2019 the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection...
TRANSCRIPT
Anim Models Exp Med. 2019;00:1â13. â|â1wileyonlinelibrary.com/journal/ame2
Received:2July2019â |â Revised:12September2019â |â Accepted:6October2019DOI:10.1002/ame2.12088
O R I G I N A L A R T I C L E
The characteristics of hDPP4 transgenic mice subjected to aerosol MERS coronavirus infection via an animal noseâonly exposure device
Xinâyan Hao â| Qi Lvâ| Fengâdi Liâ| Yanâfeng Xuâ| Hong Gao
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionâNonCommercialLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycitedandisnotusedforcommercialpurposes.©2019TheAuthors.Animal Models and Experimental MedicinepublishedbyJohnWiley&SonsAustralia,LtdonbehalfofTheChineseAssociationforLaboratoryAnimalSciences
InstituteofLaboratoryAnimalSciences,ChineseAcademyofMedicalSciences(CAMS)&ComparativeMedicineCentre,PekingUnionMedicalCollege(PUMC),KeyLaboratoryofHumanDiseaseComparativeMedicine,NationalHealthCommissionofChina(NHC),BeijingKeyLaboratoryforAnimalModelsofEmergingandReemergingInfections,Beijing,China
CorrespondenceHongGao,InstituteofLaboratoryAnimalSciences,ChineseAcademyofMedicalSciences(CAMS)&ComparativeMedicineCentre,PekingUnionMedicalCollege(PUMC),KeyLaboratoryofHumanDiseaseComparativeMedicine,NationalHealthCommissionofChina(NHC),BeijingKeyLaboratoryforAnimalModelsofEmergingandReemergingInfections,Beijing,China.Email:[email protected]
Funding informationNationalScienceandTechnologyMajorProjectsofInfectiousDisease,Grant/AwardNumber:2018ZX10734401â011
AbstractBackground: MiddleEast respiratory syndromecoronavirus (MERSâCoV),which isnotfullyunderstoodinregardtocertaintransmissionroutesandpathogenesisandlacksspecifictherapeuticsandvaccines,posesaglobalthreattopublichealth.Methods: Tosimulatetheclinicalaerosoltransmissionroute,hDPP4transgenicmicewere infectedwithMERSâCoVbyananimalnoseâonlyexposuredevice andcomâparedwith instillationâinoculatedmice.Thechallengedmicewereobservedfor14consecutivedaysandnecropsiedondays3,5,7,and9toanalyzeviralload,histopaâthology,viralantigendistribution,andcytokinesintissues.Results: MERSâCoV aerosolâinfected mice with an incubation period of 5â7 daysshowedweightlossondays7â11,obviouslunglesionsonday7,highviralloadsinthelungsondays3â9andinthebrainondays7â9,and60%survival.MERSâCoVinstilâlationâinoculatedmiceexhibitedclinicalsignsonday1,obviouslunglesionsondays3â5,continuousweightloss,0%survivalbyday5,andhighviral loadsinthelungsandbrainondays3â5.Viral antigenandhigh levelsofproinflammatorycytokinesandchemokinesweredetectedintheaerosolandinstillationgroups.Disease,lunglesion,andviralreplicationprogressionswereslowerintheMERSâCoVaerosolâinâfectedmicethanintheMERSâCoVinstillationâinoculatedmice.Conclusion: hDPP4transgenicmiceweresuccessfullyinfectedwithMERSâCoVaerâosolsviaananimalnoseâonlyexposuredevice,andaerosolâandinstillationâinfectedmice simulated the clinical symptoms of moderate diffuse interstitial pneumonia.However,thetransgenicmiceexposedtoaerosolMERSâCoVdevelopeddiseaseandlungpathologyprogressionsthatmorecloselyresembledthoseobservedinhumans.
K E Y W O R D S
animalnoseâonlyexposuredevice,hDPP4transgenicmice,intranasalinstillation,MERSâCoVaerosolinfection
2â |â ââ HAO et Al.
1â |âINTRODUC TION
MiddleEastrespiratorysyndromecoronavirus (MERSâCoV),whichwasfirstidentifiedinSaudiArabiain2012andcausesacuterespiraâtoryillness,multiorganfailure,shockandevendeath,isanimportanthighlypathogeniccoronavirusthat issimilartosevereacuterespiâratorysyndromecoronavirus (SARSâCoV)andproducessevere inâfectionswithahighmortalityrate.1â3AttheendofMay2019,therewereatotalof2428laboratoryâconfirmedcasesofMERSwith838associated deaths (caseâfatality rate: 34.5%, which is higher thanthe fatality rate of SARS) worldwide according to World HealthOrganization(WHO)statistics.4MERScaseshavebeenreportedin27countries,includingcountriesintheMiddleEast,Africa,Europe,Asia,andNorthAmericaaswellasAustralia,andcasenumbersconâtinueto increase,posingaglobal threattopublichealth. InChina,thefirstpatientinfectedwithMERSâCoVfromSouthKoreawasdiâagnosedinMay2015,5anditwillbeextremelyimportanttoprevent,control,andtreatMERSâCoVinfectionsduringanyfutureoutbreaks.Hence,effectivesmallanimalmodelsareneededtoinvestigateviralpathogenesisandevaluateMERSâCoVtherapeuticsandvaccines.
NonhumanprimateanimalmodelsofMERSâCoVinbothrhesusmacaquesandcommonmarmosetswereestablishedinpreviousreâports,6,7however,thesemodelsarelimitedbyrestrictedavailability,highcosts,experthusbandryrequirements,andethicalconcerns.8,9 Traditionalsmallanimalssuchasmice,hamsters,andferretscannotbeinfectedwithMERSâCoVowingtoabsenceofthenecessarydiâpeptidylpeptidase4(DPP4)receptorthatinteractswiththerecepâtorbindingdomainof theMERSâCoVspikeprotein (Sprotein)10â12 MERSâCoVfailstoreplicateinmice,whicharereadilyavailable,haveadefinedgeneticbackgroundandlowcostandarefrequentlyusedininfectiousdiseaseresearch,duetovariationsintheDPP4recepâtor. Previous studies showed that transgenic mice expressing thehumanDPP4 (hDPP4)receptorcouldbe infected intranasallywithMERSâCoVanddevelopedacutepneumonia.13â15Therefore,hDPP4transgenicmicewereselectedforexposuretoMERSâCoVâcontainâingaerosolsusingananimalnoseâonlyexposuredevice.
TherearetwomodesofMERSâCoVinfection,animalâtoâhumanandhumanâtoâhumantransmission.16Somereportshavefoundthatairborne transmission via the coughing and sneezing of infecteddromedarycamelsorcontactwithrespiratorysecretionsandconâsumptionofunsterilizedmilkfrominfectedcamelscansignificantlyincreasetheriskofMERSâCoVinfectioninhumans.17,18Kimetal19 discoveredextensiveviableMERSâCoVcontaminationintheairandsurroundingenvironmentinMERSisolationwards.AccordingtotheWHO,ithasbeensuggestedthathumanâtoâhumantransmission,toaverylimitedextent,iscausedbyinhalationofdropletsorairbornevirus and close contact with patients.20 The above studies havedemonstrated thatMERSâCoV has a risk of aerosol transmission.Inaddition,aerosolinhalationisthemainclinicalrouteofinfectionfor viral respiratory illnesses. There are different clinical presenâtations in animalmodelsestablishedbydifferent infection routes.ComparativestudiesusingapproacheswithdifferentperspectiveswillcontributetoadeeperunderstandingofMERS.
In this work, to simulate the aerosol transmission route forcomparison with the instillation route, hDPP4 transgenic micewereexposedtoMERSâCoVaerosolsbyananimalnoseâonlyexâposuredevice.Afterinfection,weanalyzedthemousecharacterâistics ofweight loss, survival, viral replication, tissue pathology,viral antigen distribution, and cytokine and chemokine profiles,whichprovideadditionaldata to investigate thepathogenesisofMERSâCoVâinduced disease and evaluate relevant therapeuticsandvaccines.
2â |âMATERIAL S AND METHODS
2.1â|âAnimals
SpecificpathogenâfreetransgenicC57BL/6miceexpressinghDPP4(age,6â8weeks)werepurchased from the Instituteof LaboratoryAnimal Science (ILAS), Chinese Academy of Medical Sciences(CAMS),Beijing,China.ExperimentsinvolvingMERSâCoVinfectionwerecarriedoutinanimalbiosafetylevel3(ABSLâ3)andbiosafetylevel3 (BSLâ3) laboratoriesat the ILAS,CAMS.Miceweretreatedhumanely, and all animal studies were conducted in accordancewithaprocedureapprovedbytheInstitutionalAnimalCareandUseCommittee,ILAS,CAMS(ILASâGH18001).
AllanimalswerefedunderABSLâ3conditionsfor3daysbeforethestartofthestudy.Ontwoconsecutivedayspriorto infection,each mouse was trained in an animal noseâonly aerosol device.Infectedmicewerekept in theABSLâ3 laboratory throughout thestudyandobserveddailytoensurethattheyhadenoughwaterandfood.
2.2â|âVirus and cells
MERSâCoV (human betacoronavirus 2cEMC/2012, complete geânomeGenBank:JX869059.2)wasprovidedbytheILAS,CAMS.TheviruswaspropagatedandexpandedinVeroE6cells(AmericanTypeCulture Collection, USA) cultured and passaged at 37°C and 5%CO2byroutinemethods.Purifiedandconcentratedprogenyviruseswere titratedusingVeroE6cellâbased infectivityassays,andviraltitersareexpressedinunitsof50%tissuecultureinfectiousdoseper100microliters(TCID50/100 ÎŒL).MERSâCoVstocksataconcentraâtionof106.8TCID50/100 ÎŒLwerestoredatâ80°C.
2.3â|âAnimal noseâonly aerosol exposure device
Ananimalnoseâonlyaerosolexposuredevice(INâTOXProducts)waslocated inanABSLâ3 laboratoryandcomprisedanoseâonlyexpoâsurechamberandnebulizerinsideaclassâ Ąbiologicalsafetycabinet(BSCâ Ą),acontrolbox,mouserestrainttubes,acleancompressedairtankandavacuumpump(Figure1).
The exposure device, which exposed only the mouse nose,generatedMERSâCoVaerosolparticlesof1.27±0.61ÎŒmtoinfecttransgenicmiceexpressinghDPP4andsimulatedthenaturalrouteofinfection.21,22
ââ â | â3HAO et Al.
2.4â|âInfection of hDPP4 transgenic mice
AsshowninTable1,transgenicmicewererandomlyassignedtoanaerosolgroup,an instillationgroup,anaerosolcontrolgroup,and an instillation control group, and the body weight of eachmousewasmeasuredonthedayofinfection(day0).Eachgroupcontained17mice;fivemiceineachgroupwereusedtoanalyzeclinicalsymptoms,weightlossandsurvival,andthreemiceineachgroupwererandomlychosenfornecropsyondays3,5,7,and9postinfection.
MERSâCoV virus suspensions (106.5 TCID50) and serumâfreeDulbecco'sModifiedEagleMedium(DMEM)wereseparatelyaddedtothenebulizerreservoirtoinfectexposedmiceintheaerosolandcontrol aerosol groups, respectively, for 30minutes.According to
theinstructionsoftheexposuredeviceandmouserespiratoryrate(25mL/minpermouse),thenebulizerflowratewassetto0.24L/min, the diluter flow ratewas set to 6.8 L/min, and the nebulizerpressurewassetto20PSI.Micewereanesthetizedwith1.2%tribroâmoethanol(0.2mL/10gofbodyweight,intramuscular(im))forintraânasalinoculationwith106.5TCID50ofMERSâCoVintheinstillationgroupandserumâfreeDMEMintheinstillationcontrolgroup.
2.5â|âClinical signs and sample collection
Infectedmicewereobservedfor14consecutivedaystoanalyzetheclinicalsymptomsofdisease,weightchange,andsurvival.Themicewereeuthanizedwith1.2%tribromoethanol (0.2mL/10gofbodyweight,im)whentheyreached25%weightloss.
F I G U R E 1 âTheanimalnoseâonlyinhalationexposuredevice.A,Photographoftheanimalnoseâonlyexposuredevice.B,PhotographoftheaerosolgeneratorandexposurechamberlocatedinaBSCII.C,Photographofnoseâonlyexposure
Group Material Purpose Number
Aerosol MERSâCoVaerosol Toanalyzeclinicalsigns,weightloss,andsurvival
5a
Necropsy 12b
Intranasalinstillation MERSâCoVsuspension Toanalyzeclinicalsigns,weightloss,andsurvival
5a
Necropsy 12b
Aerosolcontrol DMEMaerosol Toanalyzeclinicalsigns,weightloss,andsurvival
5a
Necropsy 12b
Instillationcontrol DMEMsuspension Toanalyzeclinicalsigns,weightloss,andsurvival
5a
Necropsy 12b
aFivemicepergroupwereobservedtorecordclinicalsymptoms,weight,andsurvivalfor14conâsecutivedayspostinfection.bThreemicefromeachgroupof12micewererandomlynecropsiedondays3,5,7,and9postinfection.
TA B L E 1 âGroupsoftransgenicmiceinfectedwithMiddleEastrespiratorysyndromecoronavirus(MERSâCoV)
4â |â ââ HAO et Al.
Ondays3,5,7,and9postinfection,threeanimalsrandomlyseâlectedfromeachgroupunderwentnecropsytoobtaintissuespeciâmensforassessingviraldistribution,associatedhistopathology,andcytokine levels using quantitative reverse transcriptionâPCR (qRTâPCR),hematoxylinandeosin(H&E)staining,immunohistochemistry(IHC),andenzymeâlinkedimmunosorbentassay(ELISA).
2.6â|âViral RNA in tissue samples
TotalviralRNAwasextractedfromtissues(lungs,brain,kidneys,spleen,liver,heart,andintestine)homogenizedusingtheRNeasyMiniKit(Qiagen)accordingtothemanufacturer'sinstructionsandfrozen at â80°C. MERSâCoV RNA copies were determined in a25.0ÎŒLmixture containing5.0ÎŒL ofRNAusing theQuantiTectProbe RTâPCR Kit (Qiagen, 204 443) with the ABI StepOnePlusâąRealâtimePCRSystem(LifeTechnologies).Theprimersandprobes for theMERSâCoVâspecific upstream E gene (upE) wereas follows: forward, 5âČâGCAACGCGCGATTCAGTTâ3âČ; reverse,5âČâGCCTCTACACGGGACCCATAâ3âČ; and fluorescent probe, 5âČâFAMâCTCTTCACATAATCGCCCCGAGCTCGâTAMRAâ3âČ. A plasâmid carrying theMERSâCOV upE gene was used as a standardcontrol.23AstandardcurvewasgeneratedforPCRusing10â107 copiesofaqualifiedstandardplasmidtocalculatecopynumbersforeachreaction.
2.7â|âHistopathology and IHC
Formalinâfixed lung, brain, and kidney samples were embeddedin paraffin wax and sectioned at an approximately 5âÎŒm thickâness. Deparaffinized and hydrated tissue sections were rouâtinely stained with H&E to examine histopathological changes.Immunohistochemical staining was performed to assess the exâpression of a viral antigen using a rabbit twoâstep detection kit(ZhongshanGoldenBridgeBiotechnologyCo.,Ltd)witharabbitpolâyclonalantiâMERSâCoVnucleoprotein(NP)antibody(SinoBiologicalInc).VisualizationwasthenperformedbyDABstainingandhemaâtoxylincounterstaining.
2.8â|âCytokine and chemokine profiles
Supernatants of tissue homogenates from infected mice (50 ”L)wereaddedtothebottomofanantibodyâcoatedplate.The levelsofinterleukin(IL)â1ÎČ,ILâ6,ILâ8,ILâ10,tumornecrosisfactor(TNF)âα,interferon (IFN)âÎł, and IFNâÎČwere assayed using ELISA kits (KeteBiotechnology Co., Ltd). Chemokine and cytokine concentrationswererecordedaspg/mLofhomogenateorng/Lofhomogenate.
2.9â|âStatistical analysis
DatawereanalyzedusingSPSS21orGraphPadPrism5.0software.Theexperimentalresultsarepresentedasthemeanplusstandarddeviation(SD).OneâwayANOVAwasusedtoassessdifferencesinbodyweight,viralload,andcytokinelevelsamongdifferentgroups.
Student'sttestwasperformedfortwoâgroupcomparisons.P <.05wasconsideredstatisticallysignificant.
3â |âRESULTS
3.1â|âClinical signs and weight loss
The infectedmice in both the aerosol and instillation groups disâplayedsignificantclinicalsymptoms,suchashuddling,hunching,rufâfledfur,weight loss,anddeath.Thereweresignificantdifferencesinweight change (P < .001) and survival (P < .0001) between theMERSâCoVinfectiongroupsandthecontrolgroups.Theincubationperiod,however,was5â7daysafteraerosolinfectionand1dayafterinstillation inoculation.AfterMERSâCoVaerosol exposure,hDPP4transgenicmice showedprofound clinical signs on days 5â7, rapidweightlossondays7â9and60%survivalbyday11(acutedeathoreuthanasiaat25%weightloss).Theintranasallyinfectedtransgenicmicedisplayedrapidweightlossondays1â5and0%survivalbyday5 (acutedeathoreuthanasia at25%weight loss).Therewere sigânificantdifferencesindiseaseprogression(P<.01)afterchallengebetween the aerosol group and the instillation group. TransgenichDPP4miceinfectedwithMERSâCoVaerosolsexhibitedmilderdisâeaseandslowerdiseaseprogressionthandidthoseinoculatedintraânasally(Figure2A,B).
Noobviousabnormalities,includingweightlossorsignsofcliniâcalillness,weredetectedintheaerosolcontrolandinstillationconâtrolgroups.TherewerenosignificantdifferencesinweightchangeorsurvivalratesbetweenmiceinoculatedwithDMEMintheabovetwocontrolgroups(P>.05;Figure2C).
3.2â|âViral load detection
BasedonqRTâPCRanalysesoftissueRNAcontents,weidentifiedhighviralloadsinthelungsandbraininmiceandasmallamountofviralRNAinothertissuesafterMERSâCoVinfectionviatheaeroâsolorinstillationroute(Figure3A,B).However,thereweresignifiâcantdifferencesinthetissueviralloadsofinfectedmicebetweenthe two groups (P < .0001). AfterMERSâCoV aerosol infection,highviralloadsweredetectedinthelungsat3â9daysandinthebrainat7â9days.Viral loadswerehigh inthe lungsandbrainofintranasallyinfectedmiceatdays3and5.Thereweresignificantdifferences (P < .0001) in the viral loads in the lungs and brainbetween the two groups at days 3 and5. The viral loads in thelungsandbrainsofthemiceintheaerosolgroupweresignificantlylowerthanthoseofthemiceintheinstillationgroup.HighlevelsofviralRNAaccumulatedmoreslowlyinthetissuesoftheMERSâCoVaerosolâexposedmicethaninthoseofthemiceinfectedinâtranasally(Figure3).
3.3â|âHistopathological assessment
Asshown inFigure4A,gross lung lesions, showing theappearanceofpulmonaryhyperemiaanddarkbrownregions,wereobserved in
ââ â | â5HAO et Al.
F I G U R E 2 âWeightchangeandsurvivalrateinmiceinfectedbytheaerosolorinstillationroute.Weightlossandsurvivalweremonitoredfor14dayspostinfection.A,Percentageofweightlossofmiceineachgroupafterinfection.B,Percentageofsurvivingmiceineachgrouppostinfection.C,Percentageofweightlossofmiceinthecontrolgroupsafterexposure.Thedataarepresentedasthemeanchange±SDforeachgroup(n=5).Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyreached25%weightloss;thesemicehada0%survivalratebyday5,whichproducednoresultsforweightlossondays7and9.Akeyindicatingthecolorcodingforthegroupsisprovidedinthefigure.*P<.05,**P<.01,***P<.001,and****P < .0001
F I G U R E 3 âqRTâPCRanalysisofmousetissuesamplescollectedafterinfectionwithMiddleEastrespiratorysyndromecoronavirus(MERSâCoV).A,ViralloadsinmousetissuesafterMERSâCoVaerosolexposure.B,ViralloadsinmousetissuesafterintranasalinfectionwithMERSâCoV.C,ViralloadsinmouselungsafterMERSâCoVinfection.D,ViralloadsinmousebrainsafterMERSâCoVinfection.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyreached25%weightloss;thesemicehada0%survivalratebyday5,sotherewerenoqRTâPCRresultsobtainedondays7and9.Thedataarepresentedasthemeanchange±SDforeachgroup(n=3).Akeyindicatingthecolorcodingofthegroupsisprovidedinthefigure.****P < .0001
6â |â ââ HAO et Al.
F I G U R E 4 âLungandbrainlesionsinmiceafterinfection.A,Grossnecropsyobservationofthelungsofinfectedmice.B,Histopathologicalchangesinthelungsofinfectedmice.Magnification:100Ă.C,Histopathologicalchangesinthebrainsofinfectedmice.Magnification:400Ă.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyresearched25%weightloss;thesemicehada0%survivalratebyday5,sonotissuelesionresultswereavailableondays7and9
ââ â | â7HAO et Al.
mice infectedwithMERSâCoVvia theaerosol inhalationor intranaâsal instillationroute,butnoobviouslesionswerefoundinothertisâsues.Therewerenoabnormalitiesinthetissuesofthenormalcontrolgroup.Itwasclearthattheappearanceofthelungsexhibitedobviouscongestionanddarkbrownregionsondays7â9intheaerosolgroup.TheMERSâCoVâintranasalmiceshowedgross lung lesionsonday3andmoreseverelunglesionsonday5.Grosslunglesionsdevelopedmoreslowlyandweremilderintheaerosolgroupthanintheinstillaâtiongroup(Figure4A).
Microscopically, challenged mice developed moderate acuteinterstitial pneumonia and brain pathology, but no pathologicalchangesweredetected inother tissues in themice. In theaerosolgroup,thelungsoftheexposedmiceshowedalveolarseptalwidenâing, inflammatorycell infiltration,andvesseldilatationandcongesâtionat3â9days,gradualdevelopmentofseverepathologicalchangesandinflammatorycellinfiltrationinperivascularregionsat5â9days,focalhemorrhagesat7â9days,andanexpandedpathologyrangeatday9(Figure4B).Dilatationandcongestionofthecerebralvesselswerenotclearlyobserveduntilday7,andfewareasofneurondeforâmationnecrosiswerefoundinthecerebralcortex,hippocampus,andthalamusbeforeday9(Figure3C).Ondays3and5afterintranasalinfection,wefoundmoderateacuteinterstitialpneumoniaandbrainlesions (Figure 4B,C). Tissue lesions, however, were milder in theaerosolgroupthanintheinstillationgroup.Furthermore,thereweresignificantdifferencesintheprogressionoflungandbrainlesionsinthetwoinfectedgroups.Tissuelesionprogressionwasslowerintheaerosolâinfectedmicethanintheinstillationâinfectedmice(Table2).
3.4â|âIHC
TheexpressionofaMERSâCoVantigenwasprimarilyevaluatedusingIHCassaysandwasfound inendothelialcellsandalveolarpneumoâcytes inthe lungsandincerebralcorticalneurons,dendrites,axons,
microgliaandthehippocampus inthebrainsofaerosolâand instillaâtionâchallengedmicebutnotincontrolmice(Figure5A,B).ProminentMERSâCoV expressionwas also observed in renal tubular epithelialcells (Figure5C).However, therewere significant differences in thetimingofvirusexpressioninthetissuesofthemicepostinfection.
AfterMERSâCoVinfection,IHCassayswitharabbitpolyclonalantiâMERSâCoV NP antibody found that viral antigens predomiânantlyappearedintrachealendothelialcellsatday3postinfectioninthelungsoftheaerosolâinfectedmiceandinbothtrachealendoâthelialcellsandpneumocytesinthelungsoftheaerosolâinfectedmiceat5â9days;thesechangeswereobservedinthelungsoftheinstillationâinfectedmiceat3and5days,respectively.Inaddition,theMERSâCoVantigenwasdiscoveredinthebrainandkidneysintheaerosolgroupat5â7daysandintheinstillationgroupat3and5days.Basedontheseresults,weconcludedthatthedistributionof the MERSâCoV antigen in the lungs, brain and kidneys afterinfectionwas slower in the aerosol group than in the instillationgroup.
3.5â|âCytokine and chemokine profiles
There were significant differences in the level of related proinâflammatorycytokineandchemokineprofiles,includingILâ1ÎČ,ILâ6,ILâ8,ILâ10,TNFâα,andIFNâÎł,betweeninfectiousgroups(theaeroâsol group and instillation group) and the control group (P < .05).Significantlyelevated levelsof ILâ1ÎČ, ILâ6, ILâ8, ILâ10,TNFâα, andIFNâÎłwerediscoveredinthelungsandbrainsofmiceintheaeroâsol groupwith increased CXCLâ1 at 3â9 days (P < .05) postchalâlengeand in thoseofmice the instillationgroupat3and5dayspostchallenge (Figure6). In the aerosol group, theexposedmiceshowedpeak ILâ10 and concentration in the lungs and ILâ10 andCXCLâ1concentrations in thebrainat5â9days, andpeakTNFâα and IFNâÎł levels in the lungs and brains with peak ILâ6 level at
TA B L E 2 âPathologicalchangesinthelungsofmiceafterMiddleEastrespiratorysyndromecoronavirusinfection
GroupAlveolar septum widtha
Interstitial inflammatory cell infiltrationb
Exudate in alveolic
Dilatation and congesâtion of vesselsd Hemorrhagee
3 d Aerosol + + â + â
Instillation +~++ + â + â
5d Aerosol + + â + â
Instillation ++~+++ ++ + ++ â
7d Aerosol ++ ++ â + â
Instillation NDf NDf NDf NDf NDf
9 d Aerosol ++ ++ â ++ +
Instillation NDf NDf NDf NDf NDf
Control â â â â â
aâ,noapparentchanges;+,mildalveolarseptumwidening;++,moderatealveolarseptumwidening;and+++,severealveolarseptumwidening.bâ,noapparentchanges;+,infiltrationofafewinterstitialinflammatorycells;and++,someinterstitialinflammatorycellinfiltration.câ,noapparentchanges;and+,asmallamountofexudateinalveoli.dâ,noapparentchanges;+,milddilatationandcongestionofvessels;and++,moderatedilatationandcongestionofvessels.eâ,noapparentchanges;and+,mildhemorrhage.fND,Notdone.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyreached25%weightloss,whichoccurredbyday5.
8â |â ââ HAO et Al.
F I G U R E 5 â Immunohistochemicalstainingofmousetissuesamplesafterinfection.A,Immunohistochemicalstainingofthelungsofinfectedmice.B,Immunohistochemicalstainingofthebrainsofinfectedmice.C,Immunohistochemicalstainingofthekidneysofinfectedmice.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyresearched25%weightloss;thesemicehada0%survivalratebyday5,sonotissuelesionresultswereavailableondays7and9
ââ â | â9HAO et Al.
7â9 days. After intranasal infection, however, the levels of ILâ1ÎČ,ILâ6,ILâ10,TNFâα,andIFNâÎłinthelungsandILâ6,ILâ8,ILâ10,andIFNâÎłinthebrainspeakedat3â5days.Thesecretionofsomecyâtokinesandchemokinesintheaerosolgroupwasslowerthanthatintheintranasalgroup(P<.05).
4â |âDISCUSSION
The dromedary camel, a natural host ofMERSâCoV, primarily deâvelopsupperrespiratorytractinfectionpostinoculationwithMERSâCoVviaanintratrachealorintranasalroute,whichfailstosimulatethesignsoflowerrespiratorytractinfectioninhumans.24MERSâCoV
naturallyinfectsrhesusmonkeysandcommonmarmosets,causingvaryingdegreesofclinicalsymptoms.AfterMERSâCoVinfectionbythe intranasal, intratracheal, oral and conjunctival routes,mildâtoâmoderate transient pneumonia occurs in rhesusmonkeyswithoutthe manifestation of severe clinical symptoms of MERSâCoV inâfection,25,26 and commonmarmosets developmoderateâtoâsevereinterstitial pneumonia or even die.6,7 Transgenic mice expressinghDPP4arepermissivetoMERSâCoVinfection,whichresultsindisâease andmortality.No animalmodels, however, fully recapitulatethehumandiseasecausedbyMERSâCoV.27,28Inthecurrentstudy,hDPP4transgenicmicewereinfectedwithMERSâCoVbytheaeroâsolorintranasalinstillationroute,andthereweresignificantdifferâencesindiseaseprogression,lunglesions,viralreplication,andvirus
F I G U R E 6 âCytokineandchemokinelevelsintissuesofmiceafterinfectionwithMiddleEastrespiratorysyndromecoronavirus(MERSâCoV).A,Postinfectioncytokineandchemokinelevelsinthelungsofmice.B,Postinfectioncytokineandchemokinelevelsinthebrainsofmice.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyresearched25%weightloss;thesemicehada0%survivalratebyday5,sonoresultswereavailableondays7and9.Theresultsrepresentthemean±SDforeachgroup(n=3).*P<.05,**P<.01,***P<.001,and****P < .0001
10â |â ââ HAO et Al.
distribution in tissues between the aerosolâ and instillationâchalâlengedmice(Table3).
AfterMERSâCoVinfection,thediseaseprogressioninthemiceintheaerosolgroupwasslowerthanthat inthemiceintheinstilâlationgroup.Sandersetalshowedthatvirusdropletsweredeposâited and concentrated in the lungs through the respiratory tractofmice inoculated intranasally, resulting in fastdiseaseonset.29,30 Correspondingly, after instillation infection with MERSâCoV, wefoundthatmicewithashortairwayandhighconcentrationofvirusdepositedinthelungsdisplayedweightlossatday1andlunglesionsat day 3, consistentwith intranasalmousemodels established byAdam,AgrawalandLietal31â33;thesemicealsoexhibited0%survivalbyday5.Previousstudiesreportedthataerosolparticlesâ€5ÎŒmpenâetratedtherespiratorytracttoreachthealveoliandwerediffuselydistributedinthelungs.30,34Inaddition,virusaerosolsenteredthebloodcirculationthroughthealveoli,andothervirusesslowlyrepliâcatedinthelungsaftermiceinhaledMERSâCoVâcontainingaerosols(particle size: 1.27 ± 0.61 ÎŒm). Compared with instillationâinocuâlatedmicewithvirusdepositioninthelungs,aerosolâexposedmicedisplayedslowerdiseaseprogressionwithan incubationperiodof
5â7days,lunglesionsonday7,continuousweightlossondays7â11,milderclinicalsigns,and60%survivalonday11.
Wefoundthattheprogressionsofvirusreplicationandlunglesionsinchallengedmicewereslowerintheaerosolgroupthanin the instillation group. Based on high viral loads in the lungsandbrainofchallengedmice,whichwasconsistentwithpreviousreports,35 andacute renal failure inMERSpatients,wecarriedout H&E staining to assess histopathological changes and imâmunohistochemical stainingwitha specific antibody to furthercharacterizeMERSâCoVexpressioninthe lungs,brain,andkidâneys.Arelativelyhighviralloadinthelowerrespiratorytractisassociatedwith severe illness in viral respiratory diseases.36,37 At 3â5 days postinfection, mice in the intranasal group, whichhadhighviralloadsinthelungsandbrainat3â5days,exhibitedacute interstitial pneumoniaandpathologicalbrain changes. Intheaerosolgroup,micedevelopedacute interstitialpneumoniaat 3â9 days and pathological brain changes at 7â9 days, whichwerecausedbyhighlevelsofvirusRNAinthelungsat3â9daysandinthebrainat7â9days,respectively.HighervirusRNAlevâelsintheinstillationgroupmightcontributetothemoresevere
ParameterMice infected with MERSâCoV aerosola
Mice infected intranasally with MERSâCoV
Incubationperiod 5â7d 1 d
Weightloss 7â11d 1â5d
Survival 60% 0%
Grosslunglesions 7â9d 3â5d
Viralload
Lungs Highlevelondays3to9 Highlevelondays3and5
Brain Highlevelondays7to9 Highlevelondays3and5
Histopathology
Lungs Moderateacuteinterstitialpneuâmoniaondays3to9
Moderateacuteinterstitialpneumoâniaondays3to5
Brain Relativelymildbrainlesionondays7and9
Brainlesionsondays3and5
MERSâCoVantigendistribution
Lungs Inbronchialendothelialcellsonday 3Inbothtrachealendothelialcellsandalveolarpneumocytesinthelungsondays5to9
Inbothtrachealendothelialcellsandalveolarpneumocytesinthelungsondays3and5
Brain Incerebralcorticalneurons,denâdrites,axons,glialcells,andthehippocampusondays5to9
Incerebralcorticalneurons,denâdrites,axons,glialcells,andthehippocampusondays3and5
Kidneys Inrenaltubularepithelialcellsondays5to9
Inrenaltubularepithelialcellsondays5to9
Cytokinesandchemokinesb
Lungs Highlevelsondays3to9,includâingCXCLâ1
Highlevelsondays3to5
Brain Highlevelsondays3to9,includâingCXCLâ1
Highlevelsondays3to5
aNoseâonlyexposure.bCytokinesandchemokinesincludeILâ1ÎČ,ILâ6,ILâ8,ILâ10,TNFâα,andIFNâÎłandCXCLâ1.
TA B L E 3 âComparisonofMiddleEastrespiratorysyndromecoronavirus(MERSâCoV)infectionofmicebytheaerosolorinstillationroute
ââ â | â11HAO et Al.
histopathological changes observed. Furthermore, IHC assaysrevealedthatMERSâCoVprimarilyreplicatedinendothelialcellsandalveolarpneumocytesinthelungsandmanycelltypesinthebrainaswellasepithelialcellsinthekidneysinchallengedmice,as evidenced by MERSâCoV expression localized in the lungs,brainandkidneys inprevious reports.32,33MERSâCoVdistribuâtioninthebrainwasslowerinaerosolâinfectedmice(at5â7days)than in instillationâinfected mice (at 3â5 days), which closelymirroredthedifferences intheprogressionsofdiseaseandtisâsue lesions. Overall, aerosolâinfected mice developed slowerprogressions of disease, viral replication, and lung lesions, andmildersymptomsthanintranasallyinfectedmice.
Middle East respiratory syndrome coronavirus infection wasassociatedwithsignificant inductionofproinflammatorycytokinesand chemokines, which substantially contributed to severe pneuâmonia.38,39AccordingtotheresultsofclinicalstudiesandpreviousreportsonMERSâCoVanimalmodels,weassayedthelevelsofeightcytokinesandchemokines:ILâ1ÎČ,ILâ6,ILâ8,ILâ10,TNFâα,IFNâÎČ,IFNâÎł,and CXCLâ1 by ELISA. Clinical studies have shown that markedlyincreased levelsof ILâ1ÎČ, ILâ6, ILâ8,TNFâα, and IFNâÎł aredetectedinpatientswithsevereMERS.38,40Previousreportshave indicatedelevated levels of ILâ1ÎČ, ILâ6, ILâ10, TNFâα, IFNâÎł, and CXCLâ1 inMERSâCoVanimalmodels.6,25,32,33Additionally,we found that theconcentrationsof these cytokines and chemokinesweremarkedlyelevated inthe lungsandbrainofaerosolâand instillationâinfectedmice.Theinductionofproinflammatorycytokinesandchemokinesin the lungs plays an important role in tissue immunopathologicalchangesandfatalpneumonia.Anelevatedpulmonaryviralloadanditsinjuriouseffectsonthepulmonarysystemarelikelyresponsiblefortheincreasedinflammatoryresponse.Itispossiblethattherelâatively robustpulmonary inflammatoryresponse inmicemayhavepromotedMERSâCoVreplication,causingincreasedpulmonaryviralloadsinthemice.36
MiddleEastrespiratorysyndromepatientsexhibitamedianinâcubationperiodof5â7days,witharangeof2â14days.41Wediscovâeredthat instillationâinoculatedmiceexhibitedclinicalsignswithin1daybutthatthe incubationperiodofaerosolâexposedmicewas5â7days,whichmorecloselyresembledtheperiodobservedinhuâmans.MERSâCoVbinds tohDPP4 receptors that areprimarilyexâpressedinthelowerrespiratorytractandalveoli,resultinginawiderangeofdiseasesymptoms inpatients, fromnosymptomstomildrespiratory illness or severe acute pneumonia, which rapidly proâgressestoacutelungdamage,multiorganfailureandevendeath.42,43
Clinically, chest radiography and chest computed tomograâphy (CT) showno lung lesions in patients in the early stages ofillness,butpneumonia is identifiedduring thecourseof thedisâease and includes patchy densities, extensive diffuse and focalalveolar space opacities, interstitial infiltrates, and consolidaâtion.44â46 Patients with severe illness progress rapidly to acuterespiratory failure andevendeath. Inone fatal case, pulmonaryhistopathologicalchangesincludedexudativeâphasediffusealveâolardamagewithdenudingof thebronchiolarepithelium,promâinent hyalinemembranes, alveolar fibrin deposit, alveolar septa,and so on.47 In thiswork,microscopic observation showed thatinfectedmice eventually developedmoderate diffuse interstitialpneumonia similar to that observed in humans withMERSâCoVinfection.Asobservedbynecropsy,aerosolâinfectedmicedevelâopedlunglesionsat7â9days,butnolunglesionsoccurredwithinthe incubationperiod, and subsequenthistopathology identifiedmildâtoâmoderate diffuse interstitial pneumonia without severehistopathologyat3â9days,whichalmostmatchedtheprogressivemoderatepneumoniaobservedinMERSpatients.However,instilâlationâinoculatedmiceshowedashorterincubationperiod(1daypostinfection)andfailedtostimulatetheprogressionofmildâtoâmoderatepneumoniaduetotheacutediseasecourse.AsshowninTable4, the incubationperiodofMERSâCoVand the induced
TA B L E 4 âComparisonsofMiddleEastrespiratorysyndromecoronavirus(MERSâCoV)âinfectedmiceandMERSpatients
Parameter MERS patientsaMice infected with MERSâCoV aerosols
Mice infected intranasally with MERSâCoV
Incubationperiod 5â7d,witharangeof2â14d 5â7d 1 d
Lunglesionprogress EarlystageshowsnoabnorâmalitiesbychestradiographyorchestCT
Within7d,nolunglesionsafternecropsy
Ondays3and5,lungdamagewithmoderatediffuseinterstiâtialpneumonia
Subsequentdevelopmentofmildpneumonia
Ondays3to5,mildalveolarseptumwideningandinflammatorycellfiltration
Progressivedevelopmentofmoderatediffuseinterstitialpneumonia
Ondays7to9,lungdamagewithmoderatediffuseinterstitialpneumonia
Afatalcase:severediffuseinterstitialpneumonia
â â
Distributionofaviralantigen Alveolarpneumocytesandendothelialcells
Alveolarpneumocytesandendothelialcells
Cytokinesandchemokines SignificantlyhighlevelsofTNFâα,IFNâÎł,ILâ1ÎČ,ILâ6,andILâ8
aMERSpatientsrefertothosewithinfectionofthelowerrespiratorytract.
12â |â ââ HAO et Al.
pulmonary pathological changes in aerosolâinfected mice weresimilartothosenotedinpatientswithrespiratorytractinfection.
Additionally, immunohistochemical staining revealed that aMERSâCoVantigenwasexpressedinalveolarpneumocytesandenâdothelialcells,thebrain,andthekidneysinchallengedtransgenicmice. Studies of a fatal case of MERSâCoV infection evidencedthat the expression of aMERSâCoV antigen was predominantlylocalizedinpneumocytesandendothelialcells,resultingincellneâcrosisandpneumocytedamage;however,noviralantigensweredetected inother tissues in the fatalcase.47Asdemonstrated inpreviousstudies,wealsodiscoveredhighviralloads,pathologicalchangesandtheexpressionofaMERSâCoVantigen in thebrainofchallengedmice;andnobrainlesions,butmultiorgandamage,wereobservedinMERSpatients.35,48,49Zhouetaldemonstratedthat human dendritic cells andmacrophageswere permissive toMERSâCoV replication, indicating that the multiorgan injury inâducedbyMERSâCoVmaybe associatedwith thedistributionofthehDPP4receptorinmanycelltypesthatarespreadthroughoutmultiple organs.38 Some studies have indicated thatMERSâCoVhascellandtissuetropisms,especiallytropismsforpneumocytesandneurons,andsynapsesmaybeoneofthestructuresbywhichvirusesdiffuse through thebrainafterMERSâCoV infection.31,35 Themechanismsunderlying thebrain lesionsanddeath inducedbyMERSâCoVinfectioninhDPP4transgenicmiceremaincomplexandcomplicatedandneedtobefurtherinvestigated.
5â |âCONCLUSION
hDPP4transgenicmiceweresuccessfullyinfectedwithMERSâCoVaerosolsbyananimalnoseâonlyexposuredevice,andaerosolâandinstillationâinfected mice all simulated the clinical symptoms ofmoderate diffuse interstitial pneumonia. Compared to instillaâtionâinfectedmice,aerosolâinfectedmicemorecloselyresembledinfected humans in terms of the progression of disease and paâthologyinthelungs,whichprovidedadditionaldataforstudyingpathogenesisandevaluatingtheefficacyofpreventiveandtheraâpeuticagentsforMERSâCoV.
ACKNOWLEDG EMENT
The current work was supported by the National Science andTechnology Major Projects of Infectious Disease (grant number2018ZX10734401â011).
CONFLIC T OF INTERE S T
None.
AUTHOR CONTRIBUTIONS
HGwastheprincipalinvestigator,designedandsupervisedthestudy,andwrotethegrantapplication.XYHperformedthemainexperiments.
XYHandQLperformedthecellexperiments.XYHandFDLconductedtheanimalexperiments.YFXcompletedthepathologyexperiments.XYHandHGconceivedtheexperiments,analyzedthedataandwrotethepaper.Allauthorsreadandapprovedthefinalmanuscript.
ORCID
Xinâyan Hao https://orcid.org/0000â0001â7664â9568
R E FE R E N C E S
1. Fehr AR, Channappanavar R, Perlman S.Middle East respiratorysyndrome: emergence of a pathogenic human coronavirus.Annu Rev Med.2017;68:387â399.
2. KimY.Nurses'experiencesofcareforpatientswithMiddleEastreâspiratorysyndromeâcoronavirusinSouthKorea.Am J Infect Control. 2018;46:781â787.
3. ChanJF,LauSK,ToKK,ChengVC,WooPC,YuenKY.MiddleEastrespiratorysyndromecoronavirus:anotherzoonoticbetacoronaviâruscausingSARSâlikedisease.Clin Microbiol Rev.2015;28:465â522.
4. MiddleEastrespiratorysyndromecoronavirus(MERSâCoV).MERSMonthlySummary,2019.https://www.who.int/emergencies/mersâcov/en/.AccessedJuly2,2019.
5. LuoCâM,WangN,YangXâL,etal.DiscoveryofnovelbatcoronaâvirusesinSouthChinathatusethesamereceptorasMiddleEastrespiratorysyndromecoronavirus.J Virol.2018;92.pii:e00116â18.
6. FalzaranoD,deWitE,FeldmannF,etal.InfectionwithMERSâCoVcauses lethal pneumonia in the commonmarmoset.PLoS Pathog. 2014;10:e1004250.
7. YuP,XuY,DengW,et al.Comparativepathologyof rhesusmaâcaqueandcommonmarmosetanimalmodelswithMiddleEastreâspiratorysyndromecoronavirus.PLoS ONE.2017;12:e0172093.
8. Skariyachan S, Challapilli SB, Packirisamy S, Kumargowda ST,SridharVS.Recentaspectsonthepathogenesismechanism,anâimalmodelsandnoveltherapeuticinterventionsforMiddleEastrespiratory syndrome coronavirus infections. Front Microbiol. 2019;10:569.
9. VergaraâAlert J, Vidal E, BensaidA, Segales J. Searching for aniâmalmodels andpotential target species foremergingpathogens:experiencegainedfromMiddleEastrespiratorysyndrome(MERS)coronavirus.One Health.2017;3:34â40.
10. vanDoremalenN,MiazgowiczKL,MilneâPriceS,etal.HostspeciesrestrictionofMiddleEastrespiratorysyndromecoronavirusthroughitsreceptor,dipeptidylpeptidase4.J Virol.2014;88:9220â9232.
11. RajVS,MouH, Smits SL, et al.Dipeptidyl peptidase4 is a funcâtionalreceptorfortheemerginghumancoronavirusâEMC.Nature. 2013;495:251â254.
12. Gardner EG, KeltonD, Poljak Z, vonDobschuetz S, Greer AL. ArapidscopingreviewofMiddleEastrespiratorysyndromecoronaâvirusinanimalhosts.Zoonoses Public Health.2019;66:35â46.
13. YaoY,LanJ,LiF,etal.Clinicalandbiologicalcharacter inmousemodelsforMiddleEastrespiratorysyndromegeneratedbytransâductionwithdifferentdosesofDPP4molecule.Bing Du Xue Bao. 2015;31:593â600.
14. AlgaissiA,AgrawalAS,HanS,etal.Elevatedhumandipeptidylpepâtidase4expressionreducesthesusceptibilityofhDPP4transgenicmice to Middle East respiratory syndrome coronavirus infectionanddisease.J Infect Dis.2019;219:829â835.
15. IwataâYoshikawa N, Okamura T, Shimizu Y, et al. Acute respiraâtoryinfectioninhumandipeptidylpeptidase4âtransgenicmiceinâfectedwithMiddleEastrespiratorysyndromecoronavirus.J Virol. 2019;93.pii:e01818â18.
ââ â | â13HAO et Al.
16. Emergenciespreparedness,response.Frequentlyaskedquestionson Middle East respiratory syndrome coronavirus (MERSâCoV).https://www.who.int/csr/disease/coronavirus_infections/faq/en/.AccessedJanuary21,2019.
17. LinQ,ChiuAP,ZhaoS,HeD.ModelingthespreadofMiddleEastrespiratory syndrome coronavirus in Saudi Arabia. Stat Methods Med Res.2018;27:1968â1978.
18. AlagailiAN,BrieseT,MishraN,etal.MiddleEastrespiratorysynâdromecoronavirusinfectionindromedarycamelsinSaudiArabia.MBio.2014;5:e00884â14.
19. KimSâH,ChangSY,SungM,etal.ExtensiveviableMiddleEastreâspiratory syndrome (MERS) coronavirus contamination in air andsurroundingenvironment inMERS isolationwards.Clin Infect Dis. 2016;63:363â369.
20. WHO. Infection prevention and control during health care forprobableorconfirmedcasesofMiddleEastrespiratorysyndromecoronavirus (MERSâCoV) infection. World Health Organization.Updated October 2019. https://www.who.int/csr/disease/coronavirus_infections/ipcâmersâcov/en/.AccessedOctober11,2019.
21. WongBA.Inhalationexposuresystems:design,methodsandoperâation.Toxicol Pathol.2007;35:3â14.
22. MarriottAC,DennisM,KaneJA,etal.InfluenzaAviruschallengemodelsincynomolgusmacaquesusingtheauthenticinhaledaerosolandintraânasalroutesofinfection.PLoS ONE.2016;11:e0157887.
23. WHO.Emergenciespreparedness,response.LaboratorytestingforMiddle East respiratory syndrome coronavirus. Interim guidance.Updated January2018. https://www.who.int/csr/disease/coronaviârus_infections/mersâlaboratoryâtesting/en/.AccessedMay1,2019.
24. Adney DR, van Doremalen N, Brown VR, et al. Replication andshedding of MERSâCoV in upper respiratory tract of inoculateddromedarycamels.Emerg Infect Dis.2014;20:1999â2005.
25. deWitE,RasmussenAL,FalzaranoD,etal.MiddleEastrespiratorysyndrome coronavirus (MERSâCoV) causes transient lower respiâratorytract infectioninrhesusmacaques.Proc Natl Acad Sci USA. 2013;110:16598â16603.
26. Cockrell AS, Johnson JC,Moore IN, et al. A spikeâmodifiedMiddleEastrespiratorysyndromecoronavirus(MERSâCoV) infectiouscloneelicitsmildrespiratorydisease in infectedrhesusmacaques.Sci Rep. 2018;8:10727.
27. BaselerL,deWitE,FeldmannH.AcomparativereviewofanimalmodelsofMiddleEastrespiratorysyndromecoronavirusinfection.Vet Pathol.2016;53:521â531.
28. DawsonP,MalikMR,ParvezF,MorseSS.Whathavewe learnedaboutMiddleEastrespiratorysyndromecoronavirusemergenceinhumans?Asystematicliteraturereview.Vector Borne Zoonotic Dis. 2019;19:174â192.
29. SandersCJ,JohnsonB,FrevertCW,ThomasPG. Intranasal influâenza infection ofmice andmethods to evaluate progression andoutcome.Methods Mol Biol.2013;1031:177â188.
30. BelserJA,GustinKM,KatzJM,MainesTR,TumpeyTM.ComparisonoftraditionalintranasalandaerosolinhalationinoculationofmicewithinfluenzaAviruses.Virology.2015;481:107â112.
31. CockrellAS,YountBL, ScobeyT, et al.Amousemodel forMERScoronavirusâinduced acute respiratory distress syndrome. Nat Microbiol.2016;2:16226.
32. Agrawal AS, Garron T, Tao X, et al. Generation of a transgenicmousemodelofMiddleEastrespiratorysyndromecoronavirusinâfectionanddisease.J Virol.2015;89:3659â3670.
33. Li K, WohlfordâLenane CL, Channappanavar R, et al. MouseâadaptedMERS coronavirus causes lethal lungdisease in humanDPP4 knockin mice. Proc Natl Acad Sci USA. 2017;114:E3119â E3128.
34. YiD,NaqwiA,PanoskaltsisâMortariA,WiedmannTS.Distributionof aerosols in mouse lobes by fluorescent imaging. Int J Pharm. 2012;426:108â115.
35. ZhaoG,JiangY,QiuH,etal.Multiâorgandamageinhumandipepâtidylpeptidase4transgenicmiceinfectedwithmiddleeastrespiraâtorysyndromeâcoronavirus.PLoS ONE.2015;10:e0145561.
36. BaselerLJ,FalzaranoD,ScottDP,etal.AnacuteimmuneresponsetoMiddleEastrespiratorysyndromecoronavirusreplicationconâtributestoviralpathogenicity.Am J Pathol.2016;186:630â638.
37. FeikinDR,AlraddadiB,QutubM,etal.AssociationofhigherMERSâCoVvirusloadwithseverediseaseanddeath,SaudiArabia,2014.Emerg Infect Dis.2015;21:2029â2035.
38. ZhouJ,ChuH,LiC,etal.ActivereplicationofMiddleEastrespiraâtorysyndromecoronavirusandaberrantinductionofinflammatorycytokinesandchemokinesinhumanmacrophages:implicationsforpathogenesis.J Infect Dis.2014;209:1331â1342.
39. Yin Y, Wunderink RG. MERS, SARS and other coronaviruses ascausesofpneumonia.Respirology.2018;23:130â137.
40. LauS,LauC,ChanKâH,etal.Delayed inductionofproinflammaâtorycytokinesandsuppressionofinnateantiviralresponsebythenovelMiddleEastrespiratorysyndromecoronavirus: implicationsforpathogenesisandtreatment.J Gen Virol.2013;94:2679â2690.
41. VirlogeuxV,ParkM,WuJT,CowlingBJ.AssociationbetweenseâverityofMERSâCoVinfectionandincubationperiod.Emerg Infect Dis.2016;22:526â528.
42. ChaMJ, ChungMJ, Kim K, Lee KS, Kim TJ, Kim TS. Clinical imâplication of radiographic scores in acuteMiddle East respiratorysyndromecoronaviruspneumonia:reportfromasingletertiaryâreâferralcenterofSouthKorea.Eur J Radiol.2018;107:196â202.
43. GaroutMA,JokhdarH,AljahdaliIA,ZeinAR,GowedaRA,HassanâHusseinA.MortalityrateofICUpatientswiththeMiddleEastreâspiratorysyndromeâcoronavirusinfectionatKingFahadHospital,Jeddah,SaudiArabia.Cent Eur J Public Health.2018;26:87â91.
44. Arabi YM,BalkhyHH,HaydenFG, et al.Middle East respiratorysyndrome.N Engl J Med.2017;376:584â594.
45. AssiriA,AlâTawfiqJA,AlâRabeeahAA,etal.Epidemiological,deâmographic,andclinicalcharacteristicsof47casesofMiddleEastrespiratorysyndromecoronavirusdiseasefromSaudiArabia:adeâscriptivestudy.Lancet Infect Dis.2013;13:752â761.
46. ChoiWS,KangCâI,KimY,etal.ClinicalpresentationandoutcomesofMiddleEastrespiratorysyndromeintheRepublicofKorea.Infect Chemother.2016;48:118â126.
47. NgDL,AlHosaniF,KeatingMK,etal.Clinicopathologic,immunoâhistochemical,andultrastructuralfindingsofafatalcaseofMiddleEastrespiratorysyndromecoronavirusinfectionintheUnitedArabEmirates,April2014.Am J Pathol.2016;186:652â658.
48. LiK,WohlfordâLenaneC,PerlmanS,etal.MiddleEastrespiratorysyndromecoronaviruscausesmultipleorgandamageandlethaldisâeaseinmicetransgenicforhumandipeptidylpeptidase4.J Infect Dis.2016;213:712â722.
49. FanC,WuXI, LiuQ,et al.AhumanDPP4âKnockinmouse's susâceptibilityto infectionbyauthenticandpseudotypedMERSâCoV.Viruses.2018;10:448.
SUPPORTING INFORMATION
Additional supporting information may be found online in theSupportingInformationsection.
How to cite this article:HaoX,LvQ,LiF,XuY,GaoH.ThecharacteristicsofhDPP4transgenicmicesubjectedtoaerosolMERScoronavirusinfectionviaananimalnoseâonlyexposuredevice. Anim Models Exp Med. 2019;00:1â13. https:// doi.org/10.1002/ame2.12088