Comparativeandtemporaltranscriptomeanalysisofpestedespetitsruminantsvirusinfectedgoatperipheralbloodmononuclearcells

SiddappaManjunatha,f

smanju712@gmail.com

BishnuPrasadMishraa

bpmishra_1@hotmail.com

BinaMishrad

binachauhanmishra@hotmail.com

AdityaPrasadSahooa

rush2aditya@gmail.com

AshokK.Tiwarib

aktiwari63@yahoo.com

KaushalKishoreRajakc

kaushalvirol@gmail.com

D.Muthuchelvanc

drchelva@gmail.com

ShikhaSaxenaa

shikhasaxenaivri@gmail.com

LakshmanSantraa

lakshman.ivri@gmail.com

AmitRanjanSahua

dramitr.sahu@gmail.com

SajadAhmadWania

wanisajad759@gmail.com

R.P.Singhd

rpsingh@dr.com

Y.P.Singhe,

ypivri@gmail.com

ArunaPandeya

arunabioinfo@gmail.com

SonamKanchana

kanchansonam@gmail.com

R.K.Singha

This is the author's manuscript of the article published in final edited form as:Manjunath, S., Mishra, B. P., Mishra, B., Sahoo, A. P., Tiwari, A. K., Rajak, K. K., … Janga, S. C. (2017). Comparative and temporal transcriptome analysis of peste des petits ruminants virus infected goat peripheral blood mononuclear cells. Virus Research, 229, 28–40. https://doi.org/10.1016/j.virusres.2016.12.014

1Introduction

rks_virology@gmail.com

GandhamRaviKumara,⁎

gandham71@gmail.com

SarathChandraJangaf,g,h,⁎⁎

scjanga@iupui.edu

aDivisionofVeterinaryBiotechnology,ICAR-IndianVeterinaryResearchInstitute,Izatnagar,Bareilly,UttarPradesh,243122,India

bDivisionofBiologicalStandardization,ICAR-IndianVeterinaryResearchInstitute,Izatnagar,243122,India

cDivisionofVirology,ICAR-IndianVeterinaryResearchInstitute(IVRI),MukteswarCampus,Nainital,Uttaranchal,263138,India

dDivisionofBiologicalProducts, (ICAR-)IndianVeterinaryResearchInstitute,Izatnagar,Bareilly,UttarPradesh,243122,India

eARISCell, (ICAR-)IndianVeterinaryResearchInstitute(IVRI),MukteswarCampus,Nainital,Uttaranchal,263138,India

fSchoolofInformaticsandComputing,IndianaUniversityPurdueUniversity,719IndianaAveSte319,WalkerPlazaBuilding,Indianapolis,IN46202,UnitedStates

gCenterforComputationalBiologyandBioinformatics,IndianaUniversitySchoolofMedicine,5021HealthInformationandTranslationalSciences(HITS),410West10thStreet,Indianapolis,IN,46202,UnitedStates

hDepartmentofMedicalandMolecularGenetics,IndianaUniversitySchoolofMedicine,MedicalResearchandLibraryBuilding,975WestWalnutStreet,Indianapolis,IN,46202,UnitedStates

⁎Correspondingauthor.

⁎⁎Correspondingauthorat:SchoolofInformaticsandComputing,IndianaUniversityPurdueUniversity,719IndianaAveSte319,WalkerPlazaBuilding,Indianapolis,IN46202,UnitedStates.

(Deletethethisline)1ARISCell,IndianVeterinaryResearchInstitute,Izatnagar,Bareilly,UttarPradesh,243122,India.

Abstract

Pestedespetitsruminanatsvirus(PPRV),amorbilliviruscausesanacute,highlycontagiousdisease–pestedespetitsruminants(PPR),affectinggoatsandsheep.Sungri/96vaccinestrainiswidelyused

formassvaccinationprograms in IndiaagainstPPRand is considered themostpotent vaccineproviding long-term immunity.However, occurrenceof outbreaksdue toemergingPPRvirusesmaybea

challenge.Inthisstudy,thetemporaldynamicsofimmuneresponseingoatperipheralbloodmononuclearcells(PBMCs)infectedwithSungri/96vaccineviruswasinvestigatedbytranscriptomeanalysis.

InfectedgoatPBMCsat48hand120hpostinfectionrevealed2540and2000differentiallyexpressedgenes(DEGs),respectively,oncomparisonwithrespectivecontrols.Comparisonoftheinfectedsamples

revealed1416DEGstobealteredacrosstimepoints.FunctionalanalysisofDEGsreflectedenrichmentofTLRsignalingpathways,innateimmuneresponse,inflammatoryresponse,positiveregulationof

signaltransductionandcytokineproduction.Theupregulationofinnateimmunegenesduringearlyphase(between2-5days)vizviz.interferonregulatoryfactors(IRFs),tripartitemotifs(TRIM)andseveral

interferon stimulatedgenes (ISGs) in infectedPBMCsand interactomeanalysis indicated induction of broad-spectrumanti-viral state.Several Transcription factors – IRF3,FOXO3andSP1 that govern

immune regulatory pathways were identified to co-regulate the DEGs. The results from this study, highlighted the involvement of both innate and adaptive immune systems with the enrichment of

complementcascadeobservedat120hp.i.,suggestiveofalinkbetweeninnateandadaptiveimmuneresponse.BasedonthetranscriptomeanalysisandqRT-PCRvalidation,aninvitromechanismforthe

inductionofISGsbyIRFsinaninterferonindependentmannertotriggerarobustimmuneresponsewaspredictedinPPRVinfection.

Keywords:PPRV;PBMCs;RNA-sequencing;Transcriptome;Protein-proteininteractionnetworks;Transcriptionfactors

PesteDesPetitsRuminants(PPR)isaviraldiseaseofeconomicimportance,causinganacute,highlycontagiousdiseaseinsmallruminants–goatsandsheep(Banyardetal.,2010).PesteDesPetitsRuminants

Virus(PPRV),thecausativeagentofPPRbelongstothegenusMorbillivirus,familyParamyxoviridae.Thediseasecausessevereeconomiclossesintermsofhighmortality(upto90%)andmorbidity(100%)affectingthe

productivityofsheepandgoatsinendemicregions(Singhetal.,2009;Banyardetal.,2010).Givenitseconomicrelevanceandseverity,thediseaseisclassifiedasaWorldOrganizationforAnimalHealth(OIE)listed

disease(Kumaretal.,2014a;Kumaretal.,2014,b).Tilltoday,onlyoneserotypeofPesteDesPetitsRuminantsVirus(PPRV)isknowntoexist,butdifferentisolatesofPPRVbelongingtofourdifferentlineages(with

fourthlineagepresentinAsia)basedonphylogeneticstudiesofpartial‘N’and‘F’genesequenceshavebeenreported(Kumaretal.,2014a,b;Albinaetal.,2013).FewrecentreportsindicatethattheAsianlineageis

nowpresentinAfricaaswell(Kwiateketal.,2011).LiveattenuatedvaccinesareusedforcontrolofPPR,whichhaveprovedtobequitesafeandprovideprotectioninnaturalhostsduringmassvaccinationprograms.

Amongtheseliveattenuatedvaccines,Nigeria75/1andSungri/96havebeenwidelyusedforthecontrolofPPRVinAfrica(Dialloetal.,1989)andIndia(Saravananetal.,2010)respectively.

Sungri/96vaccine,producedbycontinuouspassageofSungri/96strain(Goatorigin)inVerocells,iswidelyusedforvaccinationinsheepandgoatsthroughoutIndia(Singhetal.,2010).AsingledoseofaPPR

vaccinecontains∼103TCID50ofVerocell-attenuatedPPRVandisbelievedtoprovideprotectiveimmunityinsheepandgoatsforseveralyears(Kumaretal.,2014a,b).Thisrobustimmuneresponsethatresultsin

protectionofhostsaftervaccinationisattributedtostrongcell-mediatedimmunityfollowedbyhumoralimmunity,which,however,needstobeinvestigated(Kumaretal.,2014a,b;Sinnathambyetal.,2001).Even

thoughunlikely, recently field isolateofPPRV(PPRV/Nanakpur/2012)wasshownnotneutralizingagainstvaccinestrain (Sungri/96), raisingconcernsof thecross-protectionofSungri/96vaccinevirusagainstan

emergingfieldvirus(Kumaretal.,2014a,b),andmakingitallthemoreimportanttounderstandthecompletemechanismsofimmuneprotectioninducedbySungri/96PPRVvaccinestrain.

Hostdefenseresponseagainsttheinvadingvirus,startswiththerecognitionofspecific,conservedmolecularpatternsonthevirusescalledpathogenassociatedmolecularpatterns(PAMPs).TheseviralPAMPs

aredetectedbyhostpatternrecognitionreceptors(PRRs)locatedextra-cytoplasmic(toll-likereceptors–TLR3/7)orcytoplasmic(RIG-Ilikereceptors(RLRs)–RIG-I,MDA-5)inthehostcells(JensenandThomsen,

2012).TheviralsensingbytheTLRsorRLRsinitiatessignalingcascadesthatinducetheexpressionofvirus-responsivegenesandpro-inflammatorycytokines,whichinturnorchestrateinnateimmunity,chemokines,

andco-stimulatorymoleculesthatpromoteT-cellactivationandspecificimmunity,restrictingviralreplication(Akiraetal.,2006;Lazearetal.,2013).Manyvirulentviruseshaveevolveddifferentmechanismstoevade

hostdefensesystems.Ontheotherhand,vaccineviruses(liveattenuated),whicharewidelyusedformassvaccinationprograms,effectivelyinduceearlyinnateimmuneresponse,whichfurthershapestheadaptive

immunitygivingarobustimmuneresponse(Kumaretal.,2014a,b).

PPRVisbothlymphotropicandepitheliotropicinnature(Pawaretal.,2008).PeripheralBloodMononuclearCells(PBMCs)arewidelyusedasstandardinvitromodeltostudyhost-PPRVinteractionsasinother

morbillivirusinfections(Boltetal.,2002;Iwasaetal.,2010;Manjunathetal.,2015).PPRVenterslymphoidcellsthroughsignalinglymphocyteactivationmolecule(SLAM),whichiswidelyexpressedonallimmune

cells(Pawaretal.,2008).PBMCsconsistingoflymphocytes(Tcells,BcellsandNKcells),monocytesanddendriticcellsplayanimportantroleinpathogenrecognitionandinduceearlyinnateimmuneresponsefor

hostdefense.PBMCswereusedasaninvitromodeltoinvestigatetheroleofTLRs−–3and7,andcytokinesindifferentialsusceptibilityofgoatbreedsandwaterbuffalotoPPRVinfection(Dhanasekaranetal.,2014).

ThePBMCtranscriptomerepresentsnotonlytheprimaryimmunefunctionofleukocytes,butalsodisplaystranscriptomicshiftsofothertissuesandorgansduetophysiologicalandenvironmentalalterations(Liewet

al.,2006;KohaneandValtchinov,2012).Recently,transcriptomeanalysisofPBMCsinfectedwithPPRVuncoveredtranscriptionfactorsmodulatingimmuneresponse(Manjunathetal.,2015).However,dynamicsof

immuneresponsewith timewouldhelp inan improvedunderstandingof theanti-viral stateestablishedafterPPRVvaccination.Hence in thepresent study,modulationof the immuneresponsewith timeat the

transcriptomelevelingoatPBMCsinfectedwithSungri/96vaccinestrainusingRNA-Sequencingwasdetermined.TheresultsfromthisstudyindicateactivationofTLR7/10,whichstimulateseffectormolecules−–

interferonstimulatedgenesviaviainterferonregulatoryfactorsininterferonindependentmannertoprovideaneffectiveanti-viralresponseagainstPPRVinfection.

2Materialandmethods2.1Ethicsstatementandanimals

The experimental procedures in the present study were approved by Institute Animal Ethics Committee (I.A.E.CNo. F.1.53/2012-13-J.D.). Goat kids (5months old) used for blood collection were housed in appropriate

containmentfacilitieswithfeedandwateradlibitum.

2.2ViruspropagationandpurificationThevirusstrainusedinthisstudySungri/96(avaccinestrain)iswidelyusedforvaccinationthroughoutIndia.VerocellsavailableinthelaboratorywerepropagatedinEagle’sMinimumEssentialMedium(EMEM)containing

10%fetalcalfserum(FCS)at37°Cunder5%CO2.TheVerocellswereseededinto850cm2rollerculturebottlesandtheninfectedwithVerocelladaptedSungri/96vaccinevirus,at0.1multiplicityofinfection(MOI).Infectedroller

cultureswereincubatedat37°°Cunder5%CO2andmonitoredforcytopathiceffect(CPE).ThemediumwaschangedwithEMEMcontaining2%fetalcalfserum(FCS)onceeverytwodays.Whenthecellsshowed70-–80%CPE,the

viruswasharvestedbytwocyclesoffreezingandthawingandstoredat−80°°Candpurifiedbybandingonsucrosegradient(ultracentrifuged).Thepurifiedviruswastitratedbyestimating50%tissuecultureinfectivedose(TCID50)

usingVerocellsin96wellmicrotitreplate.ThepurifiedviruswastestedforitsinfectivityinVerocellsandwasusedfurtherforinfectioningoatPBMCs.

2.3ScreeningofanimalsforPPRVantibodiesGoatswerescreenedforPPRVantibodiesusingcompetitiveELISA(c-ELISA)serumneutralizationtest(SNT).CompetitiveELISAforthedetectionofPPRantibodieswascarriedoutasperthemethodof(Singhetal.,2004)

usingc-ELISAKit(IVRI,Mukteshwar).PBMCswereisolatedfrombloodcollectedfromanimalsthatshowedSNTtitre>1:8andPercentageInhibition(PI)valuelessthan40%.

2.4VirusinfectioninPBMCswithPPRVandconfirmationGoatPBMCswereisolatedusingHistopaque-1077(Sigma,USA)bydensitygradientcentrifugationandseededintofoursixwellplates.Twoplatesservedascontrolfor48hand120handtwoplateswereusedforinfection

withPPRV.PBMCsc.a1×106cellsinRPMI-1640wereaddedtoeachwellwith100IU/mlpenicillin,100μg/mlstreptomycinand10%foetalcalfserum(FCS),infectedwithPPRV(Sungri/96)at1.0MOIandincubatedat37°°Cin5%

CO2incubatorfor1hofadsorption.After1hofadsorption,thevirusinoculumwasremoved,centrifugedtocollectthelymphocytesthatwerewashedwithfreshRPMImediumandaddedbacktothewells.FreshRPMImediumwas

thenaddedtowellsandincubated.Thecontrolsample(mock-infected)ontheotherhandreceivedjusttheRPMImedium.Theinfectedcellswerecollectedat24hp.i,48hp.i,72hp.i.and120hp.i.,andviralloadwasquantified

throughNgeneexpression.Timeinterval48hp.i.waschosenasanearlytimepointinthestudytoallowincreasednumberofcellstobeinfectedwithPPRvirus,asevidentfromtheNgeneexpressionbyqRT-PCRincomparisonto

24hp.i.ThoughtheNgeneexpressionincreasedat72hp.i,weselected120hp.i.,asitsexpressionwasfoundtobehighestatthistimepoint(datanotshown).PPRVinfectionwasfurtherconfirmedbymorphologicalchanges,

reversetranscriptionpolymerasechainreaction(RT-PCR)(Manjunathetal.,2015)andFACSatboth48and120hp.i.

2.5FlowcytometryanalysisofPPRVinfectedPBMCsQuantitativemeasurementofPPRVinfectedPBMCsat48hand120hp.iwasmeasuredusingflowcytometry.Forflowcytometryanalysis,thecellswerefixedin4%paraformaldehyde(PFA),permeabilizedwith0.2%TritonX

andincubatedfor1hatroomtemperaturewithprimaryantibody(1:100)raisedagainstthewholePPRvirus(Sungri/96)ingoats.ThecellswerethenwashedwithPBSandincubatedwithanti-goatFITCconjugatedsecondaryantibody

(1:2000)for1hatroomtemperature.Afterincubation,cellswerewashedthricewithPBSandresuspendedin500μl,1XPBS.Atotalof10,000cellswereexaminedusingtheFL1channelofaflowcytometer.Numberofinfectedcells

(PPRVpositivePBMCs)at48hand120hp.i.wereanalyzedusingCellQuestsoftware(BDBiosciences).

2.6RNAextraction,librarypreparationandIlluminasequencingAnRNeasy®MiniKit(Qiagen®,USA)wasusedtoextractandpurifyRNAfromcontrolandinfectedsamplesat48hand120hp.i.,asperthemethodrecommendedbythemanufacturer.RNAconcentrationofthesampleswas

determinedbyNanodropspectrophotometer(ThermoFisherScientificInc.,USA)andthequalitywascheckedonAgilentBioanalyzer2100(AgilentTechnologies,Germany).RNAsampleswithRNAIntegrityNumber(RIN)≥8were

processedforlibrarypreparation.ThelibrarywaspreparedusingIlluminakitfollowingthemanufacturer’sprotocol.Approximately,10μgoftotalRNAfrominfectedandcontrolPBMCswasusedtoisolatemRNAusingmagneticOligo

dTbeads(Illumina)andthemRNAwaspurifiedusingmRNApurificationkit(Invitrogen).ThepurifiedmRNAfromboththesampleswasthenfragmented(100–400bp)usingdivalentcationsfor5minat94°C.Thedoublestranded

cDNAwassynthesizedusingSuperscriptDouble-strandedcDNASynthesisKit(Invitrogen,Camarillo,CA)usingrandomhexamers(N6)primer(Illumina).ThecDNAsynthesizedwasthensubjectedtoendrepairandphosphorylation

usingT4DNApolymerase,KlenowDNApolymeraseandT4polynucleotidekinase(PNK).TheendrepairedcDNAfragmentswerethenpolyadenylatedatthe3’endusingKlenowExo(3'to5'′–5′exominus,Illumina).Illuminapairedend

adapterswere then ligated to the ends of the 3′ adenylated cDNA fragments. The adapters ligated cDNAwas then enrichedwith PCR amplification using primer pairs (PE 1.0 and PE 2.0) (Illumina) catalyzed by PhusionDNA

polymerase.ThecDNAlibrariespreparedfromboththecontrolandtheinfectedsamplesweresequencedonIlluminaHiSeq2000platformaccordingtothemanufacturer’sinstructions.IlluminaSequencingwasperformedatSandor

LifeSciences,Pvt.Ltd.(Hyderabad,India).

2.7RawdatapreprocessingQualitycontrolandfilteringofthehighquality(HQ)sequencingdatawasdoneusingNGSQCtoolkit,astandaloneprogramandanopensourceapplication(PatelandJain,2012)Further,subsequentprocessingofthedatawas

performedusingprinseq-litesoftware(SchmiederandEdwards,2011)toremovereadsoflowquality(meanphredscore<25)andshortlength(<50)fordownstreamanalysis.Theaveragetotalnumberofreadswerec.a40millionper

sample.TheaveragepercentageofHQbaseswas92.68%.Furthertofindoutwhetherthesequencingreadsactuallyfallonspecifictargets(genes)andhavesufficientcoverage,TEQCpackage(Hummeletal.,2011)inRwasused.

ThecoverageandnormalizedcoverageoffivegenesatboththetimepointsisgiveninFig.1.

2.8ViraltranscriptquantificationReads from infectedsamplesatboth48hand120hp.iweremapped to thePPRVreferencegenome (GenBank:AJ849636.2)usingbowtie2.0 software (LangmeadandSalzberg,2012). Themapped reads for each viral

transcriptwerethenquantifiedbyusingCufflinksintermsoffragmentperkilobaseofexonpermillionmappedreads(FPKM)usingthePPRVgenetransferformat(GTF)filedownloadedfromNCBI.Thesixviraltranscripts(N-P-M-F-

Fig.1Coverageandnormalizedcoverageplotsoffivegene:CoverageandnormalizedcoverageplotsofIRF3,IFIT5,TRIM21,ISG15andOAS2.Thebluecolourindicatesthecontrolandtheredcolourindicatestheinfectedsample.(Forinterpretationofthe

referencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

alt-text:Fig.1

H-L)werequantifiedandthedifferentialexpressionofviralgenewasrepresentedasfoldchangebetweensamplesat48hand120hp.irespectively.

2.9Identificationofdifferentiallyexpressedgenes(DEGs)oncomparativeandtemporalanalysisandfunctionalannotationFig.2summarizes thestepsused in theanalysis.Quality filteredreads fromcontroland infectedsamples (48hand120h)weremapped to theBostaurus referencegenome.Capra hircus genome is notwell annotated

therefore thewell-definedB.taurus reference genomewas used formapping reads using TopHat version 2.0.7 (Kim et al., 2013). Bos taurus reference genomehas been previously used bymany groups for analyzing the goat

transcriptome(Liuetal.,2013;Fontanesietal.,2010).Themappedreadswerethenassembledusingcufflinks,whichquantifiesthegeneexpressionintermsofFPKM.DEGsatboththetimepoints(48hand120hp.i.)vis–a–vistheir

respectivecontrolswereidentifiedbydifferentialexpressionanalysis–referredascomparativeanalysisinthisstudyandtheDEGsbetweentheinfectedsamplesat48hand120hp.i.,wereidentifiedbydifferentialexpressionanalysis

– referredastemporalanalysisinthisstudy,usingcuffdiff2incufflinkspackage(Trapnelletal.,2012).Thesignificantly(p≤0.05)DEGswith≥±2foldchangewereusedforfunctionalenrichmentanalysisusingg:Profiler(Reimandet

al.,2011)andDatabaseforAnnotation,VisualizationandIntegratedDiscovery(DAVID)(Huangdaetal.,2009).ThecommonDEGs(243)amongallthethreeconditions(I–48hvsC–48h;I–120hvsC–120handI–48hvsI–-48hvsC-48h;

I-120hvsC-120handI-48hvsI-120h(‘I’standsforinfectedand‘C’standsforcontrol))werefunctionallyprofiledforbiologicalprocessesandKEGGpathwaysusingg:ProfilerandDAVID.TheDEGsfunctionallyannotatedtoimmune

processesandcommonDEGswerefurthersubjectedtoClueGOanalysis(Bindeaetal.,2009)forfunctionalenrichmentinKEGGandREACTOME.

2.10Protein–Pproteininteraction(PPI)networksofdifferentiallyexpressedandhighlyconnectedgenes(DEHC)PPInetworkamongalltheDEGsfromcomparativeandtemporalanalysiswasretrievedusinginteractionsavailableintheBioGRIDdatabase(Starketal.,2006)andwasfoundtobedenselyinterconnected.TheDEGswere

thennarroweddownbasedonfoldchange≥±2(Up-ordown-regulated)anddegree(calculatedusingigraphpackage(https://cran.r-project.org/web/packages/igraph/index.html))≥5forboththeconditionsandweredesignatedas

differentiallyexpressedhighlyconnected(DEHC)genes.Forcomparativeanalysisat120hp.i.,and48hp.i,2000and2540DEGswerenarroweddownto163and181DEHCgenes,respectively,andfortemporalanalysis1416DEGs

werenarroweddownto126DEHCgenes.ThePPInetworkforDEHCgeneswasvisualizedusingCytoscapesoftware(Shannonetal.,2003).

2.11Identificationoftranscriptionfactors(TFs)regulatingDEGs

Fig.2Flowchartofstepsdepictingtheanalysisoftranscriptomedata−–startingfromprocessingofrawreads,functionalenrichment,protein-proteininteractionsnetworks(PPI)toidentificationoftranscriptionfactors(TFs)thatbindtotheupstreamofthe

differentiallyexpressedhighlyconnected(DEHC)genesfromcomparativeandtemporaldataanalysis.

alt-text:Fig.2

TFs,bindingtotheupstreamofDEHCgenesfromcomparativeandtemporalanalysiswereidentifiedbyrunningMEME(Baileyetal.,2006).followedbyTOMTOM(Guptaetal.,2007)toolsfromtheMEMEpackage.Initially,

5kbupstreamofDEHCgenesforboththeconditionswereextractedfromEnsembleBioMartandthirtyoverrepresentedconservedmotifswereidentifiedusingMEME(MultipleEMforMotifElicitation)Suite.TFsbindingtothese

motifsofDEHCgenesforboththeconditions(ComparativeandTemporalanalysis)wereidentifiedusingTOMTOM.OrthologsoftheTFsinBostauruswereidentifiedusingg:orthing:Profiler.ThepresenceoftheseTFbindingsites

acrosstheDEHCgenesareisshownasaheatmapusinggeneEsoftware(BroadInstitute).

2.12ValidationofRNAsequencingdatausingQuantitativeRealtimePCR(qRT-PCR)AtotalofsixgenesofinterestfromboththeanalysesresultingfromRNAsequencingdatawereselectedforfurthervalidation.Thesegeneswereselectedkeepinginviewthepathwaythatwaspredictedinthestudy.qRT-PCR

wascarriedoutonthesamebiologicalmaterialthatwasusedinRNA-SeqexperimentonAppliedBiosystems7500Fastsystemusing2XSYBRGreenMastermix(USB,Sigma).GAPDHwasidentifiedasthestablereferencegenefrom

panelofhousekeepinggenesfornormalizationoftargetgene(s)ofinterest(SiddappaManjunathetal.,2015).TheprimersequencesforthegenesusedforvalidationaregiveninTable1.Forallgenestenfoldserialdilutionwererunin

thestudytoestimatetheefficiencyofPCR,andthepercentageefficiencyrangedbetween95and100%.Also,theexpressionofIFNs−–αandβwasalsovalidatedusingcustomTaqMangeneexpressionassaysfromLifeTechnologies.

Allthesampleswererunintriplicates.Therelativeexpressionofeachsamplewascalculatedusingthe2−DΔCTmethodwithcontrolgroupascalibrator(SchmittgenandLivak,2008).Student’stt-testwasdoneinJMP9(SASInstitute

Inc,Cary,USA)anddifferencesbetweengroupswereconsideredsignificantatp≤0.05.

Table1GenesandtheirprimersequencesusedforvalidationoftheRNA-sequencingdataandpredictionofimmunesignalingpathway.

alt-text:Table1

Genes Primersequence AccessionNumbers

IRF3 Forward:AGCGTCCCTAGCAGACAAGAReverse:CCAGGTTGAACACACCTCCT

JQ308793.1

ISG15 Forward:CAGTTCATCGCCCAGAAGATReverse:GTCGTTCCTCACCAGGATGT

XM_005690795.1

HERC5 Forward:GTATGAGGTTGGCTGGCATTReverse:CCCTGACTCCTCCAAAATCA

XM_005681669.1

IFIT3 Forward:AAGGGTGGACACTGGTCAAGReverse:AGGGCCAGGAGAACTTTGAT

XM_005698196.1

IFIT5 Forward:CTTGGAGGTGACACCAACCTReverse:CCACAGCTGCTTTGAAATGA

XM_005698239.1

IRF7 Forward:GACACGCCCATCTTTGACTTReverse:ACTGTCCAGGGAGGACACAC

XM_004019737.2

3ResultsGoats(5-–6monthsold)screened−veforPPRVantibodiesbycompetitiveELISA(c-ELISA)wereselectedforcollectionofblood.ThegoatPBMCswereinfectedwithpurifiedPPRvaccinevirus(Sungri/96)at

1.0MOIandwereobservedformorphologicalchangesfrom24hto120hp.i.Theinfectedcellsshowedballooningandclumpingofcellsfrom48hp.iwithprogressiveincreaseincytopathiceffect(CPE)at120hp.i

(Fig.3A).Nosuchchangeswereobservedinuninfectedcells.Amplificationof351bpfragmentofNgeneofPPRVininfectedgoatPBMCsat48hand120hp.i.,confirmedPPRVinfectioninPBMCs(Fig.3B).The

cells(PBMCs)infectedwithPPRVwerequantifiedfurtherbyflowcytometryusingpolyclonalserumraisedingoatsagainstPPRV(Sungri/96)vaccinevirus.ThepercentinfectedPBMCswerefoundtobe49.31%and

72.72%at48hp.iand120hp.i.,respectively(Fig.3C).TheviraltranscriptswerequantifiedusingPPRVreferencegenomeat48hand120hp.i.TheresultsshowedincreaseinexpressionofNucleoprotein(N)gene

followedbyfusion(F),Haemagglutinin(H),Large(L),Matrix(M),andPhosphoprotein(P)withtime(Fig.3D).TheseresultsratifiedprogressionofPPRVinfectionfrom48hto120hp.i.

3.1IdentificationofDEGsoncomparativeandtemporalanalysisandfunctionalannotationGeneexpressionforcomparativeanalysiswasquantifiedatboththetimepoints(48hand120hp.i.)intermsofFPKMusingcufflinks.Atotalof2540(48h)and2000(120h)genesweresignificantly(p≤0.05)differentially

expressedatafoldchange≥±±2.Atotalof797DEGswerefoundcommonlydifferentiallyexpressedatbothtimepoints.At48hp.i,amongthe2540genes,1130wereupregulatedand1410genesweredownregulated.At120hp.i.,

outof2000significantDEgenes,728geneswereupregulatedand1272genesweredownregulated.Temporaldataanalysis(I–120hvsI–-120hvsI-48h)oftheinfectedsamplesrevealed1416significantly(p≤0.05)DEGsoutofwhich

821geneswereupregulatedand595genesweredownregulatedwithfoldchange≥±±2.

Significantgeneontologytermsof2540(48hp.i.)and2000(120hp.i.)DEGsfromcomparativeanalysiswereretrievedusingg:ProfilerandDAVIDdatabases.DEGswereannotatedtoseveralgeneontology(GO)categories

belongingtothethreebranchesofontology−–biologicalprocess,molecularfunction,andcellularcomponent.Underbiologicalprocesses,significantenrichmentofDEGsat48hp.i.,wasseenforimmunesystemprocesses,cytokine

production, TLR signaling pathway, IL-6 production, regulation of T-cells, etc.Whereas, at 120 h p.i., immune systemprocess, regulation of cellular process, defense response etc.,were found to be enriched. A total of 297 (p-

value=1.20E-20)and238genes (p-value=3.19E-21)at48hand120hp.i., respectively,were involved in immunesystemprocesseswith significantenrichment.Under themolecular functioncategory, cytokineactivity,kinase

activity,cytokinereceptorbindingandchemokinereceptorbinding,wereenrichedatbothtimepoints.AnumberofDEGsatthesetimepointswerefoundtobelocalizedonthecellsurface,cellmembrane,cytosolandvesiclesunder

cellularcomponentcategory.TofurtherdefineDEGsfunctioningoatPBMCsfollowingPPRVinfectionthebiologicalpathwaysenrichedintheinfectedtranscriptomeatthesetimepointswereanalyzedusingKEGGpathwaysinDAVID

database.Atotalof597geneswith15significant(p≤0.05)categoriesat48hp.i.,and426geneswith13significantcategoriesat120hp.i.werefoundtobemappedtothereferencecanonicalpathwaysinKEGG.Themostsignificant

KEGGpathwaysrepresentedbymostoftheDEGswerecytokine-cytokinereceptorinteraction,NODlikereceptorsignalingpathway,haematopoieticcelllineageandToll-likereceptorsignalingpathwayatbothtimepoints.Inaddition,

enrichmentofcomplementcascadewasobservedat120hp.i.suggestingitasalinkbetweeninnateandadaptiveresponse.

TheDEGsfunctionallyannotatedtoimmuneprocessesbyg:Profilerat48hp.iand120hp.i.respectively,werefurthersubjectedtoClueGOanalysis.At48hp.i,TLR3/7/8signalingcascades,activationofinterferonregulatory

(IRF)pathways,cytokinesignaling,RIG-I/MDA5pathways,NF-kBactivation,ISG15antiviralsignaling,etc.,weresignificantly(p≤0.05)enriched(Fig.4A).At120hp.i.,allthesepathwayswerefoundtobemoreenrichedthan48h

p.i.asrepresentedbythesizeofthenode(pValuecorrectedwithBonferronistepdown)inadditiontoB-cellreceptorsignalingandT-cellreceptorsignalingpathways(Fig.4B).Similarly,theimmunegenesidentifiedontemporal

analysisshowedsignificant(p≤0.05)enrichmentofTLRsignalingpathway,RIGsignalingpathway,chemokinesignalingpathway,Class-IMHCpresentationandprocessing,andclassicalantibodymediatedcomplementactivation,etc.,

(Fig.4C).Theenrichmentofthesepathwaysintemporalanalysisclearlyindicatedthattheimmuneresponsewasprogressingfrominnatetoadaptivewithtime.

Fig.3Confirmationofviralinfectionandviraltranscriptquantification:(a).MorphologicalchangesininfectedPBMCsat48hand120hp.i.,showingclumpingofcells.Theclumpingofcellsincreasedat120hp.i.(b).Ngeneamplification(ampliconsize

351bp)ofPPRvirusfrominfectedPBMCsconfirminginfectionat48hand120hp.i(Lane2&3),Negativecontrol(Lane1),M-100bpLadder;(c).Flowcytometryofinfectedcellsat48hand120hp.i.,usingantibodiesraisedagainstthePPRVSungri/96

wholevirus.Thenumberofcellsinfectedincreasedwithtimefrom48hp.ito120hp.i(d).PPRviralgenetranscriptquantificationat48hand120hp.i.NgeneexpressionincreasedastheinfectionprogressedfollowedbyF,H,L,M,andPgeneexpression.

alt-text:Fig.3

Further,thefunctionalprofilingof1416DEGsfromtemporalanalysisshowedenrichmentofimmunesystem,defenseresponse,innateimmuneresponseprocessesandcytokineinteractionsetc.,indicatingthatthehostdefense

wasrespondingbyactivatingitsimmuneresponsegenesanditssignalingpathwaysastheinfectionprogressed.Inaddition,someofthebiologicalprocesseslikecelladhesion,cellmotilityandpathwaysinvolvedincelladhesion,focal

adhesions,adherensjunctions,etc.,wereenrichedindicatingthatthecytoskeletonandcelltocellcommunicationswereaffectedasPPRVinfectionprogressed.

ThecommonDEGs(243)amongcomparativeandtemporalanalysiswerealsofunctionallyenrichedusingg:ProfilerandDAVID.ThecommonDEGsalsoshowedenrichmentforbiologicalprocesseslikeimmunesystemprocess,

inflammatoryresponse,positiveregulationofsignaltransduction,cytokineproduction,MAPKcascade,andinnateimmuneresponse.TheenrichedpathwaysforKEGGshowedcytokine-cytokinereceptorinteraction,phagocytosisand

immunoglobulinproductionasimportantpathwayssuggestingtheinvolvementofbothinnateandadaptiveimmunesystems.Further,onClueGOanalysisoftheseDEGs,enrichmentofTLRsignalingpathway,RIGsignalingpathway,

Class-IMHCpresentationandprocessing,chemokinesignaling,BandT-cellreceptorsignalingpathways,alsoindicatedtheroleofbothinnateandadaptiveimmuneresponsesinPPRVinfectedPBMCs(Fig.S1inSupplementarydata).

3.2DEGsandbroadspectrumanti-viralresponseAmongtheDEGsidentifiedoncomparativeanalysis,Tolllikereceptor7(TLR7),whichisactivatedbysinglestranded(ss)RNAvirus,wasupregulatedat120hp.i.,andTLRs−–2,4,and8weredownregulatedat48h p.i.

AmongtheIRFs−–IRF7at48hp.i,andIRF3,7and9at120hp.i.,whichorchestrateandmaintainthehomeostaticmechanismofthehostdefense,wereupregulatedcomparedtotheuninfectedcontrols.IRF3andIRF7playan

essentialroleininductionofTypeIIFNs(IFN-α/β),buttheseIFNswerenotexpressedinPPRVinfectedPBMCsatbothtimepoints.Also,thebroad-spectrumanti-viralIFN-inducedproteinswithtetratricopeptiderepeats−–IFIT3and

IFIT5wereupregulatedatboth timepointsand IFN-induced transmembraneproteins−– IFITM1andIFITM2wereupregulatedat120hp.i.The tripartitemotif familyofproteins involved in regulationof innate immunesystem

Fig.4ClueGOanalysisoftheImmunegenesidentifiedfromcomparativeanalysisandtemporalanalysis.A.48hp.i.comparativeanalysisB.120hp.i.ComparativeanalysisC.Temporalanalysis.TheGOtermsarerepresentedasnodes.Thegradientfromred

togreenindicatesthepercentassociatedgenesandthediameterofthenodeisthePValuecorrectedwithBonferronistepdown.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

alt-text:Fig.4

processesthroughmodulationofPRRsignalingpathwaysandinhibitionofvirusentry,vizviz.TRIM21,TRIM34,TRIM25,TRIM5andTRIM56at48hp.i.andTRIM25,TRIM56,TRIM2andTRIM36at120hp.i.,wereallfoundtobe

upregulated.Oligoadenylatesynthetases−–OAS1Z,OAS1XandOAS1YthatrespondtoviralPAMPsbyinducingdegradationofviralandcellularRNAstoblockviral infectionswereupregulatedatbothtimepointsandOAS2was

upregulatedat120hp.i.ISG15(Interferonstimulatedgene15),whichactsascriticalanti-viralmoleculeinmanyRNAviralinfections(Influenza,hepatitisC,HIV-1,etc.)wasupregulatedatboth48hp.i(36.5folds)and120hp.i.

(36.75folds).Mx1andMx2–inhibitorsofviralentrywereupregulatedat48hp.i.,andViperin(RSAD2),anantiviraleffectorandHERC5,apositiveregulatorofinnateantiviralresponsewereupregulatedatbothtimepoints.Bcl-6

andBcl-2,whichareanti-apoptotic,werefoundtobeupregulatedat48hp.iand120hp.i.,respectively.TheotherBcl-2familyofproteinslikeBCL2L1andBCL2L2––anti-apoptotic;andBCL2L14andBCL2L15–pro-apoptotic,were

found tobedownregulated.BAG3andBIRC5(anti-apoptotic factors)were found tobeupregulatedat48hp.i.and120hp.i.All the important innate immuneproteinsvizviz. interferon regulatory factors (IRFs), tripartitemotifs

(TRIMs)andinterferonstimulatedgenes(ISGs),whichactasfirstlineofhostdefenseagainsttheinvadingviruseswereallfoundtobesignificantlyupregulatedatbothtimepoints.

AmongothersignificantlyexpressedDEGs,Vimentin(VIM),anintermediatefilamentresponsibleformaintainingcellshapeandintegrityandinvolvedinimmuneresponse,andactinbindingproteins,coroninsvizviz.CORO1B,

CORO1CandCORO2A,involvedincellcycleprogression,andsignaltransductionandapoptosis,weredownregulatedatbothtimepoints.Beta-actin,thehousekeepinggenethatisconstitutivelyexpressedinallthecellsandwidely

usedas anendogenous control in a variety of experimentswas found tobedownregulatedat 120h p.i. This ratified our previous qRT-PCR results,which showed variable expression of beta-actin in PBMCs infectedwith PPRV

(Manjunathetal.,2015).ThissuggesteddownregulationofcytoskeletalproteinsduetoPPRVinfectionintheinfectedPBMCs.

Ontemporalanalysis,TLR10,animportantinnateimmunesensorofviralinfection;interferonresponsegeneslikeIRF8,IFN-inducedtransmembraneproteins–IFITM2andIFITM3,andOAS2,whichrestrictviralreplication

andconferbasalresistancetoviruses;andcomplementC2,C1QA,C1RL,etc.,wereallupregulated.Theresultsof temporalanalysiscorroboratedwiththeresultsofcomparativeanalysis indicatingthatboth innateandadaptive

immunesystemwereactivatedtoestablishananti-viralstateasPPRVinfectionprogressed.

3.3Protein-proteininteractionnetworkofDEHCgenesBioGRIDdatabasewasusedtopredicttheinteractionsforDEHCgenesfromcomparativeandtemporalanalysis.Theprotein-proteininteractionnetwork(PPI)ofDEHCgenesat48hp.iand120hp.i.,showed299and315

interactions,respectively.ISG15,aninterferonstimulatedgene,wasfoundtobehighlyconnectedinteractingwithIFIT3,IFIT5,TRIM25,MX1,HERC5andUSP2at48h.p.i,andIRF3,IFIT3,IFIT5,HSPA1A,EIF2AK2andUSP18at

120hp.i.(Fig.5aandb).IRF3,atranscriptionalregulatorat120hp.i,wasfoundtobeconnectedtoHERC5,IKBKE,ISG15andTLR2amongtheDEHCgenes.Besidesthesegenes,IRF3wasalsoconnectedtoTRIM21amongthe

DEGs.IRF7,anotherkeytranscriptionalregulatorwasconnectedtoCREBBP,EAF1,IQSEC1,GCLM,UBC,TRAF6andTLK2at48hp.i,andSMAD3andIRF3at120hp.i.,amongtheDEGs.TheDEHCinteractionnetworkontemporal

analysisresultedin263interactions,withC2(complement)beingthehighlyconnectednodewithadegreeof49.C2waswellconnectedtoCD4,HDAC10,HDAC11,CCL2,MAP3K5,etc.Thissuggestedinvolvementofcomplementin

linkingadaptiveandinnateimmunityasthePPRVinfectionprogressedwithtime(Fig.5c).

3.4Transcriptionfactorsco-regulatingtheDEHCgenesTFsmodulatingtheexpressionofDEHCgenesfromcomparativeanalysisat48hp.iand120hp.iwereidentifiedasdescribedinmaterialsandmethods.Likewise,TFsfortheDEHCgenesfromthetemporalanalysiswerealso

identified.At48hp.i.,82TFswereidentifiedtocontrol126DEHCgenes.AmongthesepredictedTFs,atotalof6TFs(ETS1,FOXO3,GLIS2,MAFF,NKX3-1,NR2E1)at48hp.iwerefoundtobedysregulatedamongtheDEGs(Fig.

S2inSupplementarydata).Amongthese,ETS1(controlsthedifferentiationof lymphoidcellsandinductionofcytokineandchemokinegenes)andNR2E1(involvedinretinoicreceptorregulation)weredownregulatedandFOXO3

(centralregulatorofinnateimmunefunctionandregulateCD8T-cellresponseinviralinfection),GLIS2(Roleinkidneyarchitectureandfunctions),MAFF(involvedincellularstressresponse)wereupregulated.At120hp.i,72TFs

wereidentifiedbindingtotheupstreamof117DEHCgenes(Fig.S2inSupplementarydata).Outofthese,5TFs–CREB3L1,EBF1,IRF3,SP1andVDRweredysregulatedamongtheDEGsat120hp.i.CREB3L1(whichplaysan

importantroletopreventviralspreadingbyinhibitingproliferationofvirus-infectedcells),EBF1(involvedinB-cellsignaling),IRF3(importantanti-viralmolecule)andSP1(involvedinimmuneresponseandapoptosisatlaterstageof

infection)wereallupregulated,whereasVDR(VitaminDreceptor)wasfoundtobedownregulated.Intemporalanalysis,atotalof70TFswereidentifiedbindingto77DEHCgenes.AmongthepredictedTFsfortemporalanalysis,2

TFswerefoundtobedysregulatedamongtheDEGs(EGR3andNKX3-1).EGR3regulatingBandT-cellfunctioninadaptiveimmuneresponseswasupregulated,assertingtheregulationofbothhumoralandcellmediatedresponse

genesasinfectionprogressed(Fig.S3inSupplementarydata).

3.5ValidationofRNAsequencingdataqRT-PCRSixDEGs(IRF3,IRF7,IFIT3,ISG15,TRIM56andHERC5)havinganimportantroleinimmuneregulationandanti-viralresponsewerevalidatedfortheirexpressionusingrealtimeqRT-PCR(Fig.6).Theexpressionofthese

DEGswasinconcordancewithRNAsequencingresults.TheexpressionofIFNs–αandβwasfoundtobenon–significantatboththetimepoints(Fig.S4inSupplementarydata).

Fig.5Protein-ProteinInteraction(PPI)networksfordifferentiallyexpressedhighlyconnected(DEHC)genesfromcomparativeandtemporalanalysis:(a).Protein-proteininteraction(PPI)networkforDEHCgenesat48hp.ioncomparativeanalysis.(b).

Protein-proteininteractionnetworkforDEHCgenesat120hp.ioncomparativeanalysis.(c).Protein-proteininteraction(PPI)networkforDEHCgenesontemporalanalysis.Theupregulatedgenesareshowningreenanddownregulatedgenesareshownin

redwiththegradientshowingtheextentofexpression.Thesizeofthenodeindicatesconnectivity(i.e.degree).i.e.degree).(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

alt-text:Fig.5

4DiscussionInthepresentstudy,goatPBMCswereinfectedwithSungri/96vaccinevirusandharvestedat48hp.iand120hp.i.PBMCstranscriptomewasevaluatedtoidentifytheglobalgeneexpressionchangesat48h

p.i.and120hp.i,tounderstandanddelineatethemechanismofearlyimmuneprotectioninducedbythevaccinevirus.

Functionalannotationofthesignificant(p≤0.05)DEGsat48hand120hp.i(comparativeanalysis)showedenrichmentofpathways,whichwerealsoobservedtobeenrichedinotherRNAvirusinfections

reportedearlierinseveralstudies(Kuchipudietal.,2014;JinandZou,2013;Chakrabartietal.,2010;Boonetal.,2011).InnateimmunesystemisstimulatedoncetheTLRsareengagedwithPAMPs(Girardinetal.,

2002).TLRs–3,7and8aremainlyinvolvedintherecognitionoftheviralproducts(dsRNAandssRNAviruses)andsubsequentlyinitiatecellularresponsetoinfectionviaviasignalingpathwaysleadingtoearlyevents

inimmuneresponse(Alexopoulouetal.,2001;Dieboldetal.,2004).Inthepresentstudy,TLR7wasupregulated,andTLR-3&8weredownregulatedat120hp.i.,andTLRs–2,4,6and8weredownregulatedat48h

p.i.Thedownregulationofpattern-recognitionreceptors(TLRs),capableofsensingthepresenceofRNAvirus,providesamechanismbywhichtheviruscouldevadehostinnateanti-viralresponses(vanGentetal.,

Fig.6Quantitativereal-timePCRtovalidatetheRNA-seqexperiment.Foldchange(2−ΔΔCT)withcontrolasthecalibratorisrepresentedalongwiththestandarderrorofdifference.Levelsnotconnectedbysameletteraresignificantlydifferent.

alt-text:Fig.6

2011).Also,ontemporalanalysis,TLR10wasfoundtobeupregulatedastheinfectionprogressed.ItwasobservedrecentlythatactivationofTLR10byfunctionalRNA-proteincomplexof influenzavirusleadsto

robustinductionofcytokineexpression(Leeetal.,2014).Thus,inPPRVinfectionTLR7andTLR10mayplayanimportantroleintriggeringthedownstreamimmunesignalingnetworks.

ISGsare inducedbyviral infection toestablishanantiviral state,bothdirectly (by IRF3afterPAMPdetectionandPRRsignaling)and indirectly (by IFN-βproductionand IFNARsignaling)with the later

occurringinbothinfectedanduninfectedcells(Lazearetal.,2013).Theabsenceofexpressionoftype-IinterferonsinourstudysuggestedthatthestimulationofISGscouldbeIFNindependentandIRFdependent,

whichneeds further investigation.These investigationsmaybepossiblewith early and close sampling frames.This though reported in otherparamyxovirus infections (NandaandBaron,2006), is an important

observationnotreportedpreviouslyforPPRVinfection.Previously,non-structuralproteins(C,VandWproteins)ofparamyxoviruseswereshownto inhibitthe inductionoftypeI interferonsindifferentcell lines

(Boxeretal.,2009;NandaandBaron,2006).IRFsupregulatedinthepresentstudy(IRFs–3,7,8and9)werefoundtostimulateISG15bymorethan30foldsatbothtimepoints.ISG15isanearlyinduced,important

innateimmuneproteinwithbroadanti-viralactivity(Hartyetal.,2009).ThisISG15cancovalentlybecoupledtomanyhostcellularproteins(aprocessknownasISGylation),oftenmodulatingtheirfunctions(Durfee

etal.,2010).Antiviralactivityassociatedwithprotein ISGylation invitroand invivohasbeenreported forbothDNAandRNAviruses, including influenzaAandB,Sindbis,vesicularstomatitisvirus, respiratory

syncytial virus andEbola virus (Sadler andWilliams, 2008; Sen andSarkar, 2007;Osiak et al., 2005). ISG15 also conjugateswith some viral proteins, such asNS1protein of influenza, thereby inhibiting viral

replication(Zhaoetal.,2010).OtherISGslikeIFITM2andIFITM3werealsofoundtobeupregulatedinPPRVinfectedPBMCs.TheprotectiverolesofIFIT3andIFIT5werereportedinavianinfluenza(Vandervenet

al.,2012)denguevirus(Hsuetal.,2013)andotherRNAvirusinfectionsthus,reassertingtheirpossibleroleinPPRVinfection.

TRIMfamilyofproteins,positivelymodulateinnateimmunesignalingpathwaystriggeredbypatternrecognitionreceptors(Akiraetal.,2006;McNabetal.,2011;Gacketal.,2008;Tsuchidaetal.,2010).In

thisstudy,TRIM21,TRIM25andTRIM56wereupregulatedatboththetimepoints.TRIM21issignificantlyinducedandinteractswithIRF3uponRNAvirusinfectionandthehostantiviralresponsesaresignificantly

boostedorcrippledinthepresenceorabsenceofTRIM21(Yangetal.,2009).TRIM14andTRIM45werefoundtobeupregulatedintemporalanalysisasthePPRVinfectionprogressed.Recently,itwasobservedthat

TRIM14,acomponentofmitochondrialanti-viralimmunityrecruitsNF-kBessentialmodulator(NEMO)toMAVScomplexinducinginnateimmuneresponsemediatedviaRIG-Iininfluenzavirusinfectionactivating

IRF3andNF-kB(Zhouetal.,2014).TRIM14/21mayactaspossibleadaptorsbetweenPRRs(TLR/RIG-I)andactivationofIRF3/IRF7resultinginefficientcellularanti-viralresponseinPBMCsinfectedwithSungri/96

PPRVvaccinevirus.

4.1PredictedImmunesignalingpathwaytriggeredbySungri/96vaccinevirusBasedontheRNAsequencingdataanalysisandqRT-PCRvalidationofimportantimmunemolecules,animmunesignalingpathwaythatistriggeredfollowinginoculationofSungri/96PPRvaccinevirusin-vitrowaspredicted

(Fig.7).PPRvirusenterscellsthroughspecificreceptor(s)onthesurfaceofthehostcell.SLAManestablishedreceptor(Adombietal.,2011)forPPRV(foldchange>2at48hp.i),expressedat48hp.iandnotat120hp.i.,mayactas

an entry receptor in initial stages of PPRV infection. CD46 that acts as a receptor for vaccine strain of measles virus is downregulated under infection from the cell surface (Tatsuo and Yanagi, 2002). This was found to be

downregulatedat120hp.i.,inourstudy,suggestingCD46asapossiblealternateco-receptorforPPRV,apartfromSLAM,whichneedstobefurtherinvestigated.OncethePPRvirusentersthecellsthroughitsspecificreceptor(s)itis

endocytosedanddue to theacidicenvironmentof theendosomes theviralnucleicacid is released.TLR7, foundonendosomal surface recognizesnegative (−) sensesingle stranded (ss)RNAand isactivatedas indicatedby its

upregulation.TLR7onceengagedwithitsligand(ssRNAviralgenome)activatesfirstlineofdefensemoleculescalledIRFsmainlyIRF3andIRF7(Collinsetal.,2004)whichareupregulatedinPPRVinfectedPBMCs.IRFspresentin

thecytoplasmtranslocatesintothenucleusonphosphorylation,catalyzedbyIKKfamilyofkinases(Collinsetal.,2004).TheIKBKEexpressedinthepresentstudycatalyzesphosphorylationofIRFsthattranslocateintothenucleus.

Also,adaptersplayaroleinlinkingTLRactivationandIRFs(vanGentetal.,2011).TRIM14/21thataresignificantlyupregulated,actaspossibleadapter(s)inthisstudytoactivateIRFs.ThephosphorylationofIRFs,preventsthe

interactionof IRFswithPin1,whichotherwisecausesubiquitination leading tocompletedegradation (Hiscott,2007). IRFs translocated into thenucleus (IRF3and IRF9alongwithSTAT1)bind toupstream interferonstimulated

responsiveelements(ISREs)ofISGsandbringaboutthetranscriptionofISGs(Schmidetal.,2010).Onceoutsidethenucleusandtranslated,proteins−–ISGs,andIRFsactsynergistically(Zhangetal.,2013).ISG15bringsaboutthe

ISGylationofIRF3(Shietal.,2010). ISGsMx1,Mx2,OAS1X,OAS1Y,OAS1Z,RSAD2, IFIT3(ISG58), IFIT5(ISG60), IFITM2andIFITM3expressed/activatedby IRFs(IRF-3,7&9) in thepresentstudyhelp in inducingabroad-

spectrumanti-viralstate(Schneideretal.,2014;SiddappaManjunathetal.,2015).TheexpressionofIRF3,IRF7,IFIT3,ISG15,andHERC5wasvalidatedbyq-RTPCRandfoundinconcordancewiththeRNAsequencingresults.

5ConclusionInsummary,thistimepointstudyhighlightedactivationofhostTLRsandinterferonregulatoryfactors,whichfurtherresultedininductionofinterferoninducedgenesinaninterferonindependentmannerat

earlytimepoints.Anti-viralresponseinducedbySungri/96involvedbothinnateandadaptiveimmunesystemswiththeenrichmentofcomplementcascadeobservedat120hp.i.

ConflictofinterestTheauthorsdeclarenoconflictofinterest.

AcknowledgementsThisstudywassupportedinpartbyDepartmentofBiotechnology(BT/PR7729/AAQ/1/542/2013),GovernmentofIndiaandqRT-PCRwasdonepartlyfromthefundsofCentreforAgriculturalBioinfomatics

(ICAR-IASRI).

AppendixA.SupplementarydataSupplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,athttp://dx.doi.org/10.1016/j.virusres.2016.12.014.

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AppendixA.SupplementarydataThefollowingareisSupplementarydatatothisarticle:

MultimediaComponent1

Highlights

• TemporaldynamicsofimmuneresponseinPBMCsinfectedwithSungri/96vaccinestudied.

• DifferentiallyexpressedgenesreflectedenrichmentofImmuneresponsepathways.

• Theupregulationofinnateimmunegenes−–IRFsandISGsidentifiedatearlyphase.

• Broad-spectrumanti-viralstateobservedundervaccination.

• ViraltranscriptshavebeenquantifiedininfectedPBMCsfromtheRNAsequencingdata.

pleasecontactm.vs@elsevier.comimmediatelypriortoreturningyourcorrections.”Answer:correct

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