StabilityofPorcineEpidemicDiarrheaVirusonFomiteMaterialsatDifferentTemperatures YonghyanKim,VenkatramanaD.Krishna,MontserratTorremorell,SagarM.GoyalandMaximC.ͲJ.Cheeran. DepartmentofVeterinaryPopulationMedicine,CollegeofVeterinaryMedicine,UniversityofMinnesota Keypoints Ͳ TypeofmaterialandtemperaturehaveanimpactonPEDVstability. Ͳ InfectiousPEDVwasnotrecoveredfromanyfomitematerialafter2daysatroomtemperature(25ºC/77ºF). Ͳ PEDVshowedhigherstabilityonplastic,cloth,Tyvek® coveralls,aluminumfoil,Styrofoamat4ºC(39.2ºF). Ͳ ViruscouldbedetectedbyqRTͲPCRfromcontaminatedfomitesevenwheninfectivitywasnotobserved. ͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲͲ Background TransmissionofPEDVprimarilyoccursbythefecalͲoralroute,butindirecttransmissioncanoccurwhenananimalcomesincontactwithinanimateobjects (fomites)contaminatedwithfecesfromPEDVͲinfectedanimals.ThestabilityofPEDVoncontaminatedfomitesatdifferenttemperaturesispoorlyunderstood. Inanimateobjectssuchasrubberboots,gloves,coveralls,andotherequipmentareroutinelyusedonswinefarmsandhavethe potentialofbeingcontaminated withmanurefromPEDVinfectedanimals,therebyhelpingtheindirectmodeofvirustransmission.Thus,thisstudyevaluatedthesurvivalofPEDVonvarious fomitesurfacesatbothroomtemperature(RT)and4ºC. Methods 6 ® 200ʅLofviruscontaining2.1×10 TCID50/mLwasappliedonvariousfomitematerial:Styrofoam,nitrilegloves,cardboard,aluminumfoil,Tyvek coveralls,cloth, metal,rubber,andplastic.ThevirusͲcontaminatedfomiteswerethenstoredateither4ºCoratroomtemperature(RT)(25ºC).Sampleswerethentakenat0,12, 5,10,15,20and30dayspostͲcontaminationtotestforvirusstability. Results InfectiousPEDVwasrecoveredfromfomitematerialsforupto15dayspostapplicationat4ºC;only1to2logsofviruswere inactivatedduringthefirst5days postapplication. Ontheotherhand,PEDVsurvivaldecreasedprecipitouslyatRT(25ºC)within1to2Ͳdayspostapplication,losing2to4logtiterswithin24h (Figure1). Immunoplaqueassay,amoresensitivetechniquethanstandardTCID50 orviralplaqueassays,wasusedtoidentifypositivefomitesafter20daysof storageat4මC.Immunoplaqueassayallowsustodetectasfewas24focusformingunits(FFU)/mL.Theviralconcentrationsinitiallysprayed onthefomiteswas1 × 10^6.Approximately1× 10^3FFU/mLwererecoveredineluatesfromStyrofoam,metal,andplastic,representinga3Ͳlogvirusinactivationafter20days.The survivingvirusonTyvek®coverallandrubbersurfaceswasmoderatelyabovedetectionlimit(24FFU/mL). AlsotodeterminetheamountofviralRNAremainingonthematerialsquantitativeRTͲPCRwasperformed.RNAwasdetectedafter 2daysatRTandafter 20daysat4ºC.Infact,allmaterialstestedhadcyclethreshold(Ct)valuessimilartothoseofinputvirus(~16–17)indicatingthatdetectionofviralRNAbyPCR doesnotindicatetheinfectiousnatureofthesample. Discussion Thevirusremainedviableat4ºCforupto20ͲdaysonStyrofoam,metal,andplastic,althoughviraltitersdecreasedby3logsin20days.WhenstoredatRT,PEDV decreasedby4to5logswithin48h,renderingitundetectableusinginfectiousvirusassays.Thisobservationsuggeststhat thestoragetemperatureofthefomite materialhasamajorimpactonvirusstability.Itappearsthatlowstoragetemperaturedelaysdegradationofviralinfectivityonfomitematerials.Inadditionto storagetemperature,thetypeoffomitematerialmayalsoinfluencevirussurvivalandhencevirustransmission. Inthisstudy,viralRNAcopynumbersdidnotcorrelatewiththecellͲbasedinfectiousvirusassays.Inotherwords,itwaspossibletodetectviralRNAusing aqRTͲPCRbutitwasnotpossibletoprovethatthesamplewasinfectious.ItispossiblethatPEDVinfectivityisdestroyedbyfactorsthataffecttheintegrityofthe viralenvelopewithoutaffectingRNAdegradation. AlthoughweusedbothcellculturepropagatedPEDVandPEDVͲspikedfecalmaterialonfomitestomimicactualsituationinthefield,wewereunableto determinethesurvivalofthevirusinPEDVͲspikedfecalmaterial.Thiswassobecausetheeluatesfromfomitescontaminatedwithfeceswerenotsuitablefor infectiousviraltiterassayduetocytotoxicityofthefecalcontentonindicatorcells. Conclusion TheseresultsprovideanaddedunderstandingonPEDVstabilityon differentsurfacesatdifferenttemperaturesandprovidescluesonhow PEDVcouldbedisseminatedthroughfomites. Fullarticlecanbefoundatthefollowinglink: http://www.mdpi.com/2306Ͳ7381/5/1/21 Figure1.Virussurvivalonninefomitesatroomtemperature(RT)andat 4°C.PEDVwasappliedon fomitematerialsorincontrolwellsfollowed bystorageatRT(Ÿ,orangeline)orat4°C(Ŷ,blueline).Thesurviving viruswaselutedaftervarioustimeperiodsandtitrated.Decayof infectiousviruswasrapidatRTbut delayedwhenstoredat4°C.Black 2 dottedlinerepresentsdetectionlimitofTCID50 assay,whichis~2× 10 TCID50/mL.Datapresentedareaverageofthreereplicates(± SEM) obtainedfromindependent assessmentsateachtimepointindicated. Forpast MSHMPsciencepageshttps://z.umn.edu/SciencePages 3/23/2018.