2012 Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

2012·1.08 MB·English

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain
NL63
JeremyHuynh,aShimenaLi,aBoydYount,aAlexanderSmith,aLeslieSturges,bJohnC.Olsen,cJulietNagel,dJoshuaB.Johnson,d
SudhakarAgnihothram,aJ.EdwardGates,dMatthewB.Frieman,eRalphS.Baric,aandEricF.Donaldsona
DepartmentofEpidemiology,UniversityofNorthCarolina,ChapelHill,NorthCarolina,USAa;TheSaveLucyCampaign,Annandale,Virginia,USAb;Cystic
Fibrosis/PulmonaryResearchandTreatmentCenter,UniversityofNorthCarolina,ChapelHill,NorthCarolina,USAc;UniversityofMarylandCenterforEnvironmental
D
Science,AppalachianLaboratory,Frostburg,Maryland,USAd;andDepartmentofMicrobiologyandImmunology,UniversityofMarylandatBaltimore, o
Baltimore,Maryland,USAe w
n
lo
Therelationshipbetweenbatsandcoronaviruses(CoVs)hasreceivedconsiderableattentionsincethesevereacuterespiratory a
d
syndrome(SARS)-likeCoVwasidentifiedintheChinesehorseshoebat(Rhinolophidae)in2005.Sincethen,severalbats e
d
throughouttheworldhavebeenshowntoshedCoVsequences,andpresumablyCoVs,inthefeces;however,nobatCoVshave
f
r
beenisolatedfromnature.Moreover,thereareveryfewbatcelllinesorreagentsavailableforinvestigatingCoVreplicationin o
m
batcellsorforisolatingbatCoVsadaptedtospecificbatspecies.Here,weshowbymolecularclockanalysisthatalphacoronavi-

rus((cid:1)-CoV)sequencesderivedfromtheNorthAmericantricoloredbat(Perimyotissubflavus)arepredictedtosharecommon ht
t
p
ancestrywithhumanCoV(HCoV)-NL63,withthemostrecentcommonancestorbetweenthesevirusesoccurringapproxi- :
/
/
mately563to822yearsago.Further,wedevelopedimmortalizedbatcelllinesfromthelungsofthisbatspeciestodetermineif jv
thesecellswerecapableofsupportinginfectionwithHCoVs.WhileSARS-CoV,mouse-adaptedSARS-CoV(MA15),andchime- i.a
s
ricSARS-CoVsbearingthespikegenesofearlyhumanstrainsreplicatedinefficiently,HCoV-NL63replicatedformultiplepas- m
sagesintheimmortalizedlungcellsfromthisbatspecies.TheseobservationssupportthehypothesisthathumanCoVsarecapa- .o
r
bleofestablishingzoonotic-reversezoonotictransmissioncyclesthatmayallowsomeCoVstoreadilycirculateandexchange g
/
geneticmaterialbetweenstrainsfoundinbatsandothermammals,includinghumans. o
n

D
e
Batsareknowntobereservoirhostsforseveralhumanviruses, HCoV-NL63wasfirstdiscoveredin2004asanewHCoViso- ce
m
including rabies, Marburg, Nipah, Hendra, and the severe latedfroma7-month-oldbabysufferingfrombronchiolitis(48).
b
acuterespiratorysyndromecoronavirus(SARS-CoV)(5).Inad- Asimilarviruswasisolatedaroundthesametimeinsamplesde- e
r
dition,viromestudieshaveshownunprecedentednumbersofvi- rived from an 8-month-old baby with pneumonia (16). Since 3
rusespresentinthefecalsamplesofthisancientmammalianspe- 2004,thisHCoVhasbeendetectedin1.0to9.3%ofrespiratory ,
2
cies (11, 24). However, little is known about the genetic tract samples collected from several different countries (15), 0
1
architectureofmostbatspecies,thevirusvariationandgeneflow whichindicatesthatHCoV-NL63isdistributedworldwide.There 4

thatoccurthroughdifferentspecies,thepotentialofdifferentbat isnoknownreservoirforthisvirus,andlittleisknownaboutits b
y
species to support human virus replication, the differences be- evolutionary history prior to 2004, although phylogenetic evi- U
tweenthebatandhumanimmunesystems,orthepotentialofbat dencesuggeststhatHCoV-NL63hasinfectedhumansforcentu- N
I
viruses to undergo zoonotic transmission to humans and other ries,asitispredictedtohavedivergedfromHCoV-229Eapprox- V
E
mammals. imately1,000yearsago(38). R
Coronaviruses are the largest known RNA viruses; these Therearemorethan1,100speciesofbats,withbatpopulations S
I
virusescontainsingle-strandedplussensegenomesandareclas- inhabitingeverycontinentexceptAntarctica.Batsbelongtothe T
Y
sifiedinthefamilyCoronaviridae,whichisdividedintothreegen- order Chiroptera and are further divided into the suborders of
O
era, including Alphacoronavirus ((cid:1)-CoV), Betacoronavirus ((cid:2)- Yinpterochiroptera,whichincludemostlythemegabats(formerly F
CoV),andGammacoronavirus((cid:3)-CoV).FiveCoVsareknownto known as the megachiroptera) and Yangochiroptera, which in- M
causehumandisease,includingthe(cid:2)-CoVsSARS-CoV,human cludesmostofthemicrobat(formerlyknownasthemicrochirop- E
M
CoV(HCoV)-OC43,andHCoV-HKU1andthe(cid:1)-CoVsHCoV- tera)families(46).IntheUnitedStates,mostbatspeciesaresmall P
229E and HCoV-NL63 (35). Three of these HCoVs have been nocturnalbatsthatbelongtothefamilyVespertilionidae,andall H
showntoorhavebeenpredictedtohavespilledoverfromzoo- areinsectivores.InMaryland,previoussurveyshaveyieldedfecal IS
noticreservoirs,includingSARS-CoV,whichlikelyemergedfrom samplesfromthebigbrownbat(Eptesicusfuscus),thelittlebrown
the Chinese horseshoe bat (Rhinolophidae) (26), HCoV-OC43, myotis(Myotislucifugus),thenorthernlong-earedmyotis(Myotis
which probably emerged from bovine CoV (BCoV) (50), and
HCoV-229E(36),whichwaspredictedbymolecularclockanaly-
sistoshareamostrecentcommonancestor(MRCA)justover200 Received12April2012 Accepted5September2012
hundred years ago with a bat CoV found in the leaf-nosed bat Publishedaheadofprint19September2012
(Hipposideroscafferruber)batinGhana(36).Thecloselinkbe- AddresscorrespondencetoEricF.Donaldson,[email protected].
tween bat and human CoVs has led to the speculation that all Copyright©2012,AmericanSocietyforMicrobiology.AllRightsReserved.
human,andperhapsmammalian,CoVsmayhaveoriginatedin doi:10.1128/JVI.00906-12
bats(19,36,49).
12816 jvi.asm.org JournalofVirology p.12816–12825 December2012 Volume86 Number23
HCoV-NL63GrowsinTricoloredBatLungCells
septentrionalis), the eastern small-footed myotis (Myotis leibii), byPfefferleetal.(2009)(36)andusingthesame650-to800-ntfragment
thehoarybat(Lasiuruscinereus),theredbat(Lasiurusborealis), ofthereplicaseregionofseveralknownCoVstoestimatethedateofthe
and the tricolored bat (Perimyotis subflavus). In our previous mostrecentcommonancestorforARCoV.1andARCoV.2.Thereplicase
work,wehaveshownthatfiveofthesespeciesshedunique(cid:1)-CoV sequencesforARCoV.1(11),NECoV,andARCoV.2werederivedfrom
sequences (11); however, the reagents necessary to isolate these sequence reads obtained by 454 sequencing, and because NECoV and
ARCoV.1werenearlyidentical,onlytheARCoV.1sequencewasusedin
virusesanddetermineifNorthAmericanbatCoVsposeathreat
theanalysis.Ofnote,thissequenceisaportionoftheviralreplicasegene
topublichealtharecurrentlylacking.
(nsp12),whichisarguablythemostconservedregionoftheCoVgenome,
Inthisstudy,weidentifiednucleicacidsequencesthatpoten-
makingitthemostappropriatetargetformolecularclockanalysis.These
tiallyindicatethepresenceofanovel(cid:1)-CoVthatispredictedto
replicase fragment sequences were deposited in GenBank (see below).
shareanRCAwithHCoV-NL63inthetricoloredbat.Wedevel-
Mostofthesequencesweredatedinyearsbeforepresent,whichwas2011
D
opedanimmortalizedlungcelllineforthisU.S.batspeciesand whenthisstudywasconducted.UsingthedatefoundbyVijgenetal. o
showthathumanCoVsgrowinthesebatcells.Thisobservation (2005)(50)fortheHCoV-OC43andbovineCoVsequences,andfollow- w
n
suggeststhathumanCoVsarecapableofinfectingmultiplemam- ingthemethodofPfefferleetal.(2009)(36),anormalprobabilisticprior lo
malian hosts, potentially establishing zoonotic-reverse zoonotic withameanof121yearsbeforethepresenttimeandastandarddeviation a
d
cyclesthatallowCoVstomaintainviralpopulationsinmultiple of 13 years was used to calibrate the analysis (36, 50). Both the e
hosts,evolvenovelrecombinantviruseswithviralgenesderived GTR(cid:5)Gamma4(cid:5)IandtheSRD06modelsweretestedundertheas- d
f
fromhumanandanimalCoVs,andtrafficbackintohumanpop- sumptionofanuncorrelatedlognormalclockandaconstantpopulation ro
size.Inaddition,anexponentialpopulationsizeassumptionwastested m
ulations at a later date. In addition, these results suggest that
HCoV-NL63mayhaveoriginatedinbatsandcrossedthespecies usingtheSRD06model.MarkovchainMonteCarlochainsweresetto h
200,000,000iterationswithsamplingevery20,000generations.Bayesfac- tt
barriertoinfecthumansroughly563to822yearsago.Supporting p
toranalysis,whichwasusedtocomparethemodels,wasconductedusing :/
agrowingbodyofliterature,ourdatasupportthehypothesisthat theprogramTracerversion1.5(http://tree.bio.ed.ac.uk/software/tracer). /jv
cross-speciestransmissioneventsandtheemergenceandcoloni- TheSRD06modelwasfoundtobesuperiortotheGTRmodelbyalog10 i.a
zationofnewspeciesrepresentcommonfeaturesoftheCorona- Bayesfactorof240. s
m
viridae. Battissuesamples.BatsforthisstudywereobtainedfromtheSave .
o
LucyCampaign,aneducationandbatrehabilitationcenterinVirginia. r
g
MATERIALSANDMETHODS Thisorganizationspecializesinrehabilitatingbatsthataresickorinjured, /
andallofthebatsintherescuewereobservedforsymptomsofrabiesvirus o
Batsequencesfordeterminingbatphylogeny.Alloftheavailablecyto- n
chromebgenes(1)fromtheNorthAmericanbatsfoundinMarylandand onintake.Onlynonrabidbatsthatwerebadlyinjuredbeyondrecovery D
wereeuthanizedforthisstudy.Theseincludedtwobigbrownbats(EpFu) e
other bats of interest were downloaded from GenBank along with the c
samegenesfromseveralothercommonmammals.Thenucleotidegene andonetricoloredbat(PeSu).Thebatswereeuthanizedattherescue e
center,andthebatorgansweresurgicallyremovedandplacedintopro- m
sequenceswerethenalignedbyClustalX,amaximumlikelihoodtreewas b
generatedusingPhyMLwith100bootstraps,andthetreeimagewased- cessingmedium(calcium-andmagnesium-freePBSsupplementedwith e
itedandexportedusingthebioinformaticstoolsavailableintheGeneious 100mgdisodiumEDTA,1%penicillin/streptomycin,and1%gentami- r 3
softwaresuiteversion5.4.3(13). cin)(7).Organsharvestedfromeachbatincludedheart,lung,intestine, ,
Identificationofnovel(cid:1)-CoVsinNorthAmericanbats.Inourpre- brain,kidney,liver,andstomach.Theorgansinmediumweredelivered 20
viousstudies,wedemonstratedthat(cid:1)-CoVsequencesarepresentinthe onicewithin8htoourlaboratoryforprocessing. 14
fecalsamplesofeasternNorthAmericanbatspecies(11).Usingtheexact Biosafetyconsiderations.Allofthebatorgansharvestedatthebat b
procedureaspreviouslydescribed(11),weusedRoche454sequencingto rescueweretransportedtoourlabattheUniversityofNorthCarolina y
(UNC).Thetissueswerefurtherprocessedinaseparateandlockedbio- U
determinetheviralsequencespresentinbatfecalsamplesfrombigbrown
safetylevel2(cid:5)(BSL2(cid:5))roomthatrequiredallpersonneltohavereceived N
batscapturedintheSaratogaNationalHistoricalparkinNewYork(New I
aprophylacticseriesofrabiesvaccines.Allworkwasperformedunder V
EnglandCoV[NECoV])andtricoloredbatsfromtheChesapeakeand E
Ohio Canal National Historical Park in Maryland (Appalachian Ridge certified biosafety cabinets, and all personnel wore gloves and Tyvek R
CoVstrain2[ARCoV.2]).Then,weusedpreviouslyreportedprimersand aprons(FisherScientific)whenworkingwiththefreshtissue. S
protocols(11)toamplifya(cid:4)2,200-nucleotide(nt)fragmentintherepli- GenerationofprimarybatcelllinesfromNorthAmericanbats.The IT
caseregionoftheseviruses,encompassingaportionofnsp13,allofnsp14, organswereremovedfromtransportbuffer,washedwithcoldprocessing Y
andaportionofnsp15.Theamplifiedfragmentswereelectrophoresedon media,placedinspecimentubeswithfreshprocessingmedia,andthen O
F
a 1% agarose gel, and the (cid:4)2,200-nt band was excised, purified, and placedonice.Topreparethetissues,wefollowedapublishedprotocolby
M
subjectedtoSangersequencingaspreviouslydescribed(11).Thesese- Cramerietal.(2009)(7).Thetissuesfromeachorganwerefinelydissected E
quencesweredepositedintoGenBank(seebelow). usingasterilescalpelandwashedwithcoldprocessingmedia.Thedis- M
Phylogenetic and molecular clock analyses of (cid:1)-CoVs found in sectedtissueswerethentransferredtoa50-mlconicaltube,coveredwith P
NorthAmericanbats.(i)Phylogeneticanalysis.Thesequencesofthe cold0.25%trypsin(Gibco),andincubatedat4°Covernight.Thefollow- H
I
(cid:4)2,200-nt fragments of ARCoV.1, ARCoV.2, and NECoV were com- ingday,thesampleswerecentrifugedat37°Conabenchtopshakersetat S
paredtothesameregionofseveralknownCoVsequencesdownloaded 200rpmfor1h.Supernatantswerefilteredthrougha40-(cid:6)mcellstrainer
from GenBank. The sequences were aligned using ClustalX as imple- (Fisher)intoa50-mlconicaltubecontaining10mloffetalcalfserum
mentedinGeneious5.4.3(13),andthealignmentwasmanuallytrimmed (FCS)(HiClone).Thecellstrainerwasthenrinsedwithphosphate-buff-
andcorrectedtogeneratea2,321-nucleotidealignment.Amaximumlike- eredsaline(PBS)toensurethatalloftheindividualcellsweredelivered
lihoodtreewasgeneratedusingPhyMLwith100bootstraps,andthetree throughthestrainer.Next,thelargerpiecesofdissectedtissueswerein-
imagewaseditedandexportedusingthebioinformaticstoolsavailablein cubatedwith0.25%trypsin(Gibco)at37°Cfor30minontheshaker,and
theGeneioussoftwaresuiteversion5.4.3(13).Thiswasthelargestfrag- thenthesupernatantswereaddtotheFCSinthe50-mlconicaltube.This
mentavailableforallthreegenomes,andthatwasthebasisforgenerating wasrepeateduntilmostofthecellswereremovedfromthetissuescaf-
thetreeusingthesesequences. folds.Next,thecellswerepelletedat800(cid:7)gfor5mininaBeckman
(ii)Molecularclockanalysis.Molecularclockanalysiswasconducted benchtopcentrifuge.Thecellswerethenresuspendedinprimarycellcul-
usingBEASTversion1.7.1(14),followingthesameprotocolasthatused turemedium(Dulbecco’smodifiedEaglemedium[DMEM]/F12-Ham’s
December2012 Volume86 Number23 jvi.asm.org 12817
Huynhetal.
mediasupplementedwith15%FetalCloneII,1%penicillin/streptomy- B5L.ToverifySARS-CoVanditsrelatedstrainsandHCoV-NL63repli-
cin,1%nonessentialaminoacids,and1%gentamicin),transferredtoT25 cationinthePESU-B5Llungcells,weusedprimersdesignedtoamplify
flasks,andincubatedat37°Cwith5%CO .Robustculturesofprimary the subgenomic leader containing mRNA transcripts of each virus by
2
heart,lung,brain,andkidneycellswereestablished.Thecellswerefedby reverse transcriptase PCR (RT-PCR). The amplified leader-containing
removingthemediumandreplacingitwithfreshprimarymediumwith cDNAsarespecifictoeachHCoVandwouldbedetectedonlyifthevirus
15%FCSevery3to5daysuntilthecellsreachedconfluence.Theprimary wasundergoingactivetranscriptionandreplication.ForHCoV-NL63,we
cellswerethenmaintainedfor2to5passagestogeneratecellstocksthat used the primer sets and the exact RT-PCR conditions to amplify the
werecryopreservedandstoredat(cid:8)140°C. subgenomicmRNAencodingthenucleocapsid(N)geneaspreviously
Generation of immortalized bat cell lines from North American described(12).Theresultingbandswerealsoexcised,purified,andsub-
bats. Primary cells that propagated well enough to reach sufficient jectedtoSangersequencingattheUNCGenomicAnalysisCentertover-
numbersinuptothreepassageswereselectedascandidatesforim- ifytheoriginsandsequenceoftheleader-containingcDNAs.ForSARS-
D
mortalization.Cellswerepreparedin6-wellplatesandgrownto70to CoV and its related strains, we used primers designed to detect o
80%confluenceat37°Cwith5%CO inprimarycellculturemedia. subgenomictranscriptionofmultipleviralproteinsusingprimersand w
Thecellsweretheninfected,asprevio2uslydescribed(18),withlenti- reactionconditionsthathavebeenpublishedelsewhere(53).Theresult- nlo
virus vectors expressing the human telomerase reverse transcriptase ingbandfortheSARS-CoVmembranegenewasexcised,purified,and a
(hTERT) gene and the murine Bmi-1 gene (18), an oncogene that sequencedinthesamewayasdescribedfortheHCoV-NL63Ngene. de
promotesefficientself-renewingcelldivisions.AmixtureofhTERT DetectionofHCoV-NL63inPESU-B5Lbyimmunofluorescenceas- d

andBmi-1wascreatedbyplacing1.5mlofeachvectorintoa15-ml say.PESU-B5LcellswereinfectedwithHCoV-NL63atanMOIof0.7or fr
o
conical tube at vector concentrations of (cid:4)106 infectious units/ml. mock infected. Seventy-two hours postinfection, monolayers were m
Next, we added 6 (cid:6)l of Polybrene at a concentration of 8 (cid:6)g/ml to washedtwicewithcoldphosphate-bufferedsalineandfixedwitha50% h
increasetheinfectivityofthelentivirusvectors,andthemixturewas methanol-50%acetonesolution.Cellswerethenpermeabilizedwith0.1% tt
p
vortexedgentlyandstoredonice.Next,weremovedthemediumfrom TritonX-100–5%bovineserumalbumin(BSA)inPBSandblockedwith :/
the cells in the 6-well plate and washed the cells twice with PBS 5%BSAinPBS.Rabbitanti-NL63nucleocapsidsera(thekindgiftofLia /jv
(Gibco). For the cells to be immortalized, we added 1 ml of the van der Hoek) were used as the primary antibody. Unbound primary i.a
hTERT–Bmi-1–Polybrene mixture and incubated it for 3 h at37°C antibodywasremovedbywashingthreetimeswith1%BSA–0.05%Non- s
idetP-40inPBS,followedbyadditionofanAlexa546-conjugatedgoat m
with5%CO2.Forthecontrolcells,1mlofmediumwasaddedpriorto anti-rabbitIgG(Invitrogen).Afterwashingoffunboundsecondaryanti- .o
incubationunderthesameconditions.Aftertheincubation,thesu- r
body,infectedcellswerephotographedusinganinvertedmicroscopewith g
pernatants were removed from the cells and discarded into a 10% /
bleachsolution.Primarycellculturemediumwasthenaddedtoallof amercurylamplightsourceandatetramethylrhodamineisothiocyanate o
(TRITC)filter. n
thewells,andthecultureswereincubatedovernightat37°Cwith5% D
DetectionofHCoV-NL63inPESU-B5LcellsbyWesternblotting.
CO .Onthefollowingday,thecellsweretransfectedasecondtime e
2 PESU-B5LcellswereinfectedwithHCoV-NL63atamultiplicityofinfec- c
usingthesameprotocol.Thecellswerethenmonitoreddailytolook e
formorphologicalchangesinthecellsandtodetermineiftheimmor- tionof0.7ormockinfected.Seventy-twohourspostinfection,cellswere m
washedwithcoldPBSfollowedbylysisinAVlysisbuffer(20mMTris- b
talizationprocesswassuccessful.Passageswereconductedwhenthe HCl[pH7.6],150mMNaCl,0.5%deoxycholine,1.0%NonidetP-40,and e
cellsreachedconfluence,whichvariedbycelltype,withthelungcells 0.1% sodium dodecyl sulfate [SDS]). The lysates were spun down at r 3
requiring 3 to 4 days between passages. Immortalization was deter- ,
minedtobesuccessfulinthosecellsthatcontinuedtothrivebeyond 13,000rpmfor10mintopelletthenuclei.Equalvolumesof0.9%SDSand 2
10mMEDTAwereaddedtothepostnuclearsupernatants(toraisethe 0
thenumberofpassagesrequiredfortheprimarycellstoreachsenes- 1
concentrationofSDSto0.5%forvirusinactivation),andthemixturewas 4
cenceandeventualcelldeath.Todeterminetheextentoftheimmor- incubatedatroomtemperaturefor15mintoensurecompleteinactiva- b
talization, the infected cells were propagated for several additional y
tionofHCoV-NL63.Equalamountsofcelllysateswerethenloadedonto
passages, with the immortalized lung cells from the tricolored bat U
a4to12%Bis-TrisNupagegel(Invitrogen),andpostelectrophoresis,the N
showingthebestpostsenescencegrowthphenotype.Severalstocksof
proteinsweretransferredontopolyvinylidenedifluoride(PVDF)mem- I
primary and immortalized cells were cryopreserved at each passage V
andthenstoredat(cid:8)140°Cforfutureuse. bfarcatnuerseru’ssiinngstaruncXticoenlsl.bMloetmmbordanuelesw(Ienrveibtrloogcekned)awcictohr5d%inngotnoftahtedrmyamniulk- ER
InfectionofPESU-B5LcellswithHCoV-NL63andSARS-CoV.Im- S
in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) for 1 h and
mortalizedbatlungcellsfromthetricoloredbat(Perimyotissubflavus)were IT
incubatedovernightwithrabbitanti-NL63nucleocapsidsera.Unbound
seededontoa6-wellplateandgrowninprimarycellculturemediumat37°C Y
antibodywasremovedbywashinginTBS-Tfollowedbyincubationwith
with5%CO untiltheyreached70to80%confluenceandaconcentrationof O
(cid:9)3(cid:7)105ce2llsperwell.Thesupernatantswerethenremoved,andthecells awneraentviirsaubabliiztehdorusseirnagdaishPipeercroexSiudpaseer(SHigRnPa)lsWeceosntedranryblaonttcibhoedmyi.lPurmoitneiens-s F M
wereinfectedwith1mlofwild-typeHCoV-NL63(wtNL63)orHCoV-NL63
cencekit.Fortheloadingcontrol,apurifiedmousemonoclonalantibody E
expressinggreenfluorescentprotein(GFP)(NL63gfp)(12)orPBS(mock)at tobetaactin(catalogno.612656;BDTransductionTechnologies)was M
amultiplicityofinfection(MOI)of1.Fortheadsorptionperiod,thecells used,followedbystainingwithsecondaryantimouseHRPantibody(GE P
wereincubatedat32°Cwith5%CO2for1hwithagitationevery15min.At AmershamBiosciences). HI
theendoftheadsorptionperiod,anadditional2mlofculturemediumwas Nucleotidesequenceaccessionnumbers.Thesequencesdetermined S
addedtoeachwell,andtheinfectedcellsweremaintainedat32°Cwith5% in this study were deposited into GenBank under accession numbers
CO2andmonitoredfor8days.ForSARS-CoVgfp(44)andseveralrelated JX537911throughJX537914.
strains,theexperimentalinfectionswereconductedunderbiosafetylevel3
containment following the same parameters as described above, using RESULTS
mouse-adaptedSARS-CoV(MA15),aSARS-CoVstrainadaptedtobelethal
Evolutionary relatedness of North American bats. The mito-
inmice,GD03,arecombinantchimericSARS-CoVclonebearingthespike
chondrialcytochromebgenesofseveralbatandothermamma-
sequenceofa2003humanclinicalisolate,andHC/SZ/61/03,arecombinant
lianspecieswerecomparedtoshowtheevolutionaryrelatedness
chimericSARS-CoVclonebearingthespikesequenceofanearlyhuman
isolate(8,39,41,42,44).Theseinfectedcellsweremaintainedat37°Cwith ofNorthAmericanbatsfrequentlyfoundinMaryland,including
5%CO andmonitoredfor5days. the big brown bat (EpFu), the little brown myotis (MyLu), the
2
VerificationofSARS-CoVandHCoV-NL63replicationinPESU- eastern small-footed myotis (MyLe), the northern long-eared
12818 jvi.asm.org JournalofVirology
HCoV-NL63GrowsinTricoloredBatLungCells
andPeSuweremorecloselyrelatedtoeachotherthantoother
myotinespeciesfoundinMaryland(Fig.1).
Identification and characterization of novel (cid:1)-CoV se-
quences in big brown bats and the tricolored bat. Roche 454
sequencing was used to identify (cid:1)-CoV sequences in two addi-
tionalbatpopulations,includingabigbrownbatpopulationfrom
SaratogaNationalHistoricalParkinNewYork(NECoV)anda
tricoloredbatpopulationthatwassampledattheChesapeakeand
OhioNationalHistoricalParkinMaryland.RT-PCRwasusedto
amplifya(cid:4)2,200-ntregionoftheseviruses(comprisingaportion
D
of nsp13, all of nsp14, and a portion of nsp15), and these were
o
sequenced and compared to the same fragment that had been w
n
amplifiedpreviouslyfromabigbrownbatpopulationsampledin lo
MarylandandwasknownasARCoV.1(11).Otherregionsofthese a
d
genomeshavebeendeterminedby454sequencing,butthiswas e
d
the largest fragment available in common between all three se-
f
quences.Interestingly,thetwo(cid:4)2,200-ntsequencesderivedfrom ro
m
bigbrownbatswerenearlyidentical(approximately97%identical

h
at the nucleotide level), suggesting that big brown bats harbor t
t
similar(cid:1)-CoVsregardlessofgeographiclocation(Fig.2AandB). p
:
/
Incontrast,thesequencefromARCoV.2wasapproximately74% /jv
identicalatthenucleotidelevel,suggestingthatdifferentU.S.bat i.
a
FIG1Phylogenyofthemitochondrialcytochromebgenefromseveralmam- species harbor unique (cid:1)-CoV strains (Fig. 2B). Together, these s
malianspecies.ThismaximumlikelihoodtreeshowsthatNorthAmerican m
batsofthefamilyVespertilionidaearemorecloselyrelatedtothemselvesthan fragmentsofNorthAmericanbatCoVssuggestanewcladeinthe .o
to other bat or common mammalian species. EpFu, big brown bat; PeSu, Alphacoronavirusgenus(Fig.2B). rg
tricoloredbat;MyLu,littlebrownmyotis;MyLe,easternsmall-footedmyotis; Molecularclockanalysis.Duringtheannotationstage,many /
o
MySe,northernlong-earedmyotis.Thescalebarrepresentsnucleotidesubsti- oftheCoV-likesequencesfromtricoloredbatsamplesweremost n
tutions.Onlynodeswithbootstrapsupportabove70%arelabeled. closelyrelated,albeitdistantly,toHCoV-NL63(Fig.2B).There- D
e
fore, molecular clock analysis was conducted to estimate an c
e
MRCAbetweenARCoV.2andHCoV-NL63,usingasimilardata m
myotis(MySe),andthetricoloredbat(PeSu)(Fig.1).TheNorth setandthesameparametersasthoseemployedbyPfefferleetal. b
e
American bat species group in the same cluster with other bat (2009) (36). This analysis was performed using sequence frag- r

3
species,showingacloserrelationshipbetweenthesespecies.EpFu ments obtained from the highly conserved viral polymerase ,

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S
FIG2Anovel(cid:1)-CoVinthetricoloredbatiscloselyrelatedtoHCoV-NL63.Novel(cid:1)-CoVsequenceshavebeenfoundinthefecalsamplesofseveralNorth
Americanbatspecies.(A)Schematicshowingwherethetwofragments(usedforthetreesinpanelsBandC)occurintheNL63genome.(B)A(cid:4)2.2-kbfragment
ofthereplicaseregion(startingatposition16480intheschematic)wassequencedfromthreesamples,includingtwofromthebigbrownbat(NECoVand
ARCoV.1)andonefromthetricoloredbat(ARCoV.2).AmaximumlikelihoodtreecomparingthenucleotidesequencesofthesebatCoVstootherknownCoVs
indicatedthatNECoVandARCoV.1areverycloselyrelatedwhileARCoV.2issignificantlydifferentfromNECoVandARCoV.1.Thethreenovel(cid:1)-CoV
sequencesformanovelclusterinthe(cid:1)-CoVgroup.(C)Molecularclockanalysisusinga650-to800-ntportionofthehighlyconservedreplicaseregion(starting
atposition13810inpanelA)predictedthattheMRCAofbatCoVsfromtheHipposideroscafferruberbatsandHCoV-229Ewaslikelytohaveexisted212to350
yearsago(inagreementwithPfefferleetal.,2009[36]).TheMRCAforHCoV-NL63andARCoV.2waspredictedtohaveexisted563to822yearsago.NECOV
andARCoV.1wereidenticalinthisregion,soonlyARCoV.1isshown,anditclusteredwithotherbat(cid:1)-CoVs.
December2012 Volume86 Number23 jvi.asm.org 12819
Huynhetal.
TABLE1Modelsusedandhighestposteriordensities
Model Root(range)a Meansubstitutionrateb(range)
GTR(cid:5)G(cid:5)I,constant 3469(1237–5923) 1.629(cid:7)10(cid:8)4(1.034(cid:7)10(cid:8)4–2.3499(cid:7)10(cid:8)4)
SRD06,constant 5940(2424–9921) 1.76(cid:7)10(cid:8)4(1.073(cid:7)10(cid:8)4–2.570(cid:7)10(cid:8)4)
SRD06,exponential 5450(2143–9358) 1.847(cid:7)10(cid:8)4(1.078(cid:7)10(cid:8)4–2.668(cid:7)10(cid:8)4)
aValuesareyearsBCE(beforethecommonera).
bMeansubstitutionrateisthenumberofsubstitutionspersiteperyear.
(nsp12),whichisarguablythemostconservedregionoftheCoV extendedtheviabilityofthosecellsinfectedwiththeimmortaliza-
genome (Fig. 2A). Briefly, both the GTR(cid:5)Gamma 4 (cid:5) I and tionvectors.Bypassage10,theprimarycellshadreachedsenes- D
o
SRD06modelsweretestedundertheassumptionofanuncorre- cence and had stopped growing completely, while the cells that w
n
latedlognormalclockandconstantpopulationsize.Inaddition, wereimmortalizedcontinuedtogrowanddivideefficiently(Fig. lo
anexponentialpopulationsizeassumptionwastestedusingthe 3BandC).TheimmortalizedcellswerenamedPESU-B5Lcells, a
d
SRD06 model. Markov chain Monte Carlo chains were run for andthesecellshavecontinuedtoshowprolificgrowth.Atthetime e
d
200,000,000 iterations with sampling every 20,000 generations. ofwriting,wehavePESU-B5Lcellsthathaveundergoneover30
f
Bayesfactoranalysis,usedtocomparethemodels,wasconducted passagesandover100doublings.Therewerenoobservablemor- ro
m
using the program Tracer. The SRD06 model was found to be phologicalchangesinthesecellsoverpassage,althoughearlypri-

superior to the GTR model by a log Bayes factor of 240. The marycultureshadheterogenousmixturesofcelltypes.However, h
10 tt
exponentialpopulationassumptionwasfoundtobesuperiorto theimmortalizationprocessappearstohaveselectedforasingle p
:
/
theconstantassumption,butnotsignificantlyso(log10Bayesfac- fibroblast-resembling cell population (Fig. 3C). Total RNA was /jv
torof2).Theresultsoftheanalysesareshownwithhighestpos- extractedfromtheimmortalizedcells,andRT-PCRandsequenc- i.
a
teriordensities(HPD)inTable1.Interestingly,theanalysisreca- ingwereusedtodeterminethatthemurineBmi-1genewasex- s
m
pitulatedtheresultsobtainedbyPfefferleetal.(2009)(36)and pressedintheimmortalizedcells(datanotshown).
.
o
provided an estimated MCRA between ARCoV.2 and HCoV- ExperimentalinfectionofPESU-B5Llungcellswithhuman r
g
NL63ofapproximately1449CE(range,1190to1449CE)(Table CoVs.Toassesswhetherornotbatcellscouldsupportgrowthof /

o
1 and Fig. 2C). This observation suggests that ARCoV.2 and HCoVs,weinfectedthePESU-B5LcellswithHCoV-NL63,ahu- n
HCoV-NL63originatedfromthesameancestor,predictingthata man(cid:1)-CoV,andSARS-CoV,ahuman(cid:2)-CoV,usingengineered D
potential cross-species transmission event occurred about the versionsofthesevirusesthatexpressGFPduringactivereplication e
c
timethatColumbusarrivedinNorthAmerica.BecauseARCoV.1 (12,17,44,53),sothatwecouldmonitorthepresenceofapro- e
m
andNECoVwerenearlyidentical,onlyARCoV.1wasusedinthis ductive infection by fluorescence microscopy. In the case of b
analysis,andthissequenceclusteredwithother(cid:1)-CoVsofbats HCoV-NL63,wedetectedGFPfluorescenceatday2postinfection e
r

(Fig.2C). anditcontinueduntilday8,whenthecellswereharvesteddueto 3
,
Generating primary cells from sacrificed bats. Bat tissues overconfluence.Whileacytopathiceffectwasobservedinsome 2
0
usedforthisstudywereobtainedfromTheSaveLucyCampaign, cellsbyday5postinfection(Fig.4),theculturesremainedviable, 1
4
whichoperatesasabatrescueinVirginia.Batsthatweretoobadly suggestingthatmanycellswereresistanttoinfection.Theprogeny
b
damaged to be rescued were euthanized, and their organs were y

harvestedandtransportedtoUNC,wherethetissuesweresurgi- U
N
callydissectedandtrypsinized,andthefilteredprimarycellswere
I
V
platedandgrown.Onceasufficientnumberofculturedprimary E
cellswereavailable,theseweretestedforrabiesvirusbyRT-PCR. R
S
Allofthetestedbattissueswerenegativeforrabiesvirus(datanot
I
T
shown). Y
Afemaletricoloredbatthatarrivedintherescueinmid-Julyof O
2011, suffering injuries sustained from being hit by a car, pro- F

ducedthebestresults.Brain,heart,liver,lung,intestine,stomach, M
E
andkidneytissueswereobtainedfromthisbat,andprimarycells
M
were successfully generated for the brain, lung, stomach, and P
heart.Theothertissuetypesgrewinitially,andeithertheyreached H
I
senescencebeforereachingconfluenceorthereweretoofewcells S
toestablishlong-termgrowth.Thetricoloredbatlungcellsgrew
mostefficiently,andthesecellswerepassagedthreetimestogen-
erate a large stock of primary lung cells for this bat (Fig. 3A).
Stocksofthesecellswerecryopreservedat(cid:8)140°Cforfutureuse. FIG3Immortalizationoftricoloredbatlungcells,PESU-B5L.Primarytri-
coloredbatlungcellsweregrowninenrichedgrowthmediumandpassaged
Generatingimmortalizedlungscellsfromthetricoloredbat. fourtimestogenerateasuitablequantityforimmortalization.Primarycells
Primarytricoloredbatlungcellsthathadbeenpassaged3times wereimmortalizedwithlentiviralvectorscontaininghTERTandtheBmi-1
wereimmortalizedbytwoconcurrentinfectionswithtwolentivi- proto-oncogene.(A)Primarytricoloredbatlungcells.(B)PrimaryPESU-B5L
lungcellsreachedsenescencebypassage10.(C)Immortalizedcellsatpassage
rus vectors, one containing hTERT and one containing Bmi-1
10,including4passagesasprimarycellsand6passagespostimmortalization.
(18).Immortalizedandprimarycontrolcellswerethenpassaged
TheimmortalizedPESU-B5Lcellswerestillgoingstrongaftermorethan30
at3-to4-dayintervalstodetermineiftheimmortalizationprocess passages(over100doublings).
12820 jvi.asm.org JournalofVirology
HCoV-NL63GrowsinTricoloredBatLungCells
D
o
w
n
lo
a
d
e
d

f
r
o
m
FIG5VerificationofNL63replicationinPESU-B5Lcells.TotalRNAwas h
harvestedfromcellsonday5postinfectionandsubjectedtoRT-PCRusing tt
p
primersdesignedtodetectleader-containingtranscriptsoftheNgene,which :
FIG4InfectionofPESU-B5LcellswithhumancoronavirusNL63expressing wouldbepresentonlyifNL63replicated.(A)Subgenomictranscriptswere //jv
GFP.PESU-B5LcellswereinfectedwithmockorNL63gfpatanMOIof0.25 detectedinthePESU-B5LcellsinfectedwithbothwtNL63andNL63gfp.(B) i.
andmonitoredbybright-fieldandfluorescencemicroscopy.(AandB)Mock- ThesgNbandforwtNL63wasexcised,andtheDNAwaspurifiedandse- a
s
infectedcellsbybright-field(A)andfluorescence(B)microscopy.(Cthrough quencedtoverifythattheHCoV-NL63leadersequenceandthe5=endoftheN m
F)CellsinfectedwithNL63gfpvisualizedatday3postinfectionbybright-field genewerepresent.L,ladder;N,wt-NL63;G,NL63gfp;M,mock.Theleader .
o
microscopy(C)andbyfluorescencemicroscopy(D)andatday5postinfection sequencehasabeigebarbeneathit,andthe5=endoftheNgenehasagreenbar r
g
bybright-fieldmicroscopy(E)andbyfluorescencemicroscopy(F). beneathit. /

o
n

D
virionsharvestedfromthesupernatantsoftheseinfectionswere primersdesignedtodetectthesubgenomicGFPtranscript,and e
c
viable upon passage to fresh cells and formed plaques on LLC- thisresultedinthedetectionofabandinthe24-hculture,indi- e
m
MK2cells;however,thetiterwaslow((cid:10)103PFU/ml),suggesting catingthatsubgenomictranscriptionwasongoinginthesecells b
apotentialblockinviralegress.AttemptstogenerateHCoV-NL63 (Fig.7).Novirusesorreplicationwasdetecteduponpassageof e
r
plaquesinthePESU-B5Lcellswereunsuccessful. supernatantsfromthesecultures. 3
InthecaseofSARS-CoVgfp,noGFPexpressionwasdetected DetectionofreplicationofMA15andchimericSARS-CoV , 2
strainsinPESU-B5Lcells.Inadditiontoperforminginfections 0
inPESU-B5Lcellsover5daysofinfection,butGFPfluorescence 1
withSARS-CoVexpressingGFP,wealsoconductedexperimental 4
wasreadilydetectedinVeroE6cellsinfectedwiththesamevirus
infectionswithMA15andtwochimericSARS-CoVvirusesthat b
asapositivecontrolby24hpostinfection(datanotshown),indi- y
catingthatthestockofSARS-CoVgfpviruswasviable. encodedthespikeproteinsfromtwoearlyhumanisolatesknown U
asGD03andHC/SZ/61/03.Allthreeofthesevirusesreplicated N
DetectionandverificationofreplicationofHCoV-NL63in
I
subgenomicRNAsinthePESU-B5Llungcells(Fig.8),asdetected V
PESU-B5Lcells.ToverifythatHCoV-NL63replicationhadoc- E
curredinthePESU-B5Llungcells,weusedRT-PCRtoamplify byRT-PCRusingthetotalRNAofcellsharvestedat48and72h R
S
leader-containingtranscriptsofthesubgenomicNgene.TheRT-
I
T
PCRresultedinaladderofampliconsrepresentingsubgenomic Y
transcriptsthathadbeengeneratedduringthecourseofefficient O
virusreplication(Fig.5A),andtheseweresequencedtoverifythe F

presence of leader-containing transcripts during HCoV-NL63 M
E
replication(Fig.5B).Inaddition,infectionofPESU-B5Lcellsby
M
HCoV-NL63 was detected by immunofluorescence assay (IFA) P
(Fig.6A)andWesternblotting(Fig.6B),bothdirectedagainstthe H
I
nucleocapsidprotein.FortheIFA,cellsexpressingHCoV-NL63 S
nucleocapsidappearedbrightgreenunderfluorescentconditions,
whileuninfectedcellswerenotstained.Westernblottingofcell
lysatesusingtheanti-NL63Nantibodyrevealedthattheinfected
FIG6EvidenceofHCoV-NL63infectioninPESU-B5Lcells.PESU-B5Lcells
cellsstronglyexpressedaproteinofapproximately45kDa,which
wereinfectedwithHCoV-NL63atanMOIof0.8.At72hpostinfection,cells
wasconsistentwiththesizeoftheHCoV-NL63nucleocapsidpro- wereprobedforthenucleocapsidproteinofHCoV-NL63byimmunofluores-
tein. cenceassayandWesternblotting.(A)I,mock-infectedcells,fluorescencemi-
DetectionofreplicationofSARS-CoVinPESU-B5Lcells.De- croscopy;II,mock-infectedcells,bright-fieldmicroscopy;III,HCoV-NL63-
infected cells, fluorescence microscopy; IV, HCoV-NL63-infected cells,
spitethefactthatSARS-CoVgfpwasnotdetectedbyfluorescence
bright-fieldmicroscopy.(B)WesternblotofHCoV-NL63andmock-infected
in the infected cultures of PESU-B5L cells, RT-PCR was con- PESU-B5Lcelllysateswith(cid:2)-actinasaloadingcontrol.Nucleocapsid(N)
ductedonRNAharvestedfromthesecellsat24and48husing proteinappearsasadistinctbandat(cid:9)45kDa.
December2012 Volume86 Number23 jvi.asm.org 12821
Huynhetal.
FIG7EvidenceofSARS-CoVreplicationinPESU-B5Lcells.PESU-B5Lcells
wereinfectedwithSARS-CoVatanMOIof(cid:9)1,andtotalRNAwasharvested D
o
fromthecellsat24-hintervalsandsubjectedtoRT-PCRusingprimersde- w
signed to detect leader-containing transcripts of the subgenomic GFP n
(sgGFP),indicativeofSARS-CoVreplication.Subgenomictranscriptswere FIG8EvidenceofSARS-CoVreplicationinPESU-B5Lcells.PESU-B5Lcells lo
wereinfectedwiththemouse-adaptedstrainofSARS-CoV,MA15,andwith a
detectedat24hpostinfectionbutdisappearedby48hpostinfection,suggest- chimericSARS-CoVvirusesbearingthespikegenesofGD03andHC/SZ/ d
ingthatSARS-CoVreplicatesinthesecellsatalowlevel.L,Promega1-kb e
61/03atanMOIof(cid:9)1.TotalRNAwasharvestedfromthecellsat48and72h d
ladder;M,mock-infectedcells;V,SARS-CoV-infectedcells. postinfectionandsubjectedtoRT-PCRusingprimersdesignedtodetectlead- f
r
er-containing subgenomic transcripts indicative of replication. (A) Sub- o
m
genomic(sg)transcriptsforX1,envelope(E),membrane(M),andopenread-

postinfection and primers designed to detect subgenomic tran- ingframe6[ORF6(X3)]weredetectedatbothtimepointsforallthreestrains. h
t
scriptionofviralproteins.Subgenomictranscriptsweredetected Lane1,MA15at48h;lane2,MA15at72h;lane3,Invitrogen1-kbPlusladder; tp
atbothtimepointsforallthreestrains(Fig.8A).Thebandrepre- liannfeect4e,dScAeRllsS;(cid:5)laGneD70,3SAatR4S8(cid:5)hH;Cla/SnZe/651,/S0A3RaSt(cid:5)48GhD;a0n3daltan7e28h,;SlAaRneS(cid:5)6,HmCo/ScZk-/ ://jv
sentingtheMgenewasexcisedandsequencedbySangersequenc- 61/03at72h.(B)ThesgMbandsforallthreestrainswereexcised,andthe i.
a
ingforallthreeofthesestrains,andallcontainedtheappropriate DNAwaspurifiedandsequencedtoverifythattheSARS-CoVleadersequence s
sequencetoverifythepresenceoftheleadersequencecombined andthe5=endoftheMgenewerepresent.ShownhereissgMfromSARS-CoV m
withtheMgene(Fig.8Banddatanotshown).Interestingly,de- strainGD03. .o
r
g
spitethefactthatthePESU-B5LcellsinfectedwithMA15andthe /

twochimericvirusesshowedmoderateamountsofcytopathology o
n
thatoccurredpriortocellsenescence(datanotshown),wewere NorthAmericanbatshavebeenshowntoharbor(cid:1)-CoVse- D
unabletodetectanysignsofviralreplicationwhenthesuperna- quences(10,11,24,31,33);however,no(cid:1)-CoVshavebeeniso- e
c
tantsfromtheseinfectionswerepassedontofreshPESU-B5Lcells. latedfromthesebats,andinfact,nofull-lengthCoVsequences e
m
have been obtained or reported from North American bats. Of b
DISCUSSION note,inourmetagenomicsstudyofNorthAmericanbatsinthe e
r
Batsareimportantreservoirhostsforalargenumberofemerging EasternUnitedStates,weidentified(cid:1)-CoVsequencesinmultiple 3
,
virusesthatcausehumandisease.However,onlyasmallnumber bat species, including the big brown bat and the tricolored bat 2
ofspecies((cid:9)36of1,100)(5,11,24)havebeenstudiedandshown (11).Inthisstudy,wedetected(cid:1)-CoVsequencesinabigbrown 01
4
toharborRNAviruses,andmanyofthesevirusessharesequence batpopulationinNewYorkandfromatricoloredbatpopulation
b
similaritytohumanstrains.ThefactthatCoVshavebeenfoundin sampled in Maryland (Fig. 2). Interestingly, the two sequences y

multipleOldWorldandNewWorldbatspeciessuggeststhatbats derivedfrombigbrownbats(ARCoV.1andNECoV)werenearly U
may be particularly suited for maintaining CoVs in nature. In- identical in a (cid:4)2,200-nt fragment of the replicase gene, even N
I
V
cludingthefindingsinthisstudy,atleastthreehumanCoVshave though the two populations were from different regions (New E
beenlinkedtobatCoVs(26,36),andthereismountingevidence York and Maryland) (Fig. 2B). In contrast, the same sequence R
S
thatallmammalianCoVsmayhaveoriginatedfrombats(19,36, fragmentobtainedfromtricoloredbatswassignificantlydifferent
I
T
49),particularlyhumancoronaviruses. (74%identityatthenucleotidelevel),suggestingthatdifferentbat Y
Althoughtheexactnumberofinfectionsisunknown,HCoV- speciesharborCoVsthathaveadaptedspecificallytothatspecies O
NL63 has been shown to be distributed worldwide; it infects a (Fig. 2B). Whether or not a compartmentalized CoV from one F

significantnumberofchildrenandelderlypersonseachyear,and speciesismorelikelytoinfecthumans,asoccurswiththerabies M
E
HCoV-OC43and-229Eaccountfornearlyone-thirdofallcolds. virus(6,32,40),isyettobedetermined.However,itisclearthat
M
Moreover,SARS-CoVinfectedover8,000peopleworldwide,with specific bat species harbor bat SARS-like viruses that are more P
amortalityrateapproaching10%(34).Theseobservationsindi- closelyrelatedtoSARS-CoV(26). H
I
catethatCoVsareimportanthumanpathogensthatcausesignif- ThefactthatsomeoftheRoche454sequencesderivedfrom S
icantmorbidityandfrequentmortality.Thefactthatover1,000 thetricoloredbatweremorecloselyrelatedtoHCoV-NL63than
bat species have not been assessed for the presence of CoVs or any other (cid:1)-CoV suggested that this novel North American
otherRNAandDNAvirusesmayhaveenormouspublichealth (cid:1)-CoVmayberelatedtoHCoVs.Therefore,weconductedmo-
consequencesinanoutbreaksetting.Giventhattherearemore lecularclockanalysisusingBEASTtodeterminetherelatednessof
than1,100speciesofbatsandnearlyone-halfofthe36batspecies this(cid:1)-CoVtootherCoVs,byaddingittopreviousdatasetsused
thathavebeenstudiedforviruseshaveshownevidenceofCoV todetermineancestryforotherCoVs(36,50).Ofnote,ARCoV.2
sequences,theremaybehundredsofnovelcoronavirusesinbats andHCoV-NL63appeartohaveanMRCAthatoccurredjustover
throughouttheworld.Ifthisisthecase,thenthenextCoVspill- 560 years ago, based on a 650- to 800-nt portion of the highly
overfrombatstohumansisjustamatterofecologicalopportu- conserved replicase gene. This analysis was limited to this frag-
nity. mentsothatwecouldusethedataobtainedfrompreviousstudies
12822 jvi.asm.org JournalofVirology
HCoV-NL63GrowsinTricoloredBatLungCells
tocalibratetheclockandvalidateourresults.Ifthesepredictions structureandfunction(18).ThePESU-B5Lcellsimmortalizedby
arecorrect,thisobservationsuggeststhatHCoV-NL63mayhave thisapproachhaveshownnomorphologicalmodificationsover
originatedfrombatsbetween1190and1449CE.However,itis timeandappeartobeahomogenouspopulationoflungepithelial
possible that recombination between bat and human CoVs or cells.
othermammalianstrainsmayhaveoccurredinthisregion,and The isolation and growth of novel (cid:1)-CoVs from the North
therefore,moresequenceinformationisnecessarytoworkoutthe Americanandotherglobalbatpopulationscontinuetobeadiffi-
evolutionaryhistoryoftheseCoVs.Comparingthetwotreesin cultproblem.Wehaveattemptedtoisolate(cid:1)-CoVsofseveralbat
Fig.2suggeststhatrecombinationhaslikelyoccurred,asthetree speciesfromfecalsamplesthatwerepositivebyPCRforan(cid:1)-CoV
topologies are slightly different even in the nonstructural gene byinoculatingfreshlyfilteredfecalsupernatantsfromthesesam-
sequencesthatareknowntobehighlyconservedamongCoVs. plesontoavarietyofcelltypes,includingPESU-B5Llungcells.To
D
Immortalizedcelllinesareimportantreagentsforinvestigating date,wehavebeenunsuccessfulinisolatingaCoVusingthisap-
o
thesimilaritiesanddifferencesbetweenvirusesthatgrowinbats proach. Therefore, we have been using high-throughput and w
n
and other mammals. To date, only a few immortalized bat cell Sangersequencingapproachestoidentifyandfillingenomicse- lo
lineshavebeendeveloped,andthemajorityoftheseareforfruit quences that can be engineered into infectious clones that will a
d
bats,whichallowsforfurtherstudieswiththeNipahandHendra allowustostudythesevirusesinthelaboratory.Wepreviously e
d
viruses(4,7,22).Inthisstudy,wesuccessfullyimmortalizedbat used this synthetic approach to resurrect Bat CoV HKU3, the
f
cells(Fig.3)fromabatspeciesknowntoharboraunique(cid:1)-CoV SARS-likeCoVidentifiedintheChinesehorseshoebat(26),and ro
m
that appears to share common ancestry with HCoV-NL63. The showedthattheonlyblocktoreplicationofthisbatvirusinpri-

factsthatSARS-CoVandrelatedstrainsreplicate(Fig.7and8) matecellswasthe180-amino-acidportionofthespikeglycopro- h
t
t
andthatHCoV-NL63growsinthePESU-B5Limmortalizedlung teinknownasthereceptor-bindingdomain(RBD)(3). p
:
/
celllineindicatetheimportanceofdevelopingrobustreagentsto Inthispaper,wereportthefirstevidencethattraditionalhu- /jv
anumberofdifferentbatspecies.Toourknowledge,thisisthe manCoVsarecapableofgrowthinbatcells.Interestingly,both i.
a
firstbatcelllinethatsupportsgrowthofHCoVs.Wearecurrently HCoV-NL63andSARS-CoVanditsrelatedvirusesreplicatedin s
m
in the process of characterizing the innate immune response in thesecells;however,replicationoccurredovermultiplepassages
.
o
thesecells,andwehaveprimarycellsavailableforthreeadditional withHCoV-NL63.RecentstudieshavedemonstratedthatSARS- r
g
NewWorldbatspeciesthatwillbetargetedforimmortalization. CoV and HCoV-NL63 use angiotensin-1 converting enzyme-2 /

o
Thesecelllineswillbeusedtogeneratereagentsfordirectlystudy- (ACE2)asareceptorforentryintopermissivecells(25,28,37,45). n
ingCoVsinbatsandforassessingthepotentialofthesevirusesto However,crystallographystudieshaveshownthattheseHCoVs D
infecthumans.Ourdatasuggestthatthelentivirus-basedimmor- interactwithACE2usingdifferentreceptorbindingdomainsin e
c
talization system reported herein would also be successful for the spike glycoproteins of each virus to interact with different e
m
multipleNorthAmericanbatspeciesandlikelyrepresentsaro- regionsoftheACE2molecule(23,27,52).Infact,wewouldpre- b
busttoolforrapidlyestablishingcontinuouscelllinesfrombats dictthatitisdifferencesintheseinteractionsthatimpacttheabil- e
r

andotherzoonoticspecies. ityofSARS-CoVtoefficientlyreplicateuponpassageinthesecells. 3
,
Onlyasmallnumberofimmortalizedbatcelllinescurrently Interestingly, while SARS-CoVgfp replicated leader-containing 2
0
exist,andthesewerederivedbothfromOldWorldbats,including transcriptsat24h,transcriptionwasnotdetectedat48handno 1
4
theEgyptianfruitbat(Rousettusaegyptiacus)(22),thestraw-col- GFPfluorescencewasobservedinthesecultures.However,MA15
b
oredfruitbat(Eidolonhelvum)(4),andtheblackflyingfox(Ptero- and the two chimeric human SARS-CoVs bearing spike genes y

pusalecto)(7),andfromNewWorldbats,suchastheBrazilian fromtheearlystageoftheSARSepidemicappearedtoreplicate U
N
free-tailed bat (Tadarida brasiliensis) (ATCC CCL-88). In these leader-containingtranscriptsmoreefficientlyandexhibitedmore
I
V
cases,thecelllineswereimmortalizedusingthreedifferentmeth- cytopathology,indicatingthatthesevirusesgrewbetterinthese E
ods,includingthesimianvirus40(SV40)largetumorantigen(4, cells than did SARS-CoVgfp. This observation suggests that the R
S
7),theadenovirusserotype5E1AandE1Bgenesdrivenfromthe spikeproteinmaybetheprimarydeterminantforinfectivityin
I
T
promotersforhumanphosphoglyceratekinaseandthymidineki- thesecellsandthatspikeproteinsthataremoreadaptedtohuman Y
nase of herpes simplex virus (22), and hTERT (7). Initially, we ACE2,suchasSARS-CoV,arelesscapableofestablishinginfec- O
attempted to immortalize the various primary bat cells using tioninthesebatcells.However,HCoV-NL63appearedtorepli- F

hTERT, but these attempts did not result in immortalized cells catemoreefficientlythananyoftheSARS-CoVvariants,suggest- M
E
(data not shown). Interestingly, it has been shown that hTERT ing that the HCoV-NL63 interaction site on the tricolored bat
M
alone cannot immortalize primary human bronchial epithelial ACE2maybetheprimarydeterminantofinfection.Theinterac- P
(hBE)cells(30),possiblyduetosuboptimalcultureconditions. tionsbetweenPESU-B5Lcellsandvariousspikeproteinsarecur- H
I
Moreover,whilehTERTincombinationwithviraloncogeneshas rentlybeingexploredinmoredetail.Interestingly,arecentstudy S
beenusedtoimmortalizemammaliancelllines,theseimmortal- hasshownthatsignaturesofrecurrentpositiveselectioninthebat
izationshaveoftenresultedinimmortalizedcelllinesthatwere ACE2genemapalmostperfectlytoknownSARS-CoVinteraction
limited in differentiation capacity and that frequently exhibited surfaces,suggestingthatACE2utilizationprecededtheemergence
othermorphologicalandgeneticabnormalities(18).Tocircum- ofSARS-CoV-likevirusesfrombats(9).
venttheselimitations,weusedaviral-oncogene-independentap- Inaddition,thesequenceoftheorthologousACE2receptorof
proachthatwassuccessfullyusedtoimmortalizehBEcellsforthe thetricolorbathasnotbeendeterminedbutlikelypresentsthe
study of cystic fibrosis (18). The primary difference in this ap- determinants of cross-species transmission, allowing HCoVs to
proachwasthatitusedtheexpressionofthemurineBmi-1gene infectbats.Supportingthishypothesis,severalbatACE2receptors
(18),whichisaproto-oncogenethatnormallymaintainsstemcell wererecentlyshowntofunctionasreceptorsforSARS-CoVdock-
populationsbuthasalsobeenshowntorecapitulatenormalcell ingandentry,andnotably,HCoV-NL63receptorusagewasnot
December2012 Volume86 Number23 jvi.asm.org 12823
Huynhetal.
evaluatedinthesestudies(21).Ofnote,most(cid:1)-CoVsuseamino- virus to maintain viral populations in multiple hosts, exchange
peptidase N (APN) molecules as the cellular receptor, and the geneticinformationtoalterpathogenesisortransmissioncharac-
exogenousexpressionoffelineAPNincellsthatarerefractoryto teristics, and potentially evolve variants that are capable of effi-
infection has rendered these cells susceptible to most (cid:1)-CoVs cientlyinfectinghumansorothermammals.Furtherworkisnec-
(47).ThefacttheHCoV-NL63,whichisan(cid:1)-CoV,usesACE2is essarytodeterminetherisksofsuchacycle,evaluatetheimpact
anunusualfeatureofthisHCoV,althoughitisnotknowwhich thatthiscouldhaveonpublichealth,anddeterminewhetheror
receptorsthebat(cid:1)-CoVsuse.Wearecurrentlyintheprocessof notthisrepresentsageneralfeatureofthemammalianCorona-
cloning and sequencing all of the known CoV receptor ortho- viridae,whicharepredictedtohaveoriginatedfrombats.
loguesforthisbatspeciestodeterminewhichreceptor(s)isused
bySARS-CoVandHCoV-NL63andtoassesstheriskofreverse ACKNOWLEDGMENTS
D
zoonotictransmissionfromhumanstobats.Wealsoplantoeval-
ThisprojectwasfundedbyanARRAgrantthroughSERCEBno.U54- o
uatePESU-B5Linfectivityandreceptorusagebyothercoronavi- w
AI057157PIawardedtoE.F.D.,J.E.G.,andM.B.F. n
rusessuchasHCoV-229EandHKU3infutureexperiments.At- WegratefullyacknowledgetheworkofAimeeHaskew,LisaSmith, lo
temptstoobtainthesevirusesforthisstudywereunsuccessful. JennySaville,andAngelaSjollema,allofwhomparticipatedincollecting a
d
CoVs have a long history of cross-species transmission (35). batsamplesforthiswork. e
d
BCoVandHCoV-OC43arecloselyrelated,withanMRCApre-
f
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