AetiologyofviralhepatitisestablishedthatthesetwotypesofacutehepatitisarecausedbyaetiologicallydistinctagentsinfectioushepatitiswhichhasashortincubationperiodandserumhepatitiswithlongincubationTheA ID: 961631
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PostgraduateMedicalJournal(July1971)47,476-480.ThemultipleaetiologyofviralhepatitisA.J.ZUCKERMANReaderinVirologyDepartmentofBacteriologyandImmunology,LondonSchoolofHygieneandTropicalMedicine,London,WCIE7HTSummaryInfectioushepatitisisepidemiologicallyandim-munologicallydistinctfromserumhepatitis.TheAustraliaantigenisrelatedmorespecificallytoserumhepatitis.Thepossibleroleofcoronavirus-andpara-myxovirus-likeparticlesintheaetiologyofsomeinfectionsoftheliverinmanandinmarmosetsinoculatedwithhumaninfectioushepatitismaterialisdiscussedandthedifficultiesintheinterpretationofthecurrentlyavailabledataareemphasized.TherecentstudiesinMelbourneofafaecalantigenfoundinsomepatientswithinfectioushepatitisandthediscoveryofanantiseruminMilanwhichreactedwithanantigenassociatedwithepidemichepatitisarediscussed.MentionismadeoftherecentisolationinDetroit-6cellsofvirus-likeparticlesfrompatientswithinfectioushepatitis.Itisconcludedthatviralhepatitisisaninfectionofmultipleaetiologyandthatthesuccessfulcultiva-tioninvitrooftheagentoragentsofhepatitisremainstheoutstandingandmosturgentproblem.DefinitionTheusualdefinitionofviralhepatitisisacuteinflammationofthelivercausedbytwoinfectiousagents,presumablyviruses,namedvirusA,theaetiologicalagentofinfectioushepatitisorepidemichepatitisandvirusB,whichisthecauseofserumhepatitisorhomologousserumjaundice.Thedefinitionexcludesbycommonusageacuteinflam-mationoftheliverwhichmaybeinducedbywellrecognizedvirusessuchasyellowfever,adeno-viruses,cytomegalovirus,coxsackieviruses,herpessimplexvirusandothers(reviewedbyZuckerman,1970a).Itisvirtuallyimpossibletodistinguishbetweenthetwoclinicallyandpathologicallysimilarformsofviralhepatitis.Factorswhichmakesuchadifferentiationevenmoredifficultarethatserumhepatitismaybetransmittedbytheoralroute(Krugman,Giles&Hammond,1967)andCorrespondence:DrA.J.Zuckerman,LondonSchoolofHygieneandTropicalMedicine,KeppelStreet(GowerStreet),London,WC1E7HT.converselyinfectioushepatitismaybetransmittedparenterally.However,characterizationoftheinfectioustypeofhepatitisispossibleonmeticulousepidemiologicalgroundssuchasthoseoutlinedbyMosley(1970)namelyanaverageincubationperiodtotheonsetofsymptomsofapproximately30days,clearevidenceofspreadbyperson-to-personcontactoracommonsourceofinfectionandprotectionofindividualsinthesameepidemiologicalsettingbyhumanimmunoglobulininasmalldose.Indeed,referencetothenomenclatureofinfectivehepatitis(Table1),whichisbedevilledbyamultitudeofsynonymousterms,revealsaconsistentfeaturethroughout:thepredominantassociationofinfec-tioushepatitiswithanepidemicsettingandthemoresporadicnatureofserumhepatitisanditsassociationwithaparenteralmodeoftransmission.Furthermore,volunteerexperimentshaveclearlyTABLE1.TheterminologyofviralhepatitisInfectioushepatitisSerumhepatitisIcterusepidemicus-epidemicjaundiceSalvarsanjaundiceCatarrhaljaundicePost-arsphenaminejaundiceEpidemiccatarrhaljaundiceArsenotherapyhepatitisJaunissedescampsSyringejaundiceSoldatengelbsuchtPost-vaccinaljaundiceInfectiousjaundiceYellowfevervaccinehepatitisEpidemichepatitisInoculationhepatitisInfectivehepatitis(virusA)Syringe-transmittedhepatitisAcuteviralhepatitisHomologousserumjaundice(HSJ)Botkin'sdiseaseHomologousserumhepatitis(virusB)ShortincubationhepatitisTattoojaundice(MS-Iinfection)Transfusion-hepatitisInfectious(MS-I)hepatitisPost-transfusionhepatitisCommon-sourcehepatitisTransfusion-associatedhepatitisSerumhepatitis(SH)ParenteralhepatitisHippyhepatitisLongincubationhepatitis(MS-2infection)Seru
m(MS-2)hepatitis Aetiologyofviralhepatitisestablishedthatthesetwotypesofacutehepatitisarecausedbyaetiologicallydistinctagents,infectioushepatitiswhichhasashort-incubationperiodandserumhepatitiswithlong-incubation.TheAustraliaantigenThediscoveryoftheremarkableassociationbetweenviralhepatitisandaserumantigennamedtheAustraliaantigen(Blumbergetal.,1967)hasprovidedspecificserologicaltestsforaproportionofcasesofhepatitis.WhilemanyconflictingresultshavebeenreportedontheimmunologicalspecificityoftheAustraliaantigenitnowappearsthatthisantigenisrelatedmorespecificallytoserumhepatitisortypeBviralhepatitisandnotepidemichepatitis(Zuckerman,1970b,c),althoughsomedoubtsmaybeexpressedabouttheviralnatureofthisantigen(Zuckerman,1970d;Zuckerman,Taylor&Bird,1970).Coronavirusandparamyxovirus-likeparticlesinhepatitisAnticomplementaryactivitywasobservedbyPatriciaE.Taylor&A.J.Zuckermaninthecourseoftestingserabycomplement-fixationfortheAustraliaantigen.Electronmicroscopicexaminationofserafromonesuchpatientwithchronicactivehepatitisrevealedthepresenceofvirusparticlesdisplayingthetypicalmorphologicalfeaturesofamemberofthecoronavirusgroup(Zuckerman,Taylor&Almeida,1970).Allthevirusparticleswereinassociationwithantibodyandthecomple-ment-fixingantibodytitretoprototypemousehepatitisvirus3,aknowncoronavirus,was1:640.Thisobservationwasofinterestinviewoftheana-logywiththepreviousfindingsofcirculatingantigen-antibodycomplexesinsomepatientswithserumhepatitis(Taylor,Zuckerman&Brighton,1969;Almeida&Waterson,1969).Theadditionalfindingofhightitredantibodycrossingserologicallywithmousehepatitisvirusalsoraisedthepossibilitythattheseparticlesmaybeahumancounterparttothemousehepatitisvirus.Deinhardtandhisassociates(personalcommuni-cation,1970)andHolmesetal.(1970)independentlyobservedparamyxovirus-likeparticlesintheacutephaseoftwopatientswithinfectioushepatitisandinacutephaseseraofmarmosetswithexperimentallyinducedhepatitis.Normalserumspecimensandacutephaseseraofpatientswithviralhepatitisandtheseraofmarmosetswereexaminedintwoways:bycaesiumchloridedensitygradientcentrifugationandelectronmicroscopyandbytestingforinfecti-vityinmarmosets.Paramyxovirus-likeparticleswerefoundinacutephaseseraofinoculatedmarmo-setsandinacutephaseserumspecimensoftwohumanpatients,oneofwhichalsoinducedhepatitisinmarmosets.Thefractionswithadensityofabout121g/cm3wereinfectiousformarmosets.Virus-likeparticleswerenotfoundinnon-infectiousfractionsofdifferentdensities.AnAustraliaantigen-positiveserumandits120x125g/cm8and130x1-32g/cm3caesiumchloridedensitygradientfractionswerenotinfectiousformarmosets.Furtherstudiesarecur-rentlyinprogresstoconfirmandextendtheseobservationsandnofirmconclusionsshould,atthistime,bedrawnfromthesefindings.Apodacaetal.(1970)alsodescribedcorona-paramyxovirus-likeparticlesinmarmosetsinoculatedwithhumaninfectioushepatitismaterial.FiveserawereexaminedundercodebyZuckerman,Taylor&Bird(unpublishedobservations)andvirus-likeparticles(Fig.1)werefoundinthethreeacutephaseseracollectedfromtheinoculatedmarmosetsbutnotintheserumofthecontrolanimals.Hunteretal.(1970)alsofoundparamyxovirus-likeparticlesinanumberofpatientswithchronicliverdisease.Thesignificanceofcoronavirus-likeparticlesto-getherwiththestructuresusuallyassociatedwiththeAustraliaantigeninanumberofserumspecimens(Fig.2)obtainedfromdifferentpatientswithhepa-titisisimpossibletointerpretatthisstage(Zucker-man,1970c;Wright,1970).MorerecentlySirtori(1970a,b)examinedbyelectronmicroscopypercutaneousliverbiopsiesobta
inedfromthreechildrenwithinfectioushepa-titiswhoweretreatedwithazathioprine.Manyvirus-likeparticlesmeasuring80-160nmindiameterFIG.1.AnexampleoftheparticlesfoundintheserumofmarmosetsinoculatedwithinfectioushepatitismaterialattheRobertKochInstitute,WestBerlin.x88,200.(PhotomicrographsfromaseriesbyA.J.Zuckerman,PatriciaE.TaylorandR.G.Bird.)477 A.J.ZuckermanFIG.2.Serumfromapatientwithviralhepatitisshow-ingthethreetypicalmorphologicalentitiesassociatedwiththeAustraliaantigen(smallpleomorphicsphericalparticlesmeasuring20nmindiameter,tubularformsofvaryinglengths,andlargesphericalparticlesmeasuring40-45nmindiameter)aswellasalargeparticlewithradiatingclubbedprojections.x63,000.anddisplayingthemorphologyofthecorona-paramyxovirusgroupwerefoundinthecytoplasmofthehepatocytesandKupffercells.Someoftheseparticleswereshowntobebuddingfromthecellmembrane.Itisalsoofinterestthatserumspecimensfromthesethreechildrenreactedinthetwodimen-sionalmicro-Ouchterlonytechniquetogivepreci-pitinlineswithanantiserumpossessingspecificitiesforbothserumhepatitis(associatedwiththeAus-traliaantigen)andepidemichepatitis(DelPreteetal.,1970).However,alltheseobservationsarepreliminaryinnatureandtheymustbeinterpretedwithcon-siderablecautionsincetheydonot,ofcourse,provethatthesevirus-likeparticlesarespecificallyassocia-tedwithorarecausallyrelatedtoinfectioushepa-titis.Thesevirus-likestructuresmayindeedplayonlyasecondaryrole,ifany,insomeformsofhumanliverdisease.ThisviewissharedbyZucker-manandhisassociatesandbyDeinhardtandhisgroup,andbothstressthatthedataavailablesofarmerelyindicatepossiblelinesforfurtherinvestiga-tion.AfaecalantigenininfectioushepatitisFerrisetal.(1970)describedafaecalantigeninsomepatientswithinfectioushepatitis.Bacteriolo-gicallysterilesupernatantswereobtainedbyhighspeedcentrifugationofa20%suspensionoffaecescollectedfromsixpatientswithviralhepatitisinwhomtheAustraliaantigenwasnotdetectedintheserum.Thefaecalextractsweretestedbygeldiffusionagainstserafromthirty-sevenmultiply-transfusedhaemophiliacs.Twosuchseragaveprecipitinlineswithfourofthesixfaecalextracts,butnoreactionwasobtainedwithserafromthesepatientsnorfromsixpatientswithotherdiseases.Tworabbitswereimmunizedwithpositivefaecalextractswhichhasbeenpurifiedbyfractionationonsucrosegradients.Therabbitantiserawereabsorbedwithnormalhumanserumandtwicewithfaecalextractsfromnon-hepatitispatients.Singlepreci-pitinlineswereobservedwhenfaecalextractsfromninetyoutof220(409%)patientswithviralhepa-titisandfiveoutof158(3-1%)withouthepatitisweretestedagainstoneoftherabbitantisera.Positivelyreactingfaecalextractsdidnotreactwithhumanoranimalanti-Australiaantigensera.Crossandhisassociates(personalcommunication,1970)extendedtheirpreliminaryobservations,andimmuneelectronmicroscopyindicatedthepresenceoftwovirus-likeparticlesinthefaecalantigen.Oneparticlemeasures15-25nmindiameterandcloselyresemblesthesmallparticleassociatedwiththeAustraliaantigen(Almeidaetal.,1969)andtheotherisa40-45nmparticlealsofoundinmanyAustraliaantigenpositivesera(Fig.3)(Dane,Cameron&Briggs,1970;Zuckerman,1970c).Tubularformswerenotfoundinextractscontainingthefaecalantigen.Cross-serologicaltestsingelandimmuneelectronmicroscopysuggestthattherearetwoantigenicallydistincttypesofthesmallparticles,oneassociatedwiththeAustraliaantigenandtheotherassociatedwiththefaecalantigen,whereasthelargerandsomewhatmorepleomorphicFiG.3.ThetypicalmorphologyoftheAustraliaantigenparticlesfoundinserum.Notethespherical'double-shelled'particleswhichmeasu
reapproximately42nmindiameter.x63,000.478 Aetiologyofviralhepatitis479particlesappeartopossessacommongroupcomponentaswellasatype-specificcomponent.Furtherstudiesoftheseimportantobservationsareinprogress.AserumantigeninepidemichepatitisRecentlyDelPreteetal.(1970)foundahumanantiseruminMilanwhichreactedintheimmuno-diffusiontestwithserafrompatientswithlong-incubationhepatitisandalsowithshort-incubationhepatitis.Thisnewantiserumdetectedbygeldiffusionaserumantigenassociatedwithclassicalepidemichepatitis.Ahighproportionofserafromthreeepidemicsofshort-incubationhepatitis,whichwereconsistentlynegativewithanti-Australiaantigenseratestedbothbytheimmunodiffusiontechniqueandbycomplement-fixation,reactedwiththeMilanantiserum.Thusaprecipitinlinewasdemonstratedin65%of127patientsinvolvedinthreeseparateepidemicsofinfectioushepatitisandthepercentageofpositivereactionsincreasedto90%inseraobtainedduringthefirstweekofsymptoms.Theantigenwasclearedfromtheserumwithclinicalimprovement.Thisnewepidemichepatitis-associatedantigenwasfoundtoberemarkablylabile,whichisasomewhatunexpectedfindinginviewoftheknownphysicalstabilityofthehepatitisagents.Itisappreciated,ofcourse,thatthisantigenmightbeantigenicallydistinctfromthehepatitisvirusinthesamewayastheHantigenofpoliovirusisantigenicallydistinctfromtheantigenonthenativevirus.Theseobservationsareofgreatinterestsincetheymayultimatelyprovidespecificserologicalmethodsfordistinguishingbetweeninfectioushepatitisandserumhepatitis.Virus-likeparticlesininfectioushepatitisFinally,referenceismadetotheworkofW.A.RightselandhisassociateswiththehaemovirusesandinparticularwiththeAR-17agentusingtheDetroit-6cellline.Thissubjectwasrecentlyreviewedinsomedetail(Zuckerman,1970e).AttemptstoculturetheAR-17agentinthesemi-continuoushumanembryolivercelllineresultedinacyto-pathiceffectinthesecellswhichcouldnotbetransferredbeyondthefirstpassage(Zuckermanetal.,1970).Therewasnoevidenceofchromosomaldamageintheinoculatedcells.Detroit-6cellsinoculatedwiththeAR-17agentbyW.A.RightselinMemphiswereexaminedbynegativestainingandelectronmicroscopyinLondonin1969.Pleomorphicparticlesmeasuring18-23nmindiameterandarrangedincharacteristicchain-formationsandinclumpswerefoundintheinocu-latedcultures(Fig.4).Itwasimpossibletointerpretthesignificanceoftheseobservations,ifany,FIG.4.Pleomorphicparticles,measuringapproximately18-23nmindiameter,arrangedincharacteristicchainformsfoundinthesupernatantfluidofDetroit-6cellsinoculatedwiththeAR-17agentbyW.A.Rightsel.x53,300.especiallyintheabsenceofothersupportingevidence.Morerecently,Melnicketal.(1970)isolatedavirusfromtheacutephaseplasmaofavolunteerwhodevelopedhepatitis4weeksafterinoculationwithinfectioushepatitisserum.ThisagentproducedcytopathicchangesinaclonedlineofDetroit-6cells.Particlesmeasuringapproximately18nmwerefoundbyelectronmicroscopyinmaterialharvestedfromtheinfectedcellscultures.SimilarviruseswerealsoisolatedinDetroit-6cellsfromtheseraofsixotherpatientswithacuteinfectioushepatitis.Furtherworkisinprogresstoelucidatethesigni-ficanceofthesefindings.Itisbecomingapparent,however,thatviralhepatitisshouldindeedberegardedasaninfectionofmultipleaetiologyandtheproblemcouldonlyberesolvedbythesuccessfulinvitrocultivationofthenewlydescribedantigensandagentsofhepatitis.AcknowledgmentsTheoriginalworkdescribedinthispaperwasaidedbygenerousgrantsfromtheMedicalResearchCouncilandinpartbylaboratoryequipmentkindlyprovidedbyPfizerLtd,Sandwich,Kent.Table1isreproducedbykindpermissionoftheEditorofUpdate.Re
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