Wacharapluesadeeetal.VirologyJournal (2015) 12:57 DOI10.1186/s12985-015-0289-1 SHORT REPORT Open Access Diversity of coronavirus in bats from Eastern Thailand Supaporn Wacharapluesadee1*, Prateep Duengkae2, Apaporn Rodpan1, Thongchai Kaewpom1, Patarapol Maneeorn3, Budsabong Kanchanasaka3, Sangchai Yingsakmongkon1,4, Nuntaporn Sittidetboripat1, Chaiyaporn Chareesaen3, Nathawat Khlangsap2, Apisit Pidthong3, Kumron Leadprathom5, Siriporn Ghai1, Jonathan H Epstein6, Peter Daszak6, Kevin J Olival6, Patrick J Blair7, Michael V Callahan1,8 and Thiravat Hemachudha1 Abstract Background: Bats are reservoirs for a diverse range of coronaviruses (CoVs), including thosecloselyrelated to human pathogens such as Severe AcuteRespiratory Syndrome (SARS)CoV and Middle EastRespiratorySyndrome CoV.There are approximately 139 bat species reported to date in Thailand, of which two are endemic species. Due to thezoonotic potential ofCoVs, standardized surveillance efforts to characterize viral diversity inwildlife are imperative. Findings: Atotalof626batsfrom19differentbatspecieswereindividuallysampledfrom5provincesinEastern Thailandbetween2008and2013(84fecaland542rectalswabs).Samplescollected(eitherfreshfecesorrectalswabs) wereplaceddirectlyintoRNAstabilizationreagent,transportedonicewithin24hoursandpreservedat−80°Cuntil furtheranalysis.CoVRNAwasdetectedin47specimens(7.6%),from13differentbatspecies,usingbroadlyreactive consensusPCRprimerstargetingtheRNA-DependentRNAPolymerasegenedesignedtodetectallCoVs.Thirtyseven alphacoronaviruses,ninelineageDbetacoronaviruses,andonelineageBbetacoronavirus(SARS-CoVrelated)were identified.SixnewbatCoVreservoirswereidentifiedinourstudy,namelyCynopterussphinx,Taphozousmelanopogon, Hipposideroslekaguli,Rhinolophusshameli,ScotophilusheathiiandMegadermalyra. Conclusions:CoVsfromthesamegeneticlineagewerefoundindifferentbatspeciesroostinginsimilarordifferent locations.ThesedatasuggestthatbatCoVlineagesarenotstrictlyconcordantwiththeirhosts.Ourphylogeneticdata indicateshighdiversityandacomplexecologyofCoVsinbatssampledfromspecificareasineasternregionsof Thailand.FurthercharacterizationofadditionalCoVgenesmaybeusefultobetterdescribetheCoVdivergence. Keywords:Coronavirus,Bats,Diversity,Eastern,Thailand Background Syndrome (MERS) in 2012 renewed interest in bat- Following the Severe Acute Respiratory Syndrome originated CoVs. The molecular investigation in Saudi (SARS) pandemic in 2002–03, caused by the SARS cor- Arabia revealed one Taphozous perforatus bat whose onavirus (SARS-CoV), intensive surveillance has de- virus showed 100% nucleotide identity to the MERS tected a great diversity of CoVs throughout the animal virus found in the human index case [6]. Other subse- kingdom,especiallyinbats.Theinitialdiscovery ofCoVs quent studies have found MERS-related CoV lineages in bats was made in China following the SARS outbreak from a variety of bat species globally [7-11]. CoVs are [1-5]. The emergence of the Middle Eastern Respiratory divided into four genera: Alphacoronavirus and Beta- coronavirus which largely infect mammals; and Gam- macoronavirus and Deltacoronavirus which primarily *Correspondence:[email protected] infect avian species [12]. CoVs in bats are generally of 1WorldHealthOrganizationCollaboratingCentreforResearchandTraining onViralZoonoses,KingChulalongkornMemorialHospital,Facultyof the Alpha- and Betacoronavirus genus, and have been Medicine,ChulalongkornUniversity,Bangkok,Thailand identified in bats of various species from around the Fulllistofauthorinformationisavailableattheendofthearticle ©2015Wacharapluesadeeetal.;licenseeBioMedCentral.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated. Wacharapluesadeeetal.VirologyJournal (2015) 12:57 Page2of7 world. Thailand is home to 139 different bat species, of Results which two are endemic species including Hipposideros A total of 626 bats representing 19 species (Table 1) pendleburyi and Murina balaensis (new species of were sampled (84 fecal and 542 rectal swab specimens) genus Murina) [13,14], however CoV surveillance has between 2008 and 2013 from 6 locations in 5 of 7 prov- only been conducted on 25 (18%) of these species [15]. inces in Eastern Thailand. CoV RNA was detected in 47 The first report of bat CoVs in Thailand examined a (7.6%) specimens (17 fecal samples and 30 rectal swabs) total of 256 fecal specimens and discovered 28 positive from 13 different bat species. Detection rates for bat samples in H. larvatus and H. armiger [15]. Recently in CoVs were 1.6% to 45% per site in 5 of the sampling a study in Ratchaburi province,Thailand, we discovered sites (Figure 1). Phylogenetic analysis of nucleotide se- lineage C betacoronavirus in dry bat guano fertilizer, quences of 353 bp RdRp gene showed that 37 samples however thebat species was not identified as specimens were members of the Alphacoronavirus genus and 10 were collected from a mixed species roost [11]. As a re- belonged to the Betacoronavirus genus. The phylogen- sult of the risk CoVs pose to human health, ecological etic reconstruction showed 9 different clades of bat studies of CoVs in bats are warranted, particularly to CoVs (Figure 2). There were 6 clades in alphacorona- understand the baseline viral diversity circulating in virus (clades1–6), 2 inlineage D betacoronavirus(clades wildlife hosts. Here we describe a comprehensive study 7 and 8) and 1 in lineage B betacoronavirus (clade 9). of CoV diversity and prevalence among bats in Eastern The six alphacoronavirus clades were divergent, but Thailand to explore CoV infections in bat populations. were related to CoVs previously identified in bats from China, Bulgaria and Kenya [19-21]. The percent nucleo- tidesimilarity within eachcladewascalculatedandisin- Methods cluded inFigure2. Bats were captured with permission from the Depart- In our study, host restriction of CoVs was demon- ment of National Parks, Wildlife and Plant Conserva- strated in clade 4 (CoV512) and clade 6 (HKU2). Clade tion. The Institutional Animal Care and Use Committee 4 CoV from Scotophilus heathii was clustered with bat at the University of California, Davis (protocol number: CoV512 previously found in S. kuhlii from China [4]. 16048)approvedthecaptureandsamplecollection.Bats’ Clade 6 CoV (BRT55555) found in Rhinolophus shameli, species were identified in the field by experienced Thai was clustered with HKU2 (R. sinicus) described in China mammalogist (PD) based on their external morphological [22]. This is in accordance with previous studies which characteristics as described by Lekagul & McNeely have demonstrated that individual CoVs are associated [16,17]. Fresh bat fecal pellets were individually stored in with a single species or genus including Carollia, Eptesi- 0.5 ml of RNAlater® RNA Stabilization Reagent (Qiagen, cus, Miniopterus,andRhinolophusbats[4,19,20,23]. Germany) while each rectal swab was placed into 1 ml of We found evidence for species of CoV in almost every NucliSens® Lysis Buffer (bioMérieux, France), and then clade in this study being shared by different bat species storedat−80°Cuntilfurtheranalysis.Sampleswereexam- from different families. For example clades 1–3 CoVs ined using broadly reactive consensus hemi-nested Re- were found in 3 bat families, in the Miniopterus magna- verse Transcription PCR (RT-PCR) with degenerate PCR ter, M. schreibersii, M. pusillus, H. lekaguli, H. armiger primers designed to detect all CoV lineages, targeting the and T. melanopogon. Similarly, clade 5 CoVs were found RNA-dependentRNApolymerase(RdRp)gene[18].Amp- in 6 bat species (5 genera, 5 families) from 4 different lificationproductwasvisualizedusing2%agarosegelelec- sites, clade 7 CoVs were found in 3 bat species captured trophoresis. The RdRP PCR product was sequenced from the same location and clade 8 CoVs were found in directly using an automated ABI PRISM 377 model se- Cynopterus sphinx (fruit bat) and the insectivorous H. quencer or cloned using the pGEM®-T Easy Vector Sys- lekaguli (Figure 2). Further, many CoVs species were tem before sequencing. Initially, to assessclonal sequence found in a single bat species such as the H. lekaguli, R. diversity, ten individual clones from each specimen shameli, T. melanopogon, and S. heathii (Figure 2). For were sequenced. Sequences were edited using Bio-edit example, 10H. lekaguli bats roosting in the same colony program. Sixty-three CoV sequences obtained from 47 were found to harbor 2 lineage D betacoronaviruses and specimens (more than one sequence was found from 4 8alphacoronaviruses. specimens) were deposited in GenBank with accession Seven CoVs in clade 7 from S. kuhlii, S. heathii and C. numbers [KJ020577 to KJ020636, KJ652018, KJ868721 sphinx were clustered in an independent lineage. These and KJ868722]. Phylogenetic trees were constructed viruses (from 5 bats) had 99.15-100% identity of 119 based on 353 bp RdRp gene sequence, corresponding to amino acids and differed from HKU9 by 16.11-16.95% nucleotides 14,355 - 14,707 in Human CoV 229E gen- (Figure 2). Further analyses using longer gene fragments ome(GenBankaccessionno.AF304460)usingthemax- and other genes from greater number of bats are re- imum likelihood method. quiredfor confirmationofthisnovel group. Wacharapluesadeeetal.VirologyJournal (2015) 12:57 Page3of7 Table1Batspeciestestedforcoronaviruses Family Species No.ofpositive/ Samplingsite(year)† CoVclade(s)[cluster]/(no.positive) total‡(%) Pteropodidae Cynopterusbrachyotis 1/9(11.1) AA(2011*);TR(2011) 5[HKU10]/(1) Cynopterussphinx 4/14(28.6) AA(2011);RD(2008);TR(2011);CB(2012*) 7[Newcluster]/(2),8[HKU9]/(2) Eonycterisspelaea 0/11(0) AA(2011);TR(2011);CB(2012) Rousettus 0/3(0) SK(2011) amplexicaudatus Emballonuridae Taphozous 0/12(0) RD(2008/2012) longimanus Taphozous 2/123(1.6) RD(2012*/2013);CK(2012);SK(2011*/2012) 2[HKU7]/(1),5[HKU10]/(1) melanopogon Hipposideridae Hipposiderosarmiger 2/140(1.4) CK(2012);RD(2008/2012*/2013);SK(2012) 1[CoV1A/B]/(1),5[HKU10]/(1) Hipposideros 0/3 CK(2012) cineraceus Hipposideroslarvatus 1/29(3.4) CK(2012);RD(2008/2012/2013*) 9[SARS]/(1) Hipposideros 10/159(6.3) CK(2008*/2012*) 1[CoV1A/B]/(2),5[HKU10]/(6),8[HKU9]/(2) lekaguli Macroglossinae Macroglossus 0/2(0) AA(2011);TR(2011) sobrinus Megadermatidae Megadermalyra 1/2(50) RD(2012*/2013) 5[HKU10]/(1) Rhinolophidae Rhinolophus 2/20(10) CK(2012*) 5[HKU10]/(1),6[HKU2]/(1) shameli Vespertilionidae Miniopterusmagnater 6/30(20) CK(2012*) 1[CoV1A/B]/(5),2[HKU7]/(2**) Miniopteruspusillus 1/1(100) CK(2012*) 3[HKU8]/(1) Miniopterus 12/53(22.6) CK(2008*/2012) 1[CoV1A/B]/(3),2[HKU7]/(1),3[HKU8]/(8) schreibersii Myotishorsfieldii 0/4(0) CK(2012);CB(2012) Scotophiluskuhlii 2/3(66.7) CB(2012*) 7[Newcluster]/(2) Scotophilusheathii 3/8(37.5) CB(2012*) 4[CoV512]/(2),7[Newcluster]/(1) Total 47/626(7.5) ‡Sampleswere84fecaland542rectalswabs;*Apositivelocation(andyear)isindicatedbyanasterisk;FirstreportofCoVinspecies(indicatedinbold). †AA=AngAed,Chataburi;CB=Chonburi;CK=Chakan,Srakaew;RD=Rad,Chachongsao;SK=Sarika,Chantaburi;TR=Trat. **SampleNo.BRT55593(Miniopterusmagnater)contained2differentCoVspeciesbelongtoclade1and2. One specimen from M. magnater (BFE55593) was In addition, this is the first report describing the pres- foundtobeco-infectedwith2CoVspecies.Further,three ence of CoV RNA in 6 bat species including C. sphinx, individual bats from 2 species (samples no. B128 [M. T. melanopogon, H. lekaguli, R. shameli, S. heathii and schreibersii], B311 [M. schreibersii], and B55700 [C. Megaderma lyra, where the latter is the newly reported sphinx]) were found to be co-infected with multiple bat family(Megadermatidae)found toharborCoV. strainsofthesameCoVspecies.Initialsequencingshowed multiple nucleotide peaks upon direct sequencing chro- Discussion and conclusion matogram.ThesePCRproductswerecloned and10indi- Data from this study demonstrates that CoV infection in vidual clones were sequenced in order to assess clonal bats sampled in Eastern Thailand is not uncommon and sequence diversity. Analysis of 353 bp sequences revealed infection is distributed among a range of species. The the presence of sequence variants within single samples CoVs found in bats from this small region of Thailand (also known as quasispecies [24]). There were 7,4,6, and were genetically related to bat CoVs found in several 3 different sequences obtained from samples no. B128, countries from different regions of the world such as B311, BFE55593, and B55700, respectively. All sequences China, Philippines, Kenya, Spain and Bulgaria [18-21,25]. from individual samples were clustered into the same MERS-like CoV (previously found in environmentally CoVspecies (i.e. represented CoV quasispecies) except in sampled bat feces from Ratchaburi province, Western BFE55593, where 2 sequences were clustered to clade 2 Thailand [11]) was not found in this study, despite sam- andtheother4sequenceswereclusteredintoclade1. pling the bat genus Taphozous, a likely MERS-CoV Wacharapluesadeeetal.VirologyJournal (2015) 12:57 Page4of7 Figure1AreasinEasternThailandwheresampleswerecollectedwithCoV-positivebatspeciesadditionallynamed.Chakarncave(CK,Darkblue) inSrakaeoprovince;Radcave(RD,Pink)inChachoengsaoprovince;Chonburiprovince(CB,Red);AngAed(AA,Paleblue)andSarika(SK,Green) cavesinChanthaburiprovince;andTratprovince(TR,Orange).AtCKsite,batswerecaptured4times:inMay2008,July2008,January2012and May2012;RDsite,5times:May2008,July2008,January2012,May2012andJanuary2013;SKsite,2times:December2011andMay2012;AA site,1time:December2011;TRsite,1time:December2011.BetweenJanuaryandDecember2012atCBsite,batswerecapturedmonthlyat2 localswinefarms.Generally,batswerecaughtinmistnetsorharptrapsastheyemergedfromtheirroosts.Attwosites(CBandTR),batswere trappedduringthenightastheyforagednearopenorchards.ThenumberofCoVpositivebats[bracket]ineachcladeisindicatedforeachsite. **SampleNo.BRT55593(Miniopterusmagnater)contained2differentCoVspeciesbelongtoclade1and2. reservoir found in Saudi Arabia [6], in our study. Further heathii and C. sphinx) captured from the same location studies on individual bat species in Western Thailand to (unknown roost) carried similar viruses clustered in identifybat reservoirs for MERS- orSARS-likeCoVs may clade 7. The spatial overlap at feeding areas and tempor- be justified. Phylogenetic analysis revealed close corre- ary night roosts for S. kuhlii, S. heathii (insectivorous) lations between CoV/B56054 from H. larvatus and and C. sphinx (nectarivorous) may have facilitated the SARS-like CoV belonging to lineage B betacoronavirus exchange of viruses, as they may not necessarily be co- from Rhinolophus in China [1]. This finding was in ac- roosting diurnally and do not share the same direct food cordance with the previous bat CoV study in Thailand source. This data supports previous studies in Spain, [15]. However three bat species (H. lekaguli, M. lyra, China and South America, where different bat species and M. schreibersii) and 3 bat families (Pteropodidae, sampled within the same location carried similar viruses Emballonuridae, and Rhinolophidae) previously re- [4,25,26]. However, more research is needed to under- ported negative for CoV in Thailand [15], were positive stand interspecific bat behavior and transmission poten- for CoVs in our study (Table 1). tial for these species with seemingly different diet and At several of our sites, we observed many different bat foragingpatterns. species from different families roosting in the same cave Further, the H. armiger and T. melanopogon, which and subsequently were found to be harboring the same roosted at a different colony from the Miniopterus batCoVspecies.Forexample,H.lekaguliharboredCoVs bats, also harbored CoV of the same genetic lineage as ofthesamegeneticlineageasMiniopterusCoVinclades the Miniopterus CoV in clades 1 and 2 respectively. 1, and R. shameli in clade 5. Co-roosting of theses bats Similarly, HKU10-related CoVs (clade 5) were found in in an enclosed cave environment may have facilitated 5 divergent bat families including Megadermatidae, the exchange of viruses. Three bat species (S. kuhlii, S. Pteropodidae, Emballonuridae, Hipposideridae, and Wacharapluesadeeetal.VirologyJournal (2015) 12:57 Page5of7 Figure2(Seelegendonnextpage.) Wacharapluesadeeetal.VirologyJournal (2015) 12:57 Page6of7 (Seefigureonpreviouspage.) Figure2Phylogenetictreesofthecoronavirus(CoV)RNA-dependentRNApolymerase(RdRp)geneatthenucleotidelevel.Maximum-likelihoodtreeof a353bpfragmentoftheRdRpgenefrombatCoVsfoundinthisstudyarecoloredaccordingtotheirroost(Darkblue=Chakarncave,CK;Pink=Rad cave,RD;Red=Chonburiprovince,CB;Paleblue=AngAed,AA;Green=Sarikacave,SK)andpreviouslyfoundinbatsandotheranimals(black).A BulbuldeltacoronavirusHKU11-934wasusedasoutgroup.AlignmentswereconstructedusingMultipleAlignmentFastFourierTransform,MAFFT. Bootstrapvaluesweredeterminedusing1000replicatesviaMEGA5.ThetreewasvisualisedusingtheFigTreeprogram,version1.4.0.Taxaarenamed accordingtothefollowingpattern:identificationcode/strainorisolate/typicalhost/country/collectionyear/accessionnumber.Cyn_bra,Cynopterus brachyotis;Cyn_sph,Cynopterussphinx;Tap_mel,Taphozousmelanopogon;Hip_arm,Hipposiderosarmiger;Hip_lar,Hipposideroslarvatus;Hip_lek, Hipposideroslekaguli;Meg_lyr,Megadermalyra;Rhi_sha,Rhinolophusshameli;Min_mag,Miniopterusmagnater;Min_pus,Miniopteruspusillus; Min_sch,Miniopterusschreibersii;Sco_kuh,Scotophiluskuhlii;Sco_hea,Scotophilusheathii.Therewere7,4,6,and3differentsequencesobtainedfrom samplesno.B128(B128-1toB128-7),B311(B311-1toB311-4),BFE55593(BFE55593-1toBFE55593-6,andB55700(B55700-1toB44700-3),respectively. RepresentativesequenceswherethesameexactCoVspecies(>99%nucleotidesimilarity)wasfoundindifferentindividualsofthesamebatspeciesat thesamesiteshowinitalic.Clades1–6ofalphacoronaviruswerecategorizedbasedontheCoVspreviouslyreportedinChina;bat-CoV1A/ B,−HKU7,−HKU8,−CoV512,−HKU10and-HKU2,respectivelywhileclade7–8and9ofbetacoronaviruswerecategorizedbasedonHKU9and SARSCoV,respectively.Thepercentnucleotidesimilaritywithineachcladeisshowninparenthesesunderthecladename. Rhinolophidae(Figure2).TheseHKU10-positivebatspe- In conclusion, phylogenetic analysis of our study re- cies, H. lekaguli (CK site), H. armiger (RD), C. brachyotis vealed a high genetic diversity of CoVs and presence of (AA),T. melanopogon (SK), M. lyra (RD), and R. shameli cross species dissemination in bats from the Eastern re- (CK), were from 4 different sampling sites. Interestingly, gion of Thailand. Finding of new viral reservoirs and the these viruses were closely related to HKU10 CoVs found putative novel betacoronavirus lineage in this study em- in Rousettus leschenaulti and H. pomona in China, where phasizes the need for additional CoV surveillance. Our interspecies transmission between bats of different subor- data can serve as an additional dataset to the global sur- derswasalsodemonstrated[27].Furtherstudiesanddee- veillance of emerging CoVs, which may include poten- per characterization of these bat CoVs infecting different tially harmful pathogens to human health. In order to bat species may provide additional insight to their host havea complete understanding of the ecology and trans- range and the evolutionary history of their interspecies mission of CoV, a comprehensive analysis of bats across transmission. These findings indicate a greater diversity their migratory routes in Africa, Southeast Asia and and higher ecological complexity of bat CoVs in Eastern Australia shouldbe conducted. Thailandthanpreviouslyappreciated. Abbreviations Two or more different CoV clades/lineages were also CoV:Coronavirus;MERS:MiddleEastRespiratorySyndrome;SARS:Severe found circulating in the same bat species from the same AcuteRespiratorySyndrome;RT-PCR:Reversetranscription-polymerasechain site, for example C. sphinx, H. armiger, H. lekaguli, R. reaction. shameli, M. schreibersii, M. magnater, and S. heathii, Competinginterests and from different roosts for T. melanopogon (Table 1 Theauthorsdeclarethattheyhavenocompetinginterests. andFigure2).ThisCoVdiversitymaybeassociatedwith bat migration or bats from different species sharing for- Authors’contributions SWandPDparticipatedinthedesignofthestudyanddraftedthe aging sites. Previous studies also found evidence of manuscript.PD,PM,BK,CC,NK,APandKLconductedsamplingofbatfecal cross-species transmission in bats, for example Artibeus samples.TK,SY,andcarriedoutthemoleculargeneticstudies.ARandSG lituratus from Mexico [23], and R. sinicus [28] and R. participatedinthesequencealignmentanddraftedthemanuscript.TH,KJO andPJBwrotethedraftofthemanuscript.MVC,JHEandPDcarriedout leschenaultifrom China[2]. revisionofthemanuscript.Allauthorsreadandapprovedthefinal Interestingly, co-infection of divergent CoV lineages manuscript. was found in one bat (M. magnater, BFE55593), which was infected with 2 different CoV species, clustered in Authors’information TheGenBankaccessionnumbersforthecoronavirussequencesreportedin clades1(CoV1A/B)and2(HKU7)(Figure2).Thisfind- thispaperare:KJ020577–KJ020636,KJ652018,KJ868721andKJ868722. ing is similar to a previousreport from China, where co- infection of bat CoV 1B and HKU8 were detected in M. Acknowledgments ThisstudywassupportedbyaresearchgrantfromtheThailandResearch pusillus using species-specific RT-PCR assays [29]. Co- Fund(RDG5420089),theRatchadaphiseksomphotEndowmentFundof infection with different CoVs in the same host may fa- ChulalongkornUniversity(RES560530148-HR),HealthandBiomedicalScience cilitate recombination between these CoVs. Further ResearchProgrambyNationalResearchCouncilofThailand(NRCT)and HealthSystemResearchInstitute(HSRI),theResearchChairGrant,the studies of co-infection and CoV recombination within a NationalScienceandTechnologyDevelopmentAgency(NSTDA),Thailand, given bat host could improve our understanding on the theNavalHealthResearchCenter(BAA-10-93)undertheCooperative evolution of CoVs, including specific mutations or re- Agreementno.W911NF-11-2-0041,andtheUSAIDEmergingPandemic ThreatsPREDICTproject.Wegratefullyacknowledgethelocalsupportfrom combination events (e.g. involving the Spike gene), that theThaiRedCrossSociety,ChulalongkornUniversity,KasetsartUniversityand couldfacilitate spillovertonovel species. theDepartmentofNationalParksWildlifeandPlantConservation.Theviews Wacharapluesadeeetal.VirologyJournal (2015) 12:57 Page7of7 expressedinthismanuscriptarethoseoftheauthorsanddonotreflectthe 19. 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