AntiviralResearch101(2014)45–56 ContentslistsavailableatScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Review Ecology, evolution and classification of bat coronaviruses in the aftermath of SARS ⇑ Jan Felix Drexler , Victor Max Corman, Christian Drosten InstituteofVirology,UniversityofBonnMedicalCentre,Germany a r t i c l e i n f o a b s t r a c t Articlehistory: In2002/2003,anovelcoronavirus(CoV)causedapandemic,infectingmorethan8000people,ofwhom Received22July2013 nearly10%died.Thisvirus,termedsevereacuterespiratorysyndrome-CoVwaslinkedtoazoonoticorigin Revised30September2013 fromrhinolophidbatsin2005.Sincethen,numerousstudieshavedescribednovelbatCoVs,including Accepted21October2013 closerelativesofthenewlyemergingMiddleEastrespiratorysyndrome(MERS)-CoV.Inthispaperwedis- Availableonline31October2013 cussCoVgenomicpropertiesandcomparedifferenttaxonomicapproachesinlightofthetechnicaldiffi- cultiesofobtainingfullgenomicsequencesdirectlyfrombatspecimens.Wefirstpresentanoverviewof Keywords: theavailablestudiesonbatCoVs,withdetailsontheirchiropteranhosts,thencomparativelyanalyzethe Coronaviridae increaseinbatCoVstudiesandnovelgenomicsequencesobtainedsincetheSARSpandemic.Wethen Alphacoronavirus conductacomprehensivephylogeneticanalysisofthegeneraAlpha-andBetacoronavirus,toshowthat Betacoronavirus Bats batsharbourmoreCoVdiversitythanothermammalianhostsandarewidelyrepresentedinmost,but Zoonosis not all parts of the tree of mammalian CoVs. We next discuss preliminary evidence for phylogenetic Taxonomy co-segregationofCoVsandbathostsencompassingtheBetacoronaviruscladesbandd,withanemphasis onthesamplingbiasthatexistsamongbatspeciesandothermammals,thenpresentexamplesofCoVs infectingdifferenthostsontheonehandandvirusesapparentlyconfinedtohostgeneraontheother.We alsodemonstrateageographicbiaswithinavailablestudiesonbatCoVs,andidentifyacriticallackof informationfrombiodiversityhotspotsinAfrica,AsiaandLatinAmerica.Wethenpresentevidencefor a zoonotic origin of four of the six known human CoVs (HCoV), three of which likely involved bats, namelySARS-CoV,MERS-CoVandHCoV-229E;comparetheavailabledataonCoVpathogenesisinbats tothatinothermammalianhosts;anddiscusshypothesesontheputativeinsectoriginsofCoVancestors. Finally,wesuggestcautionwithconclusionsonthezoonoticpotentialofbatviruses,basedonlyongeno- micsequencedata,andemphasizetheneedtopreservetheseecologicallyhighlyrelevantanimals.This paperformspartofasymposiuminAntiviralResearchon‘‘fromSARStoMERS:10yearsofresearchon highlypathogenichumancoronaviruses’’. (cid:2)2013ElsevierB.V.Allrightsreserved. Contents 1. Introduction.......................................................................................................... 46 1.1. Coronavirusgenomicorganization................................................................................... 46 1.2. Batcoronavirusdetectionandchallengesfortheirtaxonomicclassification ................................................. 46 1.3. Increaseincoronavirusgenomicsequences ........................................................................... 48 1.4. Coronavirusphylogeny............................................................................................ 48 1.5. Batsascoronavirushostsworldwide................................................................................. 49 1.6. Associationofcoronaviruscladeswithmammalianhosts................................................................ 49 1.6.1. Promiscuousversushost-specificcoronaviruses ................................................................ 49 1.6.2. Evidenceforphylogeneticco-segregationofcoronavirusesandbathosts............................................ 49 1.7. Coronavirushostswitchesfrombats................................................................................. 49 1.7.1. Hostswitchessuggestedbyphylogenetically-relatedvirusesinhumansandbats..................................... 49 1.7.2. Mechanismsofhostswitches ............................................................................... 51 1.8. Coronaviruspathologyinbatscomparedtoothermammalianhosts....................................................... 52 ⇑ Correspondingauthor.Address:InstituteofVirology,UniversityofBonnMedicalCentre,53127Bonn,Germany.Tel.:+4922828711697;fax:+4922828719144. E-mailaddress:[email protected](J.F.Drexler). 0166-3542/$-seefrontmatter(cid:2)2013ElsevierB.V.Allrightsreserved. http://dx.doi.org/10.1016/j.antiviral.2013.10.013 46 J.F.Drexleretal./AntiviralResearch101(2014)45–56 2. Discussion............................................................................................................ 52 Acknowledgements.................................................................................................... 53 References ........................................................................................................... 53 1.Introduction proteins (nsp). A conserved sequence UUUAAAC, located around genomepositions12–14,000inalpha-andbetacoronaviruses,pro- In 2002/2003, an outbreak of severe respiratory disease oc- videsthecharacteristicribosomalslippageleadingtotranscription curred,infecting8096peopleworldwideandkilling774(9.5%)of ofORF1ab(BrianandBaric,2005;Masters,2006).Thefunctionsof them.Anovelhumancoronavirus(CoV)namedsevereacuterespi- individualnspareonlypartiallyunderstood. ratorysyndrome(SARS)-CoVwasidentifiedasthecausativeagent KnownORF1abgeneproductsinclude,amongothers,apapain- (Drostenetal.,2003).Thisviruswasmuchmorepathogenicthan likeprotease(Plpro)andmainprotease(Mpro),ahelicase,twometh- previously known human coronaviruses (HCoV) mainly causing yltransferases,aRNA-dependentRNApolymerase(RdRp)andseveral mildrespiratorydisease(vanderHoek,2007).Theclinicalaspects innateimmunityantagonists(PerlmanandNetland,2009).Down- oftheSARSepidemicandresearchontheSARS-CoVhavebeenre- streamoftheORF1ab,allCoVscontaingenescodingforthestruc- viewed in two other articles in the present series (Cheng et al., tural proteins spike, envelope, membrane and nucleocapsid and 2013;HilgenfeldandPeiris,2013). severalaccessorygenesinvariablenumberandgenomiclocation Animal CoV have been known since the late 1930s, including (Wooetal.,2009).AuniqueCoVfeatureisthediscontinuoustran- virusesthatarehighlypathogenicforlivestock,petandlaboratory scription of genes downstream of the ORF1ab from subgenomic animals,suchastransmissiblegastroenteritisvirusofswine(TGEV), mRNAs, involving interaction between characteristic transcrip- bovine CoV (BCoV), feline infectious peritonitis virus (FIPV), mouse tion-regulatorysequences(TRS)foundinthemost50-genomicre- hepatitis virus (MHV) and infectious bronchitis virus (IBV) (Saif, gion (leader TRS) and upstream of individual genes (body TRS) 2004).TheearliestdescriptionsoftwoHCoVstermedHCoV-229E (Brian and Baric, 2005; Pasternak et al., 2006; Perlman and and-OC43werealreadymadeinthe1960s(HamreandProcknow, Netland,2009;Wooetal.,2009). 1966;McIntoshetal.,1967).IntheSARSaftermath,twoadditional HCoVtermed-NL63and-HKU1weredetected(vanderHoeketal., 1.2.Batcoronavirusdetectionandchallengesfortheirtaxonomic 2004;Wooetal.,2005).In2012,ahighlypathogenicsixthHCoV, classification termed Middle East respiratory syndrome (MERS)-CoV emerged in the Arabian peninsula (de Groot et al., 2013; Zaki et al., 2012; AlmostallCoVfieldstudiesinvestigatingbatsorotheranimals HilgenfeldandPeiris,2013). arebasedonPCRassaystargetingpartsoftheORF1ab,typicallythe DuringtheSARSepidemic,firsthintspointedtoazoonoticori- RdRp. Inthese studies, PCR ampliconsizes rangefrom as littleas ginoftheSARS-CoV,withcivetsasthesuspectedsourceofhuman 121toaround404base-pairs(Anthonyetal.,2013a;daSilvaFilho infection (Guan et al., 2003; Song et al., 2005; Xu et al., 2004). et al., 2012; de Souza Luna et al., 2007; Moes et al., 2005; Tong GeneticallydiversifiedCoVsrelatedtoSARS-CoVwerethenfound et al., 2009). It should be noted that these small amplicon sizes inChineserhinolophidbats,indicatingtheseanimalsmayconsti- drastically hinder CoV phylogenetic reconstructions. Therefore, tute the animal reservoir of this novel HCoV (Lau et al., 2005; Li caution should be taken when drawing conclusions on the rela- et al., 2005). These findings and the concomitant description of tionshipswithinthesubfamilyCoronavirinae,basedonsuchsmall Ebolaviruses in Africanflyingfoxes(Leroy etal., 2005) triggered sequencefragments. researchintobatsashostsofemergingpathogens.Amongthefac- ThereareonlyfewcharacterizationsofcompleteCoVgenomes tors promoting bats as animal reservoirs for mammalian viruses frombatfeces,assummarizedinTable1.Thelackofvirusisolates are their longevity, densely packed colonies, close social interac- obtaineddirectlyfrombatschallengesthecompletegenomicchar- tion and their ability to fly (Calisher et al., 2006; Luis et al., acterization of these large and highly variable RNA viruses. The 2013). The numerous descriptions of novel viruses in bats and informationoriginatingfromthefouravailablemetagenomicstud- otheranimalshavedrasticallychangedourperceptionoftherele- iesreportingCoV sequencesoffers evenless phylogenetic power, vanceofanimalreservoirsfortheunderstandingofemergingzoo- becausetheresultinggenomicfragmentsareveryvariableingeno- noses (Drosten, 2013; Karesh et al., 2012; Morse et al., 2012). In miclocationandlength(Donaldsonetal.,2010;Geetal.,2012;Li analogy to SARS-CoV, the novel MERS-CoV may share a putative etal.,2010;Wuetal.,2012b). origin in bats (Annan et al., 2013). This review focuses on bats ToprovideorderinCoVtaxonomy,thecurrentproposalofthe andtheCoVstheyhost. International Committee for the Taxonomy of Viruses (ICTV) is basedonpairwiseaminoaciddistancesinsevenconcatenatedpar- 1.1.Coronavirusgenomicorganization tialorcompletenspdomains,encompassingabout50%oftheCoV genome. In technical analogy to an approach that has been vali- CoVs belongtothe order Nidovirales, family Coronaviridae and dated for the RNA virus family Picornaviridae (Lauber and subfamily Coronavirinae, comprising four genera termed Alpha-, Gorbalenya,2012b),aminoacididentitybelow90%wasfoundto Beta-, Gamma- and Deltacoronavirus (Adams and Carstens, 2012; be discriminatory for the designation of novel CoV species (de Perlman and Netland, 2009). Betacoronaviruses are further sepa- Grootetal.,2012).Adifferentcriterion,relyingonRdRp-grouping ratedintocladesa–d,whiletheseparationbetweenalphacoronavi- units(RGU),alsousedpairwiseaminoaciddistances,butwasre- ruscladesaandbhasbeendiscontinued(deGrootetal.,2012). strictedtoatranslated816nucleotideRdRp(nsp12)fragment.This CoV genomes are composed of one continuous RNA strand of was chosen as an amenable approach to account for the partial positive polarity, ranging between approximately 27 and 32,000 genomicsequencesgeneratedbymostPCR-basedfieldstudies.It nucleotides, constituting the largest continuous RNA genomes wasfoundthatdefinedalphacoronavirusRGUdifferedbyatleast among mammalian viruses (Woo et al., 2009). Starting from the 4.8% and betacoronavirus RGU by at least 6.3% in this RdRp frag- 50-endofthesegenomes,approximatetwo-thirdsencodesalarge ment. This allowed a surrogate criterion for species definition in open reading frame (ORF) 1, producing up to 16 nonstructural theabsenceofcompletegenomicsequences(Drexleretal.,2010; J.F.Drexleretal./AntiviralResearch101(2014)45–56 47 Table1 Studiesyieldingbatcoronavirussequences. Continent BatfamilyyieldingCoVsequences Country Reference Comments America Phyllostomidae Brazil Brandaoetal.(2008) Nofullgenomeinformationavailable Molossidae Brazil Limaetal.(2013) Phyllostomidae,Molossidae Brazil Cormanetal.(2013b) Phyllostomidae,Molossidae,Noctilionidae,Vespertilionidae Brazil Góesetal.(2013) Vespertilionidae Canada Misraetal.(2009) Phyllostomidae,Mormoopidae CostaRica Cormanetal.(2013b) Phyllostomidae,Emballonuridae,Mormoopidae,Vespertilionidae Mexico Góesetal.(2013) Phyllostomidae,Mormoopidae,Molossidae,Vespertilionidae Mexico Anthonyetal.(2013a) Phyllostomidae Panama Cormanetal.(2013b) Phyllostomidae Trinidad Carringtonetal.(2008) Vespertilionidae USA Dominguezetal.(2007) Vespertilionidae,Molossidae USA Lietal.(2010) Vespertilionidae USA Donaldsonetal.(2010) Vespertilionidae USA Osborneetal.(2011) Vespertilionidae USA Huynhetal.(2012) Europe Rhinolophidae,Vespertilionidae Bulgaria Drexleretal.(2010) SARS-relatedCoV;Fullgenome Vespertilionidae UK Augustetal.(2012) Nofullgenomeinformationavailable Vespertilionidae Germany Gloza-Rauschetal.(2008) Vespertilionidae Germany Drexleretal.(2010) Vespertilionidae Germany Drexleretal.(2011) Rhinolophidae Italy Balbonietal.(2011) Rhinolophidae Italy Balbonietal.(2012) Vespertilionidae Netherlands Reuskenetal.(2010) Vespertilionidae Netherlands Annanetal.(2013) Vespertilionidae Roumania Annanetal.(2013) Rhinolophidae Slovakia Rihtaricetal.(2010) Vespertilionidae Spain Falconetal.(2011) Vespertilionidae Ukraine Annanetal.(2013) Africa Hipposideridae Ghana Pfefferleetal.(2009) Nofullgenomeinformationavailable Nycteridae Ghana Annanetal.(2013) Pteropodidae,Hipposideridae,Vespertilionidae,Molossidae Kenya Tongetal.(2009) Pteropodidae,Megadermatidae,Vespertilionidae,Molossidae Kenya Taoetal.(2012) Hipposideridae Nigeria Quanetal.(2010) Vespertilionidae,Molossidae SouthAfrica Geldenhuysetal.(2013) Vespertilionidae SouthAfrica Itheteetal.(2013) Asia Pteropodidae Bangladesh Anthonyetal.(2013b) Nofullgenomeinformationavailable Vespertilionidae China Poonetal.(2005) Rhinolophidae,Vespertilionidae China Wooetal.(2006) Vespertilionidae China Chuetal.(2006) Pteropodidae China Lauetal.(2010b) Rhinolophidae China Yuenetal.(2012) Hipposideridae China Geetal.(2012) Rhinolophidae,Vespertilionidae China Wuetal.(2012b) Vespertilionidae Japan Shiratoetal.(2012) Emballonuridae,Pteropodidae,Rhinopomatidae,Vespertilionidae SaudiArabia Memishetal.(2013) Pteropodidae,Vespertilionidae Philippines Watanabeetal.(2010) Pteropodidae,Hipposideridae Philippines Tsudaetal.(2012) Hipposideridae Thailand Gouilhetal.(2011) Molossidae Thailand Wacharapluesadeeetal. (2013) Vespertilionidae China Chuetal.(2008) 1A/1B,HKU8;fullgenome Pteropodidae,Hipposideridae China Lauetal.(2012) HKU10;fullgenomes Rhinolophidae China Lauetal.(2007) HKU2;fullgenome Pteropodidae,Rhinolophidae,Vespertilionidae China Wooetal.(2007) HKU4,HKU5,HKU9;fullgenome Rhinolophidae,Vespertilionidae China Tangetal.(2006) SARS-relatedCoV,512,HKU4;full genome Rhinolophidae China Lauetal.(2005) SARS-relatedCoV;fullgenome Rhinolophidae China Lietal.(2005) SARS-relatedCoV;fullgenome Rhinolophidae China Renetal.(2006) SARS-relatedCoV;fullgenome Rhinolophidae China Yuanetal.(2010) SARS-relatedCoV;fullgenome Rhinolophidae China Lauetal.,2010a SARS-relatedCoV;fullgenome Rhinolophidae,Molossidae China Yangetal.(2013) SARS-relatedCoV;fullgenomes Tao et al., 2012). However, the increase in partial CoV sequences classifiable within both an RGU defined either by MERS-CoV or hascausedalossofdiscriminatorypoweroftheseRGUthresholds. by HKU5. Re-analysis of all available RdRp sequence data for the Forexample,thenovelHCoVspeciesMERS-CoV(deGrootetal., presentpaperindicatedthatthe4.8%thresholdpreviouslydeter- 2013; van Boheemen et al., 2012) and the genetically closely re- minedfor alphacoronavirusescouldbe maintained.In contrast,a lated Pipistrellus bat CoVs clearly belong to one RGU (Annan revisedlowerthresholdofatleast5.1%aminoacidsequencedis- etal.,2013;Reuskenetal.,2010).However,thesenovelbatviruses tance in this RdRp fragment was necessary to accommodate all also differ by only 5.1% from the established CoV species HKU5, betacoronavirus species included in the current ICTV proposal implying that they share sufficient RdRp sequence identity to be (deGrootetal.,2012)andthenovelpartialCoVsequences. 48 J.F.Drexleretal./AntiviralResearch101(2014)45–56 1.3.Increaseincoronavirusgenomicsequences years after SARS-CoV disappeared. The emergence of the MERS- CoVislikelytocounteractthisphenomenonintheyearstocome. Fig.1AshowstheincreaseinbatCoVsequencedepositioninto GenBank that closely followed the 2002–3 SARS pandemic. An 1.4.Coronavirusphylogeny even bigger increase of overall CoV sequence entries can be ob- served after 2009. This is likely due to both the advent of next- The rapid increase in bat CoV studies enabled hypotheses of generation sequencing techniques and changed GenBank policies bats as reservoir hosts for alpha and betacoronaviruses only a requiring host information from submitters. Fig. 1B shows that few years after the SARS epidemic (Vijaykrishna et al., 2007; theproportionofbatCoVsequencesamongthe5489overallCoV Woo et al., 2009). Fig. 2 shows the clear separation between the entries containing host information is small, compared to those mammaliangeneraAlpha-andBetacoronavirusandthebird-associ- fromhostssuchasungulates,birdsandcarnivores.Possibleexpla- ated genera Gamma- and Deltacoronavirus (Weiss and Navas- nations for this difference are likely the veterinary relevance of Martin, 2005; Woo et al., 2012) in a Bayesian phylogeny based livestockandpetCoVsandtheusageofprototypevirusesaslabo- onan816RdRpsequencefragmentofthecompletesubfamilyCor- ratory models leading to sequence entries, e.g., for BCoV, TGEV, onavirinae.Fig.3providesdetailsofthisphylogeneticanalysisfor FIPVandIBV.Still,thenumberofbatCoVentries(n=390)almost the generaAlpha- and Betacoronavirus. The large numberof deep equalledthatofHCoVs(n=460).However,thelackofhostinfor- branches leading to bat viruses (shown in red) emphasizes the mationforHCoVsprobablyintroducesabiasinthiscomparison. associationofthesetwoCoVgenerawithbathosts.Thisispartic- An analysis of published CoV research reports is shown in ularlytruefortheAlphacoronaviruscladeformerlydesignated1b, Fig.1c.TheimpactoftheSARS-epidemicin2002–3onthenumber whichincludesHCoV-229E,-NL63andtheBetacoronavirusclades ofCoVpublicationswastremendous,leadingtoanearfour-foldin- b–d. creasetoaround700publishedstudiesperyearafter2002.Simi- The relevance of bat CoVs for these genera is also reflected in larly, the identification of SARS-related CoVs in bats led to the current taxonomic proposal of the ICTV (de Groot et al., around15studiesonbatCoVsperyearafter2005.Ofnote,thisfig- 2012).Ofthe15recognizedspeciesinthesetwogenera,sixwere urealsoshowsthatscientificpublicationsbegantodecreaseafew onlyfound in bats. These viruses are shown withbat pictograms Fig.1. Coronavirusdatainpublicdatabases.PanelsAandB.DatawasbasedonaGenBanksearchusingtheterms‘‘Coronaviridae’’[ORGANISM]ANDHost[AllFields]’’inthe ‘‘Nucleotide’’database,onJune16th,2013.PanelC,PubMeddatawasretrievedfromasearchusingtheterms‘‘coronaviruses,coronavirus,coronaviridae,coronavirinae,bat, chiroptera,bats’’,onJune16th,2013. J.F.Drexleretal./AntiviralResearch101(2014)45–56 49 ampling of bats from these and other poorly studied areas will likely complete bat species coverage and further increase the knownCoVgenomicdiversity. 1.6.Associationofcoronaviruscladeswithmammalianhosts 1.6.1.Promiscuousversushost-specificcoronaviruses OnlyasmallfractionofthecurrentlyknownmammalianCoVs originates from primate, ungulate, lagomorph, carnivore and ro- denthosts.AsshowninFig.3,batsoutnumberanyothermamma- lian host in terms of virus diversity. Throughout the CoV phylogeny,examplescanbefoundofboth‘‘promiscuous’’andvery host-restrictedviruses.Theparamountexampleofapromiscuous CoV is probably Betacoronavirus 1 (the species including BCoV, HCoV-OC43andrelatedviruses),whichhasbeendetectedincows, horses,dogs,humans,waterbucks,deer,antelopes,camelsandgir- affesworldwide(Alekseevetal.,2008;Guyetal.,2000;Hasoksuz etal.,2007;Jinetal.,2007;Limetal.,2013;Majhdietal.,1997; Fig.2. PhylogeneticrelationshipsinthesubfamilyCoronavirinae.Bayesianphylog- Zhangetal.,1994).Similarly,FIPV,Caninecoronavirus(CCoV)and enyofan816-nucleotideRNA-dependentRNApolymerasefragment,asdescribed TGEVarenowincludedinasinglespeciestermedAlphacoronavirus previously(Drexleretal.,2010)ofthesubfamilyCoronavirinaeusingMrBayesV3.1 1,andMHVandRatcoronavirustogetherarenowtermedMurine (RonquistandHuelsenbeck,2003)underassumptionofaGTR+G+Isubstitution model,using2,000,000treessampledevery100steps,annotatedwithaburn-inof coronavirus(deGrootetal.,2012).Anotherexampleofanappar- 25%usingTreeAnnotatorV1.7.4andvisualizedusingFigTreeV1.4fromtheBEAST entlypromiscuousCoVistheunclassifiedbatvirusHKU10,which package(Drummondetal.,2012).Cavallyvirus(Zirkeletal.,2011)wasusedasan hasbeendetectedinthebatfamiliesHipposideridaeandPteropodi- outgroup.ValuesatdeepnodesindicatestatisticalsupportfromBayesianposterior dae(Lauetal.,2012). probabilities,scalebargeneticdistance. Most other CoVs have been confined to single host genera, inFig.3andincludeMiniopterusbatcoronavirus1,Miniopterusbat exemplified by the detection of SARS-related CoVs and several coronavirus HKU8, Rhinolophus bat coronavirus HKU2, Scotophilus alphacoronaviruses in Rhinolophus, Myotis, Miniopterus, Nyctalus bat coronavirus 512, Pipistrellus bat coronavirus HKU5, Rousettus and Carollia bat hosts, including detections of closely related batcoronavirusHKU9,andTylonycterisbatcoronavirusHKU4.Many virusesinindividualbatsseparatedbythousandsofmiles(Corman partialbatCoVRdRpsequenceswerenotincludedinFig.3,because etal.,2013b;Drexleretal.,2010;Tangetal.,2006).Similarly,Hip- onlysmallsequencefragmentsreducingthephylogeneticresolu- posideros betacoronaviruses from Thailand, Kenya, Nigeria and tion were available (Corman et al., 2013b; Drexler et al., 2010). Ghana are closely related (Gouilh et al., 2011; Pfefferle et al., Still, the sequences from these studies (detailed in Table 1) do 2009;Quanetal.,2010;Tongetal.,2009)andthebetacoronavirus not alter the overall picture of bat CoV-associated clades within HKU9hasbeendetectedindifferentspeciesofflyingfoxesinAfrica thegeneraAlpha-andBetacoronavirus. andAsia(Anthonyetal.,2013b;Lauetal.,2010b;Taoetal.,2012; FormostofthesebatCoVs,lackofcompletegenomicsequences Watanabeetal.,2010;Wooetal.,2007).Ofnote,thedetectionof preventstheirtaxonomicdesignationasspecies.Still,manyofthe both host-specific and -nonspecific mammalian CoVs parallels partialsequencesincludedinFig.3branchdeeplyinthephyloge- whatcanbeobservedintheavianCoronavirinaegenera.Forexam- netictreeandlikelyrepresentnotonlynewspecies,butevennew ple,infectiousbronchitisvirus(IBV,genusGammacoronavirus)has geneticclades.ThisisexemplifiedbytheunclassifiedAfricanHip- been detected in a wide range of birds, while the recently de- posideros betacoronaviruses (Pfefferle et al., 2009; Quan et al., scribed deltacoronaviruses appear to be more host-specific (Chu 2010;Tongetal.,2009),whichputativelyrepresentayettobede- etal.,2011). finedBetacoronaviruscladeeandunclassifiedneotropicalCarollia and Pteronotus viruses (Corman et al., 2013b) putatively corre- 1.6.2.Evidenceforphylogeneticco-segregationofcoronavirusesand spondingtoadditionalBetacoronavirusclades. bathosts Fig.4showsthat11ofthe18extantbatfamiliesalreadycon- Co-segregation of CoVs and their bat hosts is most visible for tainCoVdescriptions,includingthetwomajorbatlineagesYinpte- HKU9, the Hipposideros betacoronaviruses and the SARS-related rochiropteraandYangochiroptera(Teelingetal.,2005).Inmostbat CoV,comparedtoPteropodidae,HipposiderosandRhinolophushosts families,bothalpha-andbetacoronavirusesareknown,andthese (Figs.3and4).Astrikingcounter-exampleisthelargenumberof detections have originated from both frugivorous and insectivo- bat alphacoronaviruses that cluster together with the prototype rous bat hosts. Lack of detection in the remaining bat families is virusesHCoV-229E,-NL63andPEDV(Fig.3).Virusesfromnumer- likelydue tonon-exhaustivesampling of thealmost 1200 extant ousbathosts,togetherwithungulateandhumanviruses,arecon- bat species (Schipper et al., 2008; Simmons, 2005; Teeling et al., tainedinthispartoftheAlphacoronavirustree,andincontrastto 2005).Thisvoidmaybefilledinfuturestudies. the betacoronaviruses, the designation of clearly separated subc- ladesischallenging.However,moreworkneedstobedonetofor- 1.5.Batsascoronavirushostsworldwide mally analyze the degree of phylogenetic co-segregation in the Coronavirinaesubfamily. Fig.5showsthegeographicoriginofall53studiescharacteriz- ingnovelbatCoVsandTable1providesdetailsforthesestudies. 1.7.Coronavirushostswitchesfrombats The figure highlights that studies from all continents are now available, but there is a drastic lack of studies from resource- 1.7.1.Hostswitchessuggestedbyphylogenetically-relatedvirusesin limitedor politically unstable settings.Specifically, severalbiodi- humansandbats versityhotspotslinkedtotheemergenceofzoonoticviruses(Jones The most well-studied CoV host switches have probably oc- etal.,2008)arenotcoveredatall,includingtheCongobasin,large curredfrombatstohumans.Theforemostexampleistheparadig- parts of South-East Asia and the Neotropical ecozone. Future matichostswitchofSARS-CoVfromrhinolophidbatsintohumans 50 J.F.Drexleretal./AntiviralResearch101(2014)45–56 Fig.3. Phylogeneticrelationshipsbetweencoronavirusesandbathosts.DetailsofthephylogenyshowninFig.2forthegeneraAlpha-andBetacoronavirus.ICTVspeciesare giventotherightofcladedesignationsandbatsymbols,whenapplicable.Virusdesignationsincludestrainnames,GenBankaccessionnumbersandhostinformationasthe firstthreelettersofthelatingenusandspeciesnames.Batvirusesareshowninred.BoxesindicateAlpha-andBetacoronavirusgenera,accordingtothecoloringinFig.2. orpotentiallycivets(Balbonietal.,2011;Drexleretal.,2010;Lau However, not all rhinolophid bat species have been tested for etal.,2010a,2005;Lietal.,2005;Renetal.,2006;Rihtaricetal., SARS-relatedcoronaviruses.Forexample,only12oftheatleast19 2010; Yang et al., 2013). These human, civet and bat viruses are rhinolophidbatspeciesthatoccurinChinahavebeentestedand now officially summarized by the ICTV in one species, termed SARS-related coronavirus sequence information is only available SARS-relatedcoronavirus(deGrootetal.,2012). from 5 of these species (Lau et al., 2005; Li et al., 2005; Poon The genomic relatedness of human and bat SARS-related coro- etal.,2005;Tangetal.,2006;Wooetal.,2006,2007;Yangetal., navirusesisgreatestintheORF1ab,whileabatancestorcontaining 2013;Yuanetal.,2010).Therefore,furtherstudiesofRhinolophus thestructuralproteinsofhumanSARS-CoVhassofarnotbeende- species in Africa, Europe and Asia mayprovide more insightinto tected.BatSARS-relatedcoronavirusesfailtointeractwiththehu- the ancestral bat viruses that were the source of the emergence man SARS-CoV receptor molecule ACE2, possibly associated with ofhumanSARS-CoV. smalldeletionsintheirreceptor-bindingdomain(RBD),compared ItshouldalsobementionedthattheHipposiderosbetacoronav- tohumanSARS-CoV(Li,2013;Renetal.,2008).Inlinewiththese irusesdetectedinAfricaandAsiaareclearlydistinctfromSARS-re- differences, a bat SARS-related coronavirus synthesized by reverse lated coronaviruses. These viruses can be distinguished by both genetics was only infectious in cell culture and mice when the sequence distance-based taxonomic approaches described above. spike gene was exchanged by the human SARS-CoV homologue Additionally,thephylogeneticpositionandgenomicpropertiesof (Becker et al., 2008). Because the RBD of European rhinolophid the unclassified Hipposideros betacoronaviruses differ from SARS- batSARS-relatedcoronaviruseswasmorerelatedtothatofthehu- related coronaviruses. These genomic properties include chiefly man SARS-CoV than the RBD from Chinese bat viruses (Drexler their different viral 3’-genome ends and accessory ORFs down- et al., 2010), recombination may have played a role in the emer- streamfromthemembranegeneintheHipposiderosCoVs(Pfefferle genceofthehumanpathogenicvirus. etal.,2009;Quanetal.,2010). J.F.Drexleretal./AntiviralResearch101(2014)45–56 51 a virus termed PML/2011 from a South-African Neoromicia bat (Annanetal.,2013;Itheteetal.,2013);CoVsfromaSpanishHyp- sugosaviiandEptesicusisabellinus(Falconetal.,2011);sequences fromThaibatguano(Wacharapluesadeeetal.,2013);aCoVfrom a MexicanNyctinomopsbat(Anthonyetal.,2013a);andthefully sequencedbetacoronaviruscladecprototypebatCoVsHKU4and HKU5 from China (Woo et al., 2007). Clade c betacoronaviruses from Ghanaian Nycteris bats were more distantly related to the MERS-CoV(Annanetal.,2013).Ashort203-ntRdRpsequencefrag- ment100%identicaltotheMERS-CoVprototypestrainEMC/2012 wasdescribedinasingleSaudi-ArabianTaphozousperforatusbat, belonging to the family Emballonuridae (Memish et al., 2013). Although the high sequence identity should have facilitated characterization of other CoV genomic regions, no further virus sequencecouldbeobtainedfromthisspecimen. DetectionofcloselyrelatedCoVsindifferentbatfamiliesisrare, butmayoccur(Lauetal.,2012;Yangetal.,2013).However,evenif closely related MERS-CoV bat ancestors existed in both the dis- tantly related bat families Vespertilionidae and Emballonuridae (Fig.4),thelackoffurtherCoVsequenceinformationandthesingle detectionofashortgenomefragmentrequirefurtherconfirmation ofthisfinding.Interestingly,inthesamestudy,a202-ntsequence from a Rhinopoma hardwickii bat was detected which was 100% identical to BCoV and other Betacoronavirus 1 reference strains Fig.4. Batfamiliesinwhichcoronaviruseshavebeendetected.Phylogenyofextant (Memish et al., 2013). Again, whether clade a betacoronaviruses batfamiliesisshownaccordingto(Simmons,2005).Boxesindicatedescriptionsof alsoexistinbatsrequiresconfirmationbydetectioninmoreindi- alpha-andbetacoronavirusesaccordingtothecoloringinFig.2. vidualbatsandmoreCoVsequenceinformation. Regarding potential intermediate hosts of MERS-CoV, camels havebeenshowntohaveantibodiesagainstthisvirusathighrates andtiters(Pereraetal.,2013;Reuskenetal.,2013).Conclusionson thepassageofputativebatancestorsofMERS-CoVtohumansvia camels will only be possible upon genomic characterization of theviruseselicitingthisstrongantibodyresponse. Finally, no direct bat ancestor of HCoV-NL63 has ever been found,althoughthephylogeneticcladecontainingthisHCoVisen- closedbybatCoVs.WhiletherecentsuccessincultivatingHCoV- NL63 on immortalized bat cells may hint at some link between batsandthisvirus(Huynhetal.,2012),thebatvirusesdescribed inthatandallpreviousstudiesaregeneticallyratherdistantfrom HCoV-NL63(Cormanetal.,2013b). 1.7.2.Mechanismsofhostswitches The exactmechanismsby whichCoVs adapt to new hostsare unclear. Clearly, virus entry and innate immune responses are Fig.5. Distributionofbatcoronavirusstudies.StudiesreportingbatCoVsequences amongtheparamountobstaclestobeovercomeduringviralhost by country are indicated, with the number of studies given in blue circles and switching.ThehighdegreeofconservationoftheMERS-CoVrecep- adjusted in size accordingly. Country codes: BRA=Brazil, CAN=Canada, CRC=CostaRica,MEX=Mexico,PAN=Panama,TRI=TrinidadandTobago,USA=- torDipeptidylpeptidase4indifferenthostsisthusaworryingsce- United States of America, BGR=Bulgaria, GBR=Great Britain, GER=Germany, nario(Mulleretal.,2012;Rajetal.,2013).Generally,itisunclear ITA=Italy, NED=Netherlands, ROU=Romania, SLO=Slovenia, SPA=Spain, whetherdirectzoonotictransmissionfrombatstohumanshasoc- UKR=Ukraine, GHA=Ghana, KEN=Kenia, NGR=Nigeria, RCA=South African curred for any HCoV. Alternative scenarios involve intermediate Republic, CHN=China, JPN=Japan, PHI=Philippines, THA=Thailand. The black circles for theCentral African Republic (CAF),Gabon (GAB) and Australia(AUS) hosts,suchascarnivoresorungulates(Annanetal.,2013;Cotten indicate published sequences in GenBank, but lack of publication of the corre- et al., 2013; Enserink, 2013; Graham and Baric, 2010; Lau et al., spondingstudies.CountrieswhereCoVstudieshavebeenperformedareinblack, 2013;Songetal.,2005;Wuetal.,2012a). othersingrey. Onewaytorapidlyacquirenovelgenesthatpotentiallyfacili- tatehostswitchingisrecombinationbetweendifferentviruses.A canonical example of recombination in CoVs is FIPV type 2. The BatancestorswerealsofoundforHCoV-229EinAfricanHippo- prototype strains of this feline virus are recombinants between siderosbats(Pfefferleetal.,2009),andagrowingbodyofdataindi- FIPV type 1 and CCoV in different parts of the ORF1b and Spike catesthatbatsworldwideharbourCoVsrelatedtotheMERS-CoV genes (Herrewegh et al., 1998). Recombination has also been (Annan et al., 2013; Anthony et al., 2013a; Ithete et al., 2013; hypothesized to be involved in the emergence of the SARS-CoV Reusken et al., 2010; Wacharapluesadee et al., 2013; Woo et al., (GrahamandBaric,2010;Honetal.,2008;Lauetal.,2010a;Yuan 2007). Unfortunately, no complete genomes are available yet for etal.,2010)andHCoV-OC43genotypes(Lauetal.,2011).However, theputativebatancestorsofHCoV-229EandMERS-CoV. the observed recombination events in SARS-CoV and HCoV-OC43 PutativeMERS-CoVbatancestorshavebeenmostconsistently are restricted to genetically closely related viruses. The exact found in the bat family Vespertilionidae and the related family recombination partners giving rise to SARS-CoV have never been Molossidae. These CoVs include European Pipistrellus bat viruses; detected. 52 J.F.Drexleretal./AntiviralResearch101(2014)45–56 Another phenomenon putatively associated with CoV adapta- 2.Discussion tiontodistincthostsandcellsisexemplifiedbythevariabledele- tionsspanningover600nucleotidesintheglobularS1domainof Ten years after the SARS epidemic, there is an overwhelming theSpikegenethatisassociatedwithachangefromenteric(TGEV) bodyofevidencehighlightingtherelevanceofbatsfortheevolu- torespiratorytract(Porcinerespiratorycoronavirus,PRCV)tropism. tion of mammalian CoVs (Vijaykrishna et al., 2007; Woo et al., Whetherthe290-nucleotidedeletiondownstreamfromtheSpike 2009). From a taxonomic perspective, there is a clear need for gene in HCoV-OC43, in comparison to its likely ancestral virus the establishment of reliable criteria for these viruses, such as BCoV(Vijgenetal.,2006,2005),representsanadaptationtoahu- those established for the family Picornaviridae (Lauber and Gor- manhostremainsunknown. balenya, 2012b). The challenges observed in attempts to transfer these distance-based approaches to less well characterized virus families such as the Filoviridae (Lauber and Gorbalenya, 2012a) 1.8.Coronaviruspathologyinbatscomparedtoothermammalian highlightthatthecurrentsetofCoVtaxonomiccriteriawilllikely hosts havetobeoptimizedandthoroughlyvalidated. BoththeexistingICTVandtheRGU-basedattemptsuseasim- ThereislittleinformationontheclinicalconsequencesofCoV ilar methodological background and offer more reliable criteria infections for bat hosts. In humans, HCoVs-NL63, -OC43, -HKU1 than phylogeny alone. However, both suffer from several short- and-229Ecirculateconstantly.SARS-CoVdisappearedafewyears comings.Lossofdiscriminatorysharpnessupontheincrementin after its epidemic transmission, while the novel MERS-CoV may known CoV genetic diversity will likely limit these approaches, onlybestartingtospread(Enserink,2013).AllHCoVcauseprimar- althoughtheICTVcriteriamayprovemorerobust,duetothelarger ilyrespiratorysymptomsinhumans(HamreandProcknow,1966; genomicfragmentsincorporated.Fromatechnicalpointofview,it McIntoshetal.,1967;Peirisetal.,2003;vanderHoek,2007;van shouldbenotedthattheextensionoftheRdRpfragmentsizeused der Hoek et al., 2006; Woo et al., 2005), although the MERS-CoV forRGUdefinitionscanbechallengingwhenfieldspecimenscon- has also been associated with severe renal complications (Zaki tainlowRNAconcentrations(Cormanetal.,2013b;Drexleretal., etal.,2012).HCoVsheddinginfecesisnotuncommon(Liuetal., 2010). A similar difficultycan be observed for the ICTV proposal, 2004),andtherearesporadicreportsonCoVsinhumanswithgas- whichrequiresthecharacterizationofapproximately50%ofatyp- troenteritis,e.g.,acasereportonBCoVinapediatricpatientlead- icalCoVgenome.Themorethan20unclassifiedCoVspeciesinthe ingtothetentativedesignationHumanentericcoronavirus(Zhang currentICTVproposaldemonstratethatthistaskisnoteasilyful- et al., 1994). However, CoVs are apparently not generally related filledforviruseswhichhavenotbeengrowninculture(deGroot togastroenteritisinhumans(Esperetal.,2010). et al., 2012). Furthermore, both approaches do not incorporate Incomparison,theclinicalpictureinotheranimalsisconsider- anysequencedistance-baseddatafromgenomicregionsencoding ablymorevariable,rangingfrommildrespiratoryandgastroenter- thestructuralproteins.Besidesthetechnicalconstraintsoncharac- icsymptomstosystemicdiseasewithhepatitis,multi-organfailure terizingthesegenesthatwerestatedabove,thisislikelyalsodue anddeath(summarizedinSaif,2004).ThehighCoVconcentrations to thepoor sequence alignmentssome of these genes entail. The inbatfeces(Annanetal.,2013;Drexleretal.,2011),togetherwith highvariability,e.g.,ofthe Spikegene, complicatestheestablish- therecoveryofcoronaviralRNAfromthesmallandlargeintestine mentofcriteriaapplicabletothecompletesubfamily.Still,incor- offrugivorousbats(Watanabeetal.,2010)pointtoreplicationin porationofsomeofthemoreconservedstructuralgenes,suchas theenterictract.Thisiscompatiblebothwiththeuseofbatfecal the Envelope, Membrane or parts of the Spike and Nucleocapsid material or intestinal specimens to detect bat CoVs in almost all genes may prove helpful in carrying out this promising genome- published studies and with the high CoV RNA concentrations in basedtaxonomicapproach. the lower intestine of naturally infected hedgehogs (Corman Regarding mammalian CoV hosts, several questions remain to etal.,2013a).Althoughtherearenoapparentclinicalsignsofgas- beanswered.First,thecompleteabsenceofhost-specificCoVsin troenteritis(Watanabeetal.,2010)oranyotherdiseaseinbats,it monkeysorapesimpedesourunderstandingofanyputativeCoV shouldbenotedthatbatsappeartoraiseantibodiesagainsttheir evolution in other primates than humans. The apparently recent coronavirusesathighrates(Lauetal.,2010b,2005;Mulleretal., host switches responsible for the generation of HCoVs may indi- 2007;Tsudaetal.,2012).Whetherthiscorrelateswiththeseverity cate that even if CoVs exist in non-human primates (NHP), they ofinfectionremainsunknown. maynotberelevantfortheevolutionoftheirhumancounterparts. Fromanecologicpointofview,CoVsseemtorelyonmassive However, thecurrent lackof CoVdata fromNHPs preventsmore amplification on a population level during bat reproductive sea- definiteassertionsonthistopic. sonal cycles, potentially associated with fecal-oral transmission Second,intermediatehostsadapting batviruses to humansor (Drexler et al., 2011; Gloza-Rausch et al., 2008; Osborne et al., other mammals have neither been conclusively found for SARS- 2011).Similarphenomenahavebeenobservedforotherbat-asso- CoV, nor for any other HCoV. Despite the high seroprevalence of ciated viruses, such as the filo-, henipa-, astro- and lyssaviruses ChineseanimalhandlersagainstSARS-CoV,itisnotentirelyclear (Amman et al., 2012; Drexler et al., 2011; George et al., 2011; whethercivetsindeedadaptedSARS-CoVforinfectioninhumans Wacharapluesadee et al., 2010). Still, respiratory or vertical CoV (Graham and Baric, 2010; Guan et al., 2003). Similarly, the high transmissioncannotberuledoutatthispoint,mostlyduetothe seroprevalence of camels against MERS-CoV suggests that these necessityofshieldingtheseprotectedanimalsfromhumaninter- animals are a potential source of the initial introduction of this ference.Hypothetically,onecouldquestionwhetherinsectivorous virusintothehumanpopulation(Reuskenetal.,2013).However, batsthatrelyonafunctionallarynxforecholocationofpreycould thecurrentlackofMERS-CoVgenomicsequencesfromcamelspre- eventoleraterespiratoryinfection.Thishighlyspeculativescenario ventsdefiniteconclusionsonthisputativescenario. couldpointtoanevolutionofbat(corona-)virusestowardsinfec- Third, many mammalian orders have not been or are only tionoutsidetherespiratorytract.Alternatively,itcouldrepresenta poorly studied. This is exemplified by the single CoV detected in samplebiasduetotherapiddeathanddecayofbatssufferingfrom lagomorphs (rabbit coronavirus HKU14), which likely belongs to anyrespiratorydiseasethatimpairspredation,alikelyscenariogi- theBetacoronavirus1species,andbyMurineCoronavirus(thefor- ventheveryactivemetabolismofbats,whichforexamplerequires merMHV),theonlyknownrodentCoVspecies.WhetherCoVsex- insectivorous bats to consume several grams of insects per night ist in other mammalian hosts, and to what extent these viruses (EncarnaçãoandDietz,2006). J.F.Drexleretal./AntiviralResearch101(2014)45–56 53 contributetoourunderstandingofCoVevolution,fornowremain hostsorwithincreasedpathogenicitymayenablealternativestud- obscure. ies. These markers could include viral interferon antagonists and Finally,therecentdescriptionofdistantlyrelatednidovirusesin theRBD,bothofwhichcouldbeanalyzedinvitro,evenintheab- insectshasenabledhypothesesonaputativeinsectoriginofCoro- senceofvirusisolatesorcompletelycharacterizedgenomes. naviridaeancestors(Ngaetal.,2011;Zirkeletal.,2011).Interest- Finally,anyattempttoeradicatebatsorotherwildlifeinreac- ingly, the number of CoV descriptions from insectivorous bats is tion to virus descriptions should be strongly discouraged, as this large, and a clade c betacoronavirus was recently described from counteractstheecologicalrelevanceoftheseanimals,e.g.,forpol- insectivorous hedgehogs (Corman et al., 2013a). Insectivorous linationandpestreduction(Boylesetal.,2011;Kalkaetal.,2008). mammals, such as shrews, moles and hedgehogs from the order Eulipotyphla could thus be specifically investigated to bolster Acknowledgements hypothesesontheputativeancientinsectoriginsoftheCoVshar- boredbyinsectivorousmammalianhosts. We are grateful to Monika Eschbach-Bludau, Tobias Bleicker Ithasbeenhypothesizedpreviouslythatlargegenomesize,fre- andSebastianBrüninkfortechnicalassistance. quentrecombinationandhighmutationratemakecoronaviruses particularly prone to zoonotic host switching (Woo et al., 2009). These hallmarks require further consideration. 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