLstreptomycinand100mg/Lgentamicin.
Cellswerethencollectedfollow-inglowspeedcentrifugationandwereresuspendedinfreezingmedium(sameaswashmediumplus20%fetalbovineserum(WisentInc.
)and10%DMSO(Sigma,St-Louis,MO,USA))andslowlyfrozen,thanstoredinliquidnitrogenuntilfurtherutilization.
PAMswerecul-turedfor24hoursincompleteDMEMpriortheim-munofluorescenceassay.
Allcelllineswereculturedat37°Cin5%CO2atmosphere.
ThePRRSVstrainusedtoestablishthepermissivityoftheSJPLcellswastheMARC-145cellsadaptedIAF-KlopNorthAmericanreferencestrain[65]andtheLelystad(LV)Europeanreferencestrain[23].
ThePRRSVvirusstockswereobtainedfollowingthreecyclesoffreeze-thawofPRRSVMARC-145infectedcells.
Afterward,theviruswaspurifiedfollowinga3.
5hrsperiodofultracentrifugationona30%sucrosecushion(inaTBSsolution:50mMtrispH7.
5,150mMNaCl)usingtheSW28BeckmanCoulterrotorat83,000rela-tivecentrifugalforce(rcf).
Theviruspelletswereresus-pendedin0.
5mLofPBSandaliquotsofthevirusstockswerethenconservedat–70°Cforfutureuse.
TheProvostetal.
VirologyJournal2012,9:267Page8of14http://www.
virologyj.
com/content/9/1/267infectiousdoseofthevirusstockswascalculatedfroma96-wellmicroplateofMARC-145infectedcellsbytheKrbermethodasdescribedpreviously[67].
Virustiterswereexpressedintissuecultureinfectiousdose50%permL(TCID50/mL).
Immunofluorescenceassay(IFA)forthedetectionofPRRSVantigenThepresenceofPRRSVantigensininfectedcellswasdeterminedbyanimmunofluorescenceassay(IFA).
Briefly,cellsinfectedbyPRRSVstrainswerefixedatdif-ferenttimespost-infection(pi)witha4%paraformalde-hyde(PFA)solutionpreparedasdescribedpreviously[68].
Mock-infectedcellswereincludedasnegativecon-trols.
Afteranincubationperiodof30minutesatroomtemperature,thePFAsolutionwasremovedandcellswerewashedthreetimeswithaphosphatebuffersalinesolution(PBS).
Then,cellswereincubatedduring10minutesatroomtemperaturewithaPBSsolutioncontaining1%TritonX-100.
AfterremovingtheTritonX-100solution,thecellswerewashedthreetimeswithaPBS-Tween20solution(PBScontaining0.
02%Tween20).
Afterthepermeabilizationprocedure,cellswereincu-bated30minuteswithPBScontaining0.
2%Tween20and1%FetalBovineSerumAlbumin.
Then,theα7rabbitmonospecificantisera(aspecificanti-NPRRSVproteinantibody)[65]wasdiluted1/200inthewashingbufferandaddedtothecellsandincubatedatroomtemperaturefora30minutesperiod.
Cellswerethenwashedandincu-batedfor30minuteswiththewashingbuffercontaininga1/160dilutionofanti-rabbitspecificantiseraFITCconju-gated(Sigma-AldrichInc.
,St-Louis,USA).
Finally,cellswerevisualizedusingaDMI4000Breversefluorescencemicroscope,imageofthecellsweretakingwithaDFC490digitalcameraandtheimagewereanalyzedusingtheLeicaApplicationSuiteSoftware,version2.
4.
0(LeicaMicrosystemsInc.
,RichmondHill,Canada).
ImmunofluorescenceassayforPRRSVreceptorsCD151,CD163,andSialoadhesindetectionThepresenceofCD151,CD163andSialoadhesine(Sn)proteinsinMARC-145,SJPLandPAMcellswasdeter-minedbyanIFA.
Briefly,cellswerefixedwitha4%PFAsolutionasdescribedpreviously[68].
Afteranincuba-tionperiodof30minutesatroomtemperature,thePFAsolutionwasremovedandcellswerewashedthreetimeswithPBS.
Then,cellswereincubatedwithapermeabilizationandblockingsolution,PBSsolutioncontaining0.
1%TritonX-100,7%normalsheepserum(NSS)and5%nonfatdrymilk(NFDM),during30min-utesatroomtemperature.
Afterremovingthepermeabilization/blockingsolution,thecellswerewashedthreetimeswithaPBS.
Then,cellswereincu-batedovernightwithprimaryantibodies.
Allthoseantibodieswerediluted1/100inantibodysolutioncon-tainingPBS,0.
1%TritonX-100,1.
4%NSS,and1%NFDM.
Theantibodiesusedwere:rabbitpolyclonalanti-humanCD151(SantaCruzBiotechnology,CA,USA);mousemonoclonalanti-pigCD163(AbDSerotec,Oxford,UnitedKingdom)forPAMscellsorgoatpoly-clonalanti-humanCD163(SantaCruzBiotechnology,CA,USA)forMARC-145andSJPLcells;mousemono-clonalanti-pigCD169(synonym:Sialoadhesin,Siglec-1)(AbDSerotec,Oxford,UnitedKingdom)forPAMscellsorgoatpolyclonalanti-humanSiglec-1(SantaCruzBio-technology,CA,USA)forMARC-145andSJPLcells.
Theanti-CD163andanti-CD169polyclonalantibodiesareknowntoreactagainstseveralanimalspecies.
TheirreactivitywasconfirmedagainstthePAMscontrolposi-tivecells(datanotshown).
Thereafter,cellswerewashedthreetimesandincubatedfor60minuteswiththeanti-bodybuffercontaininga1/160dilutionofanti-rabbitspecificantiseraFITCconjugated(Sigma-AldrichInc.
,St-Louis,USA)or1/200dilutionofanti-mousespecificantiseraFITCconjugated(ICNImmunoBiological,CA,USA).
Nucleiwerestainedwith4',6-diamidino-2-pheny-lindoleDAPI(Sigma-AldrichInc.
,St-Louis,USA)asrecommendedbythemanufacturer.
Negativecontrolswereobtainedfromcellswhereonlytheprimaryanti-bodywasomitted.
Cellswerevisualizedthesamewayasdescribedabove.
VirusproductionduringmultiplecellpassagesAnamountof106MARC-145orSJPLcellswereinfectedwith0.
005MOIofIAF-Klopstrain.
Then,cellswiththeirsupernatantsweresubjectedtothreecyclesoffreeze-thawat-70°Candthevirusstocksolutionswerekeptat-70°Cforfutureuse.
Foursubsequentviralpas-sagesinMARC-145andSJPLcellsweredoneasdescribedaboveexceptthatadilutionof1/20ofthepre-viousviralstocksolutionswasusedforcellinfection.
Mock-infectedcellswereincludedascontrolsineachpassage.
Theamountofvirusproductionateachpassagewascalculatedfroma96-wellmicroplateofMARC-145infectedcellsbytheKrbermethodandtheresultswereexpressedintissuecultureinfectiousdose50%per106infectedcells(TCID50/106cells).
Virusreplicationkineticsassay105MARC-145andSJPLcellswereinfectedwithIAF-KlopandLVstrainsusinganMOIof1.
Atdifferenttimespi(0,4,9,12,18,24,48,72,96and120hrs),wholecellcultureswerecollectedandwereprocessedbylowspeedcentrifugationtoseparatethecellpellet(cellfraction)fromtheculturemedium(supernatantfraction).
Bothfractionswerestoredat-70°Cuntilused.
Afterwards,virustitrationwasperformedinMARC-145cellsasdescribedabove.
Mock-infectedcellswereProvostetal.
VirologyJournal2012,9:267Page9of14http://www.
virologyj.
com/content/9/1/267includedineachexperimentascontrols.
Allexperimentswererepeatedtwotimesintriplicate.
VirusisolationVirusisolationwasattemptedfrom22swinesamples(lungandlymphnodestissues)submittedfromOctober2007toSeptember2008totheVeterinaryvirologydiag-nosticlaboratoryoftheVeterinaryCollegeoftheUni-versitédeMontréal.
Thosesamplesoriginatedfrom3to10weeksoldanimalshousedindifferentCanadianfarmsandtheyweresubmittedfordifferentreasonssuchasPRRSVoutbreaks,porcinecircovirusassociateddiseaseoutbreaks,orothershealthproblems.
ThreeofthesubmittedsampleswerePRRSVnegativebyareal-timePCRdiagnosticassay(TetracoreInc.
,Rockville,MD,USA)andtheamountofinfectiousPRRSVcon-tainedinthe19real-timePCRpositivescaseswasdeterminedusingthesameassayasdescribedprevi-ously[67].
Forvirusisolation,about1-2cm3ofpooloftissuesampleswerehomogenizedandresuspendedin9mLofculturemediumwithoutFBS.
Then,threecyclesoffreeze-thawat-70°Cwereperformedandtissueshomogenateswerecentrifugedandthesupernatantswerefiltered(withafiltersizeof0.
2micron).
Followingthesampletreatment,1mLoffilteredsamplewasusedtoinoculatecellsandcellswereincubatedfor5days.
Then,threecyclesoffreeze-thawwereperformedat-70°Candcelllysateswerecentrifugedat4000rpmfor10min.
Supernatantsofcelllysateswerecollectedandusedforasubsequentcellinfectioncycle.
OnemLofthecelllysatesupernatantswasinoculatedtofreshlypre-paredcellculturesandcellswereincubatedfor5days.
Thisnewinfectionstepwasdoneforthreeconsecutivetimes.
Atthefourthpassage,thevirusisolationstatuswasconfirmedbythepresenceofCPEandapositiveIFAresult.
TofurthercharacterizethePRRSVstrainsthatwereisolatedfrombothcelllines,PCRproductsencom-passingtheORF5genewereobtainedfromtissuesandfourthvirusisolationcellpassages,andsequencedsubse-quently.
SequenceswereanalyzedusingtheCLUSTALWalignmentmethodoftheBioEditsequencealignmenteditorversion7.
0.
9software(IbisTherapeutics,Carlsbad,CA,USA).
ApoptosisMARC-145andSJPLcellswereinfectedwithPRRSVIAF-Klopstrainat0.
5MOIorwereincubatedwithamixofapoptoticinducers(500μg/mLactinomycinD,60nMvinblastinesulfate,100μg/mLcycloheximideand40μg/mLpuromycin2HCl;BiomolResearchLaborator-iesInc.
,Plymouthmeeting,PA,USA)aspositivecon-trols.
Cellularchangesassociatedwiththeinfectionortheinducerswerevisualizedrespectivelyat72hrspiand24hrspost-incubation,respectively,underalightmicroscope(LeicaMicrosystemsInc.
).
Atthistime,cellsweredisruptedinalysisbuffer(50mMHEPES,pH7.
4,100mMNaCl,0.
1%CHAPS,1mMDTTand100μMEDTA)for5minutesfollowedbysonication(SonifierS-450A,Branson,Danbury,CT,USA).
Then,proteinconcentrationsweremeasuredbyaBradfordassayfol-lowingthemanufacturer'sinstructions(Bio-RadLabora-toriesLtd,Mississauga,ON,Canada).
Subsequently,apoptosiswasassessedbydetectingtheactivationofpro-caspases3/7asdescribedbyGagnonetal.
(2003),withminormodifications[65].
Briefly,avolumeofcelllysatecorrespondingto50μgoftotalcellproteinwasaddedtotheassaybuffer(50mMHEPES,pH7.
4,100mMNaCl,0.
1%CHAPS,1mMDTT,100μMEDTAand10%glycerol).
Then,specificsubstrateforcaspases3/7,theAc-DEVD-AFCfluorogenicsubstrate(BiomolResearchLaboratoriesInc.
),wasaddedatafinalconcentrationof200μMandtherateoffluorescencereleasedwasmoni-toredwitha96-wellplatefluorometer(SynergyHT,Biotek,Winooski,VT,USA).
Theresultswereexpressedasrelativefluorescencereleased(relativefluorescenceunitsorRFU)persecondperμgofcelllysates.
InhibitionofPRRSVreplicationTodeterminetheamountofporcineIFNαthatisabletoinhibitthereplicationofPRRSVinpermissivecelllines,104MARC-145andSJPLcellswereincubatedovernight.
ThecellsweretheninfectedwiththePRRSVIAF-Klopstrainataninfectiousdoseof0.
5MOIinaculturemediumwithoutFBSandincubatedduring4hrs.
Theculturemediumwasthenremovedandreplacedbyacompletemedium(i.
e.
with10%FBS)withdifferentseri-allydilutedconcentrationsofporcineIFNα(PBL,NewJersey,USA)andincubatedduring5days.
Then,thede-velopmentofCPEwasmonitoredandanIFAwasper-formed.
Alltheexperimentsweredoneinduplicate.
AnalysisofcytokinemRNAsexpressionbyrealtimereversetranscriptase-quantitativePCRSJPLandMARC-145cellswereinfectedasdescribedaboveortransfectedwithPolyinosinic–polycytidylicacidpotassiumsalt(Poly(I:C)[50μg/mL](Sigma-AldrichInc.
,St-Louis,USA)asapositivecontrolforinnateim-munityinduction,usingpolyethylenimine(PEI)[1μg/μL](Sigma-AldrichInc.
,St-Louis,USA),for72hoursortreatedwith1μg/mloflipopolysaccharide(LPS)(Sigma-AldrichInc.
,St-Louis,USA)for20hours,asanIFNγin-ducer.
TotalcellularRNAwasextractedfromcellsusingTrizolreagent(Invitrogen,Burlington,ON,Canada)accordingtothemanufacturer'sprotocol.
QuantificationofRNAwasperformedwithaNanodrop(NanoDropTechnologies,Inc.
,Wilmington,Delaware,USA).
1μgoftotalRNAwasreverse-transcribedusingtheQuantiTectreversetranscriptionkit(Qiagen,Mississauga,ON,Provostetal.
VirologyJournal2012,9:267Page10of14http://www.
virologyj.
com/content/9/1/267Canada).
ThecDNAwasamplifiedusingtheSsoFastEvaGreenWSupermixkit(Bio-rad,Hercules,CA,USA).
ThePCRamplificationprogramforallcDNAconsistedofanenzymeactivationstepof3minat98°C,followedby40cyclesofadenaturingstepfor2secat98°Candanannealing/extensionstepfor5secat58°C.
TheprimersusedforamplificationofthedifferenttargetcDNAareforIFNα:F-GCCCTTTGCTTTACTGATGG;R-TCTGCTCATTTGTTTCAGGAG,IFNβ:F-TCCTGTGGCAATTGAATGG;R-AATAGCGAAGATGTTCTG-GAG,IFNγ:F-ACTCGAATGTCCAACGCAAAGCAG;R-TCGACCTCGAAACATCTGACTCCT,TNF-α:F-TCTGTCTGCTGCACTTTGGAGTGA;R-TTGAGGGTTTGCTACAACATGGGC.
Allprimersweretestedtoachieveamplificationefficiencybetween90%and110%.
Thepri-mersequenceswerealldesignedfromtheNCBIGen-BankmRNAsequencesusingweb-basedsoftwareprimerquestfromIntegratedDNAtechnologies[69].
TheBio-RadCFX-96sequencedetectorapparatuswasusedforthecDNAamplification.
Thequantificationofdiffer-encesbetweenthedifferentgroupswascalculatedusingthe2-ΔΔCtmethod.
Beta-2microglobulin(B2M)wasusedasthenormalizinggenetocompensateforpotentialdif-ferencesincDNAamounts.
TheB2Mprimersfrommon-keyoriginusedareF-GTGCTATCTCCACGTTTGAGandR-GCTTCGAGTGCAAGAGATTG.
Thenon-infectedMARC-145andSJPLcellswereusedasthecali-bratorreferenceintheanalysis.
StatisticalanalysesAtwo-wayANOVAmodel,followedbyBonferronipost-hoctests(GraphpadPRISMVersion5.
0software)wereusedtodetermineifastatisticallysignificantdifferenceexistsbetweenMARC-145andSJPLcelllinesinregardstotheamountofPRRSVproducedaftermultiplecellpassages,procaspases3/7activationandcytokinesmRNAupregulationinPRRSVinfectedcellsandinPoly(I:C)transfectedcells.
One-wayANOVAmodel,followedbyTukey'sMultipleComparisonTest(Graph-padPRISM)wereusedtodetermineifastatisticallysig-nificantdifferenceexistsbetweentreatmentswithinMARC-145orSJPLcellsinprocaspases3/7activationandtheirmRNArelativeexpressionofIFNα,IFNβ,IFNγ,andTNF-α.
Differenceswereconsideredstatisti-callysignificantwithap<0.
05.
Fortheviralreplicationkineticexperiment,thetime-courseofTCID50measuredfromthecellpelletsandsupernatantswasanalyzedwithSASVersion9.
1software.
Thefollowinglinearmixed-effectmodelforrepeatedmeasurementswassolvedusingrestrictedmaximum-likelihoodestimation[70]:Yijkl=μ+αi+βj+τk+(α·β)ij+(α·τ)ik+(β·τ)jk+(α·β·τ)ijk+R·(α·β)ijl+eijkl.
WhereYijklisthemeasuredTCID50;μisthegrandmean;cellline(αi),typeofanalyticalmatrix(βj),andsamplingtime(τk)arefixedfactors;theexperimentreplicate(Rl)isarandomeffect;andeijklistherandomerrorterm.
Asindicatedintheequationabove,thisstatisticalmodelincludedalldualandtripleinteractionsbetweenthefixed-effectfactors,andtherandom-effectfactorRlwasnestedwithincellandana-lyticalmatrix.
ThestrategyforcovariancestructuremodelingproposedbyLittelletal(2000)wasused[71].
Briefly,themodelwasestimatedfirstwithafreecovari-ancestructure.
Afterinspectingtheestimatedcovariancematrix,themodelwasestimatedanewwithmoreparsi-moniouscovariancemodels(e.
g.
,compoundsymmetry,first-orderautoregressive),whichstructureresembledthatoftheunstructuredcovariancematrix.
Thehetero-geneousfirst-orderautoregressivecovariancemodelwasselectedbecauseitwasthebestfittotheempiricalco-variancematrix,asdeterminedwiththeAkaikeinforma-tioncriterion[71].
Least-squaremeanswereusedtoassessdifferencesbetweenthetwocelllinesateachtimeandforeachtypeofanalyticalmatrix(i.
e.
,cellorsuper-natantfractions),usingBonferroni-adjustedsignificancethresholds.
Theareasunderthetime-TCID50curves(AUC)werecalculatedforeachcell*matrix*replicateinordertoobtainestimatesoftotalviralproductionforeachcelllinefollowingthe120hrsdurationoftheexperiment.
AdditionalfileAdditionalfile1:FigureS1.
SJPLandMARC-145cellsgrowthcurves.
Amountofcells(log2)wascalculatedwithahemacytometeratseedingtime(0hr),andat24,48,and72hrspost-incubation.
Doublingtimewascalculatedwithdataobtainedat24and48hrspost-incubationwiththeformula:Doublingtime(hrs)=0.
3Xincubationtime(hrs)/(logcelltimeb-logcelltimea),whereincubationtimeis24hrs,celltimeaistheamountofcellsat24hrsandcelltimebistheamountofcellsat48hrs.
Interestingly,theMARC-145cellsgrowfasterthantheSJPLcells.
Duringthisexperiment,cellssurfaceareaswerecalculatedwithImageJv.
1.
6.
0fromthreedifferentpicturesofeachcelltypeat24hrspost-incubation.
SJPLandMARC-145cellshaveasurfaceareaof4684.
41±2188.
94μm2(n=29)and3568.
96±1128.
47μm2(n=38),respectively.
StatisticalanalysesusingttestshowedthatSJPLcellshaveastatisticallywidersurfacethanMARC-145cells(P<0.
01)(datanotshown).
AbbreviationsAUC:Areasunderthetime-TCID50curves;B2M:Beta-2microglobulin;CPE:Cytopathiceffect;EAV:Equinearteritisvirus;EU:Europeangenotype;IFA:Immunofluorescenceassay;IFN:Interferon;LDV:Lactatedehydrogenase-elevatingvirus;LPS:Lipopolysaccharide;LV:Lelystadvirus;N:Nucleocapsidprotein;NA:NorthAmericangenotype;NPTr:Newborntracheaepithelialcellline;ORF:Openreadingframe;PAM:Porcinealveolarmacrophages;PBS:Phosphatebuffersalinesolution;PCV:Porcinecircovirus;PCV2:Porcinecircovirustype2;PEI:Polyethylenimine;PFA:Paraformaldehyde;Pi:Post-infection;Poly(I:C):Polyinosinic–polycytidylicacidpotassiumsalt;PRRS:Porcinereproductiveandrespiratorysyndrome;PRRSV:Porcinereproductiveandrespiratorysyndromevirus;Rcf:Relativecentrifugalforce;SJPL:St-Judeporcinelungcells;TCID50:50%tissuecultureinfectivedose;TNF-α:Tumornecrosisfactoralpha.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Provostetal.
VirologyJournal2012,9:267Page11of14http://www.
virologyj.
com/content/9/1/267Authors'contributionsCPcarriedtheqRT-PCRstudiesandtheirstatisticalanalyses,thePRRSVcellreceptorsimmunofluorescenceassay,thePAMcellsharvesting,thecellgrowthcurvesandparticipatedinthewritingofthemanuscript.
JJJcarriedoutthePRRSVmultiplecellpassagesproduction,someofthekineticstudies,andparticipatedinthewritingofthemanuscript.
CPandJJJhavecontributedequallytothismanuscript.
CLcarriedouttheIFNαinhibitionstudies,andparticipatedinthewritingofthemanuscript.
NMdidthemicroscopystudies(SJPLpermissivity),thevirusisolationefficiency,someofthekineticstudies,andparticipatedinthewritingofthemanuscript.
MELcarriedtheapoptosisstudies.
JREDCperformedthestatisticalanalysesofthevirusreplicationefficiency.
MJandCAGconceivedthestudy,realizeditsdesign,supervisedthegraduatestudentsandhelpedtodraftthemanuscript.
Inaddition,CAGconductedtheinvivoexperimentfortheharvestingofPAMcells.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsTheauthorsaregratefultoJoséeLabrieforhertechnicalsupport.
TheauthorsaregratefultoCynthiaM.
Guilbertforcriticallyreviewingthemanuscript.
ThisworkwassupportedbytheNaturalSciencesandEngineeringResearchCouncilofCanada(NSERC)discoverygrants(toCAGandMJ)andFondsderechercheduQuébecNatureettechnologies(FRQNT)teamresearchprojectprogram(toCAGandMJ).
ChantaleProvostwasarecipientofapostdoctoralfellowshipgrantfromtheCanadianSwineHealthBoard(CSHB).
Authordetails1Groupederecherchesurlesmaladiesinfectieusesduporc(GREMIP),Centrederechercheeninfectiologieporcine(CRIP),FacultédemédecinevétérinaireUniversitédeMontréal,3200rueSicotte,Saint-Hyacinthe,J2S7C6,Québec,Canada.
2GroupedeRechercheenPharmacologieAnimaleduQuébec(GREPAQ),FacultédemédecinevétérinaireUniversitédeMontréal,3200rueSicotte,Saint-Hyacinthe,QuébecJ2S7C6,Canada.
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