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48831CoronavirusesLindaJ.Saif,QiuhongWang,AnastasiaN.Vlasova,KwonilJung,andShaoboXiaoOverviewdistinct,andtheybelongtotheBetacoronavirusandDeltacoronavirusgenera,respectively.TogetherwithThefamilyCoronaviridaewithintheorderNidoviralesbovine,humanOC43,equine,andcaninerespiratoryconsistsoftwosubfamilies:(1)CoronavirinaecomprisingCoVs,pHEVisamemberoftheBetacoronavirus1spe-thegeneraAlphacoronavirus,Betacoronavirus,Gammac-cies.PDCoVismostcloselyrelatedtoothermammalianoronavirus,andDeltacoronavirusand(2)Torovirinaecom-deltacoronavirusesfromAsianleopardcatsandChineseprisingthegeneraTorovirusandBafinivirusandanferretbadgers(Maet al.2015).ForeachswineCoV,onlyunassignedgenus.asingleserotypeisrecognized.Fiveswinecoronaviruses(CoVs)havebeenidentified:CoVsareenvelopedandpleomorphic,withanoveralltransmissiblegastroenteritisvirus(TGEV)firstdescribeddiameterof60–160nmasviewedbynegativestainingin1946;porcinerespiratorycoronavirus(PRCV),aspikeelectronmicroscopy(EM)(Figure 31.2).Mosthavea(S)genedeletionmutantofTGEVisolatedin1984;por-singlelayerofclub‐shapedspikes(Sprotein)12–25nmcineepidemicdiarrheavirus(PEDV)isolatedin1977;inlength,butpHEVandsomeotherbetacoronavirusesporcinehemagglutinatingencephalomyelitisvirushaveasecondshorterlayerofsurfacespikes,thehemag-(pHEV)isolatedin1962;andporcinedeltacoronavirusglutinin‐esterase(HE)protein.(PDCoV)detectedin2012.Inaddition,aTGEV/PEDVrecombinantvirus(TGEVbackbonebutwithPEDVGenomicorganizationand geneexpression:spikegene)hasbeenidentifiedinswineinEuropeTGEVprototype(Akimkinet al.2016;Belshamet al.2016;Boniottiet al.2016),andabat‐HKU2‐likealphacoronavirushasbeenSwineCoVscontainonelarge,polyadenylated,single‐identifiedinswineinChina(Gonget al.2017;Panet al.stranded,genomicRNA(25–30kb)ofpositive‐sense2017;Zhouet al.2018).Inpigs,CoVsandtorovirusespolarity.Thegenomeorganization,replicationstrategy,(ToVs)affectavarietyoforgans,includingthegastroin-andexpressionofviralproteinsaresimilartothoseoftestinalandrespiratorytracts,theperipheralandcentralotherhumanandanimalCoVs(EnjuanesandVandernervoussystems(CNS),andthemammaryglands.MostZeijst1995;Gonzalezet al.2003;Laudeet al.1993).TheToVsandPRCVinducemainlysubclinicalinfectionsincompletegenomesofthePurdueandMillerstrainsofpigs,whereasTGEV,PEDV,PDCoV,andpHEVinfec-TGEVare28,546–28,580nucleotide(nt)longandsharetionscanresultinfatalentericornervousdiseases.96%overallidentity(Penzeset al.2001;Zhanget al.SwineCoVscomprisethreedistinctgenera –2007).MostCoVshavebuoyantdensitiesinsucroseofAlphacoronavirus,Betacoronavirus,andDeltacoronavirus1.18–1.20g/mL.Thephospholipidsandglycolipids(Figure 31.1) – andsharereplicationstrategiescommonincorporatedintothevirusenvelopearederivedfromtoCoronaviridae.TGEVandPRCVbelongtothethehostcell,andthus,theenvelopecompositionishostAlphacoronavirus1speciesthatalsocontainscloselycelldependent(EnjuanesandVanderZeijst1995).relatedCoVsofdomesticcatsanddogs.PEDVandtwoMostCoVscontainfourstructuralproteins:alargehumanCoVs(229EandNL63)areseparatespeciesinthesurfaceglycoprotein(spikeorSproteinvisibleasthesamegenusAlphacoronavirus.Thenewlyidentifiedbat‐corona;Figure 31.2),asmallmembraneprotein(E),anHKU2‐likeswineentericalphacoronavirusalsobelongsintegralmembraneglycoprotein(M),andanucleocapsidtothegenusAlphacoronavirus,butitstaxonomicnameprotein(N).However,pHEValsocontainsanHEproteinhasnotbeendefined.pHEVandPDCoVaregenetically(deGrootet al.2008).DiseasesofSwine,EleventhEdition.EditedbyJeffreyJ.Zimmerman,LockeA.Karriker,AlejandroRamirez,KentJ.Schwartz,GregoryW.Stevenson,andJianqiangZhang.©2019JohnWiley&Sons,Inc.Published2019byJohnWiley&Sons,Inc.ChapterNo.:1TitleName:c31.inddComp.by:Date:18Mar2019Time:06:57:37AMStage:WorkFlow:PageNumber:488 31Coronaviruses489100USA/Colorado/2013100USA/Iowa/18984/2013100CHN/AH2012/2012100CHN/ZMDZY/2011Non-SINDELEmergingGER/L00719/2014PEDV65100USA/OH851/2014100KOR/KNU-1406-1/2014SINDEL100KOR/virulentDR13/1999AlphaCoV100ClassicalBEL/CV777/1977100BtCoV/512/2005SeCoV/ITA/213306/2009PRCVISU-1100TGEVvirulentPurdue100100100TGEVMillerM6TGEV100TGEVH16THA/S5011/201577CHN/HKU15-44/2009100CHN/AH/200499CHN/HKU15-155/2010PDCoVDeltaCoV100CHN/HB/201494USA/OH1987/2014100KOR/KNU14-04/2014SwineHEV/VW572BetaCoVAvianIBV/Mass41VaccineGammaCoV0.05Figure31.1Phylogenetictreeofcoronaviruses.ThecompletegenomesofTGEV,PEDV,andPDCoVstrains,closelyrelatedPRCVandabatalphacoronavirus,aporcineHEVstrain,andanavianIBVstrainwereselected.MultiplesequencealignmentswereperformedusingClusterW,andaneighbor‐joiningphylogenetictreesupportedwithabootstraptestof1000replicateswasconstructedusingMEGA6.0software.Thenumberoneachbranchindicatesthebootstrapvalue.Thescalerepresentsthenucleotidesubstitutionspersite.Color‐shadedareasdenotedifferentswinevirusclusters.TheNprotein(47kDa)interactswithviralRNAtoproteinundergoconformationalchangesimportantforformahelicalribonucleoproteincomplex.Thisstruc-fusion(Maet al.2005).EntryofTGEVintothecellisture,inassociationwithMprotein,formsaninternallikelyassociatedwithcholesterol‐richmembranemicro-icosahedralcoreinTGEV.The29–36kDaMglycopro-domains(Renet al.2008),sinceexogenouscholesterolteinisembeddedintheviralenvelopeby3–4mem-rescuedvirusinfectivity.brane‐spanningregions.InTGEV,thehydrophilicN‐EpitopemappingoftheSglycoproteinofTGEVterminuswithasingleaccessibleglycosylationsiteisrevealedfourantigenicsites(A,B,C,D)(Figure 31.3).responsibleforinterferon(IFN)induction(CharleyandSiteA–B,theconservedimmunodominantepitope,isLaude1988).EpitopesonprotrudingN‐andC‐terminalrecognizedstronglybyneutralizingMAbs(Correaet al.endsoftheMproteinofTGEVbindcomplement‐1990;DelmasandLaude1990;Simkinset al.1992,1993),dependentneutralizingmonoclonalantibodies(MAbs)althoughothersites(D,C)canalsoinducevirus‐neutral-(Laudeet al.1992;Woodset al.1988).izing(VN)antibodies(DelmasandLaude1990).TheSTheTGEVSglycoprotein(220kDa)occursastrimerproteinmutationsinattenuatedTGEVstrainsorthecomplexes(DelmasandLaude1990)andfunctionsinnaturalTGEVdeletionmutantPRCVincludeaserine/virusneutralization(complementindependent),virus‐alaninemutationataminoacid(aa)585positionassoci-cellattachment,membranefusion,andhemagglutina-atedwithinductionofVNantibodies,aswellasreceptortion.ThelargedeletionintheSgeneofPRCVresultsin(aminopeptidaseN)binding(Zhanget al.2007).asmallerSprotein(170–190kDa)(Figure 31.3).DuringPorcineaminopeptidaseN(pAPN)hasbeenidentifiedfusionofTGEVwithhostcellmembranes,twohighlyastheTGEVcellreceptor(Delmaset al.1992).Therecep-conservedheptadrepeatregions(HR1andHR2)oftheStor‐bindingandmajorneutralizingsite(siteA)ontheS 490SectionIIIViralDiseases(a)(b)(c)(d)Figure31.2Electronmicrographs.(a)ATGEVparticleshowingtypicalcoronavirusmorphology.Arrowpointstotheviruspeplomersorspikes.Bar = 100nm.(b)Typicalvirus–antibodyaggregatesobservedbyimmunoelectronmicroscopyofTGEVandgnotobioticpiganti‐TGEVserum.Bar = 100nm.(c)Twoparticlesofemergingnon‐SINDELPEDVPC22Astrain,bar = 100nm.Source:Okaet al.(2014).ReproducedwithpermissionofElsevier.(d)APDCoVparticle,bar = 100nm.Source:Junget al.(2015b).proteinofTGEVarelocatedwithinthesamedomain(TGEVandPDCoV).Theoverallgenomeorganizationis(Figure 31.3)(Godetet al.1994).TGEVbindingtosialic5′UTR‐ORF1ab,S,ORF3,E,M,ORF6,N,ORF7‐3′UTR.acidresiduesonglycoproteinsoftargetcellswasproposedtoinitiateinfectionofintestinalenterocytes(Schwegmann‐Contrastsand comparisonsWesselset al.2002).TreatmentofTGEVwithsialidaseenhancedhemagglutinatingactivity(Nodaet al.1987;SevenCoVsarerelatedantigenicallyorbytheirgenomicSchultzeet al.1996).Thehemagglutinatingactivityresidessequences(EnjuanesandVanderZeijst1995):TGEV,intheN‐terminalregionoftheTGEVSprotein,aregionPRCV,caninecoronavirus(CCoV),felineinfectiousperi-thatismissingfromthePRCVSprotein;thus,determina-tonitisvirus(FIPV),felineentericcoronavirus(FECoV),tionofhemagglutinatingactivity(Schultzeet al.1996)PEDV,andhumanCoV229E.CoVswithinthespeciescouldpotentiallydifferentiatePRCVandTGEVstrains.Alphacoronavirus1(TGEV,PRCV,CCoV,FIPV,FECoV)TGEVandPEDVaswellasPDCoValsoencode1–2areantigenicallyrelated,basedoncross‐reactivityinVNaccessoryproteinsencodedbyopenreadingframeandimmunofluorescence(IF)testsandwithMAbsto(ORF)3(TGEVandPEDV),ORF6(PDCoV),andORF7theS,N,orMproteins,andallsharetheantigenicsubsite 31Coronaviruses491AcontheSprotein(EnjuanesandVanderZeijst1995).cross‐reactivitywiththeNproteinhasalsobeenreportedAsmembersofthesamespecies,theseviruseslikelyrep-forPEDV,FIPV,CCoV,TGEV,andaputativeminkCoVresenthostrangemutantsofanancestralvirusstrain(de(Haveet al.1992;Zhouet al.2010).Althoughnocross‐Grootet al.2008).TGEVandCCoVcouldbedistin-reactivitybetweenPEDVandTGEV‐relatedCoVswasguishedinvitrobytwo‐waycross‐neutralizationtestsinitiallyreported(EnjuanesandVanderZeijst,1995),andotherbiologicaldifferences(Reynoldset al.1980),one‐waycross‐reactivitybetweenTGEVMillerandsev-i.e.bothTGEVandCCoVgrowincaninekidneycellseralPEDVstrains(classicalCV777,emergingnon‐Sandafelinecellline,whereasTGEV,butneitherCCoVINDELandSINDELUSstrains)wasrecentlyconfirmednorFIPV,growsinswinecells.(Linet al.2015b).FortheSglycoproteinthatconfershostrangespecific-Invivobiologicaldifferencesinpathogenicityforneo-ity,the300aaresiduesattheN‐terminusarethemostnatalpigsareevidentamongTGEV,CCoV,andFIPVvariable.Inthisdomain,CCoVandFIPVaremoresimi-strains.WhereasvirulentFIPVcauseddiarrheaandlartoeachotherthantoTGEV(Wesselinget al.1994).intestinallesionssimilartothoseofvirulentTGEV,DifferentiationoftheTGEV‐relatedCoVsispossibleCCoVcausednoclinicalsignsandonlyslightvilloususingspecificMAbstotheSglycoproteinofTGEVthatatrophy.CCoVshedbyacutelyinfecteddogs,infectedrecognizeTGEVbutnotPRCV,FIPV,orCCoVstrainsbabypigs,andinducedserumVNantibodiestoCCoV(Callebautet al.1989;Laudeet al.1993;Sanchezet al.andTGEV(WoodsandWesley1992).However,baby1990;Simkinset al.1992,1993).pigsandpregnantgiltsinfectedwithFIPVdidnotInterestingly,outbreaksoffatalacutegastroenteritisproduceTGEVVNantibodies,butdiddevelopsomeassociatedwithTGEV‐relatedCCoVtypeII(CCoV‐II)immunitytoTGEVchallenge.werereportedinEuropeandogs(Decaroet al.2009;PRCVstrainshavebeencharacterizedandsequencedErlesandBrownlie2009).Theclosegeneticrelatedness(Brittonet al.1991;Costantiniet al.2004;Kimet al.(>96%aaidentity)inthekeyreplicasedomainssuggested2000b;Rasschaertet al.1990;Vaughnet al.1995;ZhangthattherecentlyemergedCCoV‐IIstrainsarehostrangeet al.2007).TwostrikingfeaturescharacterizethePRCVvariantsofTGEVthatinfectdogs(deGrootet al.2008).genome:(1)alargedeletion(621–681nt)neartheBasedontheSprotein,theCCoV‐IIbstrains(TGEV‐N‐terminusoftheSgeneproducingasmallerSproteinlike)(Decaroet al.2010)mayrepresentnovelrecombi-(Figure 31.3)and(2)avariableregionwithdeletionsthatnantvirusesofmixed(TGEV/CCoV)origin.compromiseORF3downstreamoftheSgene.TheseSeveralresearchersreportedthatthesevereacuteres-geneticchangesmayaccountforthealteredtissuetro-piratorysyndrome(SARS)CoVcross‐reactswithanti-pismofPRCV(Ballesteroset al.1997;Sanchezet al.bodiestoAlphacoronavirus1species(TGEV,PRCV,1999).AnoverallnucleotideandaasequenceidentityofCCoV,FeCoV)throughtheNprotein(Ksiazeket al.96–98%betweenTGEVandPRCVsuggeststhatPRCV2003;SunandMeng2004)andthatthiscross‐reactivityevolvedfromTGEV(Zhanget al.2007)andthatthismappedtotheN‐terminalregion(Vlasovaet al.2007).occurredonanumberofindependentoccasions.ThisdiscoveryledtouseofSARSCoV‐specificNpep-DiseaseoutbreakscausedbyswineCoVsareendemictidefragmentsinserologicassaystodetectSARSCoVorvariableinswine‐producingcountries.Nevertheless,antibodiesinanimalsera(Vlasovaet al.2007).One‐waythediseasesinducedbytheseCoVshaveresistederadicationS1domainS2domain15066557831449PRCVSNH2deletionRBDCOOH117297325379529538543TMDA....ACorreaetal.(1990)࢐DCB82210363371506718DelmasandLaude(1990)࢐DCABFigure31.3SchematicdiagramofTGEVSprotein.RBD,receptor‐bindingdomain(showninred);TMD,transmembranedomain(showninorange).AccordingtoCorreaet al.’s(1990)classification(lightblueboxes),siteAisdiscontinuousandlocatedaroundpositions17and297and538and543,siteBislocatedbetweenaminoacids1and325,andsiteDislocatedbetween379and529.TherelativeorderofsitesCandBandpartofsiteAisnotdetermined.AccordingtoDelmasandLaude’s(1990)classification(lightgrayboxes),SproteinantigenicsitesD,C,andA/Barelocatedbetweenaminoacids82and210,between363and371,andbetween506and718,respectively.Dottedredboxindicatesa227aadeletioninthePRCVSprotein.Source:AdaptedfromCorreaet al.(1990)andDelmasandLaude(1990). 492SectionIIIViralDiseasesefforts,andthevirusesmaycontinuetoevolveindomes-TGEVisstablewhenstoredfrozen,butlabileatticpigs,wildboars,andotherpotentialreservoir(bats)roomtemperatureorhigher.Infectiousviruspersistedorsecondaryhosts,suchascarnivores,viainterspeciesinliquidmanureslurryformorethan8weeksat5°Ctransmission.(41°F),2weeksat20°C(68°F),and24hoursat35°C(95°F)(Haaset al.1995).InrecentstudiesthatusedTGEVasasurrogateforSARSCoV(Casanovaet al.Transmissiblegastroenteritis2009),itremainedinfectiousinwaterandsewageforvirus and porcinerespiratoryseveraldaysat25°C(77°F)andforseveralweeksat4°C(39°F).coronavirusTGEVishighlyphotosensitive.Fecalmaterialcon-5taining1×10piginfectiousdoses(PID)wasinactivatedRelevancewithin6hourswhenexposedtosunlightortoultravio-TGEisahighlycontagiousentericviraldiseaseofswineletlight(Cartwrightet al.1965;Haelterman1962).characterizedbyvomiting,severediarrhea,andhighTGEVisinactivatedbyexposureto0.03%formalin,1%mortality(often100%)inpigletslessthan2weeksofage.Lysovet(phenolandaldehyde),0.01%beta‐propiolac-TGEwasfirstdescribedbyDoyleandHutchings(1946)tone,1mMbinaryethylenimine,sodiumhypochlorite,intheUnitedStatesandsubsequentlyreportedworld-NaOH,iodines,quaternaryammoniumcompounds,wide.AlthoughswineofallagesaresusceptibletoTGEVether,andchloroform(Brown1981;VanCottet al.orPRCVinfection,themortalityinTGEVand/orPRCV1993).TGEVfieldstrainsaretrypsinresistant,relativelyseropositiveherdsandinswineover5weeksofageisstableinpigbile,andstableatpH3(Laudeet al.1981),generallylow.allowingvirustosurviveinthestomachandsmallintes-TheappearanceandwidespreadprevalenceofPRCV,atine.However,propertiesofattenuatedandfieldstrainsnaturallyoccurringdeletionmutantofTGEV,lessenedofTGEVvary.theclinicalimpactofTGE(BrownandCartwright1986;Laudeet al.1993;Pensaertet al.1986,1993;PensaertPublichealth1989;Yaegeret al.2002).However,sporadicoutbreaksofseverediarrheainpigletscausedbyTGEVinTGEV/PigsarethemainspeciesnaturallysusceptibletoTGEVPRCVseronegativeherdsarestillreportedinNorthandPRCV.Noinfectionofhumanshasbeenreported.America,Europe,andAsia.Currently,TGEVandPEDVco‐circulateinAsia,Europe,andtheUnitedStates,andEpidemiologyrecently,pathogenicrecombinantTGEV/PEDVvariants(swineentericcoronavirus[SeCoV])wereidentifiedandOnaherdbasis,twoepidemiologicformsofTGEarecharacterizedinEurope(Akimkinet al.2016;Belshamrecognized:epidemicandendemic.Infectionswiththeet al.2016;Boniottiet al.2016).SeCoVthatcontainsTGEVdeletionmutantPRCVpresentadifferentpattern,PEDVSgeneonaTGEVbackbonereportedlycausesgreatlycomplicatingseroprevalencestudiesoftheepide-diseaseclinicallyindistinguishablefromthatcausedbymiologyofTGEV(Pensaert1989).TGEVandPEDV.ThisepidemiologicalsituationrequiresfrequentmonitoringanddevelopmentofreliabletoolsEpidemicversusendemicTGEfordifferentialdiagnosis(Kimet al.2001;Masudaet al.EpidemicTGEoccurswhenmostoftheanimalsina2016).herdareTGEV/PRCVseronegativeandsusceptible.Afterintroduction,thediseasespreadsrapidlytoswineofallages,especiallyduringwinter.Inappetence,vomit-Etiologying,ordiarrheaoccursinmostanimals.SucklingpigsTGEVantigencanbedemonstratedbyIFstainingintheshowmarkedclinicalsignsandrapidlydehydrate.cytoplasmofinfectedcells4–5hourspostinfectionMortalityisveryhighinpigsunder2–3weeksofagebut(Pensaertet al.1970).Maturationofvirusoccursinthedecreasesinolderpigs.Lactatingsowsoftendevelopcytoplasmbybuddingthroughtheendoplasmicreticu-anorexiaandagalactia,withreducedmilkproduction,lum,andviralparticles(65–90nmindiameter)arewhichfurthercontributestopigletmortality.observedwithincytoplasmicvacuoles(Figure 31.4a)EndemicTGEreferstothepersistenceofthevirusand(Pensaertet al.1970;Thake1968).Virusmaylinehostdiseaseinaherdperpetuatedbythecontinualorfre-cellmembranesafterexitfrominfectedcellsquentinfluxofsusceptibleswine.EndemicTGEisa(Figure 31.4b).Asimilarintracellularreplicationsce-commonsequeltoaprimaryoutbreakandoccursinnariohasbeendescribedforPEDV(Figure 31.4c).TGEVseropositiveherdsthathavefrequentfarrowingsglycoproteinsarealsoevidentonthesurfaceofinfected(Stepaneket al.1979),herdadditions,orcomminglingofSTcells(Laviadaet al.1990).susceptiblepigs.Inendemicallyinfectedherds,TGEV 31Coronaviruses493(a)(b)N(c)Figure31.4Electronmicrographs.(a)TGEVinvesiclesoftheendoplasmicreticulumofapigkidneycell(36hourspostinfection).Bar = 100nm.(b)TGEVliningthecellmembraneofapigkidneycell(36hourspostinfection).N = nucleus;bar = 200nm.(c)APEDV‐infectedVerocell.PEDVparticles(arrowheads)onthecellsurfaceandinsideavesicle(arrow)oftheinfectedVerocell.Scalebar = 200nm.For(c),Source:Okaet al.(2014).ReproducedwithpermissionofElsevier.spreadsslowlyamongadultswine(Pritchard1987).SowsEndemicTGEinsucklingorrecentlyweanedpigscanarefrequentlyimmuneandasymptomaticandwillbedifficulttodiagnoseandmustbedifferentiatedfromtransferavariabledegreeofpassivelactogenicimmunityothertypesofendemicdiarrhealpathogenscommonintotheirprogeny.Intheseherds,mildTGEVdiarrheayoungpigs,suchasPEDV,PDCoV,rotavirus,andoccurs,andmortalityisusuallyunder10–20%inpigsEscherichiacoli.EndemicTGEpersistsintheherdasfromapproximately6daysofageuntilapproximatelylongassusceptibleorpartiallyimmuneswineareexposed2weekspostweaning.Theage‐relatedeffectsareinflu-toTGEV.Itisunclearwhetherthesourceofvirusisfromencedbythemanagementsystemandthedegreeofreactivationofvirussheddingincarrierswineorreintro-passiveimmunityfromthesow.ductionofvirusintotheherdfromanexternalsource. 494SectionIIIViralDiseasesPorcinerespiratorycoronavirusofTGEVfromoneherdtoanother,sincetheycanshedPRCVisaTGEVvariantthatinfectstherespiratorytractvirusintheirfecesforvariableperiods(Haeltermanwithlimitedornosheddinginfeces(Pensaert1989).1962;McClurkinet al.1970)andvirusexcretedbydogsHowever,PRCV‐infectedpigsproduceantibodiesthatwasinfectiousforpigs(Haelterman1962;ReynoldsandneutralizeTGEV.ThefirstisolationofPRCVwasinGarwes1979).Belgiumin1984(Pensaertet al.1986).In1989,PRCVTheconcentrationofstarlings(Sturnusvulgaris)inwasdetectedintwoherdsintheUnitedStateswithnowinterinfeedingareasofswinemayfostermechanicalhistoryofTGEVvaccinationorclinicaldisease(HillspreadofTGEVamongfarms.Pilchard(1965)reportedet al.1990;Wesleyet al.1990).thatTGEVwasdetectedinthedroppingsofstarlingsforSwinepopulationdensity,distancebetweenfarms,andupto32hoursafterfeedingTGEV.Houseflies(MuscaseasoninfluencePRCVepidemiology(Have1990;domestica)havealsobeenproposedaspossiblemechan-Pensaert1989).PRCVinfectspigsofallagesbycontacticalvectorsforTGEV.TGEVantigenwasdetectedinorairbornetransmission.PRCVinfectionsareoftensub-flieswithinaswineherd,andexperimentallyinoculatedclinical.TheriskofPRCVspreadincreasesinareasoffliesexcretedTGEVfor3days(GoughandJorgensonhighswinedensity,wheretheviruscantravelseveral1983).AccordingtosurveysconductedinCentralkilometers.ThevirushasspreadrapidlyandextensivelyEurope,antibodiesagainstTGEVarealsopresentininpigsinEurope(BrownandCartwright1986;Haveapproximately30%oftheferalpigpopulation(Sedlak1990;Laudeet al.1993;vanNieuwstadtet al.1989)andet al.2008).becameendemiceveninTGEV‐freecountries(LaudeThethirdpossibilityrelatingtoTGEtransmissioniset al.1993;Pensaert1989;Pensaertet al.1993).AlimitedthedurationofTGEVsheddingandtheroleofthecar-serologicalsurveyin1995intheUnitedStatessuggestedrierpig.NasalsheddingofPRCVinexperimentallythatmanyasymptomaticherdsinIowawereseropositiveinfectedpigsoccursthrough10dayspostinfection(DPI)forPRCV(Wesleyet al.1997).(Onnoet al.1989;Wesleyet al.1990).However,howPRCVcirculatesintheherd,infectingpigsbeforethelongpigsclinicallyrecoveredfromTGEVandPRCVageof10–15weeksafterpassivelyacquiredmaternalinfectioninthefieldremaininfectiousisunknown.Oneantibodieshavedeclined.Introductionofpigsintofat-reportindicatedchronicand/orpersistentTGEVfecalteningunitsandcomminglingofPRCV‐negativeandsheddingforupto18months,suggestingapossiblerolePRCV‐positivepigsfromdiversesourcesresultinsero-forthelong‐termcarrierhogintransmittingTGEVconversiontoPRCVinpigsshortlyafterintroduction(WoodsandWesley1998).AlthoughTGEVhasbeenintomostunits.detectedintheintestinalandrespiratorytractsforperi-SusceptiblepigsexperimentallyinfectedwithPRCVodsofupto104DPI(Underdahlet al.1975),itisshedvirusfromnasalsecretionsforlessthan2weeksunknownwhetherinfectiousvirusisshedortransmit-(Onnoet al.1989;VanCottet al.1993;Wesleyet al.ted.Additionofsentinelpigstoaherdat3,4,and1990).Thereisnoevidenceforthefecal–oraltransmis-5monthsafterapreviousTGEoutbreakresultedinnosionofPRCV.PRCVpersistsinclosedbreedingfarmsbyinfectionsintheintroducedpigs,asdeterminedbyregularlyinfectingnewlyweanedpigs,eveninthepres-serologictests(Derbyshireet al.1969).enceofmaternalantibodies(Pensaertet al.1993).PRCVcanpersistintheherdthroughouttheyear,oritcanPathogenesisdisappearinsummerandreappearinthenurseryandfatteningunitsinwinter.CoincidentwiththewidespreadIntestinaland extraintestinalreplicationof TGEVdisseminationofPRCV,theseroprevalenceofTGEVinJejunalenterocytesundergomassivenecrosiswithinEuropehasdecreased,toalowprevalence(Brownand12–24hoursafterinfection,resultinginmarkedreduc-Paton1991;Pensaertet al.1993).tioninenzymaticactivity(alkalinephosphatase,lactase,etc.)inthesmallintestine.ThisdisruptsdigestionandTransmissionand reservoirscellulartransportofnutrientsandelectrolytes(includingAnepidemiologicalfeatureofTGEisitsseasonalappear-sodium),therebycausinganaccumulationofliquidinanceduringwinter.Haelterman(1962)suggestedthattheintestinallumenandacutemalabsorptivediarrheathisisbecausethevirusisstablewhenfrozenandmore(Moon1978)thatleadstosevereandfataldehydrationinlabilewhenexposedtoheatortosunlight.Thiswouldpiglets(Butleret al.1974)andlossofextravascularallowvirustransmissionbetweenherdsinwinteronprotein.Dehydrationisalsorelatedtometabolicacidosisfomitesoranimals.HeproposedatleastthreepossiblecoupledwithabnormalcardiacfunctionduetoreservoirsforTGEVbetweenseasonalepidemics:(1)pighyperkalemia.farmsinwhichthevirusspreadssubclinically,(2)hostsTheseverevillousatrophyinthejejunum(Figure 31.5aotherthanswine,and(3)carrierpigs.Thereisevidenceandb)andtoalesserextentintheileumofTGEV‐fortheexistenceofTGEVinnon‐porcinehosts.Cats,infectedpigsisoftenabsentintheproximalduodenumdogs,andfoxeshavebeensuggestedaspossiblecarriers(HooperandHaelterman1966a).Villousatrophyismore (a)(b)(c)(d)(e)Figure31.5Villiofthejejunumfromanormalpig(a)andfromaTGEV‐infectedpig(b),asviewedthroughadissectingmicroscope(approximately×10).Hematoxylinandeosin(H&E)‐stainedjejunumofanormalgnotobioticpig(17daysofage),showingnormalvilli(×80)(c)(Source:Junget al.2015b);ofaPEDV‐infectedgnotobioticpig(26daysofage)at46hourspostinoculation(atonsetofclinicalsigns),showingacutediffuse,severeatrophicjejunitis(×200)(d)(Source:Junget al.2014);andofaPDCoV‐infectedgnotobioticpig(17daysofage)at3dayspostinoculation(×40)(e)(Source:Junget al.2015b).ChapterNo.:1TitleName:c31.inddComp.by:Date:18Mar2019Time:06:57:37AMStage:WorkFlow:PageNumber:495 496SectionIIIViralDiseasessevereinnewbornpigsthanin3‐week‐oldpigs(MoonReplicationof PRCVin the respiratorytract1978),suggestinghighersusceptibilityofneonatestoPRCVhasatropismfortherespiratorytract.Itreplicates78TGEVinfection.Asimilardegreeanddistributionoftohightitersinporcinelungs(1×10–10TCID50)insmallintestinalvillousatrophyisalsoevidentforPEDVtype1and2pneumocytesandinfectsepithelialcellsof(Figure 31.5candd)andPDCoV(Figure 31.5cande).thenares,trachea,bronchi,bronchioles,alveoli,and,Mechanismstoaccountforage‐dependentsuscepti-occasionally,alveolarmacrophages(Atanasovaet al.bilitytoclinicaldiseaseincludetheslowerreplacement2008;Junget al.2007,2009;O’Tooleet al.1989;Pensaertinnewbornpigsofinfectedvillousepithelialcellsbyet al.1986).PRCVinducesnecrosisofinfectedcells,migrationofcellsfromcrypts(Moon1978).Thesenewlyincreasinginnateimmuneresponsesattheinfectionreplacedvillousenterocytesarereportedlyresistanttosites,includinghighlevelsofIFN‐αandnitricoxideinTGEVinfection,possiblyduetoinductionofinnatelungs(Junget al.2009,2010).InnatecytokinesinhibitimmunityandintestinalIFN(Abou‐YoussefandRisticinitialviralreplicationandmodulateTh1/Th2responses1972)ortheinabilityoftheregeneratingcellstosupportwiththelatterenhancingB‐cellresponses,leadingtovirusgrowth.secretionofVNantibodies.TheexposuredoseofinfectiousvirusplaysamajorVirussheddinginnasalsecretionslastedfor4–6daysroleinage‐dependentsusceptibility.TheinfectiousdoseafterexperimentalPRCVinfection.TheseverityofofTGEVneededtoinfecta6‐month‐oldmarkethogwasPRCV‐inducedpneumoniaandviralreplicationinlung4peakedat8–10DPI,coincidingwithincreasednumbers10timesgreaterthanthatneededtoinfecta2‐day‐oldpiglet(WitteandWalther1976).Moreover,theseverityofTandBcellsandfrequencyoflymphocyticinflamma-ofclinicalsignsduetoTGEVincreasedwhenpigsweretion.Thereafter,pulmonarylesionsandclinicalsignsinjectedwithasyntheticcorticosteroid,dexamethasoneresolvedconcurrentlywithincreasedVNantibodytiters(ShimizuandShimizu1979),similartodexamethasone‐(Atanasovaet al.2008;Junget al.2009).aggravatedlungpathologyinPRCVinfection(Junget al.Dependingontheexperimentalconditionsandthe2007;Zhanget al.2008),indicatingthepossibleeffectofvirusstrainsused,PRCVmaybedetectedinblood,stressonTGEV/PRCVdiseaseseverity.Inaddition,tracheobronchiallymphnodes,andoccasionallytheTGEVincombinationwithotherentericpathogens,smallintestinesofinfectedpigs.However,virusinsuchasE.coliorporcinerotavirus,causedmoresevereinfectedenterocytesdoesnotspreadtoadjacentcellsenteritisthaneitherinfectionalone(Underdahlet al.(Coxet al.1990a,b),andfecalsheddingisloworunde-1972).Likewise,PRCVrespiratoryinfectionandlungtectable.ThelimitedintestinalreplicationofPRCVlesionswereexacerbatedbypreexistingporcinerepro-mayberelatedtothedeletionintheSgene.Whenductiveandrespiratorysyndromevirus(PRRSV)infec-fecalandnasalisolatesofPRCVfromthesamepigstion(Junget al.2009;vanReethet al.1996).werecomparedgenetically,onlypointmutations,butExtraintestinalsitesforTGEVreplicationincludenotadditionaldeletions,werenotedintheSgenelungs(alveolarmacrophages)andmammarytissues(Costantiniet al.2004).(Kemenyet al.1975).OronasalinfectionofpigswithTGEVcausedpneumonia(Underdahlet al.1975).CellClinicalsignsculture‐attenuatedbutnotvirulentTGEVreplicatedinculturesofalveolarmacrophagesinvitro,suggestingaEpidemicTGEpossibleroleforthesecellsinlunginfection(Laudeet al.TypicalclinicalsignsofTGEinseronegativepigletsare1984).Moreover,TGEVwasdetectedinnasalsecretionsvomitingandprofusewatery,yellowishdiarrhea,withofinfectedpiglets(VanCottet al.1993)andlactatingrapidlossofweight,dehydration,andhighmorbiditysowsexposedtoinfectedpiglets(Kemenyet al.1975).andmortalityinpigsunder2weeksofage.TheseverityCell‐culturedstrainsofTGEVgenerallyshowedreducedofclinicalsigns,durationofdisease,andmortalityarevirulenceinpigs,withlessreplicationinthegutandinverselyrelatedtotheageofthepig.Mostpigsunderhigherlevelsofreplicationintheupperrespiratorytract7daysofagewilldiein2–7daysafteronsetofclinicalcomparedwithvirulentTGEV(Fredericket al.1976;signs.Mostsucklingpigsover3weeksofagewillsurvive,VanCottet al.1993).butmayremainstunted.ClinicalsignsofTGEinfinish-TGEVreplicatedinmammarytissuesoflactatingsowsingswineandinsowsincludeinappetence,transient(SaifandBohl1983)andinfectedsowsshedvirusinmilkdiarrhea,andvomiting.(KemenyandWoods1977).Theclinicalorepidemio-Theincubationperiodisshort,usually18hourstologicalsignificanceofmammaryglandinfectionwith3days.InfectiongenerallyspreadsrapidlythroughtheTGEVunderfieldconditionsisunclear,butagalactiaisentiregroup,andmostswineareaffectedin2–3days,oftenseeninTGEV‐infectedsowsandTGEVspreadsbutthisismorelikelytooccurinwinterthansummerrapidlyamongpigs.(Haelterman1962). 31Coronaviruses497EndemicTGEhasrevealedalterationsinthemicrovilli,mitochondria,EndemicTGEoccursinlargeherdsthatfarrowfre-endoplasmicreticulum,andothercytoplasmiccompo-quentlyandinTGEVorPRCVseropositiveherds.nents.Virusparticles,primarilyincytoplasmicvacuoles,Clinicalsignsareusuallylessseverethanthoseinseron-wereobservedinvillousenterocytesandinMcells,lym-egativepigsofthesameage.Mortalityislow,especiallyifphocytes,andmacrophagesinthedomeregionsofpigsarekeptwarm.TheclinicalsignsinsucklingpigsPeyer’spatches(Chuet al.1982;Thake1968).canresemblerotavirus,PEDV,orPDCoVdiarrhea(BohlPathologicfindingsandtheextentofvillousatrophyet al.1978;PensaertanddeBouck1978;Wanget al.arehighlyvariableinpigsfromendemicallyinfected2014a).Insomeherds,endemicTGEismanifestedpri-herds(Pritchard1987).MoxleyandOlson(1989)showedmarilyinweanedpigsandmaybeconfusedwithPEDVthatthelevelofpassiveimmunityinTGEV‐infectedpigs(Madsonet al.2014),E.coli,coccidia,orrotavirusinfec-influencedboththedegreeofvillousatrophyanditstions(Pritchard1987).segmentaldistribution.VillousatrophywasminimalinpigsnursingsowspreviouslyinfectedwithvirulentPorcinerespiratorycoronavirusTGEV,comparedwithpigsnursingseronegativesowsorExperimentally,PRCVinfectionofpigsismostlysub-sowsgivenliveattenuatedvaccines.Inpartiallypro-clinicalwithself‐limitingrespiratoryinfection.Theearlytectedpigs,villousatrophywasprimarilyintheileumantiviraleffectsofinnateimmuneresponsestoPRCVandnotthejejunum.Similarobservationswerenotedininfection,followedbycell‐mediatedandantibodypigsfromherdswithendemicTGE.responses,likelyeffectivelycontroltheinfection(Atanasovaet al.2008;Junget al.2007,2009,2010;PRCVlesionsZhanget al.2008).Clinicalsignsinclude(1)respiratorysigns(e.g.coughing,abdominalbreathing,dyspnea),(2)PRCVprimarilycausesupperandlowerrespiratorytractdepressionand/oranorexia,and(3)slightlydecreaseddisease.ThePRCV‐inducedlesionsaregenerallylimitedgrowthrates(Lanzaet al.1992;vanReethet al.1996;tothelungsandcommonlyobservedasconsolidationofWesleyandWoods1996).thelungandbronchointerstitialpneumonia,withfre-Theseverityandfrequencyofclinicalsignsareinflu-quentperibronchiolarandperivascularlymphohistio-encedbythepresenceofotherbacterialorviralpatho-cyticcuffing(Atanasovaet al.2008;Coxet al.1990a;gensintheherd.Forexample,coinfectionwithPRRSVHalburet al.1993;Jabraneet al.1994;Junget al.2007,canaltertheseverityofeitherPRCVorPRRSVinfec-2009).PRCV‐inducedbronchointerstitialpneumoniaistions.InoculationwithPRRSVfollowedbyPRCVcharacterizedby(1)thickeningofthealveolarseptabyresultedinprolongedfeverwithrespiratorydisease,infiltrationofinflammatoryleucocytes,principallymac-reducedweightgain,andprolongedseverepneumoniarophagesandlymphocytes;(2)type2pneumocyte(Junget al.2009;vanReethet al.1996).Ongoingorpre-hypertrophyandhyperplasia;(3)accumulationofexistingPRRSVinfectionsignificantlysuppressedinnatenecroticcellsandinflammatoryleucocytesinalveolarimmuneresponses(reducedIFN‐αlevelsinlungandandbronchiolarluminaduetoairwayepithelialnecrosis;bloodnaturalkiller[NK]cellcytotoxicity)duringearlyand(4)peribronchiolarorperivascularlymphohistio-PRCVinfection,whichmayexacerbatePRCVpneumo-cyticinflammation.Within10daysofPRCVinfection,nia(Junget al.2009).thevirussimultaneouslyinducesinflammatory(cellnecrotizing)andproliferative(alveolarseptalthicken-ing)chronic‐activebronchointerstitialpneumonia(JungTGEVlesionset al.2007,2009).TGEgrosslesionsareconfinedtothegastrointestinaltract.ThestomachisdistendedwithcurdledmilkandDiagnosismayhavepetechialhemorrhages(HooperandHaelterman1966b).ThesmallintestineisdistendedThecollectionandpreservationofappropriateclinicalwithyellowfluidandcurdled,undigestedmilk.Thewallspecimensisnecessaryforreliablediagnosis.Becauseisthinandtransparent,duetovillousatrophy.AmajorclinicalsignsandatrophicenteritiscausedbyTGEVarelesionofTGEismarkedlyshortenedvilliofthejejunumfrequentlyobservedinotherentericinfections(rotavi-andileum(Figure 31.5aandb),similartoPEDVandrus,PEDV,PDCoV,andcoccidia),laboratorydiagnosisPDCoVlesions(Figure 31.5c–e)(Deboucket al.1981;ofTGEmustbeaccomplishedbyoneormoreofthefol-Junget al.2015b),butusuallymoresevereandextensivelowingprocedures:detectionofviralantigenornucleicthanthatseeninrotavirusdiarrhea(Bohlet al.1978).acidsinfecesorlesions,virusisolationfromspecimens,InfectionswithsomestrainsofE.coliandcoccidiamayordetectionofTGEVantibodies.producesimilarlesions(Hornichet al.1977).DiagnosisofPRCVrequiressimilarprocedures,butTransmissionEMofTGEV‐infectedvillousenterocyteswithafocusonrespiratoryspecimens.Evaluationof 498SectionIIIViralDiseasesclinicalsigns,histologiclesions,andtissuedistributionrequirepigsintheearlystageofinfection.Asimilarviralofviralantigenmayprovideapresumptivediagnosis.antigendistributionisseeninthesmallintestineofPRCVdoesnotcausediarrheaorvillousatrophyandPEDV‐(Figure 31.6b)andPDCoV‐infectedpigsreplicatesalmostexclusivelyinrespiratorytissues(Figure 31.6c).AnexceptionforPEDVandPDCoVisthe(Pensaert1989).Thus,PRCVissuspectedifthereisanti-occasionaldetectionofviralantigensinthecryptepithe-geninlungtissues,seroconversiontoTGEV/PRCV,andlialcellsandthecolon.nosignsofentericdisease.Anenzyme‐linkedimmunosorbentassay(ELISA)usingMAborpolyclonalantibodiestoTGEVisusedtoDetectionof viralantigensornucleicacidsdetectTGEVantigensincellculture,feces,andintestinalDetectionofTGEVantigeninsmallintestinalentero-contents(Lanzaet al.1995;Sestaket al.1996,1999a;vancytesiscommonlyusedtodiagnoseTGE.EitherIFNieuwstadtet al.1988)orPRCVantigenincellculture,(Pensaertet al.1970)orimmunohistochemical(IHC)nasalswabs,orlunghomogenates(Lanzaet al.1995).(Shoupet al.1996)techniquesusingMAbagainsttheRT‐PCRorreal‐timeRT‐PCRiscurrentlyusedforhighlyconservedNproteinofTGEVmaybeusedinfro-diagnosisofTGEVanddifferentiationofTGEV,PRCV,zenorformalin‐fixedtissues(Figure 31.6a),buttheyPDCoV,andPEDV(Costantiniet al.2004;Kimet al.(a)(b)(c)Figure31.6Immunofluorescentstaining(green)of(a)TGEVantigensinalmost100%oftheilealenterocytesliningthevilliofaTGEV‐infectedpiglet.NoteabsenceofTGEVantigensinthecryptepithelialcells.(b)PEDVantigensintheenterocytesofthejejunumofapigletat67hourspostinoculationwiththeemergingnon‐SINDELPEDVstrainPC21A(37–41hoursafteronsetofclinicalsigns),indicatingthattheepithelialcellsliningatrophiedvilliarepositiveforPEDV(×200).Source:Junget al.2014.(c)PDCoVantigensinthejejunumofagnotobioticpigat3dayspostinoculationwithPDCoVstrainOH‐FD22,showingsimilarlocalizationofPDCoVantigensinthecytoplasmofvillousepithelialcells(×400). 31Coronaviruses4992000a,2001,2007;Masudaet al.2016;Ogawaet al.theseantibodiesbasedonusingMAbstoTGEVanti-2009).PRCV/TGEVdifferentiationisaccomplishedgenicsitesthatareabsentonthePRCVSproteinusingPCRprimerstargetingtheSgenedeletionregion(Bernardet al.1989;Callebautet al.1989;DelmasandinPRCVstrains.MultiplexRT‐PCRandreal‐timeRT‐Laude1990;Garweset al.1988;Sanchezet al.1990;PCRassayshavebeendevelopedforthesimultaneousSestaket al.1999b;Simkinset al.1992,1993).Blockingdetectionofmajorporcinevirusesassociatedwithdiar-ELISAsshouldonlybeappliedonaherdbasisbecauserheaincludingrotavirus,TGEV,PDCoV,andPEDVsomepigswithlowTGEVorPRCVantibodytitersmay(Masudaet al.2016;Ogawaet al.2009).Theseassaysnotbedetected(Callebautet al.1989;Sestaket al.permitdetectionofuptoninevirusesinasample.1999b;Simkinset al.1993)andtheaccuracyofcommer-Moreover,multiplexmicroarrayhybridizationwascialELISAsfordifferentiatingUSstrainsofPRCVandemployedfortherapiddifferentialdiagnosisofeightTGEVislow(Sestaket al.1999b).ELISAtestswereusedCoVsincludingTGEV(Chenet al.2005).todifferentiatenotonlybetweenTGEVandPRCVanti-bodiesbutalsobetweenTGEVandTGEV‐likeCCoVsElectronmicroscopy(EM)orclassicalCCoV‐IIantibodies(Eliaet al.2010;LopezTGEVcanbedemonstratedintheintestinalcontentset al.2009).andfecesofinfectedpigsbynegativecontrasttransmis-AriseinantibodytiterbetweenacuteandconvalescentsionEM(Figure 31.2a).ImmuneelectronmicroscopyserumsamplesprovidesretrospectiveevidenceforTGEV(IEM)hasadvantagesoverconventionalEMinbeingorPRCVinfection.TodeterminethepresenceofendemicmoresensitivefordetectingTGEVanddistinguishingitTGEorPRCV,serumsamplesfrom2‐to6‐month‐oldfromPEDV,PDCoV,andenvelopedmembranousdebris,swine(oftenfreeofpassivelyacquiredantibodiesatthisaswellasconcurrentlydetectingthepresenceofotherage)canbetestedforantibodies(Derbyshireet al.1969).entericviruses(Figure 31.2b)(Saifet al.1977).TheVNtestusingcellculture‐adaptedviruseshasbeenthemostwidelyused(Bohl1979;BohlandKumagaiVirusisolation1965).VNantibodiestoTGEVaredetectableinserumbyPrimaryandsecondarypigkidney(PK)cells(Bohland7–8DPIandpersistforatleast18months.LittleisknownKumagai1965)orcelllines(Laudeet al.1981),porcineregardingthepersistenceofVNantibodiestoPRCVthyroidcells(Witte1971),andtheMcClurkinswinetes-withinaherd.AntibodyELISAtests(Bernardet al.1989;ticle(ST)cellline(McClurkinandNorman1966)areBerthonet al.1990;Callebautet al.1989;Garweset al.recommendedfortheisolationofTGEVfromfecesor1988;Sestaket al.1999a,b;vanNieuwstadtet al.1989)gutcontentsofinfectedpigs.Distinctcytopathiceffectshavebeenreported,buttheyrequireconcentratedpuri-(CPE)maybenegligibleuponprimaryisolationoffieldfiedvirusorSorNproteinforcoatingELISAplates.strains,requiringadditionalpassages.TheCPEconsistsofenlarged,roundedcellswithaballoon‐likeappearanceImmunity(BohlandKumagai1965).FordetectingviralCPEorplaques,thesensitivityofSTcellscanbefurtherActiveimmunityto TGEVenhancedbyaddingpancreatinortrypsintocellcultureThedurationofactiveimmunityinswineafteroralmedia(Bohl1979)andusingoldercells.infectionwithvirulentTGEVhasnotbeenwellcharac-PigkidneyandSTcellsarepreferredforisolatingterized.Intestinalinfectionofbreeding‐ageswineresultsPRCVfromnasalswabfluidsorlungtissuehomogen-indetectableserumantibodiesthatpersistforatleastates.PRCV‐andTGEV‐inducedCPEaresimilar,with6monthsandpossiblyseveralyears(Stepaneket al.syncytiafrequentlyobservedasalsoreportedforPEDV1979).AlthoughserumantibodiesprovideserologicandSARSCoVgrowninVerocells(HofmannandWylerevidenceofTGEVorPRCVinfection,theyaffordlittle1988;Ksiazeket al.2003).IdentificationofcellcultureindicationofthedegreeofactiveimmunitytoTGEV.viruscanbedonebyVN,IFstaining,orIEMusingspe-SwinethathaverecoveredfromTGEareimmunetosub-cificTGEVantiserumordifferentialMAbs(Garwessequentshort‐termchallenge,presumablyduetolocalet al.1988)andRT‐PCRusingvirus‐specificprimersimmunitywithintheintestinalmucosa(Brimet al.1995;(EnjuanesandVanderZeijst1995;Kimet al.2000a;Saifet al.1994;VanCottet al.1993,1994).TheageandLaudeet al.1993).immunestatusoftheanimalatinitialinfectionandtheseverityofthechallengeinfluencethecompletenessandSerologydurationofactiveimmunity.TGEVantibodiescanbedetectedbyseveralserologicThemechanismofactiveimmunityinthegutrelatestotests.However,TGEVserologyiscomplicatedbythestimulationofthesecretoryIgA(sIgA)immunesystemfactthatbothTGEVandPRCVinduceVNantibodieswithproductionofsIgAantibodiesbyintestinalplasmathatarequalitativelyandquantitativelysimilar(Pensaertcells(Saifet al.1994;VanCottet al.1993,1994).IgA1989).AblockingELISAtestcandifferentiatebetweenTGEVantibodiesandantibody‐secretingcells(ASCs) 500SectionIIIViralDiseaseshavebeendetectedintheintestineandserumofpigsinnateimmunitymayplayaroleineitherrecoveryfromafteroral,butnotparenteralinoculationwithTGEVTGEVinfectionorresistancetoreinfectionviatherapid(Kodamaet al.1980;Saifet al.1994;VanCottet al.1993,eliminationofTGEV‐infectedepithelialcells.Some1994).Kodamaet al.(1980)proposedthatdetectionofTGEVstrainscanalsodownregulatehostimmuneIgAantibodyintheserum,presumablyintestinallyresponses.Avirulent(SHXB)butnotattenuated(STC3)derived,mightserveasanindicatorofactiveimmunitytoTGEVstrainimpairedtheabilityofporcineintestinalTGE.Enzyme‐linkedimmunospot(ELISPOT)assaywasDCsormonocyte‐derivedDCstorecognizeantigen,usedtoinvestigatethekineticsofIgAandIgGTGEVmigrate,andinduceT‐cellproliferationinvivoandinantibodyproductionbythepig’ssystemicandlocalgut‐vitro(Zhaoet al.2014).associatedlymphoidtissues(GALT).HighnumbersofIgAASCswereinducedinGALTonlybyvirulentTGEV.PRCV‐inducedactiveimmunityto TGEVIncontrast,liveattenuated(vaccine)TGEVorPRCVThedramaticdeclineinepidemicoutbreaksofTGEinstrainsinducedsignificantlyfewerIgAASCs(BerthonEuropefollowingthewidespreaddisseminationofPRCVet al.1990;Saifet al.1994;VanCottet al.1993,1994).promptedresearcherstoexamineifrespiratoryPRCVBesideslocalantibody‐mediatedimmunity,cell‐mediatedinfectioncouldinduceprotectiveintestinalimmunityimmunity(CMI)mayalsobeimportantinactiveimmu-againstTGEV.TheconsensusfromseveralstudieswasnityagainstTGEVinfections.However,onlyindirectevi-thatpriorinfectionofnursingorweanedpigswithPRCVdenceexistsconcerningtheroleofCMIinresistancetoprovidedpartialimmunityagainstTGEVchallenge,asTGEVinfection.CMIwasdemonstratedwithlympho-evidencedbyareduceddurationandlevelofviruscytesobtainedfromGALTofswineorallyinfectedwithsheddinganddiarrheainmostpigsstudied(Brimet al.virulentTGEV(Brimet al.1995;Fredericket al.1976;1995;Coxet al.1993;VanCottet al.1994;WesleyandShimizuandShimizu1979),whereasswineparenterallyWoods1996).ororonasallyinoculatedwithattenuatedTGEVorThispartialimmunitypresumablyisrelatedtothePRCV developedCMImainlyinsystemicsites.rapidincreaseinTGEVVNantibodies(Coxet al.1993;LymphoproliferativeresponsestoTGEVpersistedwithinWesleyandWoods1996)andnumbersofIgGandIgAGALT,butnotsystemiclymphocytes,foratleast110daysASCsintheintestinesofPRCV‐exposedpigsafterTGEVafteroralinfectionof6‐month‐oldswine(Shimizuandchallenge(Saifet al.1994;VanCottet al.1994).TheShimizu1979),butforonlyabout14–21daysafterinfec-alteredtissuetropismofPRCVwasalsolinkedtoashifttionofyounger(7‐to11‐day‐old)pigs(Brimet al.1995).inantibodyresponses;thatis,inTGEV‐infectedpigs,CD4ThelpercellsareinvolvedinlymphoproliferativemoreIgAASCswerefoundingut,whereasPRCVpre-responsestoTGEV(Antonet al.1995).Potentproduc-dominantlyinducedIgGASCsinthelung(VanCottet al.tionofantiviralIFN‐αbyplasmacytoiddendriticcells1994).MigrationofPRCVIgGandIgAASCsfromthe(DCs)derivedfromTGEV‐infectedswinewasobservedbronchus‐associatedlymphoidtissues(BALT)tothegutuponstimulationofthesecellsinvitrowithTGEVofthePRCV‐exposedpigsafterTGEVchallengemightantigens(Calzada‐Novaet al.2010).explaintherapidanamnesticresponseandthepartialAcorrelationbetweenlymphoproliferativeresponsesprotectioninduced(VanCottet al.1994).However,neo-andlactogenicimmunitytoTGEVwasdescribedinsowsnatalpigsrequiredatleast6–8daysafterPRCVexposurevaccinatedwithattenuatedorrecombinantTGEVtodeveloppartialimmunitytoTGEVchallenge(Wesleyvaccines(Parket al.1998).AlthoughT‐cellepitopesandWoods1996).wereidentifiedbylymphoproliferationstudiesforeachofthethreemajorproteinsofTGEV,adominantfunc-Passiveimmunityto TGEVtionalThelperepitopewasdefinedontheNproteinPassivelactogenicimmunityiscriticaltoprovidenew-(N321)(Antonet al.1995).TheN321peptide‐inducedTbornpigletswithimmediateprotectionagainstTGEVcellscollaboratedintheinvitrosynthesisofTGEVVNinfection.Circulatingpassiveantibodies,acquiredafterantibodiesspecificfortheSprotein.Maximalresponsesabsorptionofcolostralimmunoglobulin(primarilyIgG),wereinducedbynativeSproteincombinedwithrecom-protecttheneonateagainstsystemicbutgenerallynotbinantNprotein.Suchfindingshaveimportantimplica-intestinalinfection(HooperandHaelterman1966a;SaiftionsfordesignofCoVsubunitorotherrecombinantandSestak2006).MechanismsofpassiveimmunitytoCoVvaccines.TGEVinfectionshavebeenreviewed(Chatthaet al.Becauselymphocytecytotoxicitywasabsentinnew-2015;SaifandBohl1979;SaifandJackwood1990;Saifbornpigletsanddecreasedinparturientsows,itwasandSestak2006).SwinerecoveredfromTGEtransmitproposedthatalackofNKcellactivityagainstTGEV‐passiveimmunitytotheirsucklingpigsbythefrequentinfectedcellsmightcorrelatewiththeincreasedsuscep-ingestionofcolostrumormilk(lactogenicimmunity)tibilityofnewbornpigletsandparturientsowstoTGEVthatcontainsTGEVVNantibodies(Hooperandinfection(CepicaandDerbyshire1984).Thus,CMIorHaelterman1966a).Suchantibodiesinthelumenofthe 31Coronaviruses501intestineneutralizetheingestedTGEVandprotecttheantibodiesinmilk,ahallmarkofprotectionintheseandsusceptiblesmallintestinalenterocytes.Thisisaccom-otherstudies(WesleyandWoods1993)wasinductionofplishednaturallywhenpigletssuckleimmunesowsactiveimmunitytoTGEVinthesowpreventingclinicalfrequentlyorbycontinuousfeedingofantiserumtopig-diseaseoragalactia.lets.Duringthefirstweekoflactation,IgAbecomesBesidesquantitativedifferencesinthelevelsofIgAdominantinmilkandIgGdecreases.antibodiesinducedinmilkofsowsafterexposuretoTGEVIgAantibodiesinmilkarestableinthegutandTGEVorPRCV,researchershaveinvestigatedpotentialprovidethemosteffectiveprotection,butIgGantibodiesdifferencesinvirusepitopesrecognizedbythemilkIgAarealsoprotectiveifhightitersaremaintainedinmilkantibodies(DeDiegoet al.1992,1994).InTGEV‐aftervaccination(BohlandSaif1975)orbyartificialinfectedsows,antigenicsubsiteA(Aa,Ab,Ac),followedfeedingofcolostralIgGantibodies(Stoneet al.1977).byantigenicsubsiteD,wasthebestinducerofIgAanti-TGEVIgGantibodiesareproducedinthesow’smilkbodies,whileafterPRCVinfection,antigenicsiteDandafterparenteralorsystemicimmunization,whereassubsiteAbwereimmunodominant(Figure 31.3).Thus,TGEVIgAantibodiesoccurinmilkafterintestinalinfec-onlyIgArecognizingatleastantigenicsitesAandDtion.ItispostulatedthatIgAimmunocytesmigratetoconferredprotectioninvivo,whereasanyimmunoglob-themammaryglandafterantigenicstimulationintheulinisotypereactivetooneantigenicsiteneutralizedgutwheretheylocalizeandsecreteIgAantibodiesintovirusinvitro.colostrumandmilkthatplayakeyroleinpassiveintesti-nalimmunityofsucklingpigs(BohlandSaif1975;SaifPreventionand controlandBohl1979;SaifandJackwood1990;SaifandSestak2006).The“gut–mammary”immunologicaxis,firstpro-TreatmentposedinrelationtoTGEVinfectionsinswine(Bohlet al.Noantiviraldrugshavebeendevelopedfortreatmentof1972;Saifet al.1972),providedtheinitialconceptforaTGE.AfterthediscoveryofSARSCoV,studieswerecon-commonmucosalimmunesystem.Thisconceptcontin-ductedwithvarioussurrogateviruses,includingTGEV,uestobeimportantinthedesignofmaternalvaccinestodevelopanti‐CoVagents.Ortegoet al.(2007)usedthatarecapableofprovidingeffectivelactogenicimmu-TGEVdeletionmutantstoshowthatabsenceoftheEnityagainstentericpathogens.proteinblocksvirustraffickingintheendoplasmicretic-ulumandpreventsvirusmaturation.RNAinterferencePRCV‐inducedpassiveimmunityto TGEV(RNAi)targetingtheviralRNApolymerasewasstudiedTheincidenceandseverityofTGEincountrieswithinvitroasastrategytopreventTGEVinfection(ZhouPRCVhasdeclinedsincePRCVhasbecomewidespreadet al.2007).Althoughprotectiveinvitro,theresultsof(Schwegmann‐WesselsandHerrler2006).ThissuggestsanalogousinvivoexperimentswerelessconvincingthatpriorexposureofswinetoPRCVimpartspartial(Zhouet al.2010).immunitytoTGEV(Laudeet al.1993;Pensaert1989).StudiessuggestthatIFNmayactivateNKcellsinnew-PriornaturalexposureofsowstoPRCVinducedavar-bornpigs,contributingtoresistancetochallengewithiabledegreeofpassiveprotection(44–53%mortality)TGEV(LesnickandDerbyshire1988;LoewenandagainstexperimentalTGEVchallengeofsucklingpigsDerbyshire1988).Inaddition,duringafieldoutbreakof(Bernardet al.1989;PatonandBrown1990).VariableTGE,1‐to12‐day‐oldpigletstreatedorallyfor4daysprotectioninthefieldduringTGEoutbreakswasalsowith1–20IUofhumanIFN‐αhadsignificantlygreaternotedamonglittersofPRCV‐exposedsows(Pensaertsurvivalratesthanplacebo‐treatedpiglets(Cummins1989;Sanchezet al.1990).Similarvariablelevelsofpro-et al.1995).tection(30–67%mortality)werereportedafterTGEVTheonlyavailabletreatmentforTGEistoalleviatechallengeofpigletssucklingsowsthathadbeenexperi-starvation,dehydration,andacidosis.Parenteraltreat-mentallyinfectedorreinfectedwithPRCVduringpreg-mentwithfluids,electrolytes,andnutrientsareeffectivenancy(DeDiegoet al.1992;Lanzaet al.1995;Sanchezintreatingyoungpigs,butnotpracticalunderfarmcon-et al.1990;Sestaket al.1996;WesleyandWoods1993).ditions.Oraltherapywithbalancedelectrolyteorglu-Inthelattertwostudies,littermortalitywaslowestcosesolutionsiscontraindicatedinyoungpigs(Moon(range = 0–27%),andIgAandIgGmilkantibodytiters1978).Thefollowingmeasuresaresuggested:provideawerehighestinsowsmultiplyexposedtoPRCVduringwarm(above32°C[90°F]),draft‐free,dryenvironmenttwosubsequentpregnancies.Theseexperimentalfind-andprovidewaterornutrientsolutionsfreelytoTGEV‐ingsagreedwithfieldreportsthatnaturallyPRCV‐infectedpigs.SuchmeasuresreducedmortalityinpigsexposedsowsreinfectedwithPRCVduringpregnancythatwereinfectedatmorethan3–4daysofage.secretedPRCVIgAantibodiesinmilkandprovidedaAntibacterialtherapyisbeneficialin2‐to5‐week‐oldhighdegreeofprotection(0–12.5%mortality)toTGEVpigsifthereisconcurrentinfectionwithbacterialpatho-challenge(Sanchezet al.1990).BesidesPRCVIgAgens.Cross‐fosteringofinfectedorsusceptiblelitters 502SectionIIIViralDiseasesontoTGE‐immunesowswasusefulinsomefieldout-pigs,thedelayitselfcanreducemortality.Second,breakbreaks(Stepaneket al.1979).thecycleofinfectionbyeliminatingreservoirsofsuscep-tiblepigsinaunit:preventthecontinualinfluxofsus-Managementceptibleanimalsintotheherdtemporarily(alterBiosecurityfarrowingscheduleaspossible),utilizeotherfacilities,SwineintheincubativeorviralsheddingstageoftheandcreatesmallerfarrowingandnursingunitstoachievediseaseorpossiblycarrierscantransmitTGEV.Tointro-anall‐in/all‐outsystem.duceswineintoaherd,precautionsareneededtoassurethatswineoriginatefromherdsfreeofTGE,aresero-Immunoprophylaxislogicallynegative,and/orhavebeenplacedinisolationVaccinesand vaccinationsonthefarmfor2–4weeksbeforebeingaddedtotheThereareseverallicensedTGEVvaccines.Allcontainherd.AfteraTGEoutbreak,atleast4weeksshouldinactivatedorliveattenuatedTGEVandareapprovedelapsefromthelastsignofdiseasebeforeintroducingforuseinpregnantorneonatalswine.Thesevaccinessuchanimalsintoa“clean”herd.FecesfromTGEV‐andtheirefficacyhavebeenreviewed(SaifandSestakinfectedswinecanbecarriedonboots,shoes,clothing,2006)butwillbebrieflysummarized.truckbeds,feeds,andsoonandcanbeasourceofinfec-Manyvariablescomplicatetheevaluationofbothtiontootherherds,requiringstrictdisinfectionregimes,experimentalandcommercialTGEVvaccines,resultingespeciallyinwinter.inconflictingdata.TheseincludethechallengedoseandstrainofTGEV,theageofthepigatchallenge,environ-Afteronsetof TGEand endemicTGEVmentalconditions(especiallytemperature),thehealthWhenTGEoccursonafarmandpregnantanimalshavestatusandmilkingefficiencyofthevaccinatedsow,andnotyetbeenexposed,twoproceduresmayminimizetheimmunestatus(forTGEVorPRCVantibodies)oflossesofnewbornpigs:(1)Ifthesowsareduetofarrowthedamatvaccination.Ifpreviouslyinfectedsowswereinatleast2weeks,usefeedbackmethodstoorallyexposeunknowinglyusedinvaccinechallengestudies,thisthemtovirulentautogenousvirus,suchasaslurryofcouldaccountfordiscrepantresultsseeninimmunemincedintestinesofacutelyinfectedpigs,sothattheyresponsesandpigletprotection.Thispossibilitycanonlywillbeimmuneatfarrowing.(2)Ifthesowswillfarrowbeeliminatedbyusingsensitivetests(suchasVN)toinunder2weeks,attempttoprovidefacilitiesandman-measureTGEV/PRCVantibodiesandbyknowingtheagementprocedurestoavoidexposuretoTGEVuntilatherdhistoryoftestanimalssinceoccurrenceofPRCVinleast3weekspostfarrowing.herdsfurthercomplicatesTGEVvaccinestudies.SomesuccesshasbeenachievedineliminationofTGEVfromepidemicallyinfectedclosedbreederherdsTGEvaccinationof the seronegativepregnantdamwithoutdepopulationbythefollowingproceduresAvarietyofviralvaccines(virulent,attenuated,inacti-(Harriset al.1987):(1)bringinbreedingstockreplace-vated,andsubunit)androutesofadministrationmentsforthenext4–6months;(2)inthefaceofanout-(oral, intranasal,intramuscular,subcutaneous,andbreak,feedbackTGEVacutelyinfectedmincedpigletintramammary)(BohlandSaif1975;MoxleyandOlsonintestinessimultaneouslytoallpigsintheherd(includ-1989;SaifandBohl1979;SaifandJackwood1990;Saifingreplacementstock)toeliminatesusceptiblehosts,andSestak2006)havebeentestedforinductionoflac-shortenthetimethediseaseprogressesthroughthetogenicimmunity.Onlyoraladministrationoflivevir-herd,andensuremoreuniformexposurelevelsinallulentvirustopregnantsowsconsistentlystimulatedpigs;(3)maintainstrictall‐in/all‐outproductioninhighlevelsofprotectiveimmunityforthesowandfarrowingandnurseryunits;and(4)addsentinelseron-persistingTGEVIgAantibodiesinmilkthatpassivelyegativepigsabout2monthsafterclinicalsignsofTGEprotectedpiglets.disappearandmonitorthesepigsforseroconversiontoThegenerallypoorresultsfororalorintranasalvacci-TGEV.Potentialhazardsassociatedwithfeedbackcon-nationofsowsusingattenuatedTGEVstrains(MoxleytrolofTGEincludepossiblespreadofotherpathogenstoandOlson1989;SaifandBohl1979;SaifandSestakpregnantsowsandthroughouttheherd.2006)maybeattributedtothelimitedreplicationofOtherapproachestocontrolorterminateendemicmostattenuatedstrainsinthesow’sintestine(FrederickTGEincludethefollowing.First,pregnantseropositiveet al.1976).ThisresultsinlittleantigenicstimulationofsowscanbevaccinatedintramuscularlylateingestationintestinalIgAimmunocytesandcorrespondinglylittleorshortlyafterfarrowingwithliveattenuatedTGEVIgAantibodysecretioninmilk.Thus,thedilemmaisvaccinetoboostimmunity,increasemilkantibodylev-howtodevelopcommercialTGEVvaccinesthatareels,andmaintainlongerpassiveimmunityinsucklingcapableofstimulatingIgAinthegutofsows,butsuffi-pigs(SaifandSestak2006;Stepaneket al.1979).AlthoughcientlyattenuatedsoasnottoproducediseaseinthisproceduremayonlydelayonsetofTGEinexposednewbornpigs. 31Coronaviruses503ParenteralTGEVvaccinesinducedevenlowerorpassiveimmunizationwassuggestedbyfeedingtheinconsistentprotectionratesinTGEV/PRCVseronega-recombinantimmunoproteinscapableofinducingtiveswine.Theyhavetwomajordisadvantages:(1)TGEVVNantibodiestosowstoconferpassiveimmu-Vaccinatedswinedeveloplittleornogutimmunityandnitytopiglets(Bestagnoet al.2007).TheapproachmayoftengetsickwhenexposedtoTGEV,deprivingtheirbecosteffectivebyexpressingtheseproteinsinplantssucklingpigsofmilk.(2)ThelowtiterIgGandnoIgA(Mongeret al.2006).TGEVantibodiesinmilkofvaccinatedsowsfailtopro-VariouslevelsofVNantibodiesandprotectionwerevideoptimalpassiveprotectiontosucklingpigs.inducedusingeukaryoticvectorstoexpresstheTGEVSCurrentlyavailableparenterallyadministeredTGEVglycoproteinencodingtheglycosylation‐dependentanti-vaccinesmaybemoreeffectiveinboostingimmunityingenicdeterminants(sitesAandB)withorwithoutsitespregnantswinepreviouslyinfectedwithTGEVorPRCVCandD(Figure 31.3).Thebaculovirus‐orvacciniathanininitiatingimmunityinseronegativepregnantvirus‐expressedSglycoproteinofTGEVinducedlowswine.ThesevaccinesmaybeespeciallyusefulinherdstitersofVNantibodiesinserum,colostrum,andmilk,inwhichendemicTGEisaproblem(Stepaneket al.butlowornoprotection(Godetet al.1991;Huet al.1979).1985;Shoupet al.1997;Tubolyet al.1995).OnlySglyco-proteinconstructscontainingantigenicsiteAinducedTGEvaccinationof neonatalorweanedpigshighVNantibodytiters.SitesCandDinducedonlylowActiveimmunizationofsucklingorfeederpigscouldtiterVNantibodies,butinterestingly,theyprimedpigsbeimportantforcontrolofendemicinfections,espe-forsecondaryserumantibodyresponsesafterchallengeciallyinnewlyweanedpigs,inwhichTGEVinfections(Shoupet al.1997).resultinincreasedmortality.LiveattenuatedandSimilarfindingswereevidentinstudiesusingthesameinactivatedTGEVvaccineshavebeenlicensedinthebaculovirus‐expressedSconstructsadministeredIMtoUnitedStatesfororalorintraperitonealadministra-boostantibodyresponsesinsowsvaccinatedorallywithtion,respectively,shortlyafterbirth.However,theattenuatedTGEVvaccines:thepartialprotectionratespresenceofmaternalantibodiesinvaccinatedpigswerecomparablewithIMboostingwithattenuateddecreasedorcompletelysuppressed(Furuuchiet al.TGEVvaccine(Parket al.1998).Baculovirus‐expressed1978;Hesset al.1982;Lanzaet al.1995;Sestaket al.TGEVstructuralproteins(S,N,andM)coadministered1996)activeantibodyproductionfollowingoraladmin-IPwithE.colimutantLTadjuvantinducedTGEVIgAistrationofattenuatedTGEVvaccines.OtherantibodyresponsesassociatedwithreducedTGEVshed-approachesusingrecombinantTGEVproteinsdinginchallengedpigs(Sestaket al.1999b).(reviewedinnextsection)havebeenusedinattemptsRecentstudieshaveusedmolecularapproachestotoactivelyimmunizeyoungpigsagainstTGEV.developvectoredTGEVvaccinesandtesttheminpor-cineandmurinemodels.Ahumanadenovirusengi-RecombinantvaccineapproachesneeredtoexpresstheTGEVorPRCVSproteinsAmongthemajorstructuralproteinsofTGEV,theSpro-(Callebautet al.1996;Torreset al.1996;TubolyandteincontainsimmunodominantepitopesrecognizedbyNagy2001)elicitedvariableprotectionagainstTGEVVNantibodies.EpitopesforcontinuousdomainsmortalityandlittleprotectionagainstTGEVorPRCV(DelmasandLaude1990)wereincorporatedintosyn-infection.AnoralLactobacilluscasei‐basedvaccinetheticpeptidesderivedfromtheSprotein(Posthumusexpressingrepetitive(20Xand40X)peptidesoftheanti-et al.1991).However,apeptidecontainingthemajorTgenicDsiteofTGEVSproteininducedhumoralandT‐helpercellepitopederivedfromtheNproteinhasbeencell‐basedimmuneresponsesandTh17polarizationinreportedtocooperatewiththeSproteinforinvitromice(Jianget al.2014,2016).Additionally,anattenuatedinductionofTGEVantibody(Antonet al.1996).SalmonellatyphimuriumvectorexpressingTGEVNorToexpresstheTGEVS(orSepitopes),M,orNpro-MproteinsandarecombinantBacillussubtilisstrainteins,severalprokaryoticandeukaryoticsystemssuchasexpressingTGEVSproteininducedT‐cellproliferation,E.coli,Salmonella,adenovirus,vacciniavirus,poxvirus,antibodyandcytokineresponsesinmice,andantibodybaculovirus,DNAvectors,andplantswereusedresponsesinpigs,respectively(Mouet al.2016;Qing(Enjuaneset al.1992;Godetet al.1991;Gomezet al.et al.2016;Zhanget al.2016a).Yuanet al.(2015)2000;Menget al.2013;Parket al.1998;Shoupet al.1997;expressedtheAepitopeoftheSproteininswinepoxSmerdouet al.1996;Torreset al.1996;Tubolyet al.2000;virusanddemonstratedthatthisvaccineadministeredtoYuanet al.2015).Insomestudies(Torreset al.1996),butsowswasimmunogenicandprotectedpigletsagainstnotothers(Gomezet al.2000;Smerdouet al.1996;clinicaldisease.DNAplasmidsweregeneratedforPEDVTubolyet al.2000),protectiveantibodieswereinducedandTGEVforthedevelopmentofDNAvaccinesthatininoculatedanimalscorrelatingwithpartialprotectionwereimmunogenicinmice,butnottestedinpigs(Meng(Parket al.1998;Shoupet al.1997).Anovelapproachtoet al.2013). 504SectionIIIViralDiseasesAneffectiveTGEVvaccineshouldprimarilyelicitanspreadthroughoutNorthandSouthAmerica,Asia,andintestinalimmuneresponse(SaifandJackwood1990;Europe.SaifandSestak2006;VanCottet al.1993).FurtherThesituationcontinuestoevolve.Forexample,recom-improvementsofTGEVvaccinesmightbeachievedbybinantentericCoVsbetweenTGEVandPEDVhavetheuseofmucosaladjuvants/deliverysystemssuchasbeendetectedinItaly(2009–2012),inGermany(2012),immunostimulatingcomplexes(ISCOMs),vitaminA,andinEasternEurope(2016)(Akimkinet al.2016;probioticbacteria,biodegradablemicrospheres,orinfec-Belshamet al.2016;Boniottiet al.2016).tiousrecombinantTGEVclonesengineeredtoenhanceTGEVimmunogenicityandreducepathogenicityEtiology(Chatthaet al.2015;Enjuaneset al.2005).StudiesofTGEVinfectiouscDNAminigenomesindicatethatthisMorphologicandphysicochemicalpropertiesofPEDVapproachalsocanbeusedfortargeteddeliveryofimmu-(Figure 31.2c)aresimilartothoseofothermembersofnogensderivedfromotherpathogenstotheintestineorthefamilyCoronaviridae(Figure 31.2).Basedongeneticrespiratorytract.andantigeniccriteria,PEDVisincludedinthegenusAlphacoronavirustogetherwithbatcoronavirus(BtCoV)/512/2005,TGEV,PRCV,FECoV,FIPV,CCoV,andHCoV229E.BasedonphylogeneticanalysisofthePorcineepidemicdiarrheaviruscompletegenomes,theglobalPEDVstrainsaredividedintotwomajorgroups:theclassicalPEDVstrainsthatRelevancefirstemergedinthe1970sinEuropeandthePEDVIn1971,acuteoutbreaksofdiarrheainfeederandfinish-strainsappearedafter2010(Linet al.2016)(Figure 31.1).ingpigswereobservedinEngland(Oldham1972).TheTheemergingPEDVstrainsarefurtherdividedintodiseasespreadtootherEuropeancountries,andthe“non‐SINDEL”(mainlyhighlyvirulent)and“SINDEL”name“epidemicviraldiarrhea”(EVD)wasadopted.Insubgroups(Figure 31.1)becausetheformerandlatter1976,similaroutbreakswereobserved,butinswineofallcausesevereandmildPED,respectively,inthefieldandages,includingsucklingpigs(Wood1977)and,in1978,inexperimentalpigchallengestudies(see“Pathogenesis”).aCoV‐likeagentwasassociatedwiththeoutbreaksinTheSINDELstrainscontaininsertionsanddeletionspiglets(ChaseyandCartwright1978;PensaertanddesimilartotheclassicalPEDVstrainsintheS1subunitofBouck1978).ExperimentalinoculationswiththeBelgiantheSprotein(Vlasovaet al.2014).Theylikelyresultedisolate(CV777)revealeditsenteropathogenicityforpig-frommultiplerecombinationeventsbetweentheclassi-letsandgrowingpigs(DebouckandPensaert1980),andcalandemergingPEDVstrainsinAsia,perhapsrelatedthenames“porcineepidemicdiarrhea”(PED)andPEDtowidespreaduseofliveclassicalPEDVvaccinestrainsvirus(PEDV)wereadopted(Deboucket al.1982).Intheinswine.OtherminorPEDVvariantshavebeenreported,1970sand1980s,PEDVcausedwidespreadepidemicsine.g.USTC‐PC177,USA/OK10240‐8/2017,andJapaneseEurope,withseverelossesinsucklingpigs.Sincethen,TTR‐2strains,bearinglargedeletions(194–200aa)inPEDVhasbeenassociatedmoreoftenwithisolatedout-theN‐terminaldomain(NTD)oftheSprotein(Okabreaksandrecurrentdiarrheicproblemsinweanedandet al.2014;Suzukiet al.2015;Zhanget al.2018).Thefeederpigs.However,epidemicsalsooccurred,asinItaly197‐aadeletion(residues34–230)ofPC177strainin2005–2006.occurredduringVerocelladaptation,whereasthe194‐InAsia,PEDVepidemicswerefirstreportedin1982aadeletion(residues23–216)oftheTTR‐2strainandandoutbreakscontinuedthroughthe1990sand2000s.the200‐aadeletion(residues31–230)oftheOK10240‐8Thesituationchangedin2010whenPEDoutbreaksinstrainweredetectedinclinicalswinesamplesinJapanChinacausedbyhighlyvirulentPEDVstrainsresultedandtheUnitedStates,respectively.Unlikethealteredinthelossof>1millionpigletsin1year(Sunet al.tissuetropismseenforPRCV(enterictorespiratory),the2012).In2013,PEDVoutbreakswerereportedinthetwoPEDVstrains(PC177andTTR‐2)retainedtheirUnitedStates,likewisewithseverelosses(Stevensonenterictropism,butwithreducedvirulence(Linet al.et al.2013).2016;Suzukiet al.2016).TheinitialPEDVstrainsarereferredtoasclassicalAlthoughPEDVvariantshavedifferentinsertionsorPEDVstrains(Chenet al.2013).ThestrainsidentifieddeletionsintheSglycoproteinandvariationintheSgly-since2010areconsideredemergingPEDVstrains.coproteinofPEDVmayberelatedtopathogenesisandVariantsoftheemergingPEDVstrainscontaininginser-cross‐neutralizingactivity,thereappearstobeonlyonetionsanddeletionsintheSgene(“SINDEL”strains)PEDVserotype(Choudhuryet al.2016;Linet al.2016).werefirstdetectedintheUnitedStates(Linet al.2016;Thereisnocross‐neutralizationbetweenPEDVandWanget al.2014b).Bytheendof2016,theemergingTGEVorbetweenPEDVandPDCoV(Linet al.2015b;PEDVstrains,bothnon‐SINDELandSINDEL,hadMaet al.2016).However,alowdegreeofcross‐reactivity 31Coronaviruses505wasobservedbetweenPEDVandotheranimalalphac-(Puranavejaet al.2009).In2010,despitethewidespreadoronavirusantibodies.Forexample,aTGEVMAbrec-useofPEDVstrainCV777vaccines,severePEDVout-ognizedPEDVNprotein(Linet al.2015b),TGEVMillerbreaksduetonon‐SINDELstrainsoccurredinChinaantiserumreactedwithPEDVNprotein(Gimenez‐(Sunet al.2012,2016;Wanget al.2016a).Later,emerg-Lirolaet al.2017),TGEVandPRCVantiserareactedingSINDELstrainswerealsodetectedinChina(WangwithPEDVMprotein(Gimenez‐Lirolaet al.2017),andet al.2016a).minkalphacoronavirusantiserumreactedwithPEDVMSince2013,theemergingnon‐SINDELPEDVstrainsandNproteins(Haveet al.1992).havebeendetectedinotherAsiancountries/regionsVero(Africangreenmonkeykidney)cellssupporttheoutsideofChina,includingJapan(Masudaet al.2015),growthofPEDVinculturemediumsupplementedwithSouthKorea(Kimet al.2015),Vietnam(Vuiet al.2014),trypsin(Figure 31.4c).CPEconsistsofvacuolationandThailand(Cheun‐Aromet al.2015),Taiwan(Linet al.largemultinucleatedsyncytia(HofmannandWyler2014),andthePhilippines(Kimet al.2016).TheSINDEL1988).PEDValsogrowsinvariousswinecelllines,PEDVwasalsodetectedinJapanin2013(Suzukiet al.includingbladderandkidneycells(Shibataet al.2000;2015)andKoreain2014(Leeet al.2014).Wanget al.2016b),STcells(Liuet al.2015a),alveolarThefirsthighlyvirulentPEDoutbreakcausedbynon‐Smacrophagecellline3D4(ParkandShin2014),andINDELPEDVoccurredinswinefarmsintheUnitedsmallintestinalepithelialcells(IECs)(Caoet al.2015;StatesinApril2013(Stevensonet al.2013),followedbyConget al.2015).AsreviewedbyTeeravechyanet al.thedetectionofmilderPEDoutbreakscausedbytheS(2016),PEDVcanalsoreplicateinbatlungcelllineTb1‐INDELPEDVinJanuary2014(Wanget al.2014b).FromLu(Liuet al.2015a),duckIEClineMK‐DIEC(Khatri2013to2014,PEDVkilledapproximately7millionpig-2015),andhumanlivercelllineHuH‐7(Wanget al.letsintheUnitedStates.InJanuary2017,PEDVhad2016b).spreadto39USstatesandtoPuertoRico.PEDVhasalsopAPN,thecellreceptorusedbyTGEV,wasinitiallyspreadtoothercountries(e.g.CanadaandMexico)inconsideredtobetheputativereceptorofPEDVwiththeWesternHemisphere(Linet al.2016).PEDVhasnotsomesupportingevidence(Conget al.2015;Liet al.beenreportedinAfricaorAustralia.2007;NamandLee2010);however,somerecentstudiesDirectorindirectfecal–oraltransmissionisthemainarguethatpAPNmaynotbeafunctionalreceptorforrouteofPEDVtransmission.Contaminatedequipment,PEDV(Liet al.2017;Shiratoet al.2016).feedandfeedingredients,transportation,orpersonnelmayserveasvehiclesforPEDVtransmission(Deeet al.2014,2016;Schumacheret al.2016).EvidenceofPEDVPublichealthaerosoltransmissionhasbeenreportedinsome(AlonsoPEDVisonlyinfectiousforswineanddoesnotplayaet al.2014),butnototherstudies(Niederwerderet al.knownroleinpublichealth.2016).Inemergingnon‐SINDELPEDVexperimentallyinfected4‐week‐oldpigs,infectiousvirusexcretionassessedbyPEDVtransmissiontosusceptiblesentinelEpidemiologypigslasted14–16days(Crawfordet al.2015).However,ClassicalPEDVregularlycausedepidemicsinEuropeat42dayspost‐initialoralexposure,somepigsstillshedfrom1971untilthelate1980s,butreportsafter2000arePEDVRNAinfeces,illustratingdiscordancebetweenrare.AnepidemicinItaly(2005–2006)affected63herds,prolongeddetectionofPEDVRNAinfecesandthebutmortalitywaslargelyrestrictedtosucklingpigletstransmissionofinfectiousPEDVtosusceptiblepigs.(Martelliet al.2008).UntiltheemergenceofnewPEDAfteranoutbreakonabreedingfarm,PEDVcanoutbreaksin2014,PEDVwasnotconsideredimportant,becomeendemicthroughacycleofinfectionofconsecu-andtherefore,theprevalenceofclassicalPEDVinEuropetivelittersastheyloselactogenicimmunityatweaning.isunknown.ExceptforanoutbreakassociatedwiththeAlthoughastudyfromSouthKoreashowedaPEDVemergingnon‐SINDELstraininUkrainein2014,subse-infectionrateof9.75%inwildboars(Leeet al.2016a),quentoutbreaksinFrance,Germany,Belgium,Slovenia,theirroleinthemaintenanceandtransmissionofPEDVandtheNetherlandswereduetotheemergingSINDELisunknown.strains(Linet al.2016).InAsia,classicalPEDappearedinChinainthelatePathogenesis1970s,causingseriouslossesinmanyprovinces(Wanget al.2016a;Xuanet al.1984).PEDwasrecognizedinThepathogenesisofPEDisrelatedtotheageofpigsatJapanin1982(Kuwaharaet al.1988;Sueyoshiet al.1995;thetimeofinfection,virusstrainvirulence,inoculationTakahashiet al.1983)andKoreain1993(Chaeet al.routes,anddoses.2000;Hwanget al.1994;Kweonet al.1993),butisknownThePEDVpathogenesiswasfirststudiedinpigletstobepresentinIndia(Barmanet al.2003)andThailand(3daysofage)orallyinoculatedwiththeclassicalPEDV 506SectionIIIViralDiseasesCV777isolate(Coussementet al.1982;Deboucket al.TTR‐2andTC‐PC177strainsthathavealargedeletion1981)(Table 31.1).ClinicalsignswereobservedafterintheNTDoftheSproteinwasmildercomparedwith22–36hours.Viralreplicationoccurredmainlyinthethatoftheemergingnon‐SINDELPEDVstrains(Lincytoplasmofvillousepithelialcellsthroughoutthesmallet al.2016;Suzukiet al.2016).intestineasearlyas12–18hourspostinoculation(PI),TheinfectiousdosesofPEDVdifferfordifferentagespeakingat24–36hours.Infectionresultedindegenera-ofpigs:100‐to1000‐foldlessPEDVwasneededtoinfecttionofenterocytes,leadingtoareductioninthevillousyoungerpigscomparedwiththedoserequiredtoinfectheight/cryptdepth(VH/CD)ratiosfromthenormal7:13‐week‐oldpigs(Thomaset al.2015).Theinfectiousto≤4:1.ThepathogenicfeaturesofclassicalPEDVinthedoseofanemergingnon‐SINDELPEDVstrain(PC22A)smallintestineofpigletswereverysimilartothoseofwasaslowas0.1plaque‐formingunit(PFU)/pigin4‐TGEV,butsomewhatlesspronounced(Figure 31.5).day‐oldCesarean‐derivedcolostrum‐deprived(CDCD)PEDVreplicationwasalsoobservedinthecolonicepi-piglets(Liuet al.2015b).Dosesof0.1PFU/pigandtheliumwhereslightcelldegenerationwasseen1–10,000PFU/pigcauseddiarrheain40and100%pig-(Ducatelleet al.1982).Occasionally,PEDV‐positivelets,respectively.Thomaset al.(2015)comparedthecryptcellswerealsoobservedbyIHCorIFstaining,butinfectiousdosesforanotheremergingnon‐SINDELtheenterocyteregenerationcapacitywaspreservedPEDVstrain(USA/IN19338/2013)in5‐day‐oldand3‐(Deboucket al.1981;Sueyoshiet al.1995).Shibataet al.week‐oldpigs:0.056and0.56–5600TCID50/pigcaused(2000)showedthatSPFpigsinoculatedwithfieldPEDVdiarrheain25and100%ofneonatalpiglets,respectively,betweentheagesof2daysand12weeksdevelopedage‐andatleast100‐foldhigherdoses(56–5600TCID50/pig)dependentresistance.Thatis,mortalitywasonlycauseddiarrheain100%of3‐week‐oldpigs.However,observedin2‐to7‐day‐oldpiglets.Pathogenicfeaturestheinfectiousdoseforolderpigs,suchasfinisherpigs,ofPEDcausedbyclassicalPEDVstrainsdescribedinhasnotbeendetermined,butisexpectedtobehigherKoreaandJapanareverysimilartothosereportedinthanthatneededtoinfectweanedpigs,aswasobservedEurope(KimandChae2003;Sueyoshiet al.1995).forTGEV(WitteandWalther1976).Lohseet al.(2016)studiedPEDVpathogenicityin5‐DuringtheacutephaseofPEDVinfection,viralRNAweek‐oldpigsusingclassicalPEDVstrain(TCBr1/87,wasdetectedtransientlyintheserumofPEDV‐infectedP3),anemergingSINDELstraininGermany,andasucklingandweanedpigs(Chenet al.2016a;Junget al.non‐SINDELstrainintheUnitedStates.Unfortunately,2014,2015a;Lohseet al.2016;Suzukiet al.2016).PeaktheSINDELPEDVfailedtoinfectpigs.ComparedwithRNAtitersinserumwerelow(7–8log10GE/mL)com-theclassicalPEDV‐infectedpigs,thenon‐SINDELparedwithconcurrenthighpeakRNAtitersinfecesPEDV‐infectedpigshadmoresevereclinicalsignsand(11–12log10GE/mL)(Junget al.2015a).Whetherdetec-histopathologicalchanges,higherpeakviralRNAshed-tionofviralRNAinserumrepresentsinfectiousvirusdingtitersinfeces,andlongerdetectionofviralRNAinandtheroleofviremiainPEDVpathogenesisisunknown.serum.Theseresultssuggestedthattheemergingnon‐SIngeneral,PEDVRNAtitersareabout4–6log10higherINDELPEDVwasmorevirulentthanclassicalPEDV.thaninfectioustiters(PFUorTCID50)dependingondif-However,concernsrelatedtodatainterpretationincludeferentPEDVstrainsand/orthereal‐timeRT‐PCRassaysthefollowing:(1)TheBr1/87inoculumwastheVerocell(Junget al.2014;Songet al.2016;Thomaset al.2015).culture‐adaptedvirusatpassage3,whereasthenon‐SLowlevelsofPEDVRNAwerealsodetectedinotherINDELwasthewild‐typevirusfrompigsanditsinfec-tissues,suchasthelung,liver,spleen,andmuscleofpigstiousdosewasnotdetermined,sothediseaseoutcomeseuthanizedduringacutePEDVinfection(Chenet al.maybeduetodifferentinfectiousdoses.(2)Thenon‐S2016a;Lohseet al.2016;ParkandShin2014).However,INDELinoculumcontainedalowamountofrotavirusbybecausethebloodwasnotdrainedbeforecollectingeachconventionalRT‐PCR.Therefore,coinfectionwithtissue,theviralRNAwasmostlikelyfromblood,exceptPEDVandrotavirusmayinfluencediseaseseverity.forthelungs,wherePEDVantigensweredetectedbyGenerally,thepathogenesisandtheage‐dependentIHC(ParkandShin2014).Inthelaterstudy,theresearch-resistanceoftheemergingnon‐SINDELPEDVstrainsersfoundthatawild‐typeKoreanPEDVnon‐SINDELweresimilartothoseoftheclassicalPEDVstrainsstrainCNU‐091222‐01/2009replicatedinalveolar(Table 31.1)(Junget al.2014,2015a;Madsonet al.2014;macrophagesofinfectedpigs.BecausenoothershaveNiederwerderet al.2016;PensaertandMartelli2016;reportedthedetectionofPEDVinthelungs,whetherStevensonet al.2013).Comparedwiththeemergingthisisauniquecharacteristicofearlieremergingnon‐Snon‐SINDELPEDVstrains,SINDELPEDV‐infectedINDELstrains(pre‐2010)isunknownandneedstobepigletshadlowermortalityratesandlessseverehisto-investigated.Inaddition,PEDVRNAwasdetectedfrompathologicalchanges(mildervillousatrophy)andless40.8%(20/49)ofsowmilksamplesduringtheemergingantigeninthesmallintestine(Table 31.1)(Chenet al.PEDVepidemics(Sunet al.2012).TGEVreplicatedin2016a;Linet al.2015a).ThepathogenicityofPEDVthemammaryglandsofsowsinjectedintramammarily Table31.1Comparativepathogenesisof differentclustersof PEDVin experimentallyinfectedpiglets(youngerthan6daysof age).VerticallocationofLongitudinaldistributionPEDVofPEDVPigtype/age(day)atVillousatrophyOnsetofclinicalPEDVstrainInoculum/doseperpiginoculation(VH:CDratios)signs(hpi)VillousCryptD,J,ICReferencesClassicalCV777Fecalsuspension/4log10PIDCDCD/2–3Moderatetosevere22–36+++(entire)+D,J,I(cont)+Coussementet al.(1.5–4.2)(1982)SNUVR971496Cellculture(P3)/6.8log10Colostrum‐deprived/1Severe(1.1–3.3)12–36+++(entire)−D,J,I(cont)−KimandChaeTCID50(2003)Non‐SINDELIN19338Cellculture(P7)/0.056–5600Conventional/5Severe(1.2–1.7)24+++(entire)NRD,J,I(cont)NRThomaset al.(2015)TCID50PC22ACellculture(P3)/1–4log10CDCD,conventional/3–4Severe(0.8–2.3)<24+++(entire)+D,J,I(cont)+Liuet al.(2015b)PFUSINDELIowa106Fecalsuspension/10–12Conventional/4Moderatetosevere24–72++(entire)−D(patchy),J,I−Linet al.(2015)log10GE(1.4–5.4)(cont)IL20697Cellculture/5log10TCID50ConventionalwithmilkMildNR+/++NRD,J(NR),I(patchy)+Chenet al.(2016a)replacer/5Source:Thistableisadapted/updatedfromSaif(1989).ReproducedwithpermissionofTaylorandFrancis.PID,piginfectiousdose;TCID50,50%tissuecultureinfectiousdose;PFU,plaque‐formingunit;GE,genomicequivalent;CDCD,Cesarean‐derivedcolostrum‐deprived;SPF,specificpathogenfree;VH:CD,villousheight/cryptdepthratio;hpi,hours’postinoculation;D,duodenum;J,jejunum;I,ileum;cont,continuous;NR,notreported;−,+,++,and+++denotesnone,lessthan30%,30–60%,andmorethan60%ofvillousenterocytesthatwerepositiveforPEDVantigens,respectively.ChapterNo.:1TitleName:0004238433.INDDComp.by:MohamadabdulRasheethDate:06Mar2019Time:09:14:50PMStage:PrinterWorkFlow:CSWPageNumber:5070004238433.INDD50703/06/20199:15:22PM 508SectionIIIViralDiseaseswithliveTGEVduringlactation(SaifandBohl1983).LesionsWhetherPEDVreplicatesinthemammaryglandsofLesionshavebeendescribedinexperimentallyandnaturallysows,whetherthePEDVRNArepresentsinfectiousinfectedsucklingpigletsforclassicalPEDV(CoussementPEDV,orwhetherthePEDVRNAinmilkisfromtheet al.1982;KimandChae2003;Pospischilet al.1981;salivaoftheirinfectednursingpigletsisunclear.Sueyoshiet al.1995),emergingnon‐SINDEL(Chenet al.AlthoughsomePEDVstrainsmayreplicateatlowlevels2016a;Junget al.2015a;Madsonet al.2015;Stevensonet al.outsideoftheintestine,itremainsunclearwhetherit2013),andSINDELPEDVinfections(Chenet al.2016a;LincontributestoPEDVpathogenesis.et al.2015a).LesionsareconfinedtothesmallintestinethatPEDVinfectionresultsinmassivelossofenterocytesisdistendedwithwatery,yellowishfluid.Microscopically,andthemalfunctionofinfectedenterocytes,leadingtovacuolation,syncytia,andexfoliationofsmallintestinalmaldigestiveandmalabsorptivediarrhea(Coussemententerocytesoccurmainlyontheproximalvilli.Thesmallet al.1982;Deboucket al.1981;Junget al.2006).Duringintestinalvilliarereducedinlength(Figure 31.5d),andtheacutePEDVinfection,gutintegritywasreduced,leadingenzymaticactivityoftheintestineismarkedlydecreased.tolossofwaterintotheintestinallumenandhighThispathologyisverysimilartothatobservedinTGEandosmoticpressure(Annamalaiet al.2015).ThefollowingPDCoV(Figure 31.5).Nohistopathologicchangeshavebeenfactorsmaycontributetothemoresevereclinicalsigns,observedinthecolon,althoughPEDVantigenswerehighermortalityrates,andslowerrecoveryinPEDV‐detectedinvacuolatedcolonicepithelialcells(Chenet al.infectedneonatalpigletscomparedwithweanedpigs:2016a;Deboucket al.1981;Junget al.2014).1)Slowerturnoverofvillousenterocytesinneonatalpiglets(5–7days)comparedwith2–3daysin3‐week‐oldweanedpigs(Junget al.2015a;Moonet al.1975).Diagnosis2)Increasednumbersofintestinalstemcellsandprolif-Diagnosisshouldbemadebasedonbothclinicalsignsanderationofcryptcellsoccurredlater(3dayspost‐laboratorydetectionofviralRNA,viralantigens,orPEDV)inneonatalpigletsthaninweanedpigs(1dayincreasedPEDVantibodies.ForthedetectionofPEDVpost‐PEDV)(Junget al.2015a).RNA,themostwidelyusedlaboratorydiagnosticmethodis3)Deficiencyininnateimmunityinsucklingpigscom-RT‐PCR(Ishikawaet al.1997;Kimet al.2001;Kubotaet al.paredwithweanedpigs(Annamalaiet al.2015)(see1999;LiuandWang2016)orreal‐timeRT‐PCR(Kimet al.“Immunity”).2007;Wanget al.2014d;Zhanget al.2016b).Loop‐medi-atedisothermalamplification(LAMP)assays(RenandLiClinicalsigns2011;Yuet al.2015)havebeendevelopedforthedetectionPEDsharesmostclinicalfeatureswithTGEincludingofPEDVRNAbutareusedlessindiagnosticlaboratories.waterydiarrhea,vomiting,anorexia,anddepression.OnNewertechnologysuchasthespecificprimer‐independentbreedingfarms,pigsofallagesbecomesick.Morbiditymetagenomicsequencing(next‐generationsequencing)approaches100%inpiglets,butcanvaryinsows.Pigletscanbeusedtodeterminethenearlycomplete(lacking5′upto1weekofagemaydiefromdehydration,andmor-and3′ends)viralgenomesequencesfromclinicalspeci-talityrangesfrom50to100%.Olderpigsrecoveraftermens(Chenet al.2014;Marthaleret al.2013).Insituabout1week.Insows,diarrheaisvariable,andtheymayhybridizationcanbeusedtodetectPEDVRNAinfixedtis-onlyshowdepressionandanorexia.Infatteningpigs,allsues(KimandChae2000;Stadleret al.2015).pigsintheunitmayhavewateryfeceswithinaweekandDiagnosiscanbemadebydirectdemonstrationofoftenshowsevereanorexiaanddepression.PEDVand/oritsantigensusingIForIHCtestsontheThediseaseonabreedingfarmisself‐limitingandsmallintestinaltissuesofpigseuthanizedacutelynearstopswhenthepregnantsowsdeveloplactogenictheonsetofdiarrheaandpriortothedesquamationofimmunitytoprotecttheiroffspring.Theintervalenterocytes(Deboucket al.1981;Guscettiet al.1998;betweenonsetandcessationofthediseaseisgenerallyJunget al.2014;Stevensonet al.2013;Sueyoshiet al.3–4weeks,butmaybemuchlongerinlargebreeding1995)(Figure 31.6b).PEDVparticlescanbedemon-farmswithmultipleseparatedunits.AftertheacutestratedusingdirectEMorIEMoffecesofpigscollectedoutbreakhaspassed,diarrheamaypersistonthefarmacutelyafterdiarrheaonset.Virusparticlesaredifficultinweanedpigsandbecomerecurrent(Martelliet al.torecognizewhenthevirionspikesarelostornotclearly2008).PEDVmayalsobeinvolvedinamulti‐etiologicvisible.Furthermore,IEMmustbeappliedtodifferenti-diarrheasyndromeinfeederpigsappearing2–3weeksatePEDVfromTGEVandPDCoVbecausetheCoVsaftertheyenterthefatteningunits,particularlywhenhaveidenticalmorphology(Figure 31.2).thepigsoriginatefromdifferentsourcesandwhennewIsolationoffieldstrainsofPEDVfromintestinalcon-pigsarecontinuouslyaddedtothefatteningunit(vantents/homogenatesorfecesisdoneinVerocellsorinReethandPensaert1994).othercelltypes.Trypsintreatmentandblindpassages 31Coronaviruses509maybeneededbeforeCPEappears,butearlydetection11proteinshavebeenidentifiedasIFNantagonists,canbedonebyIFstaining(HofmannandWyler1988;whichincludebothORF1ab‐encodedNSproteins(nsp1,Shibataet al.2000).SuccessfulisolationofPEDVinVeronsp3,nsp5,nsp7,nsp14,nsp15,nsp16),structuralpro-cellsishigherwithintestinalcontents/homogenatesthanteins(E,M,N),andtheaccessoryproteinORF3(Dingwithfeces(Chenet al.2014;Okaet al.2014).et al.2014;Wanget al.2015;Zhanget al.2016c).Antigen‐captureELISAshavebeendevelopedforIdentificationofthevirus‐encodedIFNantagonistsanddetectionofPEDVantigensinfecesusingpolyclonalunderstandingtheirmechanismofactionmayleadtoantibodiesandMAbs(Callebautet al.1982;Carvajalnoveltherapeutictargetsandmoreeffectivevaccines.et al.1995),buttheyarenotwidelyused.PEDV‐infectedsucklingpigshadsignificantlylowerPairedserumsamplesarerequiredforserologicdiag-NKcellfrequencies,undetectableNKcellactivity,and−−+nosisofendemicPEDV.Recently,IgGandIgAantibodieslowerIFN‐γ‐producingCD3CD4CD8NKcellsintoPEDVweredetectedinoralfluids,suggestingtheymaybloodandileumcomparedwithPEDV‐infectedweanedbesuitabletomonitorpriorherdexposuretoPEDVpigs(Annamalaiet al.2015).Deficiencyininnate(Bjustrom‐Kraftet al.2016).PEDVantibodieshavebeenimmunefunctionofneonatalNKcellsmaycontributetodemonstratedwithindirectELISAsusingantigenscon-themoreseverePEDVinfectioninsucklingpigscom-sistingofcell‐cultivatedvirus(Carvajalet al.1995;paredwithweanedpigsasalsoreportedforTGEVinfec-HofmannandWyler1990;Kweonet al.1994;Thomastions(Derbyshireet al.1969).et al.2015),orSandNviralproteinsextractedfromInflammatoryresponsesplayasignificantroleintheinfectedVerocells(Knuchelet al.1992;Ohet al.2005),orpathogenesisofentericCoVs.Comparedwithsucklingexpressedinbacteriaorusingmammalianexpressionpigs,weanedpigshadadelayedproinflammatorysystems(Chenet al.2016b;Gerberet al.2014;GerberandcytokineinductionthatcoincidedwiththedelayedonsetOpriessnig2015;Houet al.2007;Okdaet al.2015;Paudelofinfection,disease,andsheddingofPEDVRNAinfeceset al.2014;Wanget al.2015).Blockingandcompetitive(Annamalaiet al.2015).Toll‐likereceptor2(TLR2),ELISAshavealsobeendevelopedforthedetectionofTLR3,andTLR9maycontributetoNF‐κBactivationinPEDVantibodiesusingMAbsorpolyclonalantibodiesasresponsetoPEDVinfectioninsmallIECsinvitro(Caocompetitiveantibodies(Carvajalet al.1995;Okdaet al.et al.2015).TheviralproteinsEandNupregulatedIL‐82015;vanNieuwstadtandZetstra1991).SerumIgGanti-expressionbyinducingendoplasmicreticulumstressbodiesagainsttheNproteinsofPEDVcanbedetectedbyandsubsequentactivationoftheNF‐κBpathway(Xu9–14DPI,withtiterspeakingaround21DPIandthenet al.2013a,b).declininggradually(Okdaet al.2015).Recently,afluores-HumoralimmuneresponsestoPEDVinfectionarecentmicrosphereimmunoassay(FMIA)wasdevelopedverysimilartothosedescribedforTGEV[reviewedin(Gimenez‐Lirolaet al.2017;Okdaet al.2015),butitisnotTGEVsection(Chatthaet al.2015;SaifandSestakwidelyusedduetotheneedforspecificequipment.The2006)].VNantibodiesaredetectableintheserum,butVNtestinVerocellsiscriticaltoassessVNantibodiestomaynotplayanimportantrolebecauseprotectionPEDV(Ohet al.2005;Okdaet al.2015;Paudelet al.2014;againstentericdiseaseisprimarilydependentontheThomaset al.2015).TheseserologicalassayshavebeenpresenceofsIgAantibodiesintheintestinalmucosawidelyusedtomonitorpriorexposuretothevirusandto(Chatthaet al.2015;Langelet al.2016).Immunitymayevaluatetheefficacyofvaccines.notbelonglasting,butarapidanamnesticresponsePEDVinfectionsmustbedifferentiatedfromTGE,uponreexposuremaypreventreoccurrenceofSeCoV,andPDCoV,whichinthecaseofacutediarrheadisease.inswineofallagescanonlybedonethroughlaboratoryAlthoughPEDoccursinpigsofallages,pigletsuptotesting.SinceSeCoVsarerecombinantsbetweenTGEV1weekofagemayexperiencehighmortalityandneedto(backbone)andPEDV(mainlySprotein),onlyassaysbeprotectedbymaternalantibodies,especiallyVNandtargetingbothTGEV(anygenesexceptforSgene)andsIgA,viacolostrumandmilkfromimmunizeddams.PEDV(Sgene)fragmentscanidentifythoseviruses.InThemechanismsoflactogenicprotectiondescribedforneonatalcolibacillosisorrotavirusdiarrhea,adultani-TGEVinfectionapplytoPEDaswell(reviewedinTGEVmalsarenotaffected,andsickpigsusuallyarebornfromsection[Chatthaet al.2015;Langelet al.2016]).giltsoryoungsows.LaboratorytechniquesmustbeusedLactogenicimmunityisinducedinsowsbyintestinaltodifferentiatePEDfromothercausesofdiarrheaininfectionwithPEDV,whichthenactivatesthegut–weanedorfeederpigs.mammary–sIgAaxis.PigsloselactogenicprotectionatweaningandsoonbecomesusceptibletoPEDVinfec-tion.Cell‐mediatedimmunitylikelyplaysaroleinviralImmunityclearance,butthereisnoexperimentaldataonthistopic.EvidenceshowsthatPEDVhastheabilitytoevadehostPEDVmaypersistonthefarminsusceptiblepigsaspartIFNresponses.Of21PEDV‐encodedproteins,atleastofrecurringweaningdiarrheaafteranacuteoutbreak. 510SectionIIIViralDiseasesPreventionand controlPreviously,PDCoVhadbeenreportedinthefecesofdomesticpigsinChinain2012(Wooet al.2012),butthePEDVishighlycontagious,andstrictsanitationandroleofthevirusasanentericpathogenwasunclearatbiosecurityarerequiredtopreventvirusentrance.Dothattime.PDCoVhasspreadnationwideintheUnitednotcomminglesourcesorgroupsofpigs;ensurefacili-States(Wanget al.2014c)andcauseddeathsamongtiesandtransportationvehiclesarethoroughlywashed,sucklingpigs(Anon2014).Experimentalstudiesverifieddisinfected,anddriedbeforepigsenter;anddonotsharethatUSPDCoVisolatesareenteropathogenicinnursingboots,clothing,orequipmentbetweendifferentagespigs,asevidentbyacute,waterydiarrheaandsevereof pigs.intestinallesions(Figure 31.5cande)(Chenet al.2015;Feedback(intentionalexposureofsowstovirususingJunget al.2015b).However,theclinicalimpactanddis-fecesorsmallintestinesfromacutelyinfectedpiglets)easeseverityofPDCoVislessthanthatofepidemicwillstimulatelactogenicimmunityinthesowherd,PEDVandTGEV(Anon2014).reduceclinicalsignsinpiglets,andshortenclinicalout-SincethePDCoVoutbreaksintheUnitedStates,ithasbreaks.Feedbackmayalsobeusedinthenursery,grower,alsobeenidentifiedonswinefarmsinCanada,Korea,orfinisherpigs,butnose‐to‐nosecontactandfecal–oralChina,Thailand,Vietnam,andLaosPDR,butinCanadaspreadwillquicklycontaminatetheentirefacility.ItandKorea,PDCoVfailedtospreadnationwide(Leeet al.shouldberecognizedthatotherpathogenspresentin2016b;Marthaleret al.2014b).DifferentialdiagnosisofclinicallyaffectedanimalscanbetransmittedviathePDCoV,PEDV,andTGEViscriticaltocontrolCoVdiar-feedbackprocess.rheasinpigfarms,especiallyintheregionswheretheseInEurope,thediseasehasbeenofinsufficienteco-CoVshaveemergedorreemerged.nomicimportancetodevelopvaccines.InChina,vari-ousinactivatedand/orattenuatedbivalent(TGEVandPEDV)ortrivalent(TGEV,PEDV,androtavirusstrainEtiologyNX)vaccinesbasedontheclassicalPEDVstrainshavePDCoVbelongstothegenusDeltacoronavirusofthebeenavailableasearlyasin1999(Maet al.1995;SunfamilyCoronaviridae.Morphologicandphysico-et al.2016;Wanget al.2016a).However,classicalPEDVchemicalpropertiesofPDCoVaresimilartothoseofvaccineswerenotefficaciousinprotectingpigsagainstothermembersinthefamilyCoronaviridaethehighlyvirulentnon‐SINDELPEDVinfection(Li(Figure 31.2).et al.2012;Sunet al.2012;Wanget al.2013)andnewerAllglobalPDCoVstrainsoverallsharehighnucleotidevaccines(e.g.abivalentinactivatedPEDVandTGEVidentities(Zhang2016)(see“Epidemiology”).However,vaccinebasedontheemergingnon‐SINDELPEDVacomprehensivegeneticanalysisofglobalstrainsstrainAJ1102)haveappearedinthemarket.InJapan,arevealedthatUS/KoreanPDCoVstrainsclusteredcommercialattenuatedPEDVvaccinebasedontheclas-together,Chinesestrainsclusteredseparately,andThaisicalPEDV83P‐5strainhasbeeninusesince1997(Satostrainsformedanothercluster(Zhang2016)(Figure 31.1).et al.2011).InKorea,attenuatedvaccinesbasedonChinesePDCoVstrainshadmultiplemutationordele-strainsKPEDV‐9(Kweonet al.1999)orDR13(SongtionsitesintheirS,NSP,or3′untranslatedregion(UTR)et al.2007)werecommercializedin1999and2004,genes,whereasthesemutationswerenotfoundintherespectively.Reportedly,notallsowsgiventhevaccinesgenomesofUSPDCoVstrains(Wanget al.2016c).developedprotectivelactogenicimmunity(Songet al.Severalinvestigatorsreportednocross‐reactivityof2015a).TwocommercialvaccinesareavailableinthePDCoVwithantibodiestoeitherPEDVorTGEV(ChenUnitedStates.Thefirstvaccine(June2014)wasdevel-et al.2015;Maet al.2015).However,anotherstudyopedusingareplication‐deficientVenezuelanequinereportedantigeniccross‐reactivitybetweenUSPDCoVencephalitisviruspackagingsystemtoexpresstheandPEDVstrains,possiblysharingatleastoneepitopePEDVSprotein(Crawfordet al.2016).Thesecondvac-ontheirNproteins(Maet al.2016).cine(September2014)isaninactivatedwholevirusvac-LLCporcinekidney(LLC‐PK)andSTcellssupple-cinebasedonanemergingnon‐SINDELPEDVstrainmentedwithexogenoustrypsinorpancreatinsupport(Crawfordet al.2016).theisolationandserialpropagationofPDCoVincellculture(Huet al.2015).TheCPEconsistedofenlargedandroundedcellsandthencellshrinkageandPorcinedeltacoronavirusdetachment.RecentstudieshavedemonstratedthatPDCoVRelevanceemployspAPNasamajorreceptorforcellularentry,InFebruary2014,acuteoutbreaksofdiarrheaassociatedalthoughitremainstobeelucidatedwhetheranotherwithPDCoVwereobservedinsowsandtheirpigletsonreceptorisinvolvedinPDCoVinfection(Liet al.2018;fiveOhiofarmsintheUnitedStates(Wanget al.2014a).Wanget al.2018;Zhuet al.2018). 31Coronaviruses511PublichealthTheThaiPDCoVstrainswerehighlysimilartoeachotherbutformedanovelphylogeneticclusterseparatedThereisnoevidencethatPDCoVisinfectiousforfromUSandChinesePDCoVs(Janetanakitet al.2016;humansorplaysaroleinpublichealth.Saeng‐Chutoet al.2017).PDCoVsidentifiedinLaoPDRweremorecloselyrelatedtoThaiPDCoVs,whereasPDCoVsdetectedinVietnamweremorecloselyrelatedEpidemiologytoUSPDCoVs(Saeng‐Chutoet al.2017).TheancestraloriginofPDCoVisunclear,butconsider-Fecal–oralisthemainPDCoVtransmissionroute.ingthatPDCoVemergedrecently,PDCoVmaybeFecesand/orvomitusandothercontaminatedfomitesincompletelyadaptedtopigs.Molecularsurveillanceinaremajortransmissionsourcesofthevirus.BasedonChinaandHongKongin2007–2011detectedDCoVsexperimentalfindings(Huet al.2016;Maet al.2015;onlyinpigsandwildbirds(Wooet al.2012).However,Zhang2016),diarrheaininfectedpigletswasobservedDCoVswerepreviouslyisolatedfromrectalswabsofforapproximately5–10days,withpersistingviralRNAsmallmammals,includingAsianleopardcatsandsheddingforupto19–28daysinfecesandforuptoChineseferretbadgers,atChineseliveanimalmarketsin42daysinoralfluids.2005–2006(Donget al.2007).TheirhelicaseandSgeneswerecloselyrelatedtothoseofPDCoV.ThedatasuggestPathogenesisthepotentialinterspeciestransmissionofDCoVsbetweenthesewildsmallmammals,pigs,andbirds.AThepathogenesisofPDCoVhasbeenstudiedinGnorrecentstudyalsorevealedthatPDCoV‐inoculatedgno-conventionalpigletsorallyinoculatedwithUSand/ortobiotic(Gn)calvesexhibitedanacuteinfectionwithoutChinesePDCoVisolatesat5–21daysofage(Chenet al.diseaseorintestinallesions,butwithpersistingfecal2015;Donget al.2016;Huet al.2016;Junget al.2015b;viralRNAsheddingandseroconversion(Junget al.Maet al.2015).Clinicalsigns(diarrheaand/orvomiting)2017).Consequently,thepotentialabilityofPDCoVandoccurredat1–3DPI.ReplicationofPDCoVisconfinedotherDCoVisolatesfrombirdsorsmallmammalstotothesmallandlargeintestinalepithelia.PDCoV‐infectdifferentspeciesshouldbeinvestigated.infectedenterocytesrapidlyundergoacutenecrosisInFebruary2014,PDCoVwasdetectedinUSswine.(Junget al.2016a),leadingtomarkedvillousatrophyinAmong42fecalorintestinalsamplescollectedfromthesmallintestine(Figure 31.5cande),butnotinthediarrheicsowsandpigletsonfiveOhiofarms,39(92.9%)largeintestine.Duringacuteinfection,PDCoVantigenswerepositiveforPDCoVbyRT‐PCR(Wanget al.2014a).aredetectedmainlyinthevillousepitheliumoftheatro-ThePDCoVOhiostrainHKU15‐OH1987hada99%phiedmid‐jejunum(Figure 31.6c)toileumand,toanucleotideidentitytothetwoprototypestrainsoflesserextent,induodenum,proximaljejunum,andPDCoV,HKU15‐44andHKU15‐155,reportedincecum/colon.Occasionally,afewPDCoVantigensareChinesepigsin2012.Duringasimilarperiod,geneticallydetectedincryptepithelialcellsofthejejunumandsimilarstrains,USA/IA/2014/8734andSDCV/USA/ileum(Junget al.2016a)andimmunecellsintheintesti-Illinois121/2014,wereidentifiedbyotherUSdiagnosticnallaminapropria,Peyer’spatches,andmesentericlaboratories(Liet al.2014;Marthaleret al.2014a).lymphnodes(Huet al.2016).Frequently,acuteviremiaAmongPDCoV‐positivepremises,coinfectionwithwithlowPDCoVRNAtitersinserumwasobservedPEDViscommon(Zhang2016).TheoriginofPDCoVin(Chenet al.2015;Huet al.2016;Maet al.2015).AfterUSswineisunknown,althoughtherewasserologicandpigsrecoveredfromclinicaldisease,largeramountsofvirologicevidencesuggestingitspresenceintheUnitedPDCoVantigensweredetectedinthegutlymphatictis-StatespriortoitsdetectioninFebruary2014(Sinhaet al.sues(Huet al.2016).PDCoVantigenswerenotdetected2015;Thachilet al.2015).inotherorgans,includingtherespiratorytractofpigsPDCoVhasalsobeenreportedinCanada,Korea,(Junget al.2016b).However,byreal‐timeRT‐PCR,mainlandChina,Thailand,Vietnam,andLaoPDR(DongPDCoVRNAcouldbedetectedinlowtomoderateet al.2015;Janetanakitet al.2016;Leeet al.2014;quantitiesinmultipleorgans,possiblyduetoviremiaLorsirigoolet al.2016;Marthaleret al.2014a;Saeng‐(Chenet al.2015;Maet al.2015).Chutoet al.2017;Songet al.2015b).TheKoreanPDCoVstrains(KUN14‐04,SL2,andSL5)hadhighnucleotideClinicalsignsidentities(98.7–99.2%)toUSPDCoVstrains(Leeet al.2014,2016b).InmainlandChina,coinfectionswithClinicalsignsofPDCoVinfectioninsucklingandolderPDCoVandPEDVwerecommon(Donget al.2015;Songpigsaresimilar,butmilder,thanthoseofPEDVandet al.2015b).ChinesePDCoVstrainshad≥98.6%nucleo-TGEVinfections.Insucklingpiglets,PDCoVinducestideidentitieswitheachotherand≥97.1%nucleotideacute,waterydiarrhea,frequentlyaccompaniedbyidentitieswiththeglobalPDCoVstrains(Zhang2016).vomiting,leadingtodehydration,lossofbodyweight, 512SectionIIIViralDiseaseslethargy,anddeath.Experimentally,theonsetofdiar-atrophicenteritisintheproximaljejunumtoileumrheacoincidedwith,orwasdetected1–2dayslaterthan,(Figure 31.5cande),occasionallyaccompaniedbymildthefirstdetectionofviralRNAinfeces(Junget al.2015b;vacuolationofthesuperficialepithelialcellsinthececumMaet al.2015).andcolon(Junget al.2015b).Novillousatrophyorhis-Diarrheaisprobablyaconsequenceofmalabsorptiontologiclesionswereevidentintheduodenum,whichduetomassivelossofabsorptiveenterocytes,resultingcoincidedwithfewPDCoVantigen‐positiveduodenalindecreasedbrushbordermembrane‐bounddigestiveepithelialcells(Chenet al.2015;Junget al.2015b).enzymes,similartoPEDVinfection(Junget al.2006).Duringacuteinfection,vacuolatedenterocytesormas-Mildvacuolationobservedintheinfectedcolonicepi-sivecellexfoliationwasseenonthetipsortheentirevillithelialcellsmayinterferewiththereabsorptionofwaterinthejejunumandileum.Atrophiedvilliwerefrequentlyandelectrolytes(Junget al.2015b).Dehydrationisalsofusedandcoveredwithadegeneratedorregeneratedexacerbatedbyvomiting.flattenedepithelium.Infiltrationofinflammatorycells,SeronegativepigsofallagesaresusceptibletoPDCoVsuchasmacrophages,lymphocytes,andneutrophils,infection.Onseronegativefarrowingfarms,morbiditywasevidentinthelaminapropria.Nolesionswereseencanreachupto100%inpigletsbutcanvaryinsows.inotherorgans.BasedonfieldobservationsinUSswinein2014(Anon2014),PDCoVinfectioncausedanumberofdeaths(upDiagnosistoa40%mortality)amongsucklingpigs.Similarly,PDCoVdiarrheaoutbreaksinbreedingfarmsinChinaThediagnosticapproachesdescribedearlierforTGEVandThailandresultedin64–80%mortalityamongsuck-andPEDValsoapplytoPDCoVdiagnosis.Laboratorylingpiglets.PDCoVinfectionismoresevereandmoretechniquesshouldbeusedtodifferentiatePDCoVinfec-likelytoresultinmortalityinpigletsascomparedwithtionfromPEDV,TGEV,androtavirusdiarrheainpigs.olderpigs.Onmanyfarms,morbidityandmortalitymayDefinitivediagnosisofPDCoVinfectionincludesdetec-beaffectedbycoinfectionswithotherentericviruses,tionofPDCoVRNAorantigensinthefecesorintestinalsuchasPEDVandrotavirus(Marthaleret al.2014b;tissuesfromdiarrheicpigs.DiagnosiscanbemadebySonget al.2015b).Thediseaseonbreedingfarmsisself‐RT‐PCRassaysthattargetaconservedregionofPDCoVlimitingandstopswhenpregnantsowsdeveloplacto-MorNgenes(Marthaleret al.2014b;Wanget al.2014a;genicimmunitytoprotecttheiroffspring.Zhanget al.2016b),IForIHCusingvirus‐specificMAbsPDCoVinfectionsharesseveralclinicalfeatureswithorpolyclonalantibodies(Chenet al.2015;Junget al.TGEVandPEDVinfections,buttheviruslikelyspreads2015b;Maet al.2015),andinsituhybridization(Jungmoreslowlyamongpigs,possiblyduetoitsloweradap-et al.2015b).Areal‐timeduplexRT‐PCRassayfordetec-tationtopigs.RelativetoPEDVinfections,PDCoV‐tionofPDCoVand/ordifferentiationofthevirusfrominfectedpigsshedlessPDCoVRNAinthefeces(JungPEDVinintestinesandfeceswasdeveloped(Zhanget al.et al.2015b),indicatinglowerreplicationofPDCoVin2016b).theintestineofpigs.ThisaspectofPDCoVinfectionmayDirectEMcanbeusedtodemonstratePDCoVparti-beacontributingfactortoitslowermortalityinnursingclesinfecescollectedfromdiarrheicpigs(Figure 31.2d),piglets,ascomparedwithPEDVinfections.butIEMusinghyperimmuneorconvalescentseraisessentialtodifferentiatePDCoVfromPEDVorTGEV(Junget al.2015b).IsolationofPDCoVfromfecesorLesionsintestinaltissueswasattemptedinLLC‐PKorSTcells,Lesionshavebeendescribedinsucklingpigletsexperi-butthesuccessratewaslowexceptforafewstrains(OH‐mentallyandnaturallyinfectedwithUS,Chinese,orFD22)(Huet al.2015).SerologicdiagnosisofPDCoVThaiPDCoVstrains(Chenet al.2015;Donget al.2016;canbeconductedbyIFA,VN,andELISAassays.IsotypesHuet al.2016;Janetanakitet al.2016;Junget al.2015b;ofPDCoVantibodiesinserumandmilkcanbequanti-Maet al.2015;Wanget al.2016c).LesionsresembletatedbyELISAusingantigensconsistingofcellculture‐thoseobservedinTGEVandPEDVinfectionsgrownvirus(Maet al.2016)orS1andNviralproteins(Figure 31.5),butareusuallylessextensive.(Okdaet al.2016;Suet al.2016;Thachilet al.2015).GrosslesionsarelimitedtothegastrointestinaltractandarecharacterizedbythinandtransparentintestinalImmunitywalls(proximaljejunumtocolon)withaccumulationoflargeamountsofyellowfluid.ThestomachisfrequentlyTheimmuneresponsesofpigstoPDCoVinfectionarefilledwithcurdledmilk.Thetransparencyandfragilitylargelyundefined,buttheyarelikelysimilartothoseofaffectedintestinesaremilder,ascomparedwithPEDVdescribedearlierforTGEVandPEDV.Huet al.(2016)andTGEVinfections.Histologicallesionsarecharacter-reportedthedevelopmentofPDCoVantibodiesinserumizedbyacute,multifocaltodiffuse,mildtosevereofPDCoV‐infectedpigs(Huet al.2016).Gnpigsorally 31Coronaviruses513inoculatedwiththeoriginalortissueculture‐grownEtiologyPDCoVstrainOH‐FD22haddetectableserumIgG,IgA,pHEVbelongstothegenusBetacoronavirusofthefam-andVNantibodiesby14DPIthatpeakedat24DPI,whenilyCoronaviridae(Figure 31.1).Thevirusagglutinatesthepigshadrecoveredfromclinicaldiseaseandfecalerythrocytesofmice,rats,chickens,andseveralothervirusshedding.WhilePDCoVinfectionisepidemic,animals.ThenaturalhostofpHEVisthepig.AlthoughyoungpigletscanbeprotectedbytransferofmaternalpHEVmayshowdifferentclinicalmanifestations,thereantibodiesviacolostrumandmilkfromimmunedams,isonlyoneserotype.Age‐relatedsusceptibilityoftheespeciallyIgAandVNantibodiesthatneutralizePDCoVpigs,possiblestraindifferencesinvirulence,andvaria-inthegut.Lactogenicimmunityisexpectedtobestronglytioninpathogenesismayinfluenceclinicalsigns.pHEVinducedinsowsbyoralinfectionwithPDCoV,whichshowsastrongtropismforneuraltissuesinpigs.thenactivatesthegut–mammarylink,asdescribedLikewise,thevirusdisplaysneurotropisminmiceandearlierforTGEV(Bohlet al.1972;Saifet al.1972).Wistarrats(Hiranoet al.2004;Yagamiet al.1993).Invitro,onlyporcinecellsaresusceptibletopHEV.Preventionand controlpHEVwasfirstisolatedinprimaryPKcellswithCPEcharacterizedbysyncytia(Greiget al.1962).pHEVwasThepreventionandcontrolmeasuresdescribedearlieralsoshownbyIFstainingtopropagateinotherporcineforTGEVandPEDVinfectionsalsoapplytoPDCoVcellcultures:adultthyroidgland,embryoniclung,andinfection.TherearenotreatmentsorvaccinestocontrolcelllinessuchasST,PK‐15,IBRS2,SK,SK‐K,andKSEK6PDCoVinfection.Preventiveortherapeuticantibioticswineembryokidney.therapycanbeimplementedifthereisconcurrentinfec-tionwithentericbacterialpathogens.Symptomatictreatmentofsucklingpigswithdiarrheaincludesintra-PublichealthperitonealadministrationofbicarbonatefluidsandfreePigsaretheonlyspeciesknowntobesusceptibletoaccesstowatertoalleviateacidosisanddehydration.IfpHEVandpHEVhasnopublichealthsignificance.mortalityissubstantialamongsucklingpiglets,feedbackmethods(intentionalexposureofpregnantsowstovirus‐positivemincedintestinesfromacutelyinfectedEpidemiologypiglets)willstimulatelactogenicimmunityandreducethehighmortalityifadministeredtosowsatleast2weeksSerologicsurveys(1960–1990)revealedthatpHEVpre‐farrowing.DuringPDCoVepidemics,high‐levelinfectioninswineoccursworldwideandisendemicinbiosecurityprocedurestoreducePDCoVtransmissionbothbreedingandfatteningswine(Pensaert2006).Theviacontaminatedfomitesareessential.presenceofpHEV,asdetectedbyisolationorserology,wasreportedinEurope,intheWesternHemisphere(UnitedStates,Canada,Argentina),inAsia(Japan,HemagglutinatingTaiwan),andinAustralia.encephalomyelitisvirus(vomitingpHEVismaintainedinswinepopulationsbyinfectingsuccessivegroupsofpigsafterreplacementorweaning.and wastingdisease)Thevirusisexcretedoronasally(Hiraharaet al.1989;PensaertandCallebaut1974)for8–10days.TransmissionRelevanceoccursvianasalsecretions,vianose‐to‐nosecontact,In1962,Greigandcoworkersisolatedaviralpathogenandaerogenically.Persistentviruscarriersarenotknownfromthebrainsofsucklingpigswithencephalomyelitistoexist.inCanada.Designatedhemagglutinatingencephalomy-Generally,pigswillonlydevelopdiseasewhentheyelitisvirus(HEV),theviruswaslaterclassifiedasaCoVbecomeinfectedoronasallypriorto3–4weeksofage(Greiget al.1971).In1969,anantigenicallyidenticalandiforiginatingfromnonimmunemothers(AppelviruswasisolatedinEnglandfromsucklingpigsshowinget al.1965).PigswithmaternallyderivedpHEVantibod-anorexia,depression,vomiting,andstunting,butwith-iesthatpreventthevirusfromreachingneuraltargettis-outsignsofencephalomyelitis(Cartwrightet al.1969).suesareclinicallyunaffectedwhenexposedtopHEVTheconditionwascalledvomitingandwastingdisease(Appelet al.1965).Pigsinfectedatlateragesnormallydo(VWD).Bothformsofthediseasewereexperimentallynotdevelopclinicaldisease.SincepHEVisendemicinreproducedbyMengelingandCutlip(1976)usingiso-mostswinepopulations,mostsowsareimmuneandlatesfromthesamefarm.pHEViswidespreadamongprotecttheiroffspringbymaternalantibodies.Thus,swine,buttheinfectionisgenerallysubclinical,althoughclinicaloutbreaksarerareandusuallyoccurinlitterssomeoutbreaksmaycauselosses(Alsop2006;Quirogafromnonimmunemothers,oftenfirst‐paritysows.Threeet al.2008).outbreaksarenotable.In2001,pHEVwasisolatedfrom 514SectionIIIViralDiseasesnewbornandearlyweanedpigswithvomitingandpos-diseaseonset,butreturnstonormalin1–2days.TheteriorparalysisonaCanadianfarm(Sassevilleet al.incubationperiodfortheappearanceofmorespecific2001).Alsop(2006)describedaclinicallydiagnosedout-signsis4–7days.Twomainclinicalmanifestationsasso-breakofVWDin2002ina650‐sowgeneticnucleusciatedwithpHEVneurotropismarepossibleinpigsherd.Quirogaet al.(2008)describedaVWDoutbreakbelow3–4weeksofage:(1)typicalVWDwithfrequentwithmotordisordersinArgentinain2006.Itoccurredinvomitingleadingtodeathorsubsequentwastingand(2)athree‐siteherdwith6,000sowswherethebreederacuteencephalomyelitiswithmotordisorders.However,stockconsistedof55%giltsandfirst‐orsecond‐paritysignsofbothclinicalformsmayoccurinthesameherdsows.duringanoutbreak.ForVWD,clinicalsignsarerepeatedretchingandvomiting.Pigsstartsuckling,butwithdrawfromthesowPathogenesisandvomitthemilk.ThepersistentvomitingandThetypeandseverityofclinicalsignsvaryandarerelateddecreasedfoodintakeresultsinconstipationandarapidtoage,possibledifferencesinvirusvirulence(Mengelingdeclineofcondition.NeonatallyinfectedpigsbecomeandCutlip1976),andthecourseofviralpathogenesis.severelydehydratedafterafewdays,exhibitdyspneaandTheprimarysiteofreplicationofpHEVinpigsisthecyanosis,lapseintocoma,anddie.Olderpigslosetheirrespiratorytract(AndriesandPensaert1980b;Hiraharaappetiteandbecomeemaciated.Theycontinuetovomit,et al.1987;Mengelinget al.1972).IFstainingrevealedalthoughlessfrequentlythanintheacutestage.Wasting,thatepithelialcellsofnasalmucosa,tonsils,lungs,andoftenwithdistensionofthecranialabdomen,maysomeunidentifiedcellsinthesmallintestinewereappear.This“wasting”statepersistsforseveralweeksinfected.Primaryreplicationmayresultinmildorsub-andmaybepostweaning,requiringeuthanasia.Duringclinicalsigns.theacutestageofVWDoutbreaks,somepigsmayshowExperimentalstudiesincolostrum‐deprivedpigletsneurologicsigns,suchasabnormalgait,dullness,trem-inoculatedoronasallywithpHEVprovidedinsightintoors,andnystagmus.pHEVpathogenesis(AndriesandPensaert1980a).FromAttheherdorfarrowingunitlevel,morbidityvariestheprimarysitesofreplication,thevirusspreadviathegreatlyandprobablydependsontheproportionofnon-peripheralnervoussystemtotheCNSviadifferentimmuneneonatallitterspresentatthetimeofinfection.pathways.OnepathwayledfromthenasalmucosaandInlitterswithoutmaternalprotection,morbidityislittertonsilstothetrigeminalganglionandthetrigeminalsen-dependentandmayapproach100%whentheinfectionsorynucleusinthebrainstem.Asecondpathwaywasoccursnearbirth.Morbiditydecreasesmarkedlywithalongthevagalnervesviathevagalsensorygangliontoincreasingageatinfection.Mortalityisvariable,butmaythevagalsensorynucleusinthebrainstem.Athirdpath-be100%inneonatallyinfectedlitters.wayledfromtheintestinalplexusestothespinalcord,IntheArgentinaoutbreak(Quirogaet al.2008),onlyalsoafterreplicationinlocalsensoryganglia.Viremiasucklingpigswereinvolved.Vomitingandwastingwerewasoflittleornoimportanceinthepathogenesisofthethemainsigns,withslightmotordisorders.Diseasedisease(AndriesandPensaert1980b).occurredin27.6%ofpigs<1weekoldanddeclinedtoIntheCNS,theinfectionstartedinwell‐definednuclei1.6%inpigs3weeksofage.InthispHEVoutbreak,anofthemedullaoblongata,butprogressedintotheentireestimated12.6%(3683)ofthesucklingpigsintheaffectedbrainstem,thespinalcord,andsometimesalsothecer-farrowingunitsdiedorwereeuthanized.Afterweaning,ebrumandcerebellum.IFstaininginthebrainwasameanof29%(15–40%)ofthepigscomingfromaffectedalwaysrestrictedtotheperikaryonandprocessesofneu-farrowingunitsshowedwasting.rons.VomitingwasinducedbyviralreplicationintheOutbreaksofthemotorencephalomyelitisdiseaseinvagalsensoryganglion(gangliondistalevagi)orbysucklingpigsmaystartwithsneezing,coughing,andimpulsestothevomitingcenterproducedbyinfectedvomiting4–7daysafterbirth.Vomitingcontinuesinter-neuronsatdifferentsites(Andries1982).Toelucidatemittentlyfor1–2days,butisrarelysevere.Insomeout-thepathogenesisofwasting,Andries(1982)suggestedbreaks,thefirstsignisacutedepressionandhuddling.thatvirus‐inducedlesionsintheintramuralplexusesofAfter1–3days,pigsexhibitvariouscombinationsofthestomachmaycontributetogastricstasisanddelayednervousdisorders.Generalizedmuscletremorsandstomachemptying.hyperesthesiaarecommon.Pigsmayhaveajerkygaitandwalkbackward,endinginadog‐sittingposition.Theybecomeweak,areunabletorise,andpaddletheirClinicalsignslimbs.Blindness,opisthotonus,andnystagmusmayalsoSneezingorcoughingmaybethefirstsignofinfectionoccur.Finally,theanimalsbecomedyspneicandliebecauseofprimarypHEVreplicationintheupperprostrateontheirsides.Inmostcases,comaprecedesrespiratorytract.Bodytemperaturecanbeelevatedatdeath. 31Coronaviruses515Morbidityandmortalityinneonatalpigsisusuallyseraaretakenneartheonsetofclinicalsigns.Pigsmay100%,butolderpigsshowamildtransientillnessindevelopantibodytitersasearlyas6–7DPI,whichoftenwhichposteriorparalysismaybethemostcommonsign.coincideswithearlydisease,makinganinterpretationofOutbreaksdescribedinTaiwan(Changet al.1993)inpairedserologymoredifficult.30‐to50‐day‐oldpigswerecharacterizedbyfever,con-DifferentialdiagnosismustbemadebetweenpHEVstipation,hyperesthesia,musculartremor,progressiveencephalomyelitis,Teschen–Talfandisease,andpseu-anteriorparesis,posteriorparesis,prostration,recum-dorabies(Aujeszky’s)disease.Inthelatterinfections,bency,andpaddlingmovementswithamorbidityof4%clinicalsignsofencephalomyelitis,includingmotorandamortalityapproaching100%.Thepigsdieddisorders,aremoresevereandmayappearinpiglets4–5daysaftertheonsetofclinicalsigns.andolderpigs.Aujeszky’sdiseaseinnon‐vaccinatedanimalsalsoinducesrespiratorysignsinolderpigsandabortionsinsows.AllthesevirusescanbegrowninPKLesionscellsandpigthyroidcells,butthetypeofCPEdiffersTheonlygrosslesionsreportedinpHEVinfectionsareandonlypHEVcauseshemadsorptionandhemaggluti-cachexia,stomachdilatation,anddistensionoftheabdo-nation.Theycanbefurtherdifferentiatedbyvirus‐meninsomechronicallyaffectedpigs(Schlenstedtet al.specifictests.1969).MicroscopiclesionsofepithelialdegenerationandImmunityinflammatorycellinfiltrationarefoundinthetonsilsand respiratorysystemofacutelydiseasedpigs(CutlipandAfterinfection,pigsdevelopdetectableprotectivecircu-Mengeling1972;Naritaet al.1989).Anonsuppurativelatingantibodies(HI,VN)topHEVin7–9days.Theencephalomyelitiswasreportedin70–100%ofpigswithdurationofantibodieshasnotbeendetermined.Thenervoussignsandin20–60%ofpigsshowingVWD.ThedurationofimmunityislessimportantinpHEVbecauselesionsarecharacterizedbyperivascularcuffing,gliosis,oftheresistancetodiseasethatdevelopswithage.andneuronaldegeneration(Alexander1962;Changet al.Neonatalpigsborntoimmunemothersarefullypro-1993;RichardsandSavan1960).Theyaremostpro-tectedbymaternallyderivedantibodiesthatpersistuntilnouncedinthegraymatteroftheponsVarolii,medullatheageof4–18(mean10.5)weeks(PaulandMengelingoblongata,andthedorsalhornsoftheupperspinalcord.1984).MicroscopicchangesinthestomachwallwerefoundonlyinpigsshowingVWD.DegenerationofthegangliaPreventionand controlofthestomachwallandperivascularcuffingwerepre-sentin15–85%ofdiseasedanimals.ThelesionswereOnmostbreedingfarms,pHEVinfectionpersistsmostpronouncedinthepyloricglandarea(Schlenstedtendemicallybypig‐to‐pigtransmissionandthroughsub-et al.1969).clinicalrespiratoryinfections.Giltsusuallycontractthevirusbeforetheirfirstfarrowingandthenprovidepro-tectiontotheiroffspringviacolostralantibodies.WhenDiagnosissowsarenotimmuneatfarrowing,(e.g.innewlypopu-Diagnosiscanbemadebyvirusisolation,IHC,orRT‐latedfarms,well‐isolatedgilts,orsmallfarmsinwhichPCR(Quirogaet al.2008).Tonsils,brainstem,andlungsthevirusisnotmaintained),infectionofpigswithinthedissectedasepticallyfromyoungacutelydiseasedpigletsfirstweeksafterbirthresultsinclinicalsigns.Promotingcanbeusedfortesting.Itisdifficulttoisolatethevirusviruscirculationinthefarmsothatgiltsareimmuneatfrompigsthathavebeensickformorethan2–3days.Forfarrowingpreventsdiseaseinpiglets.virusisolation,suspensionsareinoculatedontoprimaryOnceclinicalsignsareevident,thediseasewillrunitsPKcells,secondarypigthyroidcells,orporcinecelllines.course;spontaneousrecoveriesarerare.LittersbornpHEVisdetectedbythepresenceofsyncytia,byhemad-2–3weeksaftertheonsetofdiseaseareusuallypro-sorption,orbyhemagglutination.Oneormoreblindtectedbecausenonimmunegestatingsowsshouldhavepassagesmaybeneededsincespecimensoftencontainbecomeinfectedandimmunebyfarrowing.Pigletsbornsmallamountsofinfectiousvirus.tononimmunesowsearlyintheoutbreakcanbeAntibodiestopHEVcanbedetectedbyVNorhemag-passivelyprotectedbyparenteralinoculationwithspe-glutinationinhibition(HI)tests.TheHIandVNtestscificimmuneserumshortlyafterbirth.Hyperimmunewerealmostequallydiagnosticinswinesera,butVNisserumisnotcommerciallyavailable,butpooledserummorespecific(Sasakiet al.2003).Antibodytiterresultscollectedfromoldersows(attheslaughterhouse)shouldmustbeevaluatedcarefullybecausesubclinicalinfec-befiltersterilizedandtestedtoconfirmpresenceoftionswithpHEVareverycommon.Moreover,asignifi-pHEVantibodies.NovaccinesagainstpHEVarecantriseinantibodytitercanbedetectedonlyifacutecurrentlyavailable. 516SectionIIIViralDiseasesPorcinetorovirusBecausemostpigsbecomeinfectedwithToVspostweaning,maternalantibodiesapparentlyprovideatRelevanceleastpartialprotection.However,theimmunecorre-latesofprotectiontoporcineToVinfectionarenotTorovirus(ToV)particleswereinitiallydetectedbyEMknown.Inoneoffourfarms,itwaspostulatedthatToVinthefecesofa3‐week‐oldpigletwithdiarrheaininfectionofsucklingpigletsinthepresenceofmater-England.Subsequentstudiesrevealedahighseropreva-nalantibodiesdelayeddevelopmentofactiveimmunelence(81–100%)inadultsoryoungnursingpigletsandresponsessuchthatthesepigs,butnotpigsfromthehighdetectionrates(50–75%)amongsubclinicallyotherfarms,shedthesameToVstrainpre‐andpostinfectedweanedpigs(Kronemanet al.1998;Pignatelliweaning(Pignatelliet al.2010).Geneticallydiverseet al.2010).LatterreportsfromEurope,NorthAmerica,ToVstrainsweredetectedwithinherdsinthelatterandSouthAfricasuggestedthatToVwasendemic.InstudyandinKoreanfarms.TheporcineToVswerecontrast,inKorea,only6.4%(19of295)ofdiarrheicassociatedwithsporadicinfectionsamongdiarrheicfecesfrom3‐to45‐day‐oldpigletswerepositiveforpor-pigsfrom65Koreanfarmssurveyed(6.2%offarmscineToV(Shinet al.2010).Ofthese,about74%alsocon-positive)(Shinet al.2010).Basedonphylogenetictainedotherentericpathogens.Consequently,thelinkanalysisoftheSandNgenes,theKoreanToVstrainsbetweenporcineToVandentericdiseaseisunclear,andformeddistinctbrancheswithclusterscorrespondingtherearenoreportsconfirmingporcineToVpathogenic-tothefarmoforigin.ityorgutlesions.EtiologyDiagnosisPorcineToVrepresentsaspecieswithinthegenusMethodstopropagateporcineToVsincellcultureTorovirusofthesubfamilyTorovirinaeinthefamilyhavenotbeendescribed.Forserologicstudies,acellCoronaviridae.Thegenomicorganization,replicationculture‐adaptedequineToVhasbeenusedtoassessstrategy,andpropertiesresembleothermembersinthecross‐reactiveVNantibodiesinswine(KronemanfamilyCoronaviridae(deGrootet al.2008).Likesomeet al.1998;Pignatelliet al.2010).Recentlyanindirectbetacoronaviruses,porcineToVsalsopossessanHEELISAusingrecombinantpurifiedporcineToVNprotein.NotabledifferencesfromCoVsincludeaproteinasantigenwasdeveloped(Pignatelliet al.smallerNprotein(approximately18.7kD)andatubu-2010).Inmost,butnotallcases,therewasagoodlarnucleocapsid,leadingtodifferencesinToVparticlecorrelationbetweenELISAandVNtests.morphology(spherical,elongated,orkidneyshaped)Discrepanciesobservedcouldreflectuseofheterolo-(Kronemanet al.1998).MultipleclustersofporcinegousequineToVantigeninVN,compromisingdetec-ToVshavebeenidentifiedbasedongenesequenceanal-tionoflowtiterantibodies.ysis(deGrootet al.2008;Pignatelliet al.2010;ShinPorcineToVshavebeendetectedinfecesusinget al.2010).IEMtoidentifyantibody‐aggregatedToVparti-clesanddifferentiatethemfromotherfecalpor-cineCoVs(TGEV,PEDV,andPDCoV)(KronemanEpidemiologyand immunityet al.1998).FordetectionofToV‐specificviralBasedonserologicandsheddingdatafromclinicallyRNA,RT‐PCRandreal‐timeRT‐PCRtargetingnormalpigsorsowsinEuropeanherds,ToVswereconservedregionsoftheporcineToVNgeneorendemicin14farmstested.Highseroprevalenceratesthe3′UTRofthegenomehavebeendescribed(81%)weredetectedinsowson10Dutchfarmsbytest-(Kronemanet al.1998;Pignatelliet al.2010;Shiningforcross‐reactiveVNantibodiestoequineToVet al.2010).(Kronemanet al.1998).Similarly,100%ofsows,nurs-ing,andolderpigsinthreefarmsinSpainweresero-positiveforToVantibodiesusinganELISAbasedonPreventionand controlporcineToVNprotein(Pignatelliet al.2010).Longitudinalstudiesrevealedfecalshedding(80%)(RT‐Basedonthelimiteddataavailable,thestressoftrans-PCRorreal‐timeRT‐PCR)postweaningat4–14daysport,movement,andredistributionofpigs,evenwithinfor1–9days(Kronemanet al.1998)orat4and8weeksmultisitefarms,couldprecipitateporcineToVinfectionpostweaning(50–75%)(Pignatelliet al.2010).Inbothwithsimilarordistinctco‐circulatingstrains(Pignatellistudies,maternalantibodytiterswereinitiallyhighinet al.2010).Thus,managementpracticesapplicabletopiglets,declinedatweaning,andthenincreasedpostcontrolofotherentericCoVinfectionsshouldbeimple-infectionat11or15weeksofage.mentedforcontrolofporcineToVs. 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