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1、Vol.35,No.10 HygromycinBTherapyofaMurineCoronaviralHepatitis GEORGINAMACINTYRE,1tBERNADETTECURRY,2FREDWONG,1ANDROBERTANDERSON* DepartmentofMicrobiologyandInfectiousDiseasesandDepartmentofPathology,2UniversityofCalgary, 3330HospitalDriveN.W.,Calgary,AlbertaT2N4NI,Canada Received12April1991/Accepted22
2、July1991 Hepatitiscausedbymousehepatitisvirus(MHV-A59),amurinecoronavirus,isaccompaniedbydirect infectionandreplicationofviruswithintheliver.WedemonstrateherethattheaminoglycosidehygromycinB isabletoeliminateMHV-A59infectionfrommouseperitonealmacrophagesandculturedlivercellsinvitroand isalsoabletore
3、ducelevelsofvirusreplicationandnecroticliverfociinvivo. TheproposedselectiveentryofhygromycinBintocyto- pathicallyinfectedcells(reviewedinreference5)andthe resultantdeathofthesecellsbytheinhibitionofcellprotein synthesissuggeststhatthiscompoundmaybeusefulasan antiviraldrug.Alongtheselines,wehaveprev
4、iouslypre- sentedevidence(9)insupportoftheantiviralpropertiesof hygromycinBagainstacuteandpersistentinvitroinfections ofmousehepatitisvirus(MHV-A59).Thestudiesdescribed inthisreportattempttoexaminetheeffectofhygromycinB insystemsmorerelevanttonaturalMHVinfection.Evi- denceispresentedfrombothinvitros
5、tudies,usingmouse peritonealmacrophagesandlivercells,andinvivostudies whichsuggestthathygromycinBhasatherapeuticeffectin limitingMHVreplicationandinreducingthenumbersof hepaticlesionsproducedduringaninvivoinfection. Invitrostudieswithperitonealmacrophagesandcultured livercells.Macrophagesandlivercel
6、lsareimportantcell targetsforMHV.Sincetheinfectabilityofmousemacro- phagesoftencorrelateswithinvivosusceptibilitytoMHV (1,17),weexaminedMHV-A59infectioninculturesof peritonealmacrophages.Twostrainsofmicewereused, BALB/candA/J,bothofwhichhavebeenshowntobe susceptibletoMHV-A59infection(7,14).Starch-ac
7、tivated peritonealmacrophages(6)wereharvestedfrom4-month- oldBALB/candA/Jmicebyrepeatedwashingofthe peritonealcavitywithminimalessentialmedium(MEM) containing20%fetalcalfserum(FCS).Thetotalperitoneal cavitywashwascentrifugedat1,000 xgfor1min,andthe pelletwaswashedwithMEMsupplementedwith20%FCS. Thefi
8、nalpelletwasresuspendedinMEM(plus10%FCS), platedoutineight-wellmicrotiterslides,andincubated overnighttoallowthemacrophagestoadheretothewells. ThemonolayersweretheninfectedwithMHV-A59(multi- plicityofinfection,0.1)andweretreatedwithvarious concentrationsofhygromycinB.Immunofluorescencewas carriedout
9、on95%ethanol-5%aceticacid-treatedcultures byusingamousemonoclonalantibodydirectedagainstthe MHV-A59largeenvelopeglycoprotein(designatedS)that wasprobedwithfluoresceinisothiocyanate-conjugatedgoat anti-mouseimmunoglobulinG.Virustitersintherangeof i05to106wereobtainedbetween12and36hpostinfection (p.i.
10、)fromMHV-infectedperitonealmacrophagecultures obtainedfrombothBALB/candA/Jmice(Fig.1AandB). Thevirus-inducedcytopathiceffectfoundinbothBALB/c *Correspondingauthor. tPresentaddress:DepartmentofMicrobiologyandImmunology, McGillUniversity,Montreal,QuebecH3A2B4,Canada. andA/Jperitonealmacrophagecultures
11、wasextensive,with 100%fusionoftheA/JandBALB/cmonolayersoccurring by24hp.i.(Fig.1D)and36hp.i.(Fig.1C),respectively. ThisstudyindicatesthatperitonealmacrophagesfromA/J miceareatleastassusceptibletoMHV-A59infectionas thosefromBALB/cmiceare. HygromycinBdrasticallyreducedMHV-A59replication andvirus-induc
12、edcytopathology(toundetectablelevels)in peritonealmacrophageculturesderivedfrombothBALB/c andA/Jmice(Fig.1).Curingofthevirusinfectionin macrophagesbyhygromycinBwasconfirmedbyimmuno- fluorescence.At42hp.i.,themonolayersweretreatedfor examinationbyimmunofluorescencemicroscopyandwere scoredforthepresen
13、ceofviralantigen.Expressionofvirus proteinsinperitonealmacrophagesfrombothstrainsofmice decreasedwithincreasingconcentrationsofthedrug.Immu- nofluorescencewasnoticeablyreducedat0.1mMhygromy- cinBandwasvirtuallyundetectableatdrugconcentrations of0.75to1mM. ThesensitivityofMHVinfectiontohygromycinBwas
14、 foundtobeevenmoremarkedinculturedmouselivercells thanwasthecaseinperitonealmacrophages.Livercells wereobtainedfromBALB/cmicebytheprocedureof Wiltroutetal.(18).Briefly,liverswereperfusedwithme- dium(MEMsupplementedwith10%FCS)viathehepatic portalvein.Afterremovingthegallbladderandanyperfused areas,th
15、elivertissuewasmincedanddigestedwitha mixtureofcollagenase(0.25mg/ml),DNase(0.05mg/ml), andhyaluronidase(0.25mg/ml)inserum-freemediumfor3h atroomtemperature.Followingfiltrationthroughseveral layersofcottongauzeandcentrifugationat500 xg,the pelletedcellswereseededontocollagen-coatedslidesand cultured
16、in35-mm-diametertissueculturedishes.Cellswere theninoculatedwithMHV-A59(multiplicityofinfection,1) andwereincubatedinmediuminthepresenceorabsenceof variousconcentrationsofhygromycinBfor42hat37C.By usingimmunofluorescenceasaguidetoMHVantigen expression,levelsofviralantigenwerenoticeablyreduced at12.5,uMhygromycinBandwerenotdetectableatdrug concentrationsof50to100,uM. Macrophageandlivercellviability,whichwasmeasured bytrypanblueexclusion,andmacrophagephagocyticfunc- tion,whichwasassayedbyneutralre
