2009 Phylogenetic perspectives on the epidemiology and origins of SARS and SARS-like coronaviruses

2009·0.65 MB·English

Infection,GeneticsandEvolution9(2009)1185–1196
ContentslistsavailableatScienceDirect
Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid
Review
Phylogenetic perspectives on the epidemiology and origins of SARS and
SARS-like coronaviruses
Chi Wai Yip, Chung Chau Hon, Mang Shi, Tommy Tsan-Yuk Lam, Ken Yan-Ching Chow,
Fanya Zeng, Frederick Chi-Ching Leung*
TheSchoolofBiologicalSciences,TheUniversityofHongKong,PokfulamRoad,HongKong,China
AR TI CLE I NFO ABS TRA CT
Articlehistory: SevereAcuteRespiratorySyndrome(SARS)isarespiratorydiseasecausedbyazoonoticcoronavirus(CoV)
Received3May2009 namedSARS-CoV(SCoV),whichrapidlyswepttheglobeafteritsemergenceinruralChinaduringlate2002.
Receivedinrevisedform9August2009 TheoriginsofSCoVhavebeenmysteriousandcontroversial,untiltherecentdiscoveryofSARS-likeCoV
Accepted24September2009
(SLCoV)inbatsandtheproposalofbatsasthenaturalreservioroftheCoronaviridaefamily.Inthisarticle,
Availableonline30September2009
wefocusedondiscussinghowphylogeneticscontributedtoourunderstandingtowardstheemergenceand
transmissionofSCoV.WefirstreviewedtheepidemiologyofSCoVfromaphylogeneticperspectiveand
Keywords:
discussedthecontroversiesoveritsphylogeneticorigins.Then,wesummarizedthephylogeneticfindings
SARS
inrelationtoitszoonoticoriginsandtheproposedinter-speciesviraltransmissionevents.Finally,wealso
SARS-likecoronaviruses
discussed how the discoveries of SCoV and SLCoV expanded our knowledge on the evolution of the
Inter-speciestransmission
Evolution Coronaviridaefamilyaswellasitsimplicationsonthepossiblefuturere-emergenceofSCoV.
(cid:2)2009PublishedbyElsevierB.V.
Contents
1. Introduction.................................................................................................... 1186
1.1. Thediseaseanditsepidemiology ............................................................................. 1186
1.2. Thephylogeneticandzoonoticorigins ......................................................................... 1186
2. DissectingtheepidemiologyofSARSoutbreaksfromaphylogeneticperspective............................................. 1186
2.1. Super-spreading‘caught-in-the-act’............................................................................ 1186
2.2. Tracingthesourceoflocaloutbreaksinthelatephaseepidemic.................................................... 1186
2.3. Estimatingthetheoreticalonsetoftheepidemic................................................................. 1188
2.4. Sporadicre-emergenceinDecember2003 ...................................................................... 1188
3. ControversiesoverthephylogeneticoriginsofSCoV.................................................................... 1188
3.1. TheancienthistoryofanovelCoV ............................................................................ 1188
3.2. SCoVasanearly-splitofffromGroup2CoV:argumentsandcontentions............................................. 1188
3.3. Phylogeneticincongruenceandancientrecombination:factsandcautions ............................................ 1189
4. ZoonoticoriginsofSCoV.......................................................................................... 1189
4.1. CivetsastheimmediatesourceofSARSepidemics ............................................................... 1189
4.2. HorseshoebatsasthenaturalreservoirofSCoVandSLCoV ........................................................ 1191
4.3. ThemissinglinkbetweenSCoVsincivetsandcurrentlysampledSLCoVsinbats....................................... 1191
4.4. Estimatedtimeoftheinter-speciestransmissioneventsandtheirimplications ........................................ 1191
4.5. Adaptivemutationspotentiallyrelevanttointer-speciestransmission................................................ 1192
5. ThecontinuingstoryofCoVdiversity ............................................................................... 1192
5.1. Theexpandingdiversityandwideninghostrange................................................................ 1192
5.2. HowmuchdidrecombinationcontributetothediversityofCoV?................................................... 1193
6. Thelessonlearntandanalarmfornextzoonosis...................................................................... 1193
Acknowledgements.............................................................................................. 1193
References ..................................................................................................... 1193
* Correspondingauthorat:5N-12,KadoorieBiologicalScienceBuilding,TheSchoolofBiologicalSciences,TheUniversityofHongKong,PokfulamRoad,HongKong,China.
Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](F.-C.Leung).
1567-1348/$–seefrontmatter(cid:2)2009PublishedbyElsevierB.V.
doi:10.1016/j.meegid.2009.09.015
1186 C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196
1. Introduction aseriesofseeminglyindependentcasesandfollowedbythefirst
documented SSE in Hospital HSZ-2 in Guangdong at the end of
1.1. Thediseaseanditsepidemiology January2003(Zhongetal.,2003).Bythen,aninfectednephrologist
traveledfromGuangdongtoHongKongandinitiatedanotherSSE
Severe acute respiratory syndrome (SARS) is a highly con- inHotelMattheendofFebruary2003,leadingtotheworldwide
tagious respiratory disease caused by a previously unknown transmission of SARS and subsequent outbreaks in Hong Kong
coronavirus(CoV)namedasSARS-CoV(SCoV)(Ksiazeketal.,2003; (Ruan et al., 2003; Guan et al., 2004). The epidemic has been
Peiris et al., 2003). The major outbreak started from November dividedintothreephases(Fig.1)basedontheoccurrenceofthe
2002 as a rapid wave of epidemic from Guangdong Province of abovetwomentionedSSEs(CSMEC,2004).Theearlyphaserefers
China and spread through 29 regions around the world (WHO, totheperiodpriortotheSSEinHospitalHSZ-2whilethelatephase
2004a). The epidemic was effectively controlled under vigorous referstotheperiodaftertotheSSEinHotelM.Themiddlephase
quarantine measures and no new case was reported after July referstotheperiodbetweenthesetwoSSEs.
2003. Six months after its disappearance, SCoV re-emerged in Phylogenetic analyses enabled researchers to better under-
December 2003 as four sporadic cases in Guangdong Province, stand the transmission chains and trace the sources of viral
causingnofatalityorsecondarytransmission(WHO,2004b).SCoV epidemics, which provide important information for making
is probably one of the very few examples of zoonotic viral public health policy. Broadly speaking, phylogenies of SCoV
emergence‘caught-in-the-act’withadequatesequencessampled sequences generally agreed with the documented contact
fromdifferentphasesoftheepidemic(CSMEC,2004;Wangetal., histories, and additionally provided evidence to support some
2005a),aswellashighlyrelevantsamplesfromitszoonoticorigins uncorroboratedepidemiologicalspeculations(Zhao,2007).Phy-
(Guan et al., 2003; Song et al., 2005; Ren et al., 2006). Taking logenies reconstructed by Neighbor Joining (NJ) (CSMEC, 2004;
advantageofthewealthofthesesequencedata,theevolutionary Guanetal.,2004),MaximumLikelihood(ML)(Zhangetal.,2006;
behaviors of SCoV during the epidemic have been investigated Tang et al., 2007) and Bayesian methods (Tang et al., 2009)
extensively,e.g.chainsoftransmission,theoretictimeofepidemic consistently distinguished the late phase strains as a mono-
onset,rateofsubstitutionsandmodeofnaturalselectionactingon phyletic cluster,whichincluded the indexpatient ofthe SSEin
theviralgenome,etc.Inthefirsthalfofthisarticle,applicationsof HotelMandtheprimarycasesinVietnam,SingaporeandCanada
phylogeneticsforinvestigatingtheemergenceandtransmissionof (Guan et al., 2004), supporting the viewpoint that the SARS
SCoVwerereviewed. outbreakslaterinHongKong,aswellasthoseinotherpartsofthe
world,werelargely,ifnotcompletely,originatedfromacommon
1.2. Thephylogeneticandzoonoticorigins source. In fact, analyses of the S gene sequences suggested
multiplestrainsmighthavebeenindependentlyintroducedinto
Prior to the discovery of SCoV, members of the Coronaviridae HongKongfromGuangdongbeforetheSSEinHotelM,although
family can be unambiguously classified into three phylogenetic noneofthesestrainscontributedsubstantiallytothesubsequent
groups.Althoughinitialphylogeneticanalysesdidnotconfidently outbreaks(Tsuietal.,2003;Guanetal.,2004).Ontheotherhand,
classifySCoVasamemberofanyofthethreeexistinggroupsof the strains from early and middle phases are relatively more
CoV,futheranalysessuggestedSCoVmightshareanancientand diverse and appeared as multiple distinct clusters on the
distant ancestry with Group 2 CoVs (Snijder et al., 2003; phylogenies (CSMEC, 2004; Song et al., 2005; Wang et al.,
Gorbalenya et al., 2004). SCoVs were also isolated from small 2005b), agreeing with the epidemiological investigations that
mammals such as civets in wet markets, suggesting these SCoVshavebeencirculatinginGuangdongandcausedseemingly
mammals may have been the direct zoonotic origin of SCoV independentoutbreakspriortotheSSEinHotelM(Zhongetal.,
(Guan et al., 2003). Excitingly, wider animal surveys revealed 2003).ThisobservationfitsthemodelputforwardbyAntiaetal.
unanticipatedhighlevelsofgeneticdiversityofCoVinbats(Cui (2003)andimpliesthepossibleoccurrenceofmultiplezoonotic
etal.,2007).AgroupofCoVsnamedasSARS-likeCoVs(SLCoVs) transmissionsofphylogeneticallydistinct,butyetsimilar,SCoVs
which shares 88–92% nucleotide identity with SCoV, was also tohumanintheearlyphaseoftheepidemic.
identified from bats (Lau et al., 2005; Li et al., 2005b). These
findingsleadtothehypothesisthatbatsarethenaturalreservoirof 2.2. Tracingthesourceoflocaloutbreaksinthelatephaseepidemic
SCoVandothermembersoftheCoronaviridaefamily(Tangetal.,
2006; Vijaykrishna et al., 2007). The biological and evolutionary Theepidemiologyofsomelocaloutbreaksinthelatephasewas
aspects related to the conjectured inter-species transmission of also investigated using phylogenetics. Firstly, two major subse-
SLCoVfromitsnaturalreservoir(i.e.bats)totheintermediatehost quentoutbreaksinHongKong,theAmoyGardensoutbreak(Ng,
(i.e. civets), and finally to human, have been the center of 2003)andtheHospitalPoutbreak(TomlinsonandCockram,2003),
discussion(ShiandHu,2008).Inthesecondhalfofthisarticle,the were phylogenetically demonstrated to be directly linked to the
phylogeneticoriginsofSCoV,aswellasthediversityofCoVsinits SSE in Hotel M (Chim et al., 2003; Guan et al., 2004). Secondly,
zoonotic sources and the phylogenetic aspects of the speculated phylogenies suggested the Taiwan outbreaks were likely to be
inter-speciestransmissionevents,werereviewed. derivedfrommultiplesources(Lanetal.,2005b;Shihetal.,2005),
includingtheAmoyGardensoutbreak(Chiuetal.,2003;Lanetal.,
2. DissectingtheepidemiologyofSARSoutbreaksfroma 2005a) and the SSE in Hotel M (Yeh et al., 2004). Thirdly, the
phylogeneticperspective Singapore outbreak was traced back to two separate initial
introductions, but both were directly linked to the SSE in Hotel
2.1. Super-spreading‘caught-in-the-act’ M(Ruanetal.,2003;Vegaetal.,2004),whichlaterledtoseveral
othersubsequentoutbreakswithinSingapore(Chenetal.,2006)
EpidemiologyoftheSARSepidemichasbeenwell-documented andacaseinGermany(Liuetal.,2005a).Lastly,inBeijing,although
and one of its most intriguing characteristics is the concept of thefirstcasewasreportedapproximatelyaweekaftertheSSEin
‘‘super-spreading events’’ (SSE)(Lloyd-Smith et al., 2005), which HotelM(Bietal.,2003),phylogeneticanalysessuggestednotall
contributedsignificantlytotherapidspreadofthediseaselocally thecasesinBeijingwererelatedtotheSSEinHotelMandsomeof
andglobally(Lietal.,2004;Shenetal.,2004;Chenetal.,2006; thecaseswerelikelyoriginatedfromtheGuangdongpriorSSE(Liu
Zhongetal.,2003).Inmid-November2002,theepidemicstartedas etal.,2005c;Zhao,2007).
C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196 1187
Fig.1.AphylogenyofspikegenenucleotidesequencesfromSCoVisolatedfromhumans,civetsandraccoondogs.ThisphylogenywasmodifiedandadoptedfromLametal.
(2008a).Sequencesfromhumans,civets,raccoondogsandbatswereindicatedwithsymbols&,*,~and^,respectively.ThetreewasconstructedusingMLmethod,with
confidencesoftopologysummarizedfrom5000treessampledfromMLandNJbootstrapreplicatesandBMCMCsamples.Onlyconfidencevaluesofmajorclusterswere
shown(ML/NJ/BMCMC,intheparenthesis).Thehumanepidemiccluster(2002–2003)wasdividedintolate,earlyandmiddlephasesaccordingtoapreviousstudy(CSMEC,
2004).Accessionnumbersofthesequencesareshownwithinroundbracketsaftertheirstrainnames(inbold).Thedistanceunitwassubstitutions/site.Rp3isolatedfrom
bats(^)wasusedasanout-grouptorootthetree,andthegeneticdistanceofitsbranchisnotshown.
1188 C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196
2.3. Estimatingthetheoreticalonsetoftheepidemic the SCoVs of the 2002–2003 epidemic (Fig. 1), suggesting these
sporadic cases werelikely causedby inter-species transmissions
Robustestimationsonthetheoreticalonsetofaviralepidemic that were independent from the previous outbreak (Song et al.,
provide evidence to testify the hypotheses on viral origins, e.g. 2005;Lametal.,2008a).Theseepidemiologicalandphylogenetic
AIDS pandemic (Korber et al., 2000; Lemey et al., 2003, 2004; findings provided convincing evidence fordirect transmission of
Worobeyetal.,2004).Undertheassumptionofadequatesampling, SCoVfromcivetstohuman(discussedlaterinSection4).
thetheoreticalonsetofaviralepidemiccanbeinferredasthetime Insummary,theabovefindingsdemonstratedthevitalrolesof
of the most recent common ancestor (tMRCA) of all sampled phylogeneticsinunderstandingtheemergenceandtransmission
sequences.tMRCAcanbeestimatedusingarangeofphylogenetic of SCoV in such a short-lasting but sweeping epidemic. The
methods(Drummondetal.,2003),butallmethodsareprimarily adequacyofearlyphasesamplingandtherelativelyshortduration
basedontheassumptionofmolecularclocksofdifferentextents of the SARS epidemic make it an excellent textbook example on
(Pybus,2006).ItisnotedthatthistMRCAshouldtheoreticallybe how phylogenetic analyses can be used as an auxiliary tool in
olderthantheearliestdocumentedcase,giventhefactthatthere combinationwithclassicalepidemiologicalinvestigationstostudy
mustbeawindowperiodbetweentheemergenceandrecognition theemergenceofaviralepidemic.
of the epidemic and the length of this window period is often a
significant epidemiological interest (Stumpf and Pybus, 2002; 3. ControversiesoverthephylogeneticoriginsofSCoV
Worobeyetal.,2004).
InthecaseofSCoV,linearregressionmethodswhichcorrelate 3.1. TheancienthistoryofanovelCoV
divergencewithsamplingdateswerecommonlyusedtoestimate
thetMRCAintheearlystudies.Basedonsimplelinearregression, WhichoftheexistinggroupsofCoVisphylogeneticallyclosest
Zeng et al. (2003a) estimated the tMRCA of all human SCoVs at to SCoV? This question has been under the spotlight of most
aroundDecember2002,withthe95%credibleinterval(CI)from discussionsbecausetheanswermighthelptotracethezoonotic
September2002toJanuary2003,whichiscomparabletoalater origin of SCoV that is fundamentally important to public health.
similarstudy,withthesameestimateataroundNovember2002 Priortotheavailabilityofitscompletegenomesequences,initial
(95%CIfromJune2002toDecember2002)(CSMEC,2004).Later, phylogeneticanalysesofafragmentofORF1suggestedthatSCoV
Zhao et al. (2004) adopted three strategies to minimize possible mayrepresentanovelgroupthatisindependenttotheotherthree
errors and inferred the oldest bound of this tMRCA as early as existinggroups(Drostenetal.,2003;Ksiazeketal.,2003).Similar
spring2002.Aleastsquaremethod,evaluatedusingMonteCarlo conclusions were reached based on phylogenetic analyses on
simulations, has also been applied to estimate the onset of the multipleviralproteinsafterthecompletegenomesequenceswere
epidemicasAugusttoSeptember2002(Luetal.,2004).Whileall available(Marraetal.,2003;Rotaetal.,2003;Zengetal.,2003b).
the above studies relied on the assumption of a constant Based on the observation that SCoV appears to be genetically
evolutionary rate over all lineages, i.e. a strict molecular clock equidistant to other known CoV groups, Holmes and Enjuanes
(Pybus, 2006), enforcing a molecular clock to non-clocklike (2003)concludedthatSCoVisneitherarecenthost-rangemutant
datasets could lead to biased estimations (Yoder and Yang, ofaknownCoVnorarecentrecombinantbetweenknownCoVs,
2000). More recently, Salemi et al. (2004) applied a ML method butitprobablyevolvedseparatelyfromanancestoroftheknown
totestifytheclocklikebehaviorofadatasetoftentaxaandplaced CoVsinanunidentifiedhostforaremarkablylongperiodoftime
thistMRCAbetweenSeptemberandNovemberof2002. beforeitsemergenceinhuman.
Despite the differences in methods and datasets, the above Althoughtheaboveargumentwasgenerallywell-received,two
studies generally inferred the oldest bound of the onset of the follow-upquestionswerethenraised.Thefirstquestionis:IfSCoV
epidemicataroundmid-late2002.Theearliestdocumentedcaseof represents a lineage that anciently diverged from an ancestor of
SARSwasidentifiedon16thNovember2002(Zhongetal.,2003), theexistingCoVs,wouldthisancestorbelongtooneoftheexisting
whichisremarkablyclosetotheoldestboundsofitstheoretical CoV groups? It later led to the concept of ‘‘early split-off from
onset when compared with other well-known viral epidemics Group 2 CoV’’ for describing the phylogenetic origin of SCoV
(Stumpf and Pybus, 2002; Worobey et al., 2004). This finding (Snijderetal.,2003).Thesecondquestionis:IsSCoVan‘‘ancient-
suggeststheSCoVhasbeenquicklyrecognizedafteritsemergence recombinant’’ (if not a ‘‘recent-recombinant’’) between the
inhuman,partiallyreflectingtheexplosivemodeoftransmission ancestors of any existing CoV groups? Due to relatively high
inearlyphaseoftheepidemic. divergencebetweenSCoVandotherCoVs(Rotaetal.,2003)aswell
AfterthediscoveryofSCoVincivetsandsporadicre-emergence asthelackofarobustout-group(HolmesandRambaut,2004),the
of SCoV in December 2003, these SCoV strains have been conclusionsderivedfromvariousrecombinationanalyseswereyet
incorporated into datasets for estimation of their tMCRA (Song intricateandinconsistent(Gorbalenyaetal.,2004).Anumberof
etal.,2005;Vijaykrishnaetal.,2007;Honetal.,2008).ThistMRCA reasonswerethenproposedtoexplaintheobservedphylogenetic
represents the upper limit of the time (i.e. oldest bound) of the incongruence might not truly reflect the recombinant origin of
inter-speciestransmissionofSCoVfromcivetstohuman,whichis SCoV (Holmes and Rambaut, 2004). The following sessions
phylogenetically different from the tMRCA of all human SCoVs summarized the findings from numerous studies regarding the
discussedaboveandshouldtheoreticallybeolder.Theestimated controversialphylogeneticoriginsofSCoV.
timeofthisinter-speciestransmissioneventwillbediscussedlater
inSection4. 3.2. SCoVasanearly-splitofffromGroup2CoV:argumentsand
contentions
2.4. Sporadicre-emergenceinDecember2003
Followingtheinitialanalyses(Marraetal.,2003;Rotaetal.,2003;
InDecember2003,fourseeminglyindependentcasesofSCoV Zeng et al., 2003b), the phylogenetic position of SCoV have been
infectionwerereported(Liangetal.,2004;WHO,2004b).Allthe vigorouslyreevaluatedwithvariousapproachesthatweremainly
patientshavedirectorindirectcontacthistorywithwildanimals differentintheirchoicesofgenomeregions(Eickmannetal.,2003;
(Song et al., 2005; Wang et al., 2005a). Phylogenetic analyses of ZhuandChen,2004;Kimetal.,2006)androotingstrategies(Snijder
humanSCoVsfromthesesporadiccasesandcivetSCoVscollected etal.,2003;Gibbsetal.,2004;LioandGoldman,2004).Inspiredby
fromthesameperiodformedamonophyleticgroupdistinctfrom the positional conservation of cysteine residues between the S1
C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196 1189
regionofSCoVandGroup2CoVs,Snijderetal.(2003)constructedan 1999), has been widely used as an indicator for homologous
unrooted NJphylogenybased onthe aminoacidsequencesofS1 recombinationamongviralgenomes(Posadaetal.,2002).Several
region and concluded SCoV is ‘‘closely related’’ to Group 2 CoVs studies demonstrated phylogenetic incongruence within the
(Eickmannetal.,2003).Intwolaterstudies,concatenatedamino genome of SCoV based on a wide range of methods (Rest and
acidalignmentsofmultipleviralproteinswereusedtoconstruct Mindell, 2003; Stanhope et al., 2004; Stavrinides and Guttman,
unrooted phylogenies using various methods and the results 2004;Zhangetal.,2005b).Despitethestatisticalsignificanceofthe
consistentlydemonstratedthemonophyleticrelationshipbetween phylogeneticincongruenceobservedinthesestudies,wecouldnot
SCoVandGroup2CoVs(ZhuandChen,2004;Kimetal.,2006).In concludeaconsensuspatternofrecombinationsincetheirfindings
additiontotheseunrootedphylogenies,rootedphylogeniesbased were generally inconsistent. As an example, the potential
on ORF1 alignments reached similar conclusions that SCoV and recombination events within S gene proposed by Zhang et al.
Group2CoVsharedthelastcommonancestor(Snijderetal.,2003; (2005b) were different from those proposed by Stavrinides and
Gibbsetal.,2004;LioandGoldman,2004).However,dependsonthe Guttman(2004),andwereundetectablefromthestudyofRestand
choicesofout-groups,therootingpositionsinthesestudieswere Mindell(2003).Infact,theconsiderabledivergencebetweenSCoV
inconsistent,whichmightberelatedtothepossiblycompromised andtheexistingCoVgroupshasalreadyexcludedthepossibility
accuracy of alignment if the out-groups are too diverged to be thatSCoVisarecentrecombinantfromtheexistingCoVgroups.
aligned(HolmesandRambaut,2004). Alternativelyspeaking,ifrecombinationeventshadoccurred,they
Nonetheless,theconsensusofabovefindingsisthatSCoVcould havetobetheancientones(Bosch,2004),i.e.boththeparentsand
beclassifiedasa‘‘distant’’memberofGroup2CoVs(Gorbalenya daughter could have been evolved considerably after the
etal.,2004).Thisconclusionwasreachedprimarilybasedonthe recombination events. A simulation study demonstrated the
robustness of the monophyletic relationship between SCoV and accuracy for detecting these ancient recombination events can
Group 2 CoVs. However, considering the actual genetic distance besubstantiallydiminishediftheyweresignificantlyobscuredby
betweenSCoVandGroup2CoVs,whichiscomparabletotheinter- the subsequent post-recombination substitutions (Chan et al.,
group distance among the three CoV groups, the biological 2006).Therefore,theintricacyofthefindingsonthephylogenetic
significance of classifying SCoV as a distant member of Group 2 incongruencewithinthegenomeofSCoVmaybeareflectionofthe
CoVisrelativelylowatbest.Webelieveclassificationofanovel varying sensitivity of the detection methods on the ancient and
CoV should not be merely based on the branching orders of obscuredrecombinationevents,ifany.
phylogenies and quantitative measurement of genetic distance Tofurtherinvestigatethereportedphylogeneticincongruence
shouldalsobetakenintoaccount. withinthegenomeofSCoV,RambautandHolmesre-evaluatedthe
Ontheotherhand,reconstructingtheevolutionaryrelationships study of Stavrinides and Guttman (2004) and suggested the
between highly divergent taxa has been proven as a difficult patterns cited as evidence for recombination are more probably
phylogenetic task (Philippe and Laurent, 1998). In particular, the causedbyavariationinsubstitutionrateamonglineages(Holmes
observedbranchingorderofthedeepnodesinthehighlydivergent andRambaut,2004).Inaddition,theauthoralsostatedtheeffectof
CoVphylogeniesmaynotreflecttheirtrueevolutionaryhistory,due the long-branch attraction on the branching order of deep
to the possible influences of the long-branch attraction and rate phylogeniesmayalsobeasourceofartifact,althoughunproven.
variationamonglineages(GribaldoandPhilippe,2002;Holmesand Moreover, even if recombination did not occurred, given the
Rambaut,2004).Infact,HolmesandRambautreevaluatedthetree stochasticnatureofevolution,theauthorswouldexpecttoobserve
topologies depicting the three possible phylogenetic positions of phylogenetic incongruence among small genome fragments of a
SCoV using Kishino-Hasegawa test, which is not biased by rate set of divergent taxa like SCoV and other CoVs. Therefore, the
variations, and that demonstrated the topology with the best observedphylogeneticincongruenceamongthehighlydivergent
likelihoodisnotnecessarilysignificantlybetterthantheothertwo genomesofCoVanditspossibleindicationonancientrecombina-
topologies, even the deep nodes were well-supported by quartet tioneventsshouldbeinterpretedwithextracautions.Uptothis
puzzlingsupportvalues(HolmesandRambaut,2004).Thisfinding point,thecurrentphylogeneticevidencesupportingtherecombi-
indicatedcarefulinterpretationsareneededbeforereachingafirm nanthistoryofSCoVisweakatbest(HolmesandRambaut,2004).
conclusionfromthebranchingorderofdeepphylogenies. Puttingasidetheintricatephylogeneticevidence,thepresence
Until recently, the addition of several newly discovered CoV ofstem-loopIImotif(S2m)inthegenomeofSCoVhavealsobeen
lineages to the phylogeny broke up some of those longbranches takenasanindicationforrecombination(Marraetal.,2003).S2m
(Holmes and Rambaut, 2004), which tends to distribute the is a conserved RNA motif present in the genomes of several
convergent and parallel mutations more evenly across the tree membersofAstroviridae,Coronaviridae,andPicornaviridaefamily
and hence reduce the problem of long-branch attraction (Hillis, (Jonassenetal.,1998)whileSCoVandGroup3CoVsaretheonly
1996).InFig.2,thecloserphylogeneticrelationshipbetweenSCoV membersoftheCoronaviridaethatpossethemotif.Assumingthe
and Group 2 CoVs suggested from the previous studies is also motifinSCoVandGroup3CoVwerenotacquiredindependently,
supportedinthisrelativelywell-sampledphylogeny.Tothisend,the the explanation of the co-presence of S2m should be either, (1)
questionofwhetheritisappropriatetoclassifySCoVasadistant SCoVandGroup3CoVssharethesameancestry,or(2)SCoVhave
memberofGroup2CoVsseemstobeataxonomicproblemmore acquiredthemotiffromGroup3CoVsthroughrecombination(or
thanavirologicalone,sinceSCoVandGroup2CoVsaresodivergent vice versa). Currently, the phylogenetic data does not provide a
that they are likely to possess a substantial number of unique bettersupportforanyoftheabovescenariosbutawidersurveyfor
biological characteristics, e.g. host ranges and receptor usages thepresenceofS2minotherunknownCoVlineageswillcertainly
(Haijemaetal.,2003;Lietal.,2003).Withtherecentdiscoveriesof provide insights to the ancient evolutionary history of the
novel CoV lineages, the classification of coronaviruses has to be Coronaviridaefamily.
revisedsystematicallyinbothbiologicalandphylogeneticcontext.
4. ZoonoticoriginsofSCoV
3.3. Phylogeneticincongruenceandancientrecombination:factsand
cautions 4.1. CivetsastheimmediatesourceofSARSepidemics
Phylogeneticincongruence,whichisoftenjustifiedbyincom- SincetheearlySARScasesinGuangdongseemstoberelatedto
patibletreetopologiesfordifferentgenomeregions(Holmesetal., restaurant workers handling wild mammals, Guan et al. (2003)
1190 C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196
Fig.2.AphylogenyofallknownCoVs(n=40).ThephylogenywasconstructedbasedontheaminoacidsequencesoftheRNA-dependentRNAPolymeraseregion
(length=163a.a.).ThephylogenywasconstructedusingBEAST(DrummondandRambaut,2007)underauncorrelatedlognormallyrelaxedclockmodel(Drummondetal.,
2006).ThenumberatthenodesindicatestheBayesianposteriorprobabilitysupport(aspercentages)summarizedfromtreessampledatevery1000thstepofaBMCMCchain
of10,000,000stepswherevalueslowerthan80%werenotshown.Themeansubstitutionratewasfixedat1.0andthebranchlengthwasexpressedinunitsofsubstitutions
persite.FIPV,Felineinfectiousperitonitisvirus(AY994055);CFBGDDM9503,ChineseferretbadgerCoV2003(EU769560);PRCV,PorcinerespiratoryCoV(DQ811787);TGEV,
Transmissiblegastroenteritisvirus(NC_002306);PEDV,Porcineepidemicdiahrreavirus(NC_003436);BtCoV5122005,BatCoV512-2005(NC_009657);BatCoVHKU6,BatCoV
HKU6(DQ249224);BtCoV1A,BatCoV1A(NC_010437);BtCoV1B,BatCoV1B(NC_010436);BatCoVHKU8,BatCoVHKU8(NC_010438);HCoV229E,HumanCoV229E
(NC_002645);HCoVNL63,HumanCoVNL63(NC_005831);BatCoVHKU2,BatCoVHKU2(EF203064);HCoVOC43,HumanCoVOC43(NC_005147);BCoV,BovineCoV
(NC_003045);AntelopeCoV,SableantelopeCoV(EF424621);GiCoV,GiraffeCoV(EF424622);PHEV,Porcinehemagglutinatingencephalomyelitisvirus(NC_007732);SDAV,Rat
sialodacryoadenitisCoV(AF124990);MHV,Mousehepatitisvirus(NC_006852);HCoVHKU1,HumanCoVHKU1(NC_006577);BatCoVHKU9,BatCoVHKU9(NC_009021);
SCoV(NC_004718);Rp3,BatSLCoV(NC_009693);BatSCoVHKU3,BatSLCoVHKU3(NC_009694);Rm1,BatSLCoV(NC_009696);Rf1,BatSLCoV(NC_009695);BtCoV1332005,
BatCoV-133-2005(NC_008315);BatCoVHKU4,BatCoVHKU4(NC_009019);BatCoVHKU5,BatCoVHKU5(NC_009020);IBV,Infectiousbronchitisvirus(NC_001451);
IBVpeafowl,peafowlCoV(AY641576);IBVpartridge,partridgeCoV(AY646283);TCoV,TurkeyCoV(NC_010800);SW1,BelugawhaleCoV(NC_010646);BuCoVHKU11,Bulbul
CoV(NC_011548);ThCoVHKU12,ThrushCoV(NC_011549);MuCoVHKU13,MuniaCoV(NC_011550);ALCGXF23006,AsianleopardcatCoV2006(EF584908);CFBGXF24706,
ChineseferretbadgerCoV2006(EF584911).
surveyedthewildanimalsinalocalmarketandisolatedCoVsfrom periodformedamonophyleticcluster(Songetal.,2005;Lametal.,
Himalayan palm civets, which share 99.8% genome sequence 2008a). These findings suggested that the emergence of SCoV in
identity to the SCoVs in human. Initial phylogenetic analyses humanislikelytoberesultedfromdirecttransmissionsofSCoVs
suggested SCoVs from human and civets formed two distinct fromcivets.Inaddition,phylogeneticanalysesdemonstratedthat
clades (Guan et al., 2003), but the SCoVs from civets are theSCoVsfromthe2002–2003epidemicandthe2003–2004re-
phylogeneticallyclosertotheSCoVsoftheearly-phaseepidemic emergence are phylogenetically distinct, suggesting the inter-
thantothoseinthelate-phaseepidemic(Kanetal.,2005).These species transmission events from civets to human in the two
findingsstronglysuggestcivetsweretheimmediatesourcesofthe outbreaks might be independent (Song et al., 2005; Wang et al.,
SCoVsleadingtotheearliestSARScasesinGuangdong.Theroleof 2005a).
civetsastheimmediatezoonoticsourceoftheSARSepidemicwas However,alarge-scalesurveyofSCoVsofcivetsinmarketin
further revealed during the sporadic re-emergence of SCoV in Chinasuggestedthelackof widespreadinfectionsincivets(Kan
December2003(Wangetal.,2005a),basedontheobservationthat etal.,2005).Inaddition,thegeneticdiversityofcivetSCoVswas
the SCoVs isolated from civets and those patients of the same relatively limited and was comparable to that of human SCoVs
C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196 1191
(Song et al., 2005; Lam et al., 2008a). These findings suggested 4.3. ThemissinglinkbetweenSCoVsincivetsandcurrentlysampled
civets might not be the natural reservoir of SCoV and it only SLCoVsinbats
acquiredSCoVsshortlybeforetheemergenceofSCoVinhuman.
Based on phylogeneticanalyses using dynamichomology, Janies Although the hypothesis of horseshoe bats as the natural
etal.(2008)speculatedthatcivetsmightalsoacquiredSCoVfrom reservoirforSLCoVandSCoVisrelativelysensible,howbatsSLCoV
otherspecies,possiblyhuman,evenaftertheemergenceofSCoVin weretransmittedtocivetsisstillunexplained.Ifcivetsacquired
2002.Anexperimentalevidencesuggestingcivetsmaynotbethe SLCoV from bats shortly prior to its emergence in human as
natural reservoir of SCoV is the observed symptoms in civets proposed,thisbatSLCoVstrainshouldbegeneticallyverysimilar
experimentallyinfectedwithSCoV(Wuetal.,2005),basedonthe totheSCoVssampledfromcivets.However,basedontherelatively
factthatthenaturalreservoirhostsusuallydonotdisplaysevere distantphylogeneticrelationshipbetweenSCoVsandSLCoVs,none
signs of infection (Hudson et al., 2002). The above observations ofthecurrentlysampledSLCoVsinbatsisthedescendantofthe
lead to the speculation of another natural reservoir host, which directancestorofSCoVsinhumanandcivet(Honetal.,2008).In
harbors a diverse group of SCoV-related CoVs and transmitted particular,Liand coworkerspointedoutthatsubstantialgenetic
SCoVtocivetspriortotheemergenceofSCoVinhuman. changesintheSproteinofthecurrentlysampledSLCoVarelikely
to be necessary for the virus to infect civets or human (Li et al.,
4.2. HorseshoebatsasthenaturalreservoirofSCoVandSLCoV 2006).Therefore,thedirectancestorofSCoVsinhumanandcivets
remainselusive.
As a result of extensive searches for the natural reservoir of More recently, Hon et al. (2008) demonstrated significant
SCoV, two groups of researchers independently identified a phylogenetic discordances among different genome regions of
diverse group of CoVs from various species of horseshoe bats SLCoV strain Rp3 and speculated its potentially recombinant
(Rhinolophusspp.).TheseCoVsshared87–92%genomenucleotide origin.PhylogeneticanalysisoftheparentalregionsofRp3genome
identitywithSCoVsandformedadistinctmonophyleticcluster suggested the presence of an uncharacterized bat SLCoV lineage
withSCoVs,thereforetheywerenamedSARS-likeCoV(Lauetal., (i.e. HB-SLCoV in Fig. 3) that is phylogenetically closer to SCoVs
2005; Li et al., 2005b). The close evolutionary relationship than any of the currently sampled bat SLCoVs. Based on the
between SCoV and SLCoV is also supported by the presence of relatively high genetic diversity among the currently sampled
theS2mmotifintheir30UTR(Tangetal.,2006;ShiandHu,2008). SLCoVsinbats,theexistenceofaphylogeneticallydistinctlineage
Basedonthephylogeneticanalysesofthefourcharacterizedfull of SLCoV not yet sampled is highly possible. Thus, the authors
genomesofSLCoVinhorseshoebats,theremarkablyhighgenetic speculatedthatthedirectancestorofSCoVswasasadescendant
diversity of SLCoVs strongly suggested horseshoe bats are the derivedfromthisnotyetsampledlineage,whichcrossedfroma
natural reservoir of SCoV and SLCoV (Ren et al., 2006). This horseshoebatspeciestocivets(Honetal.,2008).
conceptisfurthersupportedbytherelativelyhighprevalenceof
SLCoV in R. sinicus (Lau et al., 2005) and the geographically 4.4. Estimatedtimeoftheinter-speciestransmissioneventsandtheir
widespreadinfectionsofSLCoVsinbatsfromdistinctlocationsin implications
China(Lietal.,2005b).Thecurrenthypothesisisthatcivetsmight
have acquired SLCoVs from horseshoe bats and transferred to Determining the time of inter-species transmission events
human,whichisconsistentwiththeobservationthatthe29-nt mighthelpustocomprehendtheviralzoonosisofthevirusfrom
deletioninORF8areretainedinbatSLCoVs,civetSCoVsandearly anevolutionarystandpoint.Theoldestboundoftheinter-species
phasehumanSCoV(Lauetal.,2005). transmission of SCoV from civets to human is theoretically
Fig.3.Atime-scaledphylogenyofSCoVandSLCoV.ThisphylogenywasmodifiedandadoptedfromHonetal.(2008).ThephylogenywassummarizedfromallMCMC
phylogeniesoftheOrf1datasetanalyzedunderaBayesianrelaxedclockmodel.Heightofthenodeswasrepresentedbythemedianofitsestimates.Thewindowperiod
betweenthecross-specieseventandtheonsetofSARSepidemicwasindicatedasadottedline.Inthetaxalabels,H,CandBrepresenthostofhuman,civetsandbats,
respectively.
1192 C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196
correspondenttothetMRCAofallhumanandcivetSCoVs.Firstly, whichiswidelyusedasameasureforselectionpressure(Yangand
Songetal.(2005)estimatedthesynonymoussubstitutionrateof Nielsen, 2002), has been employed in several studies to detect
SCoVsusinglinearregressionandplacedthistMRCAtobearound positiveselectiononviralgenesofSCoVandSLCoV.Firstly,thev
early December 2002 without providing CI. In a later study, valuesofstructuralgeneswerefoundtobehigherthanthatofthe
Vijaykrishna et al. (2007) investigated this tMRCA by applying a non-structural region, suggesting the structural proteins might
Bayesian relaxed clock model (Drummond et al., 2006) to a havebeenunderastrongerselectionpressure(Zhaoetal.,2004;
phylogeny of all representatives from the CoV family and Songetal.,2005).Moreover,thevvalueofSgeneinthestrains
estimated it at around 1999 with 95% posterior bounds of 13 from early phase is significantly larger than that of those from
years (i.e. 1990–2003). More recently, Hon et al. (2008) recon- middle and late phases (CSMEC, 2004). The above findings
sideredthistMRCAusingvariousclockmodelsandestimateditat providedpreliminaryphylogeneticevidenceforthepotentialrole
around September 2002 with 95% CIs between January and ofSproteininadaptationofSCoVfromcivetstohuman.
Decemberof2002.ThistMRCAisveryclosetotheobservedfirst Additionally,lineage-specificvintheSgenephylogeny(n=11)
caseofSARS(16thNovember2002)aswellastheestimatedonset wasestimatedusingacodon-basedgeneticalgorithm(Kosakovsky
oftheepidemic(asdiscussedearlier),suggestingSCoVsmighthave Pondetal.,2006),andasignificantlyhighervvaluewasobserved
crossed from civets to human just months before the outbreak, alongthelineageleadingtothehumancluster(Vijaykrishnaetal.,
supportingtheviewthatcivetsaretheimmediatezoonoticsource 2007).Morerecently,Tangetal.(2009)comprehensivelyanalyzed
of SCoVs in human. It also implies the civet SCoVs might have a larger dataset (n=59) using a ML branch-site codon model
adapted quickly, or alternatively speaking, a ‘by-pass’ host with (Zhangetal.,2005a),aimedatcomparingthevvaluesoflineages
onlyminimaladaptationisneeded,toestablishasustainablechain ofvariousepidemicphasesorhosts(i.e.foreground)versustherest
oftransmissioninhuman. ofthephylogeny(i.e.background).Theauthorsconcludedthatthe
On the other hand, the time of the speculated inter-species 2002 early and middle phase human lineages and the 2003
transmission of SLCoV from bats to civets has also been sporadic-re-emergence human-civet lineages are under positive
investigated. In the study described earlier (Vijaykrishna et al., selection.Despitethedifferencesinmethodologiesanddatasets,
2007),theoldestboundofthetimeofthiseventwasfirstestimated both studies demonstrated phylogenetic evidence for positive
atameanof1986witha38-yearcredibleinterval(i.e.1964–2002). selection along lineages relevant to the civets-to-human inter-
Later, Hon et al. (2008) employed the concept of estimating the speciestransmissionontheSgenephylogeny.
periodbetweentimeofdivergence(tDIV)andtMCRAproposedby On the other hand, positively selected residues on S protein
Lametal.(2008b),andspeculatedthisinter-speciestransmission havebeenidentifiedinanumberofsimilarstudies,primarilyby
event might have happened with a median of 4.08 years before applyingMLcodonmodelstosimilardatasetsbutwithdifferent
onset of the epidemic (credible intervals of 1.45–8.84 years) epidemic groupings (Zhang et al., 2005a, 2006; Shi et al., 2006).
(Fig.3).Theabovetwoestimatesarenotcontradictorysincethe Although the residues identified from these studies are not
later estimate falls within the credible intervals of the former completely overlapping, probably due to the differences in taxa
estimatebutwithanimprovedprecision.Basedonthisrelatively groupings, these sites are mainly located in the S1 subunit and
short window period between the inter-species transmission residue479isconsistentlydetectedtobeunderpositiveselection,
eventandonsetoftheepidemic,Honetal.(2008)speculatedthat supportingitsspeculatedrolesinadaptationfornewhosts(Song
civets might have acquired the ancestor of SCoV from the host et al., 2005). It should be noted that positively selectedresidues
speciesofSLCoVstrainRp3directlyandtheinvolvementofother werenotidentifiedintheSgenesfromneitherthelateepidemic
intermediate species may be unlikely. Therefore, authors sug- phase (Zhang et al., 2006) nor the SLCoV in bats (Zhang et al.,
gested more focused surveillance on the host species of SLCoV 2005a). The mode of selection relevant to the inter-species
strain Rp3 may shed light on the zoonotic origin of the direct transmission from bats to civets has not been investigated
ancestorofSCoVincivets. systematically, primarily due to the lack of bat SLCoV S gene
sequences that are phylogenetically close enough for robust
4.5. Adaptivemutationspotentiallyrelevanttointer-species analysis.
transmission
5. ThecontinuingstoryofCoVdiversity
ThehostspecificityofCoVsismainlydeterminedbythebinding
betweenthespike(S)proteinanditscellularreceptors(Haijema 5.1. Theexpandingdiversityandwideninghostrange
et al., 2003). Angiotensin I-converting enzyme 2 (ACE2) is a
functionalreceptorforSCoVsinbothhuman(Lietal.,2003)and Asaresultoftheextensiveeffortsinsearchingforthezoonotic
civets(Lietal.,2005c),andwasdemonstratedtointeractwiththe originsofSCoV,ourknowledgeonthehostrangeanddiversityof
receptorbindingdomain(RBD)intheS1subunitoftheSprotein CoVshasbeenexpandedrapidly.Forexample,inthelastcoupleof
(Wongetal.,2004).Basedontheobservedmutationswithinthe years,thenumberofavianspeciesdetectedtoharborGroup3CoVs
RBDinstrainsfromdifferentepidemicphasesandhosts,Lietal. has been doubled (Cavanagh, 2005). Furthermore, in addition to
(2005c) demonstrated that binding of S1 subunit to human and thetwonewlydiscoveredhumanCoVsintheexistingGroup1and
civet ACE2 can be significantly altered by mutating only two 2,i.e.NL63(Fouchieretal.,2004)andHKU1(Wooetal.,2005a),
residuesontheRBD(residue479and487),suggestingthesetwo respectively,anumberofdivergentCoVshavebeenidentifiedfrom
residues maycontributeto theadaptation of SCoVfrom civetto AsianLeopardCatsandChineseFerretBadgers(Dongetal.,2007),
human.Later,thisviewpointwasfurthersupportedbythelocation wild birds (Woo et al., 2009) and a beluga whale (Mihindukula-
of these two residues at the binding interface between RBD and suriya et al., 2008). These findings largely expanded the known
ACE2incrystalstructures(Lietal.,2005a;Li,2008). diversity of CoVs and the long-established classification of the
Asobservedinotherexamplesofviralhostshifts(Parrishand Coronaviridaefamilyasthreedistinctgroupsneedstoberevised
Kawaoka,2005),themoleculardeterminantsforhostspecificity, systematically. On the other hand, a number of diverse CoVs
e.g. RBD residues related to adaptation to human in the case of belongingtoGroup1and2havebeenidentifiedfromarangeofbat
SCoV, are likely to be subjected to an elevated level of selection species (Woo et al., 2006). The surprisingly high diversity of bat
pressure during the acquisition of a new host. The ratio of non- CoVs rationally leads to the hypothesis that bats are the natural
synonymous (dN) to synonymous substitution rate (dS), i.e. v, reservoirofallCoVs(Tangetal.,2006).Accordingtoatime-scaled
C.W.Yipetal./Infection,GeneticsandEvolution9(2009)1185–1196 1193
phylogeny of representative CoVs from all groups, Vijaykrishna helpthecommunitytobetterprepareforthenextzoonoticviral
et al. (2007) concluded that bats are likely to be the host of the epidemic.
ancestorforallpresentlyknownCoVlineages.Furthermore,based More importantly, while most of the attentions have been
on the results of their Bayesian coalescent analyses, the authors focusedonpreparingfortheknownpotentialpandemicslikeavian
speculatedadiversegroupofCoVsmaybeendemicinvariousbat influenza(Fauci,2006),theunanticipatedstrikeofSARSepidemic
species, with repeated introduction to other animals and occa- alarmedpublic healthofficials and researchers fortheneglected
sionalestablishmentofnewlineagesinotherspecies(Vijaykrishna possibilityofdeadlyviralemergencefromanunexpectedorigin.
et al., 2007). In fact, other than the speculated inter-species Formanyyears,CoVshavelongbeenregardedasrelatively‘‘mild’’
transmissionofCoVsfrombatstootheranimalspecies,hostsshifts viruses with broad but yet restricted host ranges. However,
ofbatCoVsbetweendifferentRhinolophusspp.werealsoproposed according to our current understanding to the diversity and
basedontheincongruencebetweenthephylogeniesofCoVsand evolutionofCoVs(Vijaykrishnaetal.,2007),atleastfivedivergent
theirhostRhinolophusspp.(Cuietal.,2007). speciesofCoVareknowntohavezoonotictransmissionintothe
Additionally, inter-species transmissions of CoVs between human population and this cross species event will be likely to
othernon-batspecieswerealsoproposed.Thefirstanimal-human continue,andthezoonosisofSCoVwasjusttheconsequenceofone
zoonotic pair of CoVs being detailedly analyzed was Bovine CoV oftheseinter-speciestransmissionevents.Althoughthepreven-
andHumanCoV-OC43(Vijgenetal.,2005).Basedonacombination tion of zoonosis from an unexpected origin seems to be
ofmolecularclockanalyses,theauthorsestimatedthetMRCAof impractical,sufficientlyflexibleandstringentsurveillancestrate-
these two CoVs at around 1890 and speculated an inter-species gies provide us an opportunity to anticipate the disease in a
transmission event from Bovine CoV to human might have population and prevent its further spreading by implementing
occurred around this period (Vijgen et al., 2005). Moreover, a appropriatecontrolmeasures.Thesporadicre-emergenceofSCoV
numberofCoVsaredocumentedtoinfectmultiplecloselyrelated in the early 2004 illustrated the importance of stringent
hostspecies,e.g.bovineCoVshasbeenisolatedfromcaptivewild surveillance in preventing the further spread of a zoonotic virus
ruminants (Alekseev et al., 2008); closely related Group 3 CoVs in the early phase. One step further, the surveillance strategies
have been isolated from various avian species (Cavanagh et al., must be adopted in a way that we could learn more about the
2002; Jonassen et al., 2005; Liu et al., 2005b). These findings diversityof potentially zoonotic viruses in their reservoirs.With
impliedtherelativelypromiscuousnatureofthehostspecificityof CoVasanexample,giventherelativelypromiscuousnatureofits
CoVs, which was best demonstrated by the evidence of inter- host specificity as discussed above, extensive and regular
species transmission of highly similar if not identical SCoVs surveillance of known CoVs in pets and agricultural animals,
betweentwodistantlyrelatedspecies,i.e.humanandcivets(Wang which are in close contact with human, should be set up. Well-
etal.,2005a). established knowledge on the diversity of potentially zoonotic
virusescertainlyacceleratestheidentificationofitsanimalorigin
5.2. HowmuchdidrecombinationcontributetothediversityofCoV? once it emerges in human. Last but not least, although the
immediate zoonotic source of SCoV seems to be eliminated by
Last but not least, the relatively high rate of homologous culling of civets in Mainland China, the uncertainties about the
recombinationhasbeenspeculatedtofacilitatetheinter-species diversityofSLCoVinbatsstillposethreatontherecurrenceSCoV
transmissionsofCoVs(Baricetal.,1995,1997).Despitethelackof (Hon et al., 2008). Therefore, we emphasize the importance of
relevantexamplestosupportthishypothesis,naturallyoccurring continuoussurveillanceonthegeneticdiversityofSLCoVinbats.
recombinationbetweenCoVstrainsofthesamespecies(Jiaetal.,
1995), as well as between strains of different CoV species, have Acknowledgements
beendocumented(Herreweghetal.,1998;Honetal.,2008;Decaro
et al., 2009), suggesting the generation of CoV diversity through This work is partially supported by Research Fund for the
recombination has been happening in the field. In fact, the two ControlofInfectiousDiseases(referencenumber06060672)from
recently discovered human CoVs were proposed to have a HongKongSARgovernmentandtheStrategicResearchThemeof
recombinant history based on the observed phylogenetic incon- InfectionandImmunology,TheUniversityofHongKong.
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