TheVeterinaryRecord,June20,1998 PapersandArticles Cellculture-grownputativebovinerespiratorytorovirus identifiedasacoronavirus L.A.H.M.Comelissen,P.A.M.vanWoensel,R.J.deGroot,M.C.Horzinek,N.Visser, H.F.Egberink VeterinaryRecord(1998)142,683-686 Aputativebovinerespiratorytorovirus(BRTV)waspropagat- edinbovinefetaldiploidlungandhumancolonictumour cells,andfringedpleomorphicparticlesweredetectedinthe culturesupernatantsbyelectronmicroscopy.Antiseradirect- edagainstabovine(Bredastrain)andequine(Bernestrain) torovirusfailedtoreactwithBRTV-infectedcellsin immunofluorescenceassaysanddidnotneutraliseBRTV.No toroviralRNAwasfoundinthesupernatantsofinfectedcells bymeansofareversetranscriptase-polymerasechainreaction withtorovirus-specificprimers.Ontheotherhand,bovine coronavirus-specificantiseraandmonoclonalantibodiesdid neutralisethecytopathiceffects,andcoronaviralantigenwas detectedintheculturesbyimmunofluorescence.Furthermore, bovinecoronavirusRNAwasdetectedinthesupernatantsof BRTV-infectedcellsafternucleicacidamplification.Itiscon- cludedthatthecytopathicBRTVisolateisacoronavirus. ATTEMPTStopropagatetorovirusesincellculturehavebeen unsuccessful,withthenotableexceptionofasingleisolationfrom thehorse(Weissandothers1983).WhenVanopdenboschand others(1991,1992a)reportedthein vitroadaptationofatorovirus fromtherespiratorytractofacalfwithpneumonia,itwasgreeted asanimportantfinding:boththeisolationofabovinetorovirus anditsinferredpathogenicpropertiesareofveterinaryrelevance. TorovirusesformagenusinthefamilyCoronaviridae(Pringle 1992)oftherecentlyestablishedorderNidovirales(deVriesand others1997).Virionsappearaspleomorphic,envelopedparticles withasingleordouble(Comelissenandothers1997)fringeof spikes(peplomers)attheirsurfaceandacharacteristictubularnucle- ocapsidofhelicoidalsymmetrythatcanconferadoughnut-shapeto thevirion(Weissandothers1983).Becauseofitsgrowthincell culture,theBemestrainoftheequinetorovirushasbeenstudied extensively(SnijderandHorzinek1993,deVriesandothers1997). Torovirusesinfecthorses,cattle,pigsandpossiblyhumanbeings andcats(Woodeandothers1982,Beardsandothers1984,Weiss andothers1984,Scottandothers1987,Muirandothers1990, KoopmansandHorzinek1994,Kronemanandothers1998).The highprevalenceofseropositiveanimalsindicatesthattheinfection L.A.H.M.Cornelissen,DVM,DepartmentofLargeAnimalMedicineand Nutrition,R.J.deGroot,PhD,M.C.Horzinek,DVM,PhD,H.F.Egberink, DVM,PhD,VirologyUnit,DepartmentofInfectiousDiseasesand Immunology,FacultyofVeterinaryMedicine,UtrechtUniversity,3584CL Utrecht,TheNetherlands P.A.M.vanWoensel,BSc,N.Visser,PhD,IntervetInternationalBV,5830 AABoxmeer,TheNetherlands iswidespreadinhorseandcattlepopulations(Weissandothers 1984,Brownandothers1987,1988,Woode1987,Koopmansand others 1989).Noclinicaldiseasehasbeenassociatedwithtorovirus infectioninhorses,butthebovinetorovirusisanentericpathogen causingmildtoprofusediarrhoeainexperimentallyornaturally infectedyoungcalves(Woodeandothers1982,Koopmansandoth- ers1990)andpossiblyinadultcattle(Koopmansandothers1991b, Scottandothers1996).Somereportshavesuggestedthattorovirus infectionsarealsoresponsibleforupperrespiratorytractillnessin calves(Woodeandothers1982,1985,Vanopdenboschandothers 1992a,b,KoopmansandHorzinek1994). Vanopdenboschandothers(1992a)recoveredaputativeisolate ofbovinerespiratorytorovirus(BRTV)fromacalfthathaddied fromneonatalpneumonia(Vanopdenboschandothers1991).By usingtorovirusantiserainanimmunofluorescenceassaythe authorsdetectedstainedcellsintissuesectionsofthelungandtra- chea.Inoculationofalunghomogenateintoculturesofdifferent celltypeswasfollowedbyacytopathiceffect(cpe),and torovirus-likeparticleswereobservedbyelectronmicroscopy. Theinvitrocultivationofabovinetoroviruswouldnotonly allowthedetailedmolecularcharacterisationofasecond torovirus,butwouldalsofacilitatetheproductionofantigenfor diagnosticassays.Moreover,iftheviruswerefoundtobe pathogenic,itwouldjustifythedevelopmentofavaccine.This paperdescribesattemptstoconfirmthepublisheddataandto characterisethecytopathogenicagentpresentintheBRTVisolate. Thestudieswereconductedindependentlyintwolaboratories, andindicatethatthecytopathogenicvirusreplicatingincellcul- tureisacoronavirus. Materialsandmethods Viruses,cellsandantisera TheBRTVisolateprovidedbyVanopdenboschhad beenpas- sagedsixoreighttimesinMadin-DarbyBovineKidney(MDBK) cells(BRTVP6andP8,respectively).TheMebusstrainofthe bovinecoronaviruswasusedthroughoutthestudy.Bovinefetal diploidlung(BFDL)cellswereobtainedfromtheID-DLO,Lelystad, andallothercelllineswerepurchasedfromtheAmericaType CultureCollection.ThecellsweremaintainedinDulbeccosmod- ifiedEaglesmedium(DMEM;GibcoLaboratories)orGlasgows MEMsupplementedwith10percentfetalcalfserum(Fcs)and antibiotics.AfieldserumsampledfromacowservedastheBRTV referenceserum(BaBRTV,Vanopdenboschandothers1992a);sera fromgnotobioticcalvesinfectedexperimentallywithbovinecoro- navirus(GC78)orbovinetorovirusstrainBreda2(GC76)werepro- videdbyG.Woode(Woodeandothers1982).Therabbitimmune serumagainstbovinecorona