Longitudinal study of age-specific pattern of coronavirus infection in Lyle’s flying fox (Pteropus lylei) in Thailand


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Longitudinal study of age-specific pattern of coronavirus infection in Lyle’s flying fox (Pteropus lylei) in Thailand

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Title:
Longitudinal study of age-specific pattern of coronavirus infection in Lyle’s flying fox (Pteropus lylei) in Thailand
Series Title:
Virology Journal
Creator:
Wacharapluesadee, Supaporn
Duengkae, Prateep
Chaiyes, Aingorn
Kaewpom, Thongchai
Rodpan, Apaporn
Yingsakmongkon, Sangchai
Petcharat, Sininat
Phengsakul, Patcharakiti
Maneeorn, Pattarapol
Hemachudha, Thiravat
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Coronavirus ( local )
Chiroptera ( local )
Pteropus ( local )
Thailand ( local )
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serial ( sobekcm )

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Abstract:
Bats are natural reservoirs for several highly pathogenic and novel viruses including coronaviruses (CoVs) (mainly Alphacoronavirus and Betacoronavirus). Lyle’s flying fox (Pteropus lylei)‘s roosts and foraging sites are usually in the proximity to humans and animals. Knowledge about age-specific pattern of CoV infection in P. lylei, prevalence, and viral shedding at roosts and foraging sites may have an impact on infection-age-structure model to control CoV outbreak. P. lylei bats were captured monthly during January–December 2012 for detection of CoV at three areas in Chonburi province; two human dwellings, S1 and S2, where few fruit trees were located with an open pig farm, 0.6 km and 5.5 km away from the bat roost, S3. Nested RT-PCR of RNA-dependent RNA polymerase (RdRp) gene from rectal swabs was used for CoV detection. The strain of CoV was confirmed by sequencing and phylogenetic analysis. CoV infection was found in both juveniles and adult bats between May and October (January, in adults only and April, in juveniles only). Of total rectal swab positives (68/367, 18.5%), ratio was higher in bats captured at S1 (11/44, 25.0%) and S2 (35/99, 35.4%) foraging sites than at roost (S3) (22/224, 9.8%). Juveniles (forearm length ≤ 136 mm) were found with more CoV infection than adults at all three sites; S1 (9/24, 37.5% vs 2/20, 10%), S2 (22/49, 44.9% vs 13/50, 26.0%), and S3 (10/30, 33.3% vs 12/194, 6.2%). The average BCI of CoV infected bats was significantly lower than uninfected bats. No gender difference related to infection was found at the sites. Phylogenetic analysis of conserved RdRp gene revealed that the detected CoVs belonged to group D betacoronavirus (n = 64) and alphacoronavirus (n = 4). The fact that CoV infection and shedding was found in more juvenile than adult bats may suggest transmission from mother during peripartum period. Whether viral reactivation during parturition period or stress is responsible in maintaining transmission in the bat colony needs to be explored.
Original Version:
Virology Journal, Vol. 15 (2018-02-20).

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k26.5210 ( USFLDC: LOCAL Handle )

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RESEARCHOpenAccess Longitudinalstudyofage-specificpatternofcoronavirusinfectioninLyle’sflyingfox(Pteropuslylei)inThailandSupapornWacharapluesadee1* ,PrateepDuengkae2,AingornChaiyes2,ThongchaiKaewpom1,ApapornRodpan1,SangchaiYingsakmongkon3,SininatPetcharat1,PatcharakitiPhengsakul2,PattarapolManeeorn4andThiravatHemachudha1AbstractBackground:Batsarenaturalreservoirsforseveralhighlypathogenicandnovelvirusesincludingcoronaviruses(CoVs)(mainlyAlphacoronavirusandBetacoronavirus).Lyle’sflyingfox(Pteropuslylei)‘sroostsandforagingsitesareusuallyintheproximitytohumansandanimals.Knowledgeaboutage-specificpatternofCoVinfectioninP.lylei,prevalence,andviralsheddingatroostsandforagingsitesmayhaveanimpactoninfection-age-structuremodeltocontrolCoVoutbreak.Methods:P.lyleibatswerecapturedmonthlyduringJanuary–December2012fordetectionofCoVatthreeareasinChonburiprovince;twohumandwellings,S1andS2,wherefewfruittreeswerelocatedwithanopenpigfarm,0.6kmand5.5kmawayfromthebatroost,S3.NestedRT-PCRofRNA-dependentRNApolymerase(RdRp)genefromrectalswabswasusedforCoVdetection.ThestrainofCoVwasconfirmedbysequencingandphylogeneticanalysis.Results:CoVinfectionwasfoundinbothjuvenilesandadultbatsbetweenMayandOctober(January,inadultsonlyandApril,injuvenilesonly).Oftotalrectalswabpositives(68/367,18.5%),ratiowashigherinbatscapturedatS1(11/44,25.0%)andS2(35/99,35.4%)foragingsitesthanatroost(S3)(22/224,9.8%).Juveniles(forearmlength136mm)werefoundwithmoreCoVinfectionthanadultsatallthreesites;S1(9/24,37.5%vs2/20,10%),S2(22/49,44.9%vs13/50,26.0%),andS3(10/30,33.3%vs12/194,6.2%).TheaverageBCIofCoVinfectedbatswassignificantlylowerthanuninfectedbats.Nogenderdifferencerelatedtoinfectionwasfoundatthesites.PhylogeneticanalysisofconservedRdRpgenerevealedthatthedetectedCoVsbelongedtogroupDbetacoronavirus(n=64)andalphacoronavirus(n=4).Conclusions:ThefactthatCoVinfectionandsheddingwasfoundinmorejuvenilethanadultbatsmaysuggesttransmissionfrommotherduringperipartumperiod.Whetherviralreactivationduringparturitionperiodorstressisresponsibleinmaintainingtransmissioninthebatcolonyneedstobeexplored.Keywords:Coronavirus,Chiroptera,Pteropus,ThailandBackgroundCoronaviruses(CoVs)areclassifiedintofourgenera:Alphacoronavirus(CoV),Betacoronavirus(CoV),Gam-macoronavirus,andDeltacoronavirus[1].CoVsinfectwidevarietyofmammalsandbirds,causingupperandlowerrespiratory,hepatic,entericandneurologicalill-nesseswithvaryingseverity.BatCoVs(BtCoVs)arelikelythegenesourceofCoVandCoV,whileavianCoVsaresourcesofGammacoronavirus,andDeltacoronavirus[2].AlthoughthereissinglelineageinCoV,CoVsarefur-therseparatedintofourlineages(A–D)[3].LineageACoV,includingbovineCoVs,humanCoV(HCoV)-OC43andrelatedviruses,havebeendetectedinvariousmam-malssuchascows,horses,deer,antelopes,camels,gi-raffes,waterbucks,dogs,andhumansworldwide,butnotinbats.LineagesB-DCoVshavebeendetectedinbatsworldwide[4].Currently,sixCoVstrainsareknowntocausehumaninfection;fourCoVscausemildrespiratoryillness, *Correspondence:spwa@hotmail.com1ThaiRedCrossEmergingInfectiousDiseases-HealthScienceCentre,WorldHealthOrganizationCollaboratingCentreforResearchandTrainingonViralZoonoses,ChulalongkornHospital,FacultyofMedicine,ChulalongkornUniversity,Bangkok,ThailandFulllistofauthorinformationisavailableattheendofthearticle ©TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.Wacharapluesadeeetal.VirologyJournal (2018) 15:38 https://doi.org/10.1186/s12985-018-0950-6

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includingtwoCoVs:HCoV-NL63andHCoV-229E,andtwoCoVs:HCoV-HKU1andHCoV-OC43[5].TheothertwoCoVscausesevererespiratorytractinfectionwithhigh-fatalityrates,suchassevereacuterespiratorysyn-drome(SARS)andMiddleEastrespiratorysyndrome(MERS),belongingtolineagesBandC,respectively.BatrelatedMERS-CoVsphylogenetically-relatedtohumanshavebeenpreviouslydiscoveredinTylonycterispachypus(BtCoV-HKU4)andPipistrellusabramus(BtCoV-HKU5)inHongKong,in2006[6],Neoromiciacapensis(Neo-CoV)fromSouthAfrica,in2011[7],andPipistrelluscf.hesperidus(PREDICT/PDF-2180CoV)fromUganda,in2013[8].SARS-likeBtCoVwasinitiallyidentifiedfromthegenusRhinolophusin2005,aftertheSARSoutbreakinhumansin2002–2003,andidentificationofvirusinpalmcivets(Pagumalarvata)fromliveanimalmarketinGuangdong,Chinain2003[9,10].BtCoVshavebeenidentifiedinmanyinsectivorousandfrugivorous(familyPteropodidae)batsonmanyconti-nents:America,Europe,Africa,andAsia[4].Differentspe-ciesofPteropodidaehavebeenidentifiedasamajorsourceoflineageDCoV(HKU9)inAfrica(Rousettusaegyptia-cus,Kenya[11],PteropusrufusandEidolondupreanum,Madagascar[12]),andAsia(R.leschenaulti,China[13],Cynopterusbrachyotis,Philippines[14],Ptenochirusjagori,Philippines[15],Pteropusgiganteus,Bangladesh[16],Cynopterussphinx,Thailand[17].Thailandishometo146batspecies(125insectivorousand21frugivorous)[18].TheprevalenceanddiversityofBtCoVsinThailandhasbeenstudiedinthelastdecade[17,19,20].CoVswerefoundin11insectivorousbatspe-ciesandin2frugivorousbatspecies.However,datafromPteropusbatshavebeenlackingdespitePteropusbeingthebiggestcolonyofPteropodidaeinThailand.Threespe-cies(P.lylei,P.vampyrusandP.hypomelanus)arereser-voirsofNipahvirus(NiV)inThailand[21].TheprevalenceofNiVRNAinurineofP.lyleihasbeensea-sonallydetectedduringthemonthsofMayandJune[22].P.lylei(Lyle’sflyingfox(LFF))rangesfromYunnaninChina,andextendstoCambodia,Thailand,andVietnam[23].Upto20colonieshavebeenidentifiedinThailand[24]andthelargestknowncolonycomprisesofabout10,000individuals[22].Itsharesforagingareaswithotherfrugivorousbatsinfruittrees,fromwhichthefruitsarealsosharedbyhumans.Moreover,treesinpopulatedtemplegroundsandcultivatedlandarecom-monroostingsitesforLFF.Thus,consumptionofpar-tiallyeatenfruit,uncookedmeat,orcontactwithsaliva,urineorfaeces,whichcanbecontaminatedwithbatvi-ruses,posesariskofviraltransmissionfromLFFtohumansordomesticanimals.Thepotentialforemergenceofzoonoticvirusesintothehumanpopulationdependsontheprevalenceofthevirusinitshostspecies,hostrangemutationswithinviralquasispecies,andthedegreetowhichthereservoirhostinteractswithhumans[25].Tobetterunderstandtheprevalence,persistence,phylogeny,andpotentialforinteractionwithhumans,herewedescribeacomprehen-sivelongitudinalstudytodetectCoVinLFF,andfactorsinfluencinginfectivity.Batrectalswabswerecollectedmonthlyfromtheirroostingareaandfromtwohumandwellings(foragingsites)nearby.Individualbatswereweighedandforearm(FA)lengthsweremeasuredforfurthercharacterizationonitsbodycompositionindex(BCI).Ourresultsdemonstratedforthefirsttimethat-and-CoVsareendemicallycirculatinginLFFsinThailand,andthatageandBCIaresignificantlydifferentbetweeninfectedanduninfectedbats.MethodsStudysitesLFFswerecapturedfromthelargestcolonyinThailand(totalpopulationofaround10,000bats)[22]atChonburiprovince(Luangtemple,13,830,018.9”N,101809054.9E,6masl)inCentralThailand.Batsweresampledfromthreedifferentsites:twohumandwellings(batforagingareas)situatedatameandistanceof0.6km(S1)and5.5km(S2withasmallopen-systempigfarm,40pigs)fromabatroost,andthebatroost(S3).Samplingwascar-riedoutunderprotocolsapprovedandpermittedbytheDepartmentofNationalParks,WildlifeandPlantConser-vation,Thailand(No.0909.204/2686)andtheAnimalUseProtocolNo.1473001approvedbyChulalongkornUniver-sityAnimalCareandUseCommittee.BatcaptureandsamplecollectionLFFswerecapturedmonthlyduringJanuary–Decem-ber2012fromthethreesites,S1–3(Table1).AtS1andS2,batswerecaptured2nightspermonth,wherethenetsweresetinthelateevening,andcapturedanimalswereremovedimmediately.AtS3,10–20batswerecap-turedusingmist-netsonthesamenightsasS1andS2.Batswerenoteuthanized,andtheywerereleasedaftermeasurementsweretakenandsampleswerecollected.Batswereidentifiedmorphometricallyandspecies,sex,reproductivestatus,FAlengthandbodymasswerede-termined.Rectalswabwascollectedfromeachindivid-ualbatandimmediatelyputintoLysisbuffer(bioMérieux,SA,France).Thesamplesweretransportedtolaboratoryonicewithin48handstoredat-80oCuntilfurtheranalysis.NucleicacidextractionandCoVRNAdetectionTotalnucleicacidwasextractedfrom1mlofsuspendedrectalswabusingeasyMAG®platform(bioMérieux,SA,France).Elutionvolumewas50l.Hemi-nestedReverseTranscriptionPCR(RT-PCR)wasperformedusingbroadlyreactiveconsensusPCRprimersforCoV,Wacharapluesadeeetal.VirologyJournal (2018) 15:38 Page2of10

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targetingtheRNA-dependentRNApolymerase(RdRp)gene.Atotalof5lofextractednucleicacidwasaddedto50lofreactionmixtureofOneStepRT-PCRkit(QIA-GEN,Hilden,Germany),permanufacturer’sinstructions,andreactedwitheachforwardprimerandreverseprimer[14].Hemi-nestedPCRamplificationswereperformedusing2loffirstamplificationproductand48lofreac-tionmixturecontaining1.0unitofPlatinumTaqDNApolymerasein2.5mMMgCl2,400MdNTPs,0.6Mofsecondforwardprimerand0.6MofthesamereverseprimerasthefirstroundofRT-PCR.Amplificationprod-uctof434bpwasvisualizedusing2%agarosegelelectro-phoresis.AllpositivePCRproductswerefurthersequencedforconfirmationandstraincharacterization.SequencingandphylogeneticanalysisTheRdRpPCRproductsweregelpurifiedusingtheNucleoSpin®GelandPCRClean-upkit(MACHEREY-NAGELGmbH&Co.KG),andsequenceddirectlyusinganautomatedABIPRISM377DNAsequencer.Whenmultipeakswereshowninchromatogramatsamepos-itionfromdirectsequencing,PCRproductswereclonedusingthepGEM®-TEasyVectorSystemandtheLigaFast™RapidDNALigationSystem(Promega)beforesequen-cing.Fivecolonieswerepickedupforsequencing.Se-quenceswerecleanedusingBio-editprogramandalignedwithreferencesequencescollectedfromGenBank.Align-mentswereperformedusingMultipleAlignmentusingFastFourierTransform(MAFFT)[26].Phylogenetictreeswerecreatedbasedon357and299bpRdRpgenese-quenceusingthemaximumlikelihoodmethod.Bootstrapvaluesweredeterminedusing1000replicatesviaRaxml-GUI1.3withoutgroup(BulbulCoV/HKU11–934/Pycno-notusjocosus/CHN/2007/FJ376619)usingtheGTRIsubstitutionmodel[27].Thephylogenetictreewasvisua-lisedusingtheFigTreeprogram,version1.4.2[28].StatisticalanalysisWeconsideredtherelativelevelofCoVinfectioninvar-iablesofbat.WeusedChi-squareandFisher’sexactteststodeterminetheprevalencepatternofCoVbyexaminingwhethercuesrecordedineachkindofvari-ables(location,sex,ageandseason)differedfromex-pected.AllstatisticaltestswerecompletedinRstatisticcomputing(version3.2.2)withp<0.05interpretedasbeingstatisticallysignificant.Thebodyconditionindex(BCI)wasdefinedasbodymassdividedbyFAlength.ToassessdifferencesinBCIbetweenCoVinfectedanduninfectedbats,ANOVAwithTukey’stestforpair-wisecomparisonswasusedforanalysis.ResultsSamplecollectionOnlytheP.lyleispecies(LFF)wasincludedinthisstudy.Atotalof367bats(220maleand147female)werecap-turedandsampled.TotalnumberofcapturedbatsfromsitesS1,S2andS3were44,99,and224respectively(Table1).FAlength(136mm)wasusedtodistinguishbetweenjuvenileandadult[29].BodymassandFAlengthsweredeterminedfor352bats(95.9%).FAlengthsofjuveniles(n=96)rangedfrom79.23–136.0mm,andinadults(n=256)rangedfrom136.47–170.0mm.Bodymassofjuvenilesrangedfrom124.0–307.0g,whileadultsrangedfrom212.0–658.0g.TheBCIinjuvenilesandadultsrangedfrom1.08–2.32and1.43–4.27respectively.TheratioofjuvenileandpupperadultbatscapturedfromsitesS1(24:20,1.2)andS2(49:50,0.9)weresimilar,butalowerratiowasfoundatsiteS3(30:194,0.15)(Table1).Numberofbatstrapped/capturedeachmonthvariedbe-tween14and46bats;minimumof10wascapturedeachmonthattheirroost(S3)ascontrol.JuvenilebatswerenotcapturedinJanuarythroughtoMarchfortesting,astheratioofjuvenileandadultbatsinnaturalpopulationis Table1CoVpositivebats,categorizedbypossibleinfluentialfactors-collectionsite,age,sexandBCI(367bats)PossibleinfluentialfactorCoVPCRresults:No.Positive/no.oftested(%)S1(house)S2(pigfarm)S3(roost)ALLSITESAgeaJuvenile(J)9/24(37.5)22/49(44.9)10/30(33.3)41/103(39.8)Adult(A)2/20(10.0)13/50(26.0)12/194(6.2)27/264(10.2)Total11/44(25.0)35/99(35.4)22/224(9.8)68/367(18.5)Ratio(J:A)24:20=1.2049:50=0.9830:194=0.15103:264=0.39SexMale6/27(22.2)16/51(31.4)14/142(9.9)36/220(16.4)Female5/17(29.4)19/48(39.6)8/82(9.8)32/147(21.8)Total11/44(25.0)35/99(35.4)22/224(9.8)68/367(18.5)MeanBCIb(352bats)Allbats1.831.942.902.50Positive1.631.762.151.86Negative1.892.042.992.65aJ=juvenilebat(forearmlength136mm)A=adultbat(forearmlength>136mm)bMeanBCIiscalculatedfrom352batsthoseFAlengthandbodymassweremeasuredWacharapluesadeeetal.VirologyJournal (2018) 15:38 Page3of10

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lowduetotheLFF’sbreedingcycle,whichisonceayearfromNovembertoFebruary[22].Thusnew-bornsarede-liveredinFebruary/March,andweaningjuvenilebatsaremostlyobservedinMay.Ofthe147femalescaptured,18wereatactivebreedingageevidentbyeitherlactating(n=10,whichthespecimenswereavailablefortestingfrom9pups),beingpregnant(n=4)orhavingenlargednipplesindicativeofpreviouslactation(n=4).Virusdetection-prevalenceSixtyeightof367(18.5%)rectalswabsfromLFFwerepositiveforCoVbyfamilywideCoVPCR[14].AsshowninTable1,CoVRNApositivebatswerefoundin16.4%(36/220)malebats,and21.8%(32/147)infemalebats.Therewasnosignificantdifferenceintherateofinfectivitywhencomparingsex(p>0.05).ThenumberofCoVpositivejuvenileandadultbatsfromallthreesites(n=367bats)were39.8%(41/103)and10.2%(27/264),respectively(Table2).CoVpositivejuvenilebatsfromsitesS1-S3were37.5%(9/24),44.9%(22/49),and33.3%(10/30),respectively.CoVpositiveadultbatsfromsitesS1-S3were10%(2/20),26.0%(13/50),and6.2%(12/194),respectively(Table1).StatisticalanalysisshowedthatCoVinfectivitybetweenjuvenileandadultbatssignificantlydiffered(p<0.01),indicatingthatCoVinfectionfavouredjuvenileLFFs.Noneoftherectalswabsfromthe18pregnantorlac-tatingadultfemalebatstestedpositiveforCoV.Interest-ingly,threeattachedpubs,butnottheirmothers,fromatotalof9pairswerefoundpositiveforCoVRNA.BCIofthetwopupswithavailabledatawere1.69(211gbodymass/125mmFA)and1.42(170gbodymass/120mmFA),whichwerelowerthantheuninfectedmeanforjuvenilebats(1.72)(Table3).TheprevalenceofCoVinfectioninbatsfromsitesS1-S3were25.0%(11/44),35.4%(35/99)and9.8%(22/224),respectively(Table1).StatisticalanalysisshowedthatthenumberofCoVinfectedbatsatthedifferentsitesdifferedsignificantly(p<0.01).Frequencyofviraldetectionwashigheratthesetwoforagingsitesthantheroost(2=36.31,p<0.001).However,theprevalenceofCoVinfec-tioninjuvenilebatsfromthe3siteswassimilar,37.5%,44.9%,and33.3%,respectively(Table1).Ageandcondi-tionsofbatswhichmayreflecttheirphysicalhealthandfitnessmayinfluencetheselectionofforagingsiteandtheirvulnerabilitytoinfection.Duringthesameyearofstudy,trackedbatsfromthiscolonymostlyforagedinfarmland,plantations,andgardenswiththemaximumlin-eardistancesfrom2.2–23.6kmbetweendayroostsandforagingareas[30].TemporaldynamicsofviralsheddingCombiningdatafromallsites,theCoVpositivebatswerefoundin8of12months,exceptFebruary,March,Novem-berandDecember.HighprevalentseasonswerefromMaytoAugust,withhighestinJune(14/29,48.3%).HighestprevalenceinjuvenileswerefoundinMay(13/21,61.9%),andinadultsinJune(3/9,33.3%).Therewashigherpreva-lenceofCoVinfectionamongjuvenilethanadultbatsdur-ingApril–October(Table2).InJanuary,4CoVpositiveadultbatswerefoundatS3.IndividualBCIoftheonefe-maleand3malebatswere2.83,1.77,1.84,2.75,respect-ively,whichwerelowerthanthemeanBCIinuninfectedadultbats(2.88)(Table3).Weanalysedthemonthlypreva-lenceofCoVinfectivityinjuvenileandadultbats,andcombined(Table2).TherewassignificantdifferenceintheseasonalprevalenceofCoVinfectionandsheddinginadults(p<0.05),butnotinjuvenilebatsorcombined. Table2NumberofbatsPCR-positiveforcoronavirusbymonthandageafromSI,S2andS3MonthJuvenileAdultTotalTestedPositive(%)TestedPositive(%)TestedPositive(%)January00(0)144(28.6)144(28.6)February00(0)140(0)140(0)March00(0)200(0)200(0)April122(16.7)340(0)462(4.3)May2113(61.9)183(16.7)3916(41.0)June2011(55.0)93(33.3)2914(48.3)July217(33.3)257(28.0)4614(30.4)August94(44.4)265(19.2)359(25.7)September123(25.0)233(13.0)356(17.1)October41(25.0)232(8.7)273(11.1)November30(0)330(0)360(0)December10(0)250(0)260(0)Total10341(39.8)26427(10.2)36768(18.5)aJuvenilebat:forearmlength136mmWacharapluesadeeetal.VirologyJournal (2018) 15:38 Page4of10

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BCI–InfectedbatsTherewassignificantdifferencebetweenFAandbodymassofCoVpositivebatscomparedtouninfectedbats(pvalue<0.01).BCIanalysiswasperformedonbatswithcompletedataofFAandbodymass(352bats).ThemeanBCIofcap-turedbatsinthestudyvariedeachmonth(Fig.1).BCIoftotaltestedbats(Fig.1)anduninfectedbats(Fig.2)showsimilarseasonality.ThelowestmeanBCIofbothtotalnumberoftestedbatsandCoV-positivebatswerefoundinJune(mean2.05and1.62respectively)(Figs.1and2)whenCoVinfection(48.28%)wasmostprevalent(Table2).Inuninfectedbats,thelowestmeanBCIwasfoundinJuly(2.12)ratherthanJune(2.44)(Fig.2).Fromthisstudy,theCoVinfectedbatshadsignificantlylowermeanBCIthanuninfectedbats,1.86and2.65respectively(pvalue<0.01)(Table3).TheBCIofCoVinfectedbatsvariedbetween1.11and3.36(mean1.86)whilsttheBCIofuninfectedbatswasbetween1.08and4.27(mean2.65).TheBCIofunin-fectedjuvenilebatswasbetween1.08and2.32(mean1.72),whilsttheBCIofuninfectedadultbatsvariedbetween1.43and4.27(mean2.88).TheBCIofCoVinfectedjuvenilebatswasbetween1.11and2.26(mean1.61),whilsttheBCIofCoVinfectedadultbatsvariedbetween1.52and3.36(mean2.23).ThemeanBCIofinfectedjuvenileandadultbatsweresignificantlylowerthanuninfectedjuvenileandadultbatsrespectively,(pvalue<0.01,pvalue<0.01re-spectively)(Fig.3).Therewasstatisticallysignificantdiffer-enceinthemeanbodymass(pvalue<0.01)andmeanFAlength(pvalue<0.01)betweenCoVinfectedandunin-fectedbats(Table3).PhylogeneticanalysesSixty-eightCoVsequencesweredepositedinGenBankwithaccessionMG256395-MG256474andMG333996-MG333999.Phylogeneticanalysisof357bpofRdRpgeneusingraxmlGUIprogramrevealedthat64of68de-tectedCoVsbelongedtoCoVgenus,roostingwithHongKongstrain,BtCoVHKU9(R.lechenaulti,EF065513)andKenya(R.aegyptiacus,GU065422),whiletheother4belongedtogroup1ACoV(Fig.4a).The Table3Rangeandmeanofforearm(FA),bodymass,andbodyconditionindex(BCI)ofbatsinthisstudy(352bats)classifiedbyageand/orCoVinfectionstatusBatcharacteristicsFA(mm)Range(mean)BodyMass(g)Range(mean)BCIRange(mean)CoVpositivebats117–160.28(135.14)141–538(255.19)1.11–3.36(1.86)CoVnegativebats79.23–170.0(147.55)124–658(396.47)1.08–4.27(2.65)CoVpositivejuvenilebats117–136(128.25)141–295(206.75)1.11–2.26(1.61)CoVnegativejuvenilebats79.23–135.82(128.82)124–307(221.79)1.08–2.32(1.72)CoVpositiveadultbats136.67–160.28(145.34)212–538(326.96)1.52–3.36(2.23)CoVnegativeadultbats136.47–170(151.51)215–658(439.19)1.43–4.27(2.88)TotalJuvenilebats79.23–136.0(128.58)124–307(215.52)1.08–2.32(1.67)TotalAdultbats136.47–170(150.86)212–658(427.35)1.43–4.27(2.81) Fig.1Bodyconditionindices(BCI)of352batscapturedinthestudyfromJanuarytoDecember2012.Batswerecapturedmonthlyatthreesites(S1-S3).Numbersinbracketsindicatesamplesizefrom3sites.Boxesdepictthe25thand75thpercentiles,lineswithinboxesmarkthemedian,redspotandnumberrepresentmean,whiskersrepresentsminimumandmaximumvalues,andcirclesindicatesoutliersWacharapluesadeeetal.VirologyJournal (2018) 15:38 Page5of10

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CoVsfromthisstudyclusteredinthesamecladeandshared95.5–100%nucleotideidentitywitheachother(98.3–100%identityof118aminoacids).Twoindividualbats(BRT55709andBRT55734)werefoundtobeco-infectedwithmultiplestrainsofthesameCoVspecies(differenceof1–2aminoacids).TheseviruseshadaminoacidsdifferingfromtheHKU9BtCoV,groupDCoVrefer-encestrainby11.7–14.2%.TheyformedadifferentcladetootherCoVsfromthesamebatgenus(Pteropus)fromMadagascar’sP.rufus(Fig.4b).However,theywereinthesamecladewithCoVsfromdifferentbatspeciescapturedatthesamesitewiththisstudy;Cynopterussphinx,Scoto-philusheathii,andScotophiluskuhlii(GenbankaccessionnumbersKJ868722,KJ020607,KJ020608,respectively)[17].DiscussionThisisthefirstlongitudinalstudyofCoVinfectioninwildbatsinThailand,where367LFFbatswerecapturedmonthlyforoneyearatoneroostingsiteandtwo Fig.2Bodyconditionindices(BCI)ofbatstestednegative(gray)andpositive(brown)inthestudy.BatswerecapturedmonthlyfromJanuarytoDecember2012atthreesites.Rectalswabsfrom352batsweretestedforCoVbyPCR.Numbersinbracketsindicatesamplesizefrom3sites.Boxesdepictthe25thand75thpercentiles,lineswithinboxesmarkthemedian,spotandnumberrepresentmean,whiskersrepresentsminimumandmaximumvalues,andcirclesindicatesoutliers Fig.3Bodyconditionindices(BCI)ofbatstestednegative(gray)andpositive(brown)inthestudy.Rectalswabsfrom352batsweretestedforCoVbyPCR.Forearmlength136mmwasusedtoclassifybatsasjuvenile.Numbersinbracketsindicatesamplesize.Boxesdepictthe25thand75thpercentiles,lineswithinboxesmarksthemedian,whiskersrepresentsminimumandmaximumvalues,andcirclesindicatesoutliersWacharapluesadeeetal.VirologyJournal (2018) 15:38 Page6of10

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foragingsitesclosetothebatroost.Onefourthofbatswerejuvenile,and59.9%weremale.Theratiosbetweencapturedjuvenileandadultbatsweredifferentatthebatroostandforagingsites.Only13%ofjuvenile(30/224)batswerecapturedatthebatroostintheyearofthestudy,whereashalfofthejuvenilebatswerecapturedfrombothforagingsites(24/44,49/99,fromS1andS2respectively).Themaximumlineardis-tancesbetweenroostsandforagingareasofLFFatthissitevariedfrom2.2–22.3km[30].Foragingsitesnearroost,evenwithlimitedfoodsources,maybepracticalforyoungorunhealthybatsthatareunabletoflyfar. LineageLineage Lineage LineageHKU 10CoV512HKU7 CoV1AHKU8 HKU2 Betacoronaviruse Ce B eAe DAlphacoronavirus2 ClustePteropClustePteropClustePterop ClusteHKU9r 4 us, Thailand r 3 us,Madagascar 2 us,Madagascaer 1 , Hong Kong ar 2 ar1ab Fig.4Maximumlikelihoodphylogenetictreesofcoronavirus(CoV)generatedusing357(a-BatCoVs)and299(b-LineageDCoVs)nucleotidesoftheRdRpgenesequencesofCoVsfromThailand;inLFF(thisstudy-blue),otherbatspeciesfrompreviousstudyinThailand[15](green),fromMadagascar’sPteropusrufus[17](b,pink)andreferencestrainsofCoVsgroup(black).OnlytherepresentativesequencesofLFFCoVswereusedforanalysis.TheraxmlGUI1.3andtheGTRIsubstitutionmodelwith1000bootstrapwereusedforgeneratingbothphylogenetictrees.TreeswerevisualizedusingFigTree1.4.2.BuCoV/HKU11–934/Pyc_joc/CHN/2007/FJ376619wasusedasanoutgroupfortreeAWacharapluesadeeetal.VirologyJournal (2018) 15:38 Page7of10

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CoVRNAwasdetectedinapproximately18%ofallbatssampled,whichisinthesamerangeasthestudyinChina(16%,[31];15.8%,[32]),andGermany(9.8%,[33]).TheprevalenceofCoVinfectioninPteropusbats(P.rufus)fromMadagascarwassimilartothisstudy(17.1%,13/76)[12].Ontheotherhand,theprevalenceinthisstudywashigherthanthetwopreviousstudiesinThailandbyWacharapluesadeeetal.(6.7%,47/626)[17]andGouilhetal.(10.5%,28/265)[20].Thismaybetheresultofabiasfromthecrosssectionalstudyofthesetwopreviousstudiesoranindicationofdifferenceinprevalencerateindifferentbatspecies.Ratiosofcapturedbatgendersinthisstudywereroughlysimilaratforagingsites.Attheroost,malebatswerepredominantlycaptured.CoVinfectionwasnotcorrelatedwithsexofbat,neitherattheroostnorattheforagingsites.ThisfindingissimilartothestudiesfromGermany[33]andColorado,USA[25].Inourstudy,CoVinfectionwasfoundtobeassociatedwithyoungerages;39.8%ofjuvenilebatsversus10.2%adultbatswerepositiveforCoVRNA.SimilarfindingshavebeenreportedfromthestudyininsectivorousbatsfromUSA(19%juvenileversus9%adultbatspositiveforCoV)[25]andVespertilionidbatsinGermany(23.7%juvenileversus15.9%sub-adultversus8.5%adultbatspositiveforCoV)[33].ThesefindingssupportthehypothesisthatyoungbatsmaybemoresusceptibletoCoVinfection,andservestopropagateandplayanim-portantroleinmaintainingtheviruswithinbatcolonies.ThedivergenceinrateofCoVinfectionfromdifferentstudysites(Table1)waslikelytobeinfluencedbytheageandbodyconditionofbats.Threeof9unweanedpupswereCoVRNApositive,whiletheirmothersandalllactatingfemalecarryingpupswerenegativeforCoV.Itmaybepossibletherewasaplacentaltransmission,afterwhichtheviruswasthenclearedfromadultfemalebats.Anotherpossibilityisthattheunweanedbatsacquiredinfectionfromcon-taminatedsecretionofotherbatshangingfromthesametree.However,thestudybyGloza-Rauschetal.2008[33],where54of178(30%)ofstudiedfemalebatswerelactating,foundhigherrateofCoVinfectioninlactatingbats(22.4%)thaninnon-lactatingbats(9.7%)whichsupportsthefirstscenario.Itistobenotedthatlimitednumberoflactatingbatswereincludedinourstudy(9of147,6.1%).Targetingmother-puppairsinfuturestud-ieswouldberequiredtoconfirmthevertical(placental)transmissionofCoVinLFF.Seasonalprevalencewasmostlyrelatedtothenumberofjuvenilebatscapturedfortestingineachmonth(Table2),exceptinJanuarywhenallfourCoVpositivebatswereadult.Notably,thesepositiveadultbatshadlowerBCI(2.83,1.77,1.84,2.75)thanthemeanunin-fectedadultbats(2.88).Threeofthe4infectedadultbatshadlowerbodymass(444,429,258,276g)thanmeanuninfectedadultbats(439g).Themeanbodymassofinfectedbatswassignificantlylowerthaninun-infectedbats(Table3).ThisissimilartothestudywhereHipposiderospomonabatsinHongKongwithHKU10CoVinfectionhadlowerbodymassthanuninfectedbats,eventhoughtheyappearedtobehealthy[34].Thesebatsseemedtobeinpoorcondition,servingastheothergroupinadditiontojuvenilebatsthatfurthermaintainedtheviruswithinthepopulation.SixtyeightCoVsweredetectedfromthisstudy,form-ing2geneticallydistinctstrains.SixtyfourbelongedtoCoV(SARS-relatedgroup)withrelativelyclosehom-ologytothereferencevirus,BtCoV-HKU9[6].FourbelongedtoCoV,andtheirsequencesrelatedtoCoVspreviouslydetectedininsectivorousbatsinThailandsuchasH.lekaguli,H.armigerandTaphozousmelano-pogon[17].Thissupportsthepossibilityofinterspeciestransmission,ratherthanvirus-hostspecificsharing,be-tweenbatsofdifferentsuborder(PteropusinPteropodi-dae,HipposideridaeandEmballonuridae)thatdonotsharefood,foragingsites,orroosts,similartotheearlierHKU10CoVstudybetweenR.leschenaultiandH.po-monabats[15].TheevolutionofCoVsindifferenthostspecies-ordershouldbefurtherstudiedinordertounderstandtherouteofspilloverandtransmission.Batsfromdifferentspecies-genusthatshareforagingsitesmayalsoshareinfectionsandparticularCoVstrains,forexampleCoVfromLFF(thisstudy),C.sphinx,S.Heathii,andS.kuhlii[17](Fig.4a-b).CoVfromsamebatgenusindifferentgeographicregiondisplayeddistinctclusters(Fig.4b),forexampleP.rufusfromMadagascar(cluster2–3)[12]andLFFfromthisstudy(cluster4).ThisdemonstratedtheCoVinclinesinterspeciessharingra-therthanvirus-hostspecificsharing.GiventhemobilityofLFFinThailand,wherethemax-imumlineardistancebetweendayroostsandforagingareasforLFFis23.6km[29],anditstendencyforshar-inghabitatwithothercolonies,thedetectedstrainsofCoVsfromthisstudymaybefoundinLFFsallovertheregion.ThehighprevalenceofCoVinthisstudysug-gestscirculationofinfectionwithinthebatcolony.StudyofCoVdiversityfromotherLFFcolonyinThailandandregionisrequiredtoimproveourunder-standingoftheevolutionandspilloverpatternsofCoV.ConclusionsOurstudyfoundthatCoVtransmissioninLFFoccurredthroughouttheyearatabaselinelevel,andthemonthssurroundingthebirthingseason(May–August)repre-sentedtimesofincreasedinfectionamongjuveniles.TheCoVprevalenceinLFFrelatedmostlytotheageofbatratherthanlocation,sexorseason.TheinterspeciestransmissionofCoVamongdifferentbatgenusorfamilyWacharapluesadeeetal.VirologyJournal (2018) 15:38 Page8of10

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demonstratedthepossibilityofspilloverandthepoten-tialforemergenceofzoonoticvirusesintothehumanpopulation.Thisdataprovidesthefirstlong-termmoni-toringofCoVcirculationinnatureandidentifieseco-logicaldrivers.Therelationshipbetweenanimalageandinfectivitytootherbatspeciesshouldbefurtherinvesti-gatedtoconfirmthisphenomenon.AdditionalstudiesonCoVdiversityamongPteropusbatspeciesinThailandandneighbouringcountries,aswellasaspectsofthevirus-hostinteractionareneededtounderstandtheorigins,evolution,maintenancepatterns,dispersalandzoonoticpotentialofCoVacrosstheregion.AbbreviationsANOVA:Analysisofvariance;BCI:Bodycompositionindex;bp:Basepairs;BtCoV:Batcoronavirus;CoV:Coronavirus;DNA:Deoxyribonucleicacid;FA:Forearm;LFF:Lyle’sflyingfox;MERS:MiddleEastrespiratorysyndrome;NiV:Nipahvirus;nt:nucleotides;PCR:PolymeraseChainReaction;RdRp:RNA-dependentRNApolymerase;RNA:ribonucleicacid;RT-PCR:ReverseTranscription-PolymeraseChainReaction;S1:Studysite1–humandwelling(0.6kmfrombatroost);S2:Studysite2–humandwelling,smallopen-pigfarm(40pigs)(5.5kmfrombatroost);S3:Studysite3–Batroost;SARS:Severeacuterespiratorysyndrome;CoV:Alphacoronavirus;CoV:BetacoronavirusAcknowledgementsWegratefullyacknowledgethelocalsupportfromtheThaiRedCrossSociety,ChulalongkornUniversity,KasetsartUniversityandtheDepartmentofNationalParksWildlifeandPlantConservation.WewouldalsoliketothankSiripornGhaiforherintensiveeditingofthemanuscript.FundingThisstudywassupportedbyaresearchgrantfromtheThailandResearchFund(RDG5420089);theRatchadaphiseksomphotEndowmentFundofChulalongkornUniversity(RES560530148-HR);RatchadaphiseksomphotFundofFacultyofMedicine,ChulalongkornUniversity;HealthandBiomedicalScienceResearchProgrambyNationalResearchCouncilofThailandandHealthSystemResearchInstitute(P-13-50154);theResearchChairGrant(P-13-01091);ClusterandProgramManagementOffice(CPMO)(P-15-50535);theNationalScienceandTechnologyDevelopmentAgency(NSTDA),Thailand;andtheUSAIDEmergingPandemicThreatsPREDICT2project(07–306–7119-52272).AvailabilityofdataandmaterialsTheGenBankaccessionnumbersforcoronavirussequencesreportedinthispaperare:MG256395-MG256474(CoV)andMG333996-MG333999(CoV)for357bppartialRdRpgenefromPteropuslylei.Authors’contributionsSWandTHparticipatedinthedesignofthestudyanddraftedthemanuscript.PD,TK,SY,SN,PP,andPMconductedsamplingofbatspecimens.TKcarriedoutthemoleculargeneticstudies.ACandARparticipatedinthesequencealignmentanddataanalysis.Allauthorsreadandapprovedthefinalmanuscript.EthicsapprovalandconsenttoparticipateBatspecimenswerecollectedwithpermissionfromtheDepartmentofNationalParks,WildlifeandPlantConservation(No.0909.204/2686)andtheAnimalUseProtocolNo.1473001approvedbyChulalongkornUniversityAnimalCareandUseCommittee.ConsentforpublicationNotApplicable.CompetinginterestsTheauthorsclaimnoconflictofinterestinthepublicationofthisinformation.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1ThaiRedCrossEmergingInfectiousDiseases-HealthScienceCentre,WorldHealthOrganizationCollaboratingCentreforResearchandTrainingonViralZoonoses,ChulalongkornHospital,FacultyofMedicine,ChulalongkornUniversity,Bangkok,Thailand.2FacultyofForestry,KasetsartUniversity,Bangkok,Thailand.3FacultyofVeterinaryMedicine,KasetsartUniversity,Bangkok,Thailand.4DepartmentofNationalParks,WildlifeandPlantConservation,Bangkok,Thailand.Received:21November2017Accepted:15February2018 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