Anim Models Exp Med. 2019;00:1–13.  | 1wileyonlinelibrary.com/journal/ame2

Received:2July2019  |  Revised:12September2019  |  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 nose‐only exposure device

Xin‐yan Hao  | Qi Lv | Feng‐di Li | Yan‐feng Xu | Hong Gao

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution‐NonCommercialLicense,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:gaoh@cnilas.org

Funding informationNationalScienceandTechnologyMajorProjectsofInfectiousDisease,Grant/AwardNumber:2018ZX10734401‐011

AbstractBackground: MiddleEast respiratory syndromecoronavirus (MERS‐CoV),which isnotfullyunderstoodinregardtocertaintransmissionroutesandpathogenesisandlacksspecifictherapeuticsandvaccines,posesaglobalthreattopublichealth.Methods: Tosimulatetheclinicalaerosoltransmissionroute,hDPP4transgenicmicewere infectedwithMERS‐CoVbyananimalnose‐onlyexposuredevice andcom‐paredwith instillation‐inoculatedmice.Thechallengedmicewereobservedfor14consecutivedaysandnecropsiedondays3,5,7,and9toanalyzeviralload,histopa‐thology,viralantigendistribution,andcytokinesintissues.Results: MERS‐CoV aerosol‐infected mice with an incubation period of 5‐7 daysshowedweightlossondays7‐11,obviouslunglesionsonday7,highviralloadsinthelungsondays3‐9andinthebrainondays7‐9,and60%survival.MERS‐CoVinstil‐lation‐inoculatedmiceexhibitedclinicalsignsonday1,obviouslunglesionsondays3‐5,continuousweightloss,0%survivalbyday5,andhighviral loadsinthelungsandbrainondays3‐5.Viral antigenandhigh levelsofproinflammatorycytokinesandchemokinesweredetectedintheaerosolandinstillationgroups.Disease,lunglesion,andviralreplicationprogressionswereslowerintheMERS‐CoVaerosol‐in‐fectedmicethanintheMERS‐CoVinstillation‐inoculatedmice.Conclusion: hDPP4transgenicmiceweresuccessfullyinfectedwithMERS‐CoVaer‐osolsviaananimalnose‐onlyexposuredevice,andaerosol‐andinstillation‐infectedmice simulated the clinical symptoms of moderate diffuse interstitial pneumonia.However,thetransgenicmiceexposedtoaerosolMERS‐CoVdevelopeddiseaseandlungpathologyprogressionsthatmorecloselyresembledthoseobservedinhumans.

K E Y W O R D S

animalnose‐onlyexposuredevice,hDPP4transgenicmice,intranasalinstillation,MERS‐CoVaerosolinfection

2  |     HAO et Al.

1  | INTRODUC TION

MiddleEastrespiratorysyndromecoronavirus (MERS‐CoV),whichwasfirstidentifiedinSaudiArabiain2012andcausesacuterespira‐toryillness,multiorganfailure,shockandevendeath,isanimportanthighlypathogeniccoronavirusthat issimilartosevereacuterespi‐ratorysyndromecoronavirus (SARS‐CoV)andproducessevere in‐fectionswithahighmortalityrate.1‐3AttheendofMay2019,therewereatotalof2428laboratory‐confirmedcasesofMERSwith838associated deaths (case‐fatality rate: 34.5%, which is higher thanthe fatality rate of SARS) worldwide according to World HealthOrganization(WHO)statistics.4MERScaseshavebeenreportedin27countries,includingcountriesintheMiddleEast,Africa,Europe,Asia,andNorthAmericaaswellasAustralia,andcasenumberscon‐tinueto increase,posingaglobal threattopublichealth. InChina,thefirstpatientinfectedwithMERS‐CoVfromSouthKoreawasdi‐agnosedinMay2015,5anditwillbeextremelyimportanttoprevent,control,andtreatMERS‐CoVinfectionsduringanyfutureoutbreaks.Hence,effectivesmallanimalmodelsareneededtoinvestigateviralpathogenesisandevaluateMERS‐CoVtherapeuticsandvaccines.

NonhumanprimateanimalmodelsofMERS‐CoVinbothrhesusmacaquesandcommonmarmosetswereestablishedinpreviousre‐ports,6,7however,thesemodelsarelimitedbyrestrictedavailability,highcosts,experthusbandryrequirements,andethicalconcerns.8,9 Traditionalsmallanimalssuchasmice,hamsters,andferretscannotbeinfectedwithMERS‐CoVowingtoabsenceofthenecessarydi‐peptidylpeptidase4(DPP4)receptorthatinteractswiththerecep‐torbindingdomainof theMERS‐CoVspikeprotein (Sprotein)10‐12 MERS‐CoVfailstoreplicateinmice,whicharereadilyavailable,haveadefinedgeneticbackgroundandlowcostandarefrequentlyusedininfectiousdiseaseresearch,duetovariationsintheDPP4recep‐tor. Previous studies showed that transgenic mice expressing thehumanDPP4 (hDPP4)receptorcouldbe infected intranasallywithMERS‐CoVanddevelopedacutepneumonia.13‐15Therefore,hDPP4transgenicmicewereselectedforexposuretoMERS‐CoV‐contain‐ingaerosolsusingananimalnose‐onlyexposuredevice.

TherearetwomodesofMERS‐CoVinfection,animal‐to‐humanandhuman‐to‐humantransmission.16Somereportshavefoundthatairborne transmission via the coughing and sneezing of infecteddromedarycamelsorcontactwithrespiratorysecretionsandcon‐sumptionofunsterilizedmilkfrominfectedcamelscansignificantlyincreasetheriskofMERS‐CoVinfectioninhumans.17,18Kimetal19 discoveredextensiveviableMERS‐CoVcontaminationintheairandsurroundingenvironmentinMERSisolationwards.AccordingtotheWHO,ithasbeensuggestedthathuman‐to‐humantransmission,toaverylimitedextent,iscausedbyinhalationofdropletsorairbornevirus and close contact with patients.20 The above studies havedemonstrated thatMERS‐CoV has a risk of aerosol transmission.Inaddition,aerosolinhalationisthemainclinicalrouteofinfectionfor viral respiratory illnesses. There are different clinical presen‐tations in animalmodelsestablishedbydifferent infection routes.ComparativestudiesusingapproacheswithdifferentperspectiveswillcontributetoadeeperunderstandingofMERS.

In this work, to simulate the aerosol transmission route forcomparison with the instillation route, hDPP4 transgenic micewereexposedtoMERS‐CoVaerosolsbyananimalnose‐onlyex‐posuredevice.Afterinfection,weanalyzedthemousecharacter‐istics ofweight loss, survival, viral replication, tissue pathology,viral antigen distribution, and cytokine and chemokine profiles,whichprovideadditionaldata to investigate thepathogenesisofMERS‐CoV‐induced disease and evaluate relevant therapeuticsandvaccines.

2  | MATERIAL S AND METHODS

2.1 | Animals

Specificpathogen‐freetransgenicC57BL/6miceexpressinghDPP4(age,6‐8weeks)werepurchased from the Instituteof LaboratoryAnimal Science (ILAS), Chinese Academy of Medical Sciences(CAMS),Beijing,China.ExperimentsinvolvingMERS‐CoVinfectionwerecarriedoutinanimalbiosafetylevel3(ABSL‐3)andbiosafetylevel3 (BSL‐3) laboratoriesat the ILAS,CAMS.Miceweretreatedhumanely, and all animal studies were conducted in accordancewithaprocedureapprovedbytheInstitutionalAnimalCareandUseCommittee,ILAS,CAMS(ILAS‐GH18001).

AllanimalswerefedunderABSL‐3conditionsfor3daysbeforethestartofthestudy.Ontwoconsecutivedayspriorto infection,each mouse was trained in an animal nose‐only aerosol device.Infectedmicewerekept in theABSL‐3 laboratory throughout thestudyandobserveddailytoensurethattheyhadenoughwaterandfood.

2.2 | Virus and cells

MERS‐CoV (human betacoronavirus 2cEMC/2012, complete ge‐nomeGenBank:JX869059.2)wasprovidedbytheILAS,CAMS.TheviruswaspropagatedandexpandedinVeroE6cells(AmericanTypeCulture Collection, USA) cultured and passaged at 37°C and 5%CO2byroutinemethods.Purifiedandconcentratedprogenyviruseswere titratedusingVeroE6cell‐based infectivityassays,andviraltitersareexpressedinunitsof50%tissuecultureinfectiousdoseper100microliters(TCID50/100 μL).MERS‐CoVstocksataconcentra‐tionof106.8TCID50/100 μLwerestoredat−80°C.

2.3 | Animal nose‐only aerosol exposure device

Ananimalnose‐onlyaerosolexposuredevice(IN‐TOXProducts)waslocated inanABSL‐3 laboratoryandcomprisedanose‐onlyexpo‐surechamberandnebulizerinsideaclassⅡbiologicalsafetycabinet(BSCⅡ),acontrolbox,mouserestrainttubes,acleancompressedairtankandavacuumpump(Figure1).

The exposure device, which exposed only the mouse nose,generatedMERS‐CoVaerosolparticlesof1.27±0.61μmtoinfecttransgenicmiceexpressinghDPP4andsimulatedthenaturalrouteofinfection.21,22

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2.4 | Infection 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.

MERS‐CoV virus suspensions (106.5 TCID50) and serum‐freeDulbecco'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%tribro‐moethanol(0.2mL/10gofbodyweight,intramuscular(im))forintra‐nasalinoculationwith106.5TCID50ofMERS‐CoVintheinstillationgroupandserum‐freeDMEMintheinstillationcontrolgroup.

2.5 | Clinical signs and sample collection

Infectedmicewereobservedfor14consecutivedaystoanalyzetheclinicalsymptomsofdisease,weightchange,andsurvival.Themicewereeuthanizedwith1.2%tribromoethanol (0.2mL/10gofbodyweight,im)whentheyreached25%weightloss.

F I G U R E 1  Theanimalnose‐onlyinhalationexposuredevice.A,Photographoftheanimalnose‐onlyexposuredevice.B,PhotographoftheaerosolgeneratorandexposurechamberlocatedinaBSCII.C,Photographofnose‐onlyexposure

Group Material Purpose Number

Aerosol MERS‐CoVaerosol Toanalyzeclinicalsigns,weightloss,andsurvival

5a

Necropsy 12b

Intranasalinstillation MERS‐CoVsuspension Toanalyzeclinicalsigns,weightloss,andsurvival

5a

Necropsy 12b

Aerosolcontrol DMEMaerosol Toanalyzeclinicalsigns,weightloss,andsurvival

5a

Necropsy 12b

Instillationcontrol DMEMsuspension Toanalyzeclinicalsigns,weightloss,andsurvival

5a

Necropsy 12b

aFivemicepergroupwereobservedtorecordclinicalsymptoms,weight,andsurvivalfor14con‐secutivedayspostinfection.bThreemicefromeachgroupof12micewererandomlynecropsiedondays3,5,7,and9postinfection.

TA B L E 1  GroupsoftransgenicmiceinfectedwithMiddleEastrespiratorysyndromecoronavirus(MERS‐CoV)

4  |     HAO et Al.

Ondays3,5,7,and9postinfection,threeanimalsrandomlyse‐lectedfromeachgroupunderwentnecropsytoobtaintissuespeci‐mensforassessingviraldistribution,associatedhistopathology,andcytokine levels using quantitative reverse transcription‐PCR (qRT‐PCR),hematoxylinandeosin(H&E)staining,immunohistochemistry(IHC),andenzyme‐linkedimmunosorbentassay(ELISA).

2.6 | Viral RNA in tissue samples

TotalviralRNAwasextractedfromtissues(lungs,brain,kidneys,spleen,liver,heart,andintestine)homogenizedusingtheRNeasyMiniKit(Qiagen)accordingtothemanufacturer'sinstructionsandfrozen at −80°C. MERS‐CoV RNA copies were determined in a25.0μLmixture containing5.0μL ofRNAusing theQuantiTectProbe RT‐PCR Kit (Qiagen, 204 443) with the ABI StepOnePlus™Real‐timePCRSystem(LifeTechnologies).Theprimersandprobes for theMERS‐CoV‐specific upstream E gene (upE) wereas follows: forward, 5′‐GCAACGCGCGATTCAGTT‐3′; reverse,5′‐GCCTCTACACGGGACCCATA‐3′; and fluorescent probe, 5′‐FAM‐CTCTTCACATAATCGCCCCGAGCTCG‐TAMRA‐3′. A plas‐mid carrying theMERS‐COV upE gene was used as a standardcontrol.23AstandardcurvewasgeneratedforPCRusing10‐107 copiesofaqualifiedstandardplasmidtocalculatecopynumbersforeachreaction.

2.7 | Histopathology and IHC

Formalin‐fixed lung, brain, and kidney samples were embeddedin paraffin wax and sectioned at an approximately 5‐μm thick‐ness. Deparaffinized and hydrated tissue sections were rou‐tinely stained with H&E to examine histopathological changes.Immunohistochemical staining was performed to assess the ex‐pression of a viral antigen using a rabbit two‐step detection kit(ZhongshanGoldenBridgeBiotechnologyCo.,Ltd)witharabbitpol‐yclonalanti‐MERS‐CoVnucleoprotein(NP)antibody(SinoBiologicalInc).VisualizationwasthenperformedbyDABstainingandhema‐toxylincounterstaining.

2.8 | Cytokine and chemokine profiles

Supernatants of tissue homogenates from infected mice (50 µL)wereaddedtothebottomofanantibody‐coatedplate.The levelsofinterleukin(IL)‐1β,IL‐6,IL‐8,IL‐10,tumornecrosisfactor(TNF)‐α,interferon (IFN)‐γ, and IFN‐βwere assayed using ELISA kits (KeteBiotechnology Co., Ltd). Chemokine and cytokine concentrationswererecordedaspg/mLofhomogenateorng/Lofhomogenate.

2.9 | Statistical analysis

DatawereanalyzedusingSPSS21orGraphPadPrism5.0software.Theexperimentalresultsarepresentedasthemeanplusstandarddeviation(SD).One‐wayANOVAwasusedtoassessdifferencesinbodyweight,viralload,andcytokinelevelsamongdifferentgroups.

Student'sttestwasperformedfortwo‐groupcomparisons.P <.05wasconsideredstatisticallysignificant.

3  | RESULTS

3.1 | Clinical signs and weight loss

The infectedmice in both the aerosol and instillation groups dis‐playedsignificantclinicalsymptoms,suchashuddling,hunching,ruf‐fledfur,weight loss,anddeath.Thereweresignificantdifferencesinweight change (P < .001) and survival (P < .0001) between theMERS‐CoVinfectiongroupsandthecontrolgroups.Theincubationperiod,however,was5‐7daysafteraerosolinfectionand1dayafterinstillation inoculation.AfterMERS‐CoVaerosol exposure,hDPP4transgenicmice showedprofound clinical signs on days 5‐7, rapidweightlossondays7‐9and60%survivalbyday11(acutedeathoreuthanasiaat25%weightloss).Theintranasallyinfectedtransgenicmicedisplayedrapidweightlossondays1‐5and0%survivalbyday5 (acutedeathoreuthanasia at25%weight loss).Therewere sig‐nificantdifferencesindiseaseprogression(P<.01)afterchallengebetween the aerosol group and the instillation group. TransgenichDPP4miceinfectedwithMERS‐CoVaerosolsexhibitedmilderdis‐easeandslowerdiseaseprogressionthandidthoseinoculatedintra‐nasally(Figure2A,B).

Noobviousabnormalities,includingweightlossorsignsofclini‐calillness,weredetectedintheaerosolcontrolandinstillationcon‐trolgroups.TherewerenosignificantdifferencesinweightchangeorsurvivalratesbetweenmiceinoculatedwithDMEMintheabovetwocontrolgroups(P>.05;Figure2C).

3.2 | Viral load detection

BasedonqRT‐PCRanalysesoftissueRNAcontents,weidentifiedhighviralloadsinthelungsandbraininmiceandasmallamountofviralRNAinothertissuesafterMERS‐CoVinfectionviatheaero‐solorinstillationroute(Figure3A,B).However,thereweresignifi‐cantdifferencesinthetissueviralloadsofinfectedmicebetweenthe two groups (P < .0001). AfterMERS‐CoV aerosol infection,highviralloadsweredetectedinthelungsat3‐9daysandinthebrainat7‐9days.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.HighlevelsofviralRNAaccumulatedmoreslowlyinthetissuesoftheMERS‐CoVaerosol‐exposedmicethaninthoseofthemiceinfectedin‐tranasally(Figure3).

3.3 | Histopathological assessment

Asshown inFigure4A,gross lung lesions, showing theappearanceofpulmonaryhyperemiaanddarkbrownregions,wereobserved in

     |  5HAO et Al.

F I G U R E 2  Weightchangeandsurvivalrateinmiceinfectedbytheaerosolorinstillationroute.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  qRT‐PCRanalysisofmousetissuesamplescollectedafterinfectionwithMiddleEastrespiratorysyndromecoronavirus(MERS‐CoV).A,ViralloadsinmousetissuesafterMERS‐CoVaerosolexposure.B,ViralloadsinmousetissuesafterintranasalinfectionwithMERS‐CoV.C,ViralloadsinmouselungsafterMERS‐CoVinfection.D,ViralloadsinmousebrainsafterMERS‐CoVinfection.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyreached25%weightloss;thesemicehada0%survivalratebyday5,sotherewerenoqRT‐PCRresultsobtainedondays7and9.Thedataarepresentedasthemeanchange±SDforeachgroup(n=3).Akeyindicatingthecolorcodingofthegroupsisprovidedinthefigure.****P < .0001

6  |     HAO et Al.

F I G U R E 4  Lungandbrainlesionsinmiceafterinfection.A,Grossnecropsyobservationofthelungsofinfectedmice.B,Histopathologicalchangesinthelungsofinfectedmice.Magnification:100×.C,Histopathologicalchangesinthebrainsofinfectedmice.Magnification:400×.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyresearched25%weightloss;thesemicehada0%survivalratebyday5,sonotissuelesionresultswereavailableondays7and9

     |  7HAO et Al.

mice infectedwithMERS‐CoVvia theaerosol inhalationor intrana‐sal instillationroute,butnoobviouslesionswerefoundinothertis‐sues.Therewerenoabnormalitiesinthetissuesofthenormalcontrolgroup.Itwasclearthattheappearanceofthelungsexhibitedobviouscongestionanddarkbrownregionsondays7‐9intheaerosolgroup.TheMERS‐CoV‐intranasalmiceshowedgross lung lesionsonday3andmoreseverelunglesionsonday5.Grosslunglesionsdevelopedmoreslowlyandweremilderintheaerosolgroupthanintheinstilla‐tiongroup(Figure4A).

Microscopically, challenged mice developed moderate acuteinterstitial pneumonia and brain pathology, but no pathologicalchangesweredetected inother tissues in themice. In theaerosolgroup,thelungsoftheexposedmiceshowedalveolarseptalwiden‐ing, inflammatorycell infiltration,andvesseldilatationandconges‐tionat3‐9days,gradualdevelopmentofseverepathologicalchangesandinflammatorycellinfiltrationinperivascularregionsat5‐9days,focalhemorrhagesat7‐9days,andanexpandedpathologyrangeatday9(Figure4B).Dilatationandcongestionofthecerebralvesselswerenotclearlyobserveduntilday7,andfewareasofneurondefor‐mationnecrosiswerefoundinthecerebralcortex,hippocampus,andthalamusbeforeday9(Figure3C).Ondays3and5afterintranasalinfection,wefoundmoderateacuteinterstitialpneumoniaandbrainlesions (Figure 4B,C). Tissue lesions, however, were milder in theaerosolgroupthanintheinstillationgroup.Furthermore,thereweresignificantdifferencesintheprogressionoflungandbrainlesionsinthetwoinfectedgroups.Tissuelesionprogressionwasslowerintheaerosol‐infectedmicethanintheinstillation‐infectedmice(Table2).

3.4 | IHC

TheexpressionofaMERS‐CoVantigenwasprimarilyevaluatedusingIHCassaysandwasfound inendothelialcellsandalveolarpneumo‐cytes inthe lungsandincerebralcorticalneurons,dendrites,axons,

microgliaandthehippocampus inthebrainsofaerosol‐and instilla‐tion‐challengedmicebutnotincontrolmice(Figure5A,B).ProminentMERS‐CoV expressionwas also observed in renal tubular epithelialcells (Figure5C).However, therewere significant differences in thetimingofvirusexpressioninthetissuesofthemicepostinfection.

AfterMERS‐CoVinfection,IHCassayswitharabbitpolyclonalanti‐MERS‐CoV NP antibody found that viral antigens predomi‐nantlyappearedintrachealendothelialcellsatday3postinfectioninthelungsoftheaerosol‐infectedmiceandinbothtrachealendo‐thelialcellsandpneumocytesinthelungsoftheaerosol‐infectedmiceat5‐9days;thesechangeswereobservedinthelungsoftheinstillation‐infectedmiceat3and5days,respectively.Inaddition,theMERS‐CoVantigenwasdiscoveredinthebrainandkidneysintheaerosolgroupat5‐7daysandintheinstillationgroupat3and5days.Basedontheseresults,weconcludedthatthedistributionof the MERS‐CoV antigen in the lungs, brain and kidneys afterinfectionwas slower in the aerosol group than in the instillationgroup.

3.5 | Cytokine and chemokine profiles

There were significant differences in the level of related proin‐flammatorycytokineandchemokineprofiles,includingIL‐1β,IL‐6,IL‐8,IL‐10,TNF‐α,andIFN‐γ,betweeninfectiousgroups(theaero‐sol group and instillation group) and the control group (P < .05).Significantlyelevated levelsof IL‐1β, IL‐6, IL‐8, IL‐10,TNF‐α, andIFN‐γwerediscoveredinthelungsandbrainsofmiceintheaero‐sol groupwith increased CXCL‐1 at 3‐9 days (P < .05) postchal‐lengeand in thoseofmice the instillationgroupat3and5dayspostchallenge (Figure6). In the aerosol group, theexposedmiceshowedpeak IL‐10 and concentration in the lungs and IL‐10 andCXCL‐1concentrations in thebrainat5‐9days, andpeakTNF‐α and IFN‐γ levels in the lungs and brains with peak IL‐6 level at

TA B L E 2  PathologicalchangesinthelungsofmiceafterMiddleEastrespiratorysyndromecoronavirusinfection

GroupAlveolar septum widtha

Interstitial inflammatory cell infiltrationb

Exudate in alveolic

Dilatation and conges‐tion of vesselsd Hemorrhagee

3 d Aerosol + + — + —

Instillation +~++ + — + —

5d Aerosol + + — + —

Instillation ++~+++ ++ + ++ —

7d Aerosol ++ ++ — + —

Instillation NDf NDf NDf NDf NDf

9 d Aerosol ++ ++ — ++ +

Instillation NDf NDf NDf NDf NDf

Control — — — — —

a—,noapparentchanges;+,mildalveolarseptumwidening;++,moderatealveolarseptumwidening;and+++,severealveolarseptumwidening.b—,noapparentchanges;+,infiltrationofafewinterstitialinflammatorycells;and++,someinterstitialinflammatorycellinfiltration.c—,noapparentchanges;and+,asmallamountofexudateinalveoli.d—,noapparentchanges;+,milddilatationandcongestionofvessels;and++,moderatedilatationandcongestionofvessels.e—,noapparentchanges;and+,mildhemorrhage.fND,Notdone.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyreached25%weightloss,whichoccurredbyday5.

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F I G U R E 5   Immunohistochemicalstainingofmousetissuesamplesafterinfection.A,Immunohistochemicalstainingofthelungsofinfectedmice.B,Immunohistochemicalstainingofthebrainsofinfectedmice.C,Immunohistochemicalstainingofthekidneysofinfectedmice.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyresearched25%weightloss;thesemicehada0%survivalratebyday5,sonotissuelesionresultswereavailableondays7and9

     |  9HAO et Al.

7‐9 days. After intranasal infection, however, the levels of IL‐1β,IL‐6,IL‐10,TNF‐α,andIFN‐γinthelungsandIL‐6,IL‐8,IL‐10,andIFN‐γinthebrainspeakedat3‐5days.Thesecretionofsomecy‐tokinesandchemokinesintheaerosolgroupwasslowerthanthatintheintranasalgroup(P<.05).

4  | DISCUSSION

The dromedary camel, a natural host ofMERS‐CoV, primarily de‐velopsupperrespiratorytractinfectionpostinoculationwithMERS‐CoVviaanintratrachealorintranasalroute,whichfailstosimulatethesignsoflowerrespiratorytractinfectioninhumans.24MERS‐CoV

naturallyinfectsrhesusmonkeysandcommonmarmosets,causingvaryingdegreesofclinicalsymptoms.AfterMERS‐CoVinfectionbythe intranasal, intratracheal, oral and conjunctival routes,mild‐to‐moderate transient pneumonia occurs in rhesusmonkeyswithoutthe manifestation of severe clinical symptoms of MERS‐CoV in‐fection,25,26 and commonmarmosets developmoderate‐to‐severeinterstitial pneumonia or even die.6,7 Transgenic mice expressinghDPP4arepermissivetoMERS‐CoVinfection,whichresultsindis‐ease andmortality.No animalmodels, however, fully recapitulatethehumandiseasecausedbyMERS‐CoV.27,28Inthecurrentstudy,hDPP4transgenicmicewereinfectedwithMERS‐CoVbytheaero‐solorintranasalinstillationroute,andthereweresignificantdiffer‐encesindiseaseprogression,lunglesions,viralreplication,andvirus

F I G U R E 6  CytokineandchemokinelevelsintissuesofmiceafterinfectionwithMiddleEastrespiratorysyndromecoronavirus(MERS‐CoV).A,Postinfectioncytokineandchemokinelevelsinthelungsofmice.B,Postinfectioncytokineandchemokinelevelsinthebrainsofmice.Miceintheinstillationgroupdiedacutelyorwereeuthanizedwhentheyresearched25%weightloss;thesemicehada0%survivalratebyday5,sonoresultswereavailableondays7and9.Theresultsrepresentthemean±SDforeachgroup(n=3).*P<.05,**P<.01,***P<.001,and****P < .0001

10  |     HAO et Al.

distribution in tissues between the aerosol‐ and instillation‐chal‐lengedmice(Table3).

AfterMERS‐CoVinfection,thediseaseprogressioninthemiceintheaerosolgroupwasslowerthanthat inthemiceintheinstil‐lationgroup.Sandersetalshowedthatvirusdropletsweredepos‐ited and concentrated in the lungs through the respiratory tractofmice inoculated intranasally, resulting in fastdiseaseonset.29,30 Correspondingly, after instillation infection with MERS‐CoV, wefoundthatmicewithashortairwayandhighconcentrationofvirusdepositedinthelungsdisplayedweightlossatday1andlunglesionsat day 3, consistentwith intranasalmousemodels established byAdam,AgrawalandLietal31‐33;thesemicealsoexhibited0%survivalbyday5.Previousstudiesreportedthataerosolparticles≤5μmpen‐etratedtherespiratorytracttoreachthealveoliandwerediffuselydistributedinthelungs.30,34Inaddition,virusaerosolsenteredthebloodcirculationthroughthealveoli,andothervirusesslowlyrepli‐catedinthelungsaftermiceinhaledMERS‐CoV‐containingaerosols(particle size: 1.27 ± 0.61 μm). Compared with instillation‐inocu‐latedmicewithvirusdepositioninthelungs,aerosol‐exposedmicedisplayedslowerdiseaseprogressionwithan incubationperiodof

5‐7days,lunglesionsonday7,continuousweightlossondays7‐11,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 im‐munohistochemical stainingwitha specific antibody to furthercharacterizeMERS‐CoVexpressioninthe lungs,brain,andkid‐neys.Arelativelyhighviralloadinthelowerrespiratorytractisassociatedwith severe illness in viral respiratory diseases.36,37 At 3‐5 days postinfection, mice in the intranasal group, whichhadhighviralloadsinthelungsandbrainat3‐5days,exhibitedacute interstitial pneumoniaandpathologicalbrain changes. Intheaerosolgroup,micedevelopedacute interstitialpneumoniaat 3‐9 days and pathological brain changes at 7‐9 days, whichwerecausedbyhighlevelsofvirusRNAinthelungsat3‐9daysandinthebrainat7‐9days,respectively.HighervirusRNAlev‐elsintheinstillationgroupmightcontributetothemoresevere

ParameterMice infected with MERS‐CoV aerosola

Mice infected intranasally with MERS‐CoV

Incubationperiod 5‐7d 1 d

Weightloss 7‐11d 1‐5d

Survival 60% 0%

Grosslunglesions 7‐9d 3‐5d

Viralload

Lungs Highlevelondays3to9 Highlevelondays3and5

Brain Highlevelondays7to9 Highlevelondays3and5

Histopathology

Lungs Moderateacuteinterstitialpneu‐moniaondays3to9

Moderateacuteinterstitialpneumo‐niaondays3to5

Brain Relativelymildbrainlesionondays7and9

Brainlesionsondays3and5

MERS‐CoVantigendistribution

Lungs Inbronchialendothelialcellsonday 3Inbothtrachealendothelialcellsandalveolarpneumocytesinthelungsondays5to9

Inbothtrachealendothelialcellsandalveolarpneumocytesinthelungsondays3and5

Brain Incerebralcorticalneurons,den‐drites,axons,glialcells,andthehippocampusondays5to9

Incerebralcorticalneurons,den‐drites,axons,glialcells,andthehippocampusondays3and5

Kidneys Inrenaltubularepithelialcellsondays5to9

Inrenaltubularepithelialcellsondays5to9

Cytokinesandchemokinesb

Lungs Highlevelsondays3to9,includ‐ingCXCL‐1

Highlevelsondays3to5

Brain Highlevelsondays3to9,includ‐ingCXCL‐1

Highlevelsondays3to5

aNose‐onlyexposure.bCytokinesandchemokinesincludeIL‐1β,IL‐6,IL‐8,IL‐10,TNF‐α,andIFN‐γandCXCL‐1.

TA B L E 3  ComparisonofMiddleEastrespiratorysyndromecoronavirus(MERS‐CoV)infectionofmicebytheaerosolorinstillationroute

     |  11HAO et Al.

histopathological changes observed. Furthermore, IHC assaysrevealedthatMERS‐CoVprimarilyreplicatedinendothelialcellsandalveolarpneumocytesinthelungsandmanycelltypesinthebrainaswellasepithelialcellsinthekidneysinchallengedmice,as evidenced by MERS‐CoV expression localized in the lungs,brainandkidneys inprevious reports.32,33MERS‐CoVdistribu‐tioninthebrainwasslowerinaerosol‐infectedmice(at5‐7days)than in instillation‐infected mice (at 3‐5 days), which closelymirroredthedifferences intheprogressionsofdiseaseandtis‐sue lesions. Overall, aerosol‐infected 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 pneu‐monia.38,39AccordingtotheresultsofclinicalstudiesandpreviousreportsonMERS‐CoVanimalmodels,weassayedthelevelsofeightcytokinesandchemokines:IL‐1β,IL‐6,IL‐8,IL‐10,TNF‐α,IFN‐β,IFN‐γ,and CXCL‐1 by ELISA. Clinical studies have shown that markedlyincreased levelsof IL‐1β, IL‐6, IL‐8,TNF‐α, and IFN‐γ aredetectedinpatientswithsevereMERS.38,40Previousreportshave indicatedelevated levels of IL‐1β, IL‐6, IL‐10, TNF‐α, IFN‐γ, and CXCL‐1 inMERS‐CoVanimalmodels.6,25,32,33Additionally,we found that theconcentrationsof these cytokines and chemokinesweremarkedlyelevated inthe lungsandbrainofaerosol‐and instillation‐infectedmice.Theinductionofproinflammatorycytokinesandchemokinesin the lungs plays an important role in tissue immunopathologicalchangesandfatalpneumonia.Anelevatedpulmonaryviralloadanditsinjuriouseffectsonthepulmonarysystemarelikelyresponsiblefortheincreasedinflammatoryresponse.Itispossiblethattherel‐atively robustpulmonary inflammatoryresponse inmicemayhavepromotedMERS‐CoVreplication,causingincreasedpulmonaryviralloadsinthemice.36

MiddleEastrespiratorysyndromepatientsexhibitamedianin‐cubationperiodof5‐7days,witharangeof2‐14days.41Wediscov‐eredthat instillation‐inoculatedmiceexhibitedclinicalsignswithin1daybutthatthe incubationperiodofaerosol‐exposedmicewas5‐7days,whichmorecloselyresembledtheperiodobservedinhu‐mans.MERS‐CoVbinds tohDPP4 receptors that areprimarilyex‐pressedinthelowerrespiratorytractandalveoli,resultinginawiderangeofdiseasesymptoms inpatients, fromnosymptomstomildrespiratory illness or severe acute pneumonia, which rapidly pro‐gressestoacutelungdamage,multiorganfailureandevendeath.42,43

Clinically, chest radiography and chest computed tomogra‐phy (CT) showno lung lesions in patients in the early stages ofillness,butpneumonia is identifiedduring thecourseof thedis‐ease and includes patchy densities, extensive diffuse and focalalveolar space opacities, interstitial infiltrates, and consolida‐tion.44‐46 Patients with severe illness progress rapidly to acuterespiratory failure andevendeath. Inone fatal case, pulmonaryhistopathologicalchangesincludedexudative‐phasediffusealve‐olardamagewithdenudingof thebronchiolarepithelium,prom‐inent 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 withMERS‐CoVinfection.Asobservedbynecropsy,aerosol‐infectedmicedevel‐opedlunglesionsat7‐9days,butnolunglesionsoccurredwithinthe incubationperiod, and subsequenthistopathology identifiedmild‐to‐moderate diffuse interstitial pneumonia without severehistopathologyat3‐9days,whichalmostmatchedtheprogressivemoderatepneumoniaobservedinMERSpatients.However,instil‐lation‐inoculatedmiceshowedashorterincubationperiod(1daypostinfection)andfailedtostimulatetheprogressionofmild‐to‐moderatepneumoniaduetotheacutediseasecourse.AsshowninTable4, the incubationperiodofMERS‐CoVand the induced

TA B L E 4  ComparisonsofMiddleEastrespiratorysyndromecoronavirus(MERS‐CoV)‐infectedmiceandMERSpatients

Parameter MERS patientsaMice infected with MERS‐CoV aerosols

Mice infected intranasally with MERS‐CoV

Incubationperiod 5‐7d,witharangeof2‐14d 5‐7d 1 d

Lunglesionprogress Earlystageshowsnoabnor‐malitiesbychestradiographyorchestCT

Within7d,nolunglesionsafternecropsy

Ondays3and5,lungdamagewithmoderatediffuseintersti‐tialpneumonia

Subsequentdevelopmentofmildpneumonia

Ondays3to5,mildalveolarseptumwideningandinflammatorycellfiltration

Progressivedevelopmentofmoderatediffuseinterstitialpneumonia

Ondays7to9,lungdamagewithmoderatediffuseinterstitialpneumonia

Afatalcase:severediffuseinterstitialpneumonia

— —

Distributionofaviralantigen Alveolarpneumocytesandendothelialcells

Alveolarpneumocytesandendothelialcells

Cytokinesandchemokines SignificantlyhighlevelsofTNF‐α,IFN‐γ,IL‐1β,IL‐6,andIL‐8

aMERSpatientsrefertothosewithinfectionofthelowerrespiratorytract.

12  |     HAO et Al.

pulmonary pathological changes in aerosol‐infected mice weresimilartothosenotedinpatientswithrespiratorytractinfection.

Additionally, immunohistochemical staining revealed that aMERS‐CoVantigenwasexpressedinalveolarpneumocytesanden‐dothelialcells,thebrain,andthekidneysinchallengedtransgenicmice. Studies of a fatal case of MERS‐CoV infection evidencedthat the expression of aMERS‐CoV antigen was predominantlylocalizedinpneumocytesandendothelialcells,resultingincellne‐crosisandpneumocytedamage;however,noviralantigensweredetected inother tissues in the fatalcase.47Asdemonstrated inpreviousstudies,wealsodiscoveredhighviralloads,pathologicalchangesandtheexpressionofaMERS‐CoVantigen in thebrainofchallengedmice;andnobrainlesions,butmultiorgandamage,wereobservedinMERSpatients.35,48,49Zhouetaldemonstratedthat human dendritic cells andmacrophageswere permissive toMERS‐CoV replication, indicating that the multiorgan injury in‐ducedbyMERS‐CoVmaybe associatedwith thedistributionofthehDPP4receptorinmanycelltypesthatarespreadthroughoutmultiple organs.38 Some studies have indicated thatMERS‐CoVhascellandtissuetropisms,especiallytropismsforpneumocytesandneurons,andsynapsesmaybeoneofthestructuresbywhichvirusesdiffuse through thebrainafterMERS‐CoV infection.31,35 Themechanismsunderlying thebrain lesionsanddeath inducedbyMERS‐CoVinfectioninhDPP4transgenicmiceremaincomplexandcomplicatedandneedtobefurtherinvestigated.

5  | CONCLUSION

hDPP4transgenicmiceweresuccessfullyinfectedwithMERS‐CoVaerosolsbyananimalnose‐onlyexposuredevice,andaerosol‐andinstillation‐infected mice all simulated the clinical symptoms ofmoderate diffuse interstitial pneumonia. Compared to instilla‐tion‐infectedmice,aerosol‐infectedmicemorecloselyresembledinfected humans in terms of the progression of disease and pa‐thologyinthelungs,whichprovidedadditionaldataforstudyingpathogenesisandevaluatingtheefficacyofpreventiveandthera‐peuticagentsforMERS‐CoV.

ACKNOWLEDG EMENT

The current work was supported by the National Science andTechnology Major Projects of Infectious Disease (grant number2018ZX10734401‐011).

CONFLIC T OF INTERE S T

None.

AUTHOR CONTRIBUTIONS

HGwastheprincipalinvestigator,designedandsupervisedthestudy,andwrotethegrantapplication.XYHperformedthemainexperiments.

XYHandQLperformedthecellexperiments.XYHandFDLconductedtheanimalexperiments.YFXcompletedthepathologyexperiments.XYHandHGconceivedtheexperiments,analyzedthedataandwrotethepaper.Allauthorsreadandapprovedthefinalmanuscript.

ORCID

Xin‐yan Hao https://orcid.org/0000‐0001‐7664‐9568

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsection.

How to cite this article:HaoX,LvQ,LiF,XuY,GaoH.ThecharacteristicsofhDPP4transgenicmicesubjectedtoaerosolMERScoronavirusinfectionviaananimalnose‐onlyexposuredevice. Anim Models Exp Med. 2019;00:1–13. https:// doi.org/10.1002/ame2.12088