Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus


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Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus

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Title:
Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus
Series Title:
mBio
Creator:
Anthony, S. J.
Gilardi, K.
Menachery, V. D.
Goldstein, T.
Ssebide, B.
Mbabazi, R.
Navarrete-Macias, I.
Liang, E.
Wells, H.
Hicks, A.
Petrosov, A.
Byarugaba, D. K.
Debbink, K.
Dinnon, K. H.
Scobey, T.
Randell, S. H.
Yount, B. L.
Cranfield, M.
Johnson, C. K.
Baric, R. S.
Lipkin, W. I.
Mazet, J. A. K.
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mBio
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Bats ( local )
Evolutionary Source ( local )
Mers ( local )
Middle East Respiratory Syndrome ( local )
Coronavirus ( local )
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serial ( sobekcm )

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Abstract:
The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae. Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.
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mBio, Vol. 8, no. 2 (2017-04-04).

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FurtherEvidenceforBatsasthe EvolutionarySourceofMiddleEast RespiratorySyndromeCoronavirusS.J.Anthony,a,b,cK.Gilardi,dV.D.Menachery,eT.Goldstein,dB.Ssebide,fR.Mbabazi,fI.Navarrete-Macias,aE.Liang,a,cH.Wells,aA.Hicks,aA.Petrosov,aD.K.Byarugaba,g,hK.Debbink,eK.H.Dinnon,eT.Scobey,iS.H.Randell,jB.L.Yount,eM.Cran“eld,d,eC.K.Johnson,dR.S.Baric,eW.I.Lipkin,a,bJ.A.K.MazetdCenterforInfectionandImmunity,MailmanSchoolofPublicHealth,ColumbiaUniversity,NewYork,New York,USAa;DepartmentofEpidemiology,MailmanSchoolofPublicHealth,ColumbiaUniversity,NewYork, NewYork,USAb;EcoHealthAlliance,NewYork,NewYork,USAc;OneHealthInstituteandKarenC.Drayer WildlifeHealthCenter,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,California,USAd; DepartmentofMicrobiologyandImmunology,UniversityofNorthCarolinaSchoolofMedicine,ChapelHill, NorthCarolina,USAe;GorillaDoctors,c/oMGVP,Inc.,Davis,California,USAf;MakerereUniversityWalterReed Project,Kampala,Ugandag;MakerereUniversity,CollegeofVeterinaryMedicine,Kampala,Ugandah; DepartmentofEpidemiology,UniversityofNorthCarolinaSchoolofMedicine,ChapelHill,NorthCarolina, USAi;DepartmentofCellBiologyandPhysiologyandMarsicoLungInstitute/CysticFibrosisCenter,University ofNorthCarolinaSchoolofMedicine,ChapelHill,NorthCarolina,USAj ABSTRACT TheevolutionaryoriginsofMiddleEastrespiratorysyndrome(MERS) coronavirus(MERS-CoV)areunknown.Currentevidencesuggeststhatinsectivorous batsarelikelytobetheoriginalsource,asseveral2cCoVshavebeendescribed fromvariousspeciesinthefamily Vespertilionidae .Here,wedescribeaMERS-like CoVidenti“edfroma Pipistrelluscf.hesperidus batsampledinUganda(strain PREDICT/PDF-2180),furthersupportingthehypothesisthatbatsaretheevolutionary sourceofMERS-CoV.PhylogeneticanalysisshowedthatPREDICT/PDF-2180isclosely relatedtoMERS-CoVacrossmuchofitsgenome,consistentwithacommonancestry;however,thespikeproteinwashighlydivergent(46%aminoacididentity),suggestingthatthetwovirusesmayhavedifferentreceptorbindingproperties.Indeed, severalaminoacidsubstitutionswereidenti“edinkeybindingresiduesthatwere predictedtoblockPREDICT/PDF-2180fromattachingtotheMERS-CoVDPP4receptor.Toexperimentallytestthishypothesis,aninfectiousMERS-CoVcloneexpressing thePREDICT/PDF-2180spikeproteinwasgenerated.Recombinantvirusesderived fromtheclonewerereplicationcompetentbutunabletospreadandestablishnew infectionsinVerocellsorprimaryhumanairwayepithelialcells.Our“ndingssuggestthatPREDICT/PDF-2180isunlikelytoposeazoonoticthreat.Recombinationin theS1subunitofthespikegenewasidenti“edastheprimarymechanismdriving variationinthespikephenotypeandwaslikelyoneofthecriticalstepsintheevolutionandemergenceofMERS-CoVinhumans. IMPORTANCE Globalsurveillanceeffortsforundiscoveredvirusesareanimportant componentofpandemicpreventioninitiatives.Thesesurveyscanbeusefulfor“ndingnovelvirusesandforgaininginsightsintotheecologicalandevolutionaryfactorsdrivingviraldiversity;however,“ndingaviralsequenceisnotsuf“cienttodeterminewhetheritcaninfectpeople(i.e.,posesazoonoticthreat).Here,we investigatedthespeci“czoonoticriskofaMERS-likecoronavirus(PREDICT/PDF-2180) identi“edinabatfromUgandaandshowedthat,despitebeingcloselyrelatedto MERS-CoV,itisunlikelytoposeathreattohumans.Wesuggestthatthisapproach constitutesanappropriatestrategyforbeginningtodeterminethezoonoticpotentialofwildlifeviruses.ByshowingthatPREDICT/PDF-2180doesnotinfectcellsthatReceived 4March2017 Accepted 9March 2017 Published 4April2017 Citation AnthonySJ,GilardiK,MenacheryVD, GoldsteinT,SsebideB,MbabaziR,NavarreteMaciasI,LiangE,WellsH,HicksA,PetrosovA, ByarugabaDK,DebbinkK,DinnonKH,Scobey T,RandellSH,YountBL,Cran“eldM,Johnson CK,BaricRS,LipkinWI,MazetJAK.2017.Further evidenceforbatsastheevolutionarysourceof MiddleEastrespiratorysyndromecoronavirus. mBio8:e00373-17. https://doi.org/10.1128/ mBio.00373-17 . Editor StaceySchultz-Cherry,St.Jude Children'sResearchHospital Copyright ©2017Anthonyetal.Thisisan open-accessarticledistributedundertheterms ofthe CreativeCommonsAttribution4.0 Internationallicense . AddresscorrespondencetoS.J.Anthony, sja2127@cumc.columbia.edu. R.S.B.,W.I.L.,andJ.A.K.M.contributedequallyto thiswork. RESEARCHARTICLEcrossm March/April2017Volume8Issue2e00373-17 ® mbio.asm.org 1

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expressthefunctionalreceptorforMERS-CoV,wefurthershowthatrecombination waslikelytobethecriticalstepthatallowedMERStoemergeinhumans. KEYWORDS bat,MERScoronavirus,spike,Uganda,zoonoses I n2012,MiddleEastrespiratorysyndrome(MERS)emergedinSaudiArabia.Clusters offatalpneumoniainadultsweredeterminedtobecausedbyanovellineageC betacoronavirus(2cCoV),termedMERS-CoV( 1 ).Thiswasthe“rst2cCoVknownto causediseaseinhumansandatthetimeofitsdiscoverywasmostcloselyrelatedto twoknownbatcoronaviruses( 2 ),raisingthepossibilitythatbatswereareservoirand sourceforthevirus.Concurrently,epidemiologistsidenti“edanassociationbetween MERSinfectionsinpatientsandtheircontactwithdromedarycamels( 3 , 4 ).MERS-CoV wassubsequentlydetectedincamelsatafarmlinkedtotwohumancasesinQatar( 5 ) andincamelsinEgypt( 6 ),followedbysurveysthatdemonstratedwidespreadexposuretothevirusintheMiddleEastandinNorthandEastAfricaasearlyasthe1980s ( 7…10 ).Itisnowclearthatcamelsplayanimportantroleinthetransmissionof MERS-CoVtopeople( 11 ),withseroprevalencehighestamongthosewhohavehad contactwithcamels( 12 ). WhilecamelsarethoughttobeimportantforthetransmissionofMERS-CoV,bats arewidelyconsideredtobetheevolutionarysourceofthevirus.Several2cCoVshave nowbeendescribedinbats,includingHKU4from Tylonycterispachypus ( 13 ),HKU5from Pipistrellusabramus ( 13 ),andtherecentlyidenti“edNeoCoVfrom Neoromiciacapensis ( 14 ).NeoCoVistheclosestrelativeyetdiscovered(85%identicaltoMERS)andshares suf“cientgeneticsimilarityinthereplicasegenestobeconsideredpartofthesame viralspecies( 15 );however,despitebeingcloselyrelatedacrossmuchofthegenome, theS1subunitofthespikegeneishighlydivergentasaresultofapriorrecombination event.Recombinationinthespikegeneisparticularlysigni“cantbecausethederived proteinisresponsibleforhostreceptorrecognitionandmembranefusion( 16 )andthus iscentralindetermininghostspeci“city.TheS1subunitcontainsthereceptorbinding domainandthereforehasaspeci“croleinde“ninghosttropism( 17 ).Otherprocesses arealsoimportant,suchastheactivationofthespikeproteinbyhostproteases( 18 ), buttheabilityofS1tobindwithahostreceptorisacriticalstepintheemergence pathway„anditcanbequicklyalteredbyasinglerecombinationevent.Thesequence variationintheS1regionofMERS-CoVandNeoCoVcouldthereforeindicatedifferences inhostbindingpreferences. Predictingtheinteractionsofvirusbindingdomainswithaparticularhostreceptor (forexample,thehumanMERS-CoVreceptorDPP4)ispossiblethroughtheuseof structuralmodelingandthegenerationofinfectiousclones.Protein-proteininteractionscanbemodeledusingarelatedhomologouscomplex( 19 , 20 )whilereverse geneticstrategiescantestthepermissivenessofhumanorotherprimatecellsfor infectiousclonesexpressingthenovelreceptorbindingdomainsorcompletespike glycoprotein( 21…24 ).Pseudotypedlentivirussystemshavealsobeenused,forexample, toshowthatDPP4isthereceptorforHKU4butnotforthecloselyrelatedHKU5( 25 , 26 ). Andwhilepseudotypesarenotalwaysaccuratepredictorsofspikeglycoprotein function( 23 ),these“ndingsindicatethatmultiplecell-entrystrategiescouldexistfor 2cvirusesandthatnotallMERS-likeCoVsposeanequalriskofzoonoticemergence. Here,weinvestigatedthereceptorbindingpropertiesofanewstrainofMERS-like CoVfoundinabatfromUganda.Thisvirus(PREDICT/PDF-2180)sharesthesame putativeS1subunitrecombinationthatwasobservedinNeoCoV,allowingustoalso considerwhetherthespikerecombinationwascriticalfortheemergenceofMERS-CoV inhumans. RESULTS Samplingandsitecharacterization. Abat(identi“er[ID]OTBA03-20130220)was trappedon20February2013intheNkuringoareaofKisoroDistrict,insouthwestern Uganda(latitude 1.12,longitude29.68)( Fig.1 ).ThisareaisanestablishedsettlementAnthonyetal. ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 2

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ofvillagescomprisingapproximately15,000inhabitantsadjacenttothesouthwestern boundaryofBwindiImpenetrableNationalPark.Communitiesincludesubsistence farmersgrowingsmallcrops,withsomemembersworkinginsidethenationalparkor supportingtourism-relatedbusinesses.Livestock,includingcattle,pigs,sheep,goats, andpoultry,arepresentinthevillageandareraisedonasmallscaleprimarilyforlocal consumption. Thesampledbatweighed3.0gandhadaforearmlengthof25mm( Fig.1 ).Itwas identi“edas Pipistrelluscf.hesperidus basedon95%sequenceidentityinthecytochrome b (Cytb)gene.Thecytochromeoxidasesubunit1(CO1)wasalsosequenced, butnocorrespondingCO1sequencesfor P.hesperidus wereavailableinGenBankfor comparison.WethereforereliedontheCytbsequenceforspeciesidenti“cation. Discoveryandsequencecharacterization. Theoralswab,rectalswab,andwhole bloodofbatOTBA03-20130220wereassayedforthepresenceofcoronavirusby consensusPCR(cPCR).Twoseparateassayswereused,eachtargetingadifferentregion oftheORF1bRNA-dependentRNApolymerase(RdRp).Bandsoftheexpectedsizewere ampli“edfromtherectalswab(PDF-2180)bybothassaysandcon“rmedtorepresent viralproductsbytraditionalSangerdideoxysequencing.Bothfragmentsshowed 98%aminoacidsequenceidentitytoMERS-CoV,promptingfurthercharacterization ofthevirus.Theoralswabandbloodwerenegative. Thenear-full-lengthgenome(identi“edasPREDICT/PDF-2180)wasassembledfrom 100-nucleotide(nt)Illuminasingle-endreadsatanaveragedepthof26 .Onlythe5 = and3 = noncodingregionswereleftincomplete.Theorderofallpredictedopenreading frames(ORFs)wasconsistentwithMERS-CoVandwiththerecentlydescribedNeoCoV (KC869678)identi“edinabatfromSouthAfrica.Similarly,thehexanucleotidetranscriptionregulatorysequence(AACGAA)wasconservedandfoundinthesameposition asbothMERSandNeoCoVupstreamofeachpredictedORF. Acrossthefullgenome,thesequencehad86.5%aminoacididentitytoMERS-CoV and91%toNeoCoV;however,considerablevariationwasobservedindifferentgenes. FIG1 Mapshowingthedistributionof Pipistrellushesperidus (basedonInternationalUnionfor ConservationofNature[IUCN]data)andthelocationofthebatsampledforthestudy.UgandaMERS-LikeVirusinBats ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 3

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Aminoacididentitycouldbeashighas97%tobothMERS-CoVandNeoCoVinORF1b oraslowas45%toMERS-CoVinsubunit1ofthespikeprotein.Forthefullspike protein,identitywas94%toNeoCoVand63%toMERS-CoV.Percentsequenceidentity ofthespikeprotein(subunits1and2)toother2cvirusesisshownin Tables1and2 , respectively.Basedonthecurrentcriteriaforspeciesdemarcationestablishedbythe InternationalCommitteefortheTaxonomyofViruses( 90%aminoacidsequence identityinthereplicaseproteins),PREDICT/PDF-2180sharessuf“cientgeneticidentity toMERS-CoVtobeconsideredamemberofthe MERS-likeCoronavirus species. Phylogeneticanalysis. Maximumlikelihoodphylogeneticreconstructionsshowed thatPREDICT/PDF-2180ismostcloselyrelatedtoNeoCoV( Fig.2 ).Thetwoviruseswere basalorformedsistercladestoMERS-CoVinallgenesexceptsubunit1ofthespike.The full-genomealignmentwasscannedforrecombinationusingsevendifferentalgorithms(RDP,GENECONV,Bootscan,MaxChi,Chimaera,SiScan,and3seq)implemented inRDPv4.46.AsinglerecombinationeventwasdetectedwithinthespikegenebyRDP, Bootscan,MaxChi,Chimaera,SiScan,and3seq(Bonferroni-corrected P of 0.001), suggestingthattheincongruentphylogeniesobservedbetweenspikesubunit1and therestofthegenomearetheresultofrecombination.AttemptstodatethedivergenceofthesetwovirusestoestimatetheminimumŽnumberofyearssincethis recombinationwerepreventedbyevidenceofstrongnegative(purifying)selection acrossthegenome( Fig.2 ).Giventhatpurifyingselectioncanconfoundtruephylogeneticdepth,wefeltthatattemptstoestimatethenumberofyearstocommonancestry wereinappropriateandwouldresultinarti“ciallyrecentŽdates. ZoonoticpotentialofPREDICT/PDF-2180. Thehighgeneticvariabilityinsubunit 1suggeststhathumanandbatstrainsofMERShavedifferentreceptorbinding properties.Toinvestigatethis,wemodeledthespeci“caf“nityofthePREDICT/PDF2180spikeproteinforthehumanMERS-CoVreceptorDPP4( 27 ).Weutilizedthecrystal structureoftheMERS-CoVspikebindingdomainincomplexwithDPP4tocreatea homologymodelforthecomparableregionofthePREDICT/PDF-2180spike( Fig.3 ). Previousworkhasdemonstrated11speci“caminoacidresiduesinMERS-CoVthatTABLE1 Pairwiseaminoacidsequenceidentityofsubunit1ofspikeproteinof2cCoVs Accessionno. and/orisolate %identityforsubunit1(%identityforreceptorbindingdomain) NeoCoV PDF2180 EriCoV/ 2012/174 EriCoV/ 2012/216 BtCoV/ 133HKU4HKU5-1HKU5-5SC2013 EMC2012 AlHasa1 NRCHKU205 KC869678 , NeoCoV Predict/ PDF-2180 91.0(93.1) KC545383 , EriCoV/2012/ 174 54.7(59.7)54.3(60.4) KC545386 , EriCoV/2012/ 216 54.9(59.7)54.4(60.4)99.9(100) DQ648794 , BtCoV/133 45.1(42.5)45.5(42.5)43.9(50.0)44.0(50.0) EF065505 , BatCoV HKU4 45.1(41.8)45.7(41.8)44.3(50.0)44.4(50.0)95.6(96.2) EF065509 , BatCoV HKU5-1 47.8(46.3)47.7(47.0)45.8(53.7)45.9(53.7)57.7(63.4)58.4(63.4) EF065512 , BatCoV HKU5-5 47.4(45.5)47.8(46.3)44.6(50.7)44.7(50.7)56.3(61.8)56.7(61.8)90.1(93.9) KJ473821 , SC2013 45.5(42.5)46.2(44.0)45.8(55.2)45.9(55.2)59.7(57.5)59.7(56.7)63.7(73.9)64.1(73.1) KC667074 , EMC-2012 43.5(40.3)44.4(41.0)43.9(47.8)44.1(47.8)61.2(64.9)61.1(63.4)56.5(63.4)56.7(61.1)61.4(61.2) KF186567 , Al-Hasa1 43.5(40.3)44.4(41.0)43.9(47.8)44.1(47.8)61.4(65.6)61.2(64.1)56.5(63.4)56.7(61.1)61.4(61.2)99.9(99.2) KJ477102 , NRC-HKU205 43.7(39.6)44.4(40.3)43.8(47.0)43.9(47.0)61.1(64.9)61.0(63.4)56.3(62.6)56.4(60.3)61.4(60.4)99.1(98.5)99.2(99.2) Anthonyetal. ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 4

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facilitatebindinginteractionswiththehumanDPP4( 28 ).Oftheseresidues,onlyoneis conservedforPREDICT/PDF-2180.Todeterminewhetherthebindinginteractionsmay beconservedbetweenDPP4andPREDICT/PDF-2180regardlessofthedifferencesin aminoacidresiduesatthesepositions,weanalyzedthepredictedinteractionsbetween PREDICT/PDF-2180andDPP4,comparedtoMERS-CoVandDPP4.Overall,wefounda globalreductioninpredictedhydrogenbondinginteractionsintheDPP4-PREDICT/ PDF-2180bindinginterfacecomparedwithDPP4-MERS-CoV( Fig.3 ).WhiletheinteractionsinconservedresidueY499weremaintained,DPP4interactionswithPREDICT/ PDF-2180residues501,502,510,511,513,539,and542weredisrupted.Theinteraction betweenDPP4Y322andMERSD510isabolishedinthePREDICT/PDF-2180prediction, whereD510isreplacedbyK510.Thisisachargechangefromnegativetopositive. Interestingly,achangefromR511inMERStoD511inPREDICT/PDF-2180facilitatesa potentialinteractionwithY322toreplacethehydrogenbondlostwithK510.Regardless,duetothepredictedlossofthemajorityoftheDPP4bindinginteractions,the modelpredictsthatPREDICT/PDF-2180willnotbindtoDPP4. Tocon“rmtheseresults invitro ,arecombinantMERS-CoVcDNAclonewasconstructedcontainingthePREDICT/PDF-2180spikegeneinthecontextofthefull-length MERS-CoVbackbone.Thechimericvirusmaintainstheentireectodomainofthe PREDICT/PDF-2180spikewiththeexceptionofthe“rst20aminoacidsofthe5 = end, whichweretakenfromwild-typeMERS-CoV.Similarly,thetransmembranedomains (TMDs)andcytoplasmictailofthechimericvirususedthewild-typeMERS-CoVsequenceinordertominimizeincompatibilityinvirionformation.Followingtransfection intoVerocells,PCRampli“cationofleader-containingtranscriptsforalloftheexpected nestedsubgenomic(sg)mRNAs(includingthesgspikemRNA)con“rmedreplicationof therecombinantvirus( Fig.4 ).However,subsequentpassagesbysupernatanttransfer touninfectedmonolayersfailedtoreproducetheinfection,suggestingthatthe PREDICT/PDF-2180spikeproteinisunabletomediatecellentryinVerocellsasseen withwild-typeMERS-CoV( Fig.4 ).TABLE2 Pairwiseaminoacidsequenceidentityofsubunit2ofspikeproteinof2cCoVs Accessionno. and/orisolate %identity Al-Hasa1 EMC2012 NRCHKU205NeoCoV PDF2180 EriCoV/ 2012/174 EriCoV/ 2012/216 BtCoV/ 133HKU4HKU5-1HKU5-5SC2013 KF186567 , Al-Hasa1 KC667074 , EMC-2012 99.7 KJ477102 , NRC-HKU205 98.698.7 KC869678 , NeoCoV 84.784.984.4 Predict/ PDF-2180 85.585.784.997.6 KC545383 , EriCoV/2012/174 67.867.867.870.270.4 KC545386 , EriCoV/2012/216 68.368.368.370.570.796.9 DQ648794 , BtCoV/133 72.972.972.573.773.369.068.2 EF065505 , BatCoVHKU4 73.073.072.773.873.569.068.398.4 EF065509 , BatCoVHKU5-1 71.271.270.974.373.666.966.679.679.1 EF065512 , BatCoVHKU5-5 71.771.771.474.573.966.966.679.979.497.9 KJ473821 , SC2013 73.573.573.272.571.767.666.881.080.581.882.0 UgandaMERS-LikeVirusinBats ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 5

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SupernatantfromthetransfectedVerocells(passage0[P0])wasalsousedtoinfect primaryhumanairwayepithelial(HAE)cells,whichwerederivedfromlungdonorswith nopreexistingchronicdisease.Thesewell-differentiatedprimarycellsaregrownonan air-liquidinterfaceandrepresentanimportantmodelforviralinfectionofthehuman FIG2 PREDICT/PDF-2180andNeoCoVareancestraltoMERS-CoV.Maximumlikelihoodphylogeneticreconstructionsof2ccoronaviruses(nucleotide)showth at PREDICT/PDF-2180andNeoCoVareconsistentlybasalto,orformsistercladeswith,MERS-likeCoV(human/camelstrains),exceptinsubunit1ofthesp ike protein.HumanOC43istheoutgroup.Allgeneswereshowntobeunderpurifyingselection().Anthonyetal. ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 6

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lung.Severalcoronavirusesshowimprovedreplicationinthesepolarizedprimary respiratorycellscomparedtostandardcelllines.Usingwild-typeMERS-CoVasa control,primaryHAEcellcultureswereinfectedwithpassage0fromthePREDICT/PDF2180-MERSchimericcloneandshowednoevidenceofviralreplication( Fig.5A ). Similarly,viralRNAexpressionanalysisindicatednoevidenceofreplicationfollowing infectionwiththePREDICT/PDF-2180chimericvirus( Fig.5B ).Incontrast,wild-type FIG3 ThespikeproteinofPREDICT/PDF-2180ishighlydivergent.(A)AnucleotideidentitySimplot showsthatPREDICT/PDF-2180andNeoCoVarecloselyrelatedtoMERS-CoVacrossmuchofthegenome butarehighlydivergentinsubunit1ofthespikeprotein,suggestingthattheymayhavedifferent receptorbindingproperties.(B)Variationinkeyaminoacidbindingresidues( * )andmodelingtohuman DPP4bothsuggestthatPREDICT/PDF-2180isunabletobindtoDPP4.UgandaMERS-LikeVirusinBats ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 7

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MERS-CoVinducesrobustreplicationasmeasuredbyplaqueassayandviral-leadercontainingtranscripts.Together,theresultsindicatethatthePREDICT/PDF-2180spike isnotlikelytoef“cientlyreplicateinthehumanairwaywithoutfurtheradaptation. DISCUSSION ThediscoveryofPREDICT/PDF-2180inUgandaaddstothegrowingnumberof groupCbetacoronavirusesthathavenowbeenidenti“edinbats.Theseinclude NeoCoVfromSouthAfrica( 15 ),Mex_CoV-9fromMexico( 29 ),BatCoV/KW2Efrom Thailand( 30 ),P.pipi/VM314fromtheNetherlands( 31 ),H.sav/206645-40fromItaly( 32 ), andBetaCoV/SC2013,HKU4,andHKU5,allfromChina( 33 ).Collectively,theseexamples demonstratethattheMERS-relatedCoVsarehighlyassociatedwithbatsandare geographicallywidespread. FIG4 UgandaspikeproteindoesnotpermitentryintoVerocells.(A)GenomeorganizationofMERS-CoVencodingthe Ugandaspikeglycoprotein.(Bi)Reversetranscription-PCRdetectionofleader-containingnestedsubgenomicmRNAs encodingthenucleocapsidtranscript,Etranscript,andORF5andORF4atranscripts(p0,RNA-transfectedcells;p1,passage 1;p2,passage2).(Bii)Reversetranscription-PCRampli“cationofleader-containingmRNA2containingtheUgandaSgene. Notethelossoftheleader-containingtranscriptsinp1andp2,demonstratingthelossofinfectivityassociatedwith insertionoftheUgandaSgene.Ladder,1kb. FIG5 PDF-2180spikeunabletomediateinfectionofprimaryhumanairwaycultures.(A)Primaryhuman airwayepithelial(HAE)cellsgrownonanair-liquidinterfacewereinfectedwithwild-typeMERS-CoV (blackbars)orpassage0ofPDF-2180/MERSchimericCoV(redbars)andassayedbyplaqueassayonVero cells.ND,nonedetected.(B)Reversetranscription-PCRdetectionofleader-containingnestedsubgenomicmRNAsencodingthenucleocapsidtranscript,Etranscript,andORF5andORF4atranscripts followinginfection.Ladder,1kb;WT,wildtype.Anthonyetal. ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 8

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Thegroup2cvirusesappeartohaveaparticular,thoughnotexclusive,association withvespertilionidbats,whichformahighlydiverseandwidelydistributedfamily withinthe Microchiroptera .NeoCoV,SC2013,HKU4,HKU5,H.sav/206645-40,P.pipi/ VM314,andPREDICT/PDF-2180wereallfoundinspeciesbelongingtothisfamily.Ifthe fulldiversityof2cvirusesre”ectsthenumberofvespertilionidspeciesdescribed( n 475species),thereispotentialforasubstantialdiversityofMERS-relatedvirusestobe circulatinginbats. OurdatasuggestthatPREDICT/PDF-2180cannotinfecthumansandisnotlikelyto poseathreattohumanhealth,atleastinitscurrentform.Thespikeproteinofthisvirus isdistinctfromtheMERS-CoVspike,sharingonly46%aminoacididentity,andit appearsunabletoentercellsthatexpressthefunctionalreceptorusedbyMERS-CoV (DPP4)„oranyotherreceptorexpressedbyeitherprimateVerocellsorhumanairway epithelialcells.Importantly,failuretoassembleandreleaseviralparticlesfromthe initialinfectioncouldalsoexplainourresults;however,wesuggestthatreceptor incompatibilityismorelikelygiventhestepstakentominimizeparticledisruption(see MaterialsandMethods).Theseresultssuggestthatadaptationofthespikewouldbe requiredtopermitPREDICT/PDF-2180replicationinhumanairways.Whilewedidnot examinethespeci“cbindingpropertiesoftherelatedvirusNeoCoV,thehighamino acidsequenceidentitywithPREDICT/PDF-2180indicatesthatitsharesasimilarphenotypeandismostlikelyalsorefractoryforhumaninfections. OurdatasuggestthatRNArecombinationisthemechanismthatunderliesthe observeddifferenceinreceptorbinding.Recombinationcanoccurathighfrequency duringmixedcoronavirusinfection,allowingdifferentvirallineagestoexchange speci“cfunctionalmotifsorevenentiregenes( 22 , 34 , 35 ).Phylogeneticincongruence wasnotedinsubunit1ofthespikeprotein,andbreakpointswereobservedinthis sameregionbymultiplerecombinationdetectionalgorithms.Itisalsoparsimonious withthehighpurifyingselectionobservedacrossthegenomeof2cviruses(which arguesagainstreceptoradaptationviadriftorselection)andwithpreviousreports citingrecombinationinassociationwithhostswitchingforothercoronaviruses( 36…38 ). GiventhattherecombinationisobservedinbothPREDICT/PDF-2180andNeoCoV,we supporttheprevioussuggestionbyCormanetal.( 15 )thatitwastheMERS-CoVthat acquiredanewspike.GivenalsothatthePREDICT/PDF-2180spikedoesnotuseDPP4 andisseeminglynotcompetentforhumaninfection,wefurthersuggestthatthe recombinationeventwasthecriticalfactordrivingtheemergenceofMERS-CoV. Whatislesscleariswhetherthisrecombinationoccurredinbatsoranintermediate host.Lineage2cstrainsthatuseDPP4havebeenreportedinbats( 25 , 26 ),andthere isalsoevidenceofpositiveselectioninthebatDPP4thatwouldindicatetheexistence ofalargediversityof(as-yet-unknown)DPP4-competentstrains( 39 ).Justasdetailed metagenomicsstudieshaverevealedthepresenceofseveralsevereacuterespiratory syndrome(SARS)-likebatCoVsthatcanusethehumanangiotensinconvertingenzyme 2receptorand/orreplicateef“cientlyinhumancells( 23 , 24 , 40…42 ),itseemslikelythat subsetsofdiverseMERS-CoV-likebatcoronaviruseswillalsoexistwhicharepreprogrammedtoef“cientlyusethehumanDPP4receptor.Thiswouldsupportthehypothesisthattherecombinationoccurredinbats;however,theMERS-CoVspikeseemsto haveadaptedandacquiredapreferenceforhumanDPP4overthebathomologue( 26 , 43 )makingitdif“culttoconcludewithcertaintythattheMERS-CoVspikehasbat origins.Increasedsurveillancewillberequiredtounderstandthefulldiversityofspike phenotypescirculatinginbatsorinintermediatehostssuchascamels. Inrecentyears,globalsurveillanceeffortssuchastheUSAIDEmergingPandemic ThreatsPREDICTprogramhaveadvancedourunderstandingoftheviraldiversitythat existsinwildlife( 44 ).Whilethisknowledgecanbeusefulforprovingtheexistenceof novelviruses( 29 , 30 , 45…49 ),quantifyingoverallviraldiversity( 45 , 46 ),andmeasuring infectionprevalencewithinapopulation,itdoesnotprovideinformationontheir speci“czoonoticthreat.Giventhatnosinglecorrelateofpathogenicityorvirulencehas beendeterminedforanyviralfamily( 50 , 51 )andthatitisnotpossibletodeterminerisk throughphylogeneticdataalone( 51 ),theapproachusedhereisanimportanttoolinUgandaMERS-LikeVirusinBats ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 9

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characterizingthezoonoticpotentialofviralsequencesdetectedinwildlife.Doingso onalargescale(forexample,aspartofprojectslikeUSAIDPREDICT)willalsoprovide criticalinformationonhostandgeographicvariationinkeyviraltraits,likepotential hosttropism,whicharecurrentlymissingfrommostrisk-basedmodelsforecastinghot spotsofdiseaseemergence. MATERIALSANDMETHODS Sampling. Abat(IDOTBA03-20130220)wastrappedon20February2013intheNkuringoareaof KisoroDistrictinsouthwesternUganda.Thebatwascaughtwithamistnet(3.8-mmmesh;Avinet,Inc.) accordingtoestablishedprotocolsandwasreleasedunharmedpostsampling.Standardmorphometric measurements(weightandforearmlength)andphotographswereobtainedtoaidspeciesidenti“cation, whichwascon“rmedbyDNAbarcodingofthecytochrome b (Cytb)andcytochromeoxidasesubunit1 (CO1)mitochondrialDNAgenes( 52 ).Approximately200lofwholebloodwascollectedintoEDTA.Oral andrectalswabswerealsocollectedinduplicate(oneintoviraltransportmediumandonedry). Specimenswerestoredtemporarilyongelpacksandfrozeninliquidnitrogeninthe“eldwithi n4hof collectionandthentransferredto 80°Cforstorageuntiltesting.SamplesweretransferredtotheCenter forInfectionandImmunityatColumbiaUniversityforviraldiscoveryandcharacterization. CoronavirusdiscoverybyconsensusPCR. Totalnucleicacid(TNA)wasextractedusingtheRoche MagNAPure96platformaccordingtothemanufacturersinstructions.TNAwasreversetranscribedinto cDNAusingSuperScriptIII(Invitrogen)accordingtothemanufacturersinstructions.Twobroadly reactiveconsensusPCRassaystargetingpartialandnonoverlappingregionsofthecoronavirusORF1b (containingtheRdRp)wereperformed( 53 , 54 ).Bandsoftheexpectedsizewereexcisedfrom1% agarose,clonedintoStrataclonePCRcloningvector,andsequencedtocon“rmdetection. Sequencingandbioinformaticprocessing. TotalRNAextractwasDNasetreated(DNaseI;Ambion, LifeTechnologies,Inc.)andreversetranscribedusingSuperScriptIII(Invitrogen,LifeTechnologies,Inc.) withrandomhexamerprimers.ThecDNAwasRNaseHtreatedbeforesecond-strandsynthesiswith Klenowfragment(3 = to5 = exonuclease)(NewEnglandBiolabs).Theresultingdouble-strandedcDNAwas shearedto200-bp(average)fragmentsusingaCovarisfocusedultrasonicatorE210,accordingtothe manufacturersstandardsettings,andusedforlibraryconstructionusingtheKapaHyperlibrary preparationkit(KapaBiosystems,Roche),againaccordingtothemanufacturersinstructions.The“nal librarywasquanti“edusinganAgilentBioanalyzer2100andpooledtoallocate20millionreadsonthe IlluminaHiSeq2500platform. TheQ30-“lteredFastQ“leswereusedtogeneratequalitycontrolreportsusingPRINSEQsoftware (v0.20.2)( 55 )andwerefurther“lteredandtrimmed.Hostbackgroundlevelsweredeterminedby mappingthe“lteredreadsagainstabatreferencedatabaseusingBowtie2mapper(v2.0.6, http://bowtie -bio.sourceforge.net )( 56 ).Thehost-subtractedreadswere denovo assembledusingMIRAassembler (v4.0)( 57 ).ContigsanduniquesingletonsweresubjectedtohomologysearchusingMegaBlastagainst theGenBanknucleotidedatabase.Sequencesthatshowedpoorornohomologyatthenucleotidelevel werescreenedbyBLASTXagainsttheviralGenBankproteindatabase.ViralsequencesfromBLASTX analysisweresubjectedtoanotherroundofBLASTXhomologysearchagainsttheentireGenBank proteindatabasetocorrectforbiasedEvaluesandtaxonomicmisassignments.Thegenomeof PREDICT/PDF-2180wasmappedwithBowtie2againstthe“ltereddatasettovisualizedepthand coverageinIntegratedGenomicsViewer. Geneticandphylogeneticanalyses. SequenceswereanalyzedandeditedusingGeneious(version 6.0.3).FullgenomeandindividualgenesequenceswerealignedwithClustalW,andmaximumlikelihood phylogenetictreeswereconstructedinPAUP * (500bootstraps).Modelsofnucleotidesubstitutionwere selectedusingjModelTest.NucleotidesequencesimilaritybetweenMERS-likeviruseswasassessedusing Simplotv3.5.1( 58 )withaslidingwindowsizeof500bp,astepsizeof50nucleotides,and1,000 bootstrapreplicatesusinggap-strippedalignmentsandtheF84(maximumlikelihood)distancemodel. Thefull-genomealignmentwasscannedforrecombinationusingsevendifferentalgorithms(RDP, GENECONV,Bootscan,MaxChi,Chimaera,SiScan,and3seq)implementedinRDP(v4.46)( 59 ). Structuralmodeling. PredictedbindingdifferencesbetweenDPP4andeitherMERSorUgandawere determinedbystructuralanalysis.ThecrystalstructuredemonstratingtheinteractionsbetweenDPP4 andMERSspikebindingdomainhaspreviouslybeenreported( 28 ),andthecrystalstructureisPDBID 4KR0 .WecreatedahomologymodeloftheregionoftheUgandaspikeproteinhomologoustotheMERS spikebindingdomainbasedonthe 4KR0 structureinassociationwithDPP4.We“rstalignedtheamino acidsequencesfor 4KR0 (28)andtheUgandaspikeusingClustalOmega( 60 ).WethenusedMODELLER ( 61 )tocreatepredictedstructuralcoordinatesfortheUgandaspikebasedonthecoordinatesof 4KR0 . BecauseMODELLERrequiresthetwosequencestobethesamelength,weintroducedgapsinthe sequenceswhereappropriatetomaintainthebestsequenceidentitybetweenthe2aminoacid sequences.NumberingisbasedonMERS-CoVaminoacidresidues.Wethenimportedthepredicted crystalstructureforUgandaandtheknownDPP4-MERSstructureintoPyMOL( 62 )forvisualizationand comparativeanalysis.HydrogenbondinginteractionswerepredictedbyselectingtheknownDPP4and 4KR0 orthehomologousDPP4andUgandainteractionsitesandusingthe“ndpolarinteractionsŽ functionwithinPyMOL. GenerationofaMERS-CoVrecombinantvirus. Previously,wereportedtheisolationofrecombinantMERS-CoVthatwasderivedfromacDNAclone( 63 ).ToreconstituteaMERSgenomeexpressingthe PREDICT/PDF-2180CoVspike,newEandFplasmidswereorderedsynthetically(Bio-Basic)tocontainthe PREDICT/PDF-2180spikeectodomain;theseplasmidswerenamedMERS-UgandaEandF.MERSORF1Anthonyetal. ® March/April2017Volume8Issue2e00373-17 mbio.asm.org 10

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andORF2overlap,sotomaintainafunctionalreplicasesequenceandsignalsequenceforspike,the“rst 20aminoacidsoftheMERSspikewereretainedandthePREDICT/PDF-2180sequencewasfusedinframe downstreamoftheMERS-CoVSglycoproteinsignalpeptidasedomainbeginningatits24thaminoacid. Inshort,thesequenceoftheMERSspikecodingforaminoacids21to1306wasreplacedwiththe sequenceofthePREDICT/PDF-2180spikecodingforaminoacids24to1298,sothatfollowingprocessing,anintactspikeglycoproteinwasexpressedduringvirusinfection.TheEandFplasmidswere sequenceveri“edpriortotheassemblyoffull-lengthrecombinantDNAs. TheMERSAthroughFinserts(containingtheUgandaSgene)wererestrictiondigested,resolvedon 0.8%agarosegels,visualized,excised,andpuri“edusingaQIAquickgelextractionkit(Qiagen).TheMERS AtoFinsertsweremixedandligatedovernightat4°C,phenol-chloroformextracted,andprecipitated underisopropylalcohol.Full-lengthT7transcriptsweregenerated invitro asdescribedbythemanufacturer(Ambion;mMessagemMachine)withcertainmodi“cations( 63 ).ForMERS-CoVNtranscripts, 1gofplasmidDNAcontainingtheNgene(ampli“edusingforwardprimer5 = -ATTTAGGTGACACTAT AGATGGCATCCCCTGCTGCACC-3 = andreverseprimer5 = -TTTTTTTTTTTTTTTTTTTTTTCTAATCAGTGTTAACA TCAATCATTGG-3 = )wastranscribedbySP6RNApolymerasewitha4:1ratioofcapanalogtoGTP.RNA transcriptswereaddedto800lofVerocellsuspension(8.0 106cells)inanelectroporationcuvette, andfourelectricalpulsesof450Vat50FweredeliveredwithaGenePulserIIelectroporator(Bio-Rad). ThetransfectedVerocellswereallowedtorecoverfor10minatroomtemperatureandthenincubated at37°Cfor2to4daysina75-cm2”ask.VirusprogenywerethenpassagedseveraltimesinVerocells orprimaryhumanairwayepithelialcellsfor48htodetectviableviruses.Allvirusesweremaintained underbiosafetylevel3(BSL3)conditionswithredundantfans,andpersonnelusedpoweredair-purifying respirators(PAPRs)andTyveksuits. Todetectleader-containingRNAs,intracellularRNAfromwildtypeandrecombinantMERS-CoVUganda(rMERS-CoV-Uganda)wasreversetranscribedwithaprimeratthe3 = endofthegenomeand cDNAwasisolatedforPCRusingareverseprimerlocatedinORF5andaforwardprimerlocatedinthe leaderRNAsequenceatthe5 = endofthegenome(5 = -CTATCTCACTTCCCCTCGTTCTC-3 = ).Leadercontainingampliconsweresequencedaspreviouslydescribed( 64 ).ThecDNAproductswereseparated andvisualizedin0.8%agarosegels. Viruses,cells,andinfection. Wild-typeandchimericCoVswereculturedonVeroE6cells,grownin Dulbeccomodi“edEaglemedium(DMEM)(Gibco,Carlsbad,CA)and5%fetalcloneserum(HyClone, SouthLogan,UT)alongwithantibiotic-antimycotic(anti-anti;Gibco,Carlsbad,CA).GrowthcurvesinVero andprimaryhumanairwayepithelialcellswereperformedaspreviouslydescribed( 65 , 66 ).Humanlungs wereprocuredunderUniversityofNorthCarolinaatChapelHillInstitutionalReviewBoard-approved protocols. Biosafetyandbiosecurity. ReportedstudieswereinitiatedaftertheNIHandtheUniversityofNorth CarolinaInstitutionalBiosafetyCommitteeapprovedtheexperimentalprotocol(projecttitle,Generating InfectiousClonesofBatSARS-likeCoVs;labsafetyplanID,20167715;scheduleGID,19982). Accessionnumber(s). Thenear-completegenomesequenceforPREDICT/PDF-2180hasbeendepositedinGenBankunderaccessionnumberKX574227. ACKNOWLEDGMENTS WethankNancySimmons(AmericanMuseumofNaturalHistory)andCarlosZambrana(EcoHealthAlliance)forhelpwithspeciesidenti“cation.WealsothankKomalJain forbioinformaticassistance(ColumbiaUniversity)andBernardErima,TitusTugume, andRaymondMayanja(allfromMakerereUniversity)forprocessingsamples. ThisstudywasmadepossiblebythesupportoftheAmericanpeoplethroughthe UnitedStatesAgencyforInternationalDevelopment(USAID)EmergingPandemic ThreatsPREDICTproject(cooperativeagreementnumberGHN-A-OO-09-00010-00)and bysupportfromtheNationalInstitutesofAllergyandInfectiousDisease(AI109761and AI110700)andAging(K99AG049092).Humanairwayepithelialcellcultureswere supportedbytheNationalInstituteofDiabetesandDigestiveandKidneyDiseases (DK065988). 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