comparative and temporal transcriptome analysis of peste
TRANSCRIPT
Comparativeandtemporaltranscriptomeanalysisofpestedespetitsruminantsvirusinfectedgoatperipheralbloodmononuclearcells
SiddappaManjunatha,f
BishnuPrasadMishraa
BinaMishrad
AdityaPrasadSahooa
AshokK.Tiwarib
KaushalKishoreRajakc
D.Muthuchelvanc
ShikhaSaxenaa
LakshmanSantraa
AmitRanjanSahua
SajadAhmadWania
R.P.Singhd
Y.P.Singhe,
ArunaPandeya
SonamKanchana
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
GandhamRaviKumara,⁎
SarathChandraJangaf,g,h,⁎⁎
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.
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