2017 Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice

2017·2.12 MB·English

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Mouse-adapted MERS coronavirus causes lethal lung P
disease in human DPP4 knockin mice
KunLia,ChristineL.Wohlford-Lenanea,RudragoudaChannappanavarb,Jung-EunParkc,JamesT.Earnestc,
ThomasB.Baird,AmberM.Batese,Kim A.Brogdene,HeatherA.Flahertyf,TomGallagherc,DavidK.Meyerholzg,
StanleyPerlmana,b,andPaulB.McCrayJr.a,b,1
aDepartmentofPediatrics,PappajohnBiomedicalInstitute,UniversityofIowa,IowaCity,IA52242;bDepartmentofMicrobiology,UniversityofIowa,Iowa
City,IA52242;cDepartmentofMicrobiologyandImmunology,LoyolaUniversityChicago,Maywood,IL60153;dIowaInstituteofHumanGenetics,
UniversityofIowa,IowaCity,IA52242;eCollegeofDentistry,UniversityofIowa,IowaCity,IA52242;fDepartmentofVeterinaryPathology,IowaState
University,Ames,IA50011;andgDepartmentofPathology,UniversityofIowa,IowaCity,IA52242
EditedbyDianeE.Griffin,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,MD,andapprovedMarch2,2017(receivedforreviewNovember21,
2016)
The Middle East respiratory syndrome (MERS) emerged in Saudi further processed into multiple nonstructural proteins. In
Arabia in 2012, caused by a zoonotically transmitted coronavirus contrast, conserved S,E,M, and N structuralproteinsare trans-
(CoV). Over 1,900 cases have been reported to date, with ∼36% latedfromseparatesubgenomicmRNAs.
fatality rate. Lack of autopsies from MERS cases has hindered un- EpidemiologicstudieshaveestablishedthatMERSiszoonoticin
derstandingofMERS-CoVpathogenesis.Asmallanimalmodelthat origin,withevidenceforacloselyrelatedvirusindromedarycamels
developsprogressivepulmonarymanifestationswheninfectedwith on the Arabian Peninsula and throughout Africa (3–5). Spread
MERS-CoVwouldadvancethefield.Asmicearerestrictedtoinfec- from camels to people is documented (6), as is person-to-person
tionatthelevelofDPP4,theMERS-CoVreceptor,wegeneratedmice spread among health care workers in hospital settings (7). The ES
withhumanizedexons10–12ofthemouseDpp4locus.Uponinocu- transmission of MERS-CoV from person to person is inefficient, ENC
lationwithMERS-CoV,humanDPP4knockin(KI)micesupportedvirus butthiscouldchangewithvirusevolution(8,9)andifthenumbers SCI
replicationinthelungs,butdevelopednoillness.After30serialpas- ofso-called“superspreaders”increased(10).TheMERS-CoVhas AL
C
sagesthroughthelungsofKImice,amouse-adaptedvirusemerged also been detected in people with mild, influenza-like illnesses, DI
E
(MERSMA)thatgrewinlungstoover100timeshighertitersthanthe thosewithadengue-likeillness,andinthosewithoutobviousdis- M
starting virus. A plaque-purified MERSMA clone caused weight loss ease signs or symptoms (11–14). Although human-to-human or
andfatalinfection.Virusantigenwasobservedinairwayepithelia, zoonoticspreadofMERShasnotreachedepidemicorpandemic
pneumocytes,andmacrophages.Pathologicfindingsincludeddiffuse levels,itspotentialtospreadbetweenpersonswasdemonstratedin
alveolardamagewithpulmonaryedemaandhyalinemembranefor- healthcare settings in the Middle East (7) and by an outbreak in
mation associated with accumulation of activated inflammatory SouthKoreacausedbyasingleinfectedindividual(15).
monocyte–macrophages and neutrophils in the lungs. Relative to A lack of autopsy studies from MERS fatalities has hindered
theparentalMERS-CoV,MERSMAvirusescontained13–22mutations, understandingofMERS-CoVpathogenesis(16).Nonehavebeen
including several within the spike (S) glycoprotein gene. S-protein reportedfromSaudiArabiaorSouthKorea.Tobetterunderstand
mutations sensitized viruses to entry-activating serine proteases
the pathogenesis of MERS-CoV lung disease and aid the devel-
andconferredmorerapidentrykinetics.RecombinantMERS bear-
MA opmentofvaccinesandtherapies,weengineeredamousemodel
ingmutantSproteinsweremorevirulentthantheparentalvirusin
of MERS. Dipeptidyl peptidase 4 (DPP4, CD26), a type II
hDPP4KImice.ThehDPP4KImouseandtheMERS providetoolsto
MA transmembraneectopeptidase,isthereceptorfortheMERS-CoV
investigatediseasecausesanddevelopnewtherapies.
(17), and the key amino acids responsible for binding the virus
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emergingpathogen interferon viruspathogenesis spikeprotein CD26
Significance
MiddleEastrespiratorysyndrome(MERS)wasrecognizedasa
Middle East respiratory syndrome, caused by a zoonotically
significant illness on the Saudi Arabian peninsula in mid-
transmitted coronavirus (MERS-CoV), has a high mortality
2012 and the causative agent was rapidly identified as a novel
coronavirus (CoV) (1). MERS has a high mortality (∼36%) asso- (∼36%).BecauseoflimitedautopsydataontissuesfromMERS
fatalities,asmallanimalmodelcanprovideanimportanttoolto
ciatedwithseverelungdiseasethatcanprogresstoacuterespiratory
betterunderstandthedisease.Wehumanizedthemouselocusof
distresssyndrome(ARDS).Sinceitsemergence,theWorldHealth
thevirusreceptorDPP4,preservingnativeDPP4expression.After
Organization (WHO) has been notified of 1,905 laboratory-
inoculatinghDPP4knockinmicewithMERS-CoV,therewasvirus
confirmed cases of MERS-CoV infection in 27 countries, result-
replicationwithoutdisease.Wethengeneratedamouse-adapted
ing in at least 677 related deaths (WHO website, www.who.int/
MERS-CoVbyserialpassageinhDPP4knockinmice.Theresultant
emergencies/mers-cov/en/; February24, 2017). MERS-CoV, sim-
virus causes fatal lung disease that includes diffuse alveolar
ilar to the severe acute respiratory syndrome (SARS)-CoV that
damage and immune dysregulation. Here, we characterize the
caused an epidemic in 2003, has been a serious cause for global
pathologicfeaturesofthemodelandelucidatekeyaspectsofthe
concernduetoitshighfatalityrate.Ofnote,theMERS-CoVhas
immunopathologyandfactorscontributingtovirulence.
agenomicstructuresimilartothatofotherCoVsandcontainsa
30-kbpositive-strandRNAgenomethatencodesfor11ORFsthat
areordered5′to3′:ORF1a,ORF1b,spike(S),ORF3,ORF4a/b, Authorcontributions:K.L.,C.L.W.-L.,R.C.,T.G.,S.P.,andP.B.M.designedresearch;K.L.,
C.L.W.-L.,R.C.,J.-E.P.,J.T.E.,T.B.B.,A.M.B.,K.A.B.,H.A.F.,andD.K.M.performedresearch;
ORF5, envelope (E), membrane (M), nucleocapsid (N), and K.L.,C.L.W.-L.,R.C.,J.-E.P.,J.T.E.,T.B.B.,H.A.F.,T.G.,D.K.M.,S.P.,andP.B.M.analyzed
ORF8b (1, 2). ORF1a and -1b are predicated to encode non- data;andK.L.,T.G.,S.P.,andP.B.M.wrotethepaper.
structural proteins. The subgenomic mRNAs of MERS-CoV Theauthorsdeclarenoconflictofinterest.
encode five unique accessory proteins, designated 3, 4a/b, 5, ThisarticleisaPNASDirectSubmission.
and8b,whicharefoundonlyintheviruslineagethatincludes 1Towhomcorrespondenceshouldbeaddressed.Email:[email protected].
HKU4 and HKU5 (1). Like other CoVs, the long genomic
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
mRNAofMERS-CoVencodesreplicasepolyproteinsthatare 1073/pnas.1619109114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1619109114 PNASEarlyEdition | 1of10
spikeglycoproteinhavebeenidentified(18).Wereplacedmouse Plaque-Purified MERS CausesFatalLungDiseaseinDPP4-KIMice.
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Dpp4exons10–12,encodingtheresiduesrequiredforvirusbind- Because CoV, like all RNA viruses, exist as quasispecies, we fur-
ing,withhumanexons10–12.Invivoserialpassageofthehuman therpurifiedMERS plaques.Sixofnineplaque-purifiedisolates
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MERS-CoV[ErasmusMedicalCenter(EMC)/2012]inthehuman wereevaluatedinKImiceata105pfuinoculumandeachcaused
DPP4 receptor knockin (KI) mice yielded a mouse-adapted virus fataldisease(SIAppendix,Fig.S2).Serialpassageofcoronaviruses
(MERS ) that causes a fatal pulmonary disease phenotype as- may result in gene deletions (26) and we observed that deletions
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sociated with diffuse alveolar damage and a severe inflammatory hadoccurredinmostofourplaques.SequencingoftheORF3and
response,includinganincreasednumberofCD11b+Ly6Chiinflam- 4a/4b genes revealed that plaque 6 virions included a population
matory monocyte–macrophages (IMMs). Sequencing of plaque- without deletions in these regions (Dataset S1). Therefore, we
purifiedMERSMAclonesrevealedseveralmutationsthatemerged selected virus from plaque 6 as a representative MERSMA. To
withinvivopassage,includingaclusterinthespike(S)glycoprotein ensurecompleteclonalisolation,weperformedtwomorerounds
codingsequence.ArecombinantMERS-CoVwiththeseS-protein ofplaquepurification.Fig.2C–FshowsthatKImiceinfectedwith
mutationswasmorevirulentinDPP4KImicethanparentalMERS- asingletripleplaque-purifiedvirus,termed“clone6.1.2,”exhibited
CoV.ThehumanDPP4KImouseinfectedwithMERSMArevealed dose-dependentfataldisease.Wecomparedtheoutcomesofmale
severalimportant diseasecorrelates,attributed tothevirus andits (Fig. 2 C and D) and female (Fig. 2 E and F) mice to several
cellentry,andtothehostanditsimmunologicalresponse. inocula of MERS clone 6.1.2 and observed similar outcomes.
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Aninoculumof5×103pfuwaslethalinbothsexes.
Results LungvirustitersinKImiceinfectedwithclone6.1.2were∼1–2
Generation of a Mouse with Knockin of Human DPP4 Exons 10–12. log higher compared with those challenged with the EMC/Vero
WhereashumanDPP4has26exons,theresiduesencodedbyexons parental virus from 1 to 5 days postinoculation (d.p.i.) (Fig. 3A).
10–12comprisethecriticalMERS-CoVreceptor(18,19).Weand
Virus was readily detected in serum of clone 6.1.2-infected mice,
others showed previously that changing the exon 10–12 codons
butrarelyinmiceinfectedwithEMC/Vero(Fig.3B).Noviruswas
from mouse to human conferred high-affinity binding of the
detectedinthebrainorotherorgans.WealsoexaminedviralRNA
MERSSprotein(20–23).Usingaplasmid-mediatedgenetargeting
distribution by PCR and identified signal in lung at 1 and 3 d.p.i.
strategy (SI Appendix, Fig. S1), a congenic C57BL/6 mouse was
(Fig.3C).Alow-levelvirusgenomicRNAsignalwasalsodetected
developed by replacing mouse Dpp4 exons 10–12 with the corre-
in the liver at 1 d.p.i. only. Of note, the EMC/Vero virus yielded
spondinghumanDPP4codons(hDPP4-KI).Thetargetedfounders
more progeny than clone 6.1.2 virus in human cells (Fig. 3D),
were expanded by breeding and offspring characterized by geno-
suggesting that mouse adaptation did not enhance infection in
typingforthepresenceofhumanDPP4sequenceasdescribedin
humancells.
Materials and Methods. For the studies reported, we used mice
We compared the tissue histology of KI mice infected with
homozygousforhDPP4.ToidentifytheMERS-CoVreceptordis-
EMC/Vero or MERS clone 6.1.2. At 1 and 3 d.p.i. we noted
tribution in the mouse lung, we localized DPP4 in uninfected MA
similarmildinflammatorycellinfiltratesinperivascularregionsand
hDPP4-KI mice. As shown in Fig. 1A, airway epithelial cells had
alveolarseptaofmiceinfectedwithbothviruses.By4and5d.p.i.,
mild to moderate DPP4 immunoreactivity. In the alveolus,
lung tissue from mice infected with EMC/Vero virus was notable
DPP4immunostainingwasmostoftenseeninalveolarmacrophages
for multifocal mononuclear cell infiltrates (Fig. 4A, arrowheads),
with less common immunostaining of alveolar epithelia, and the
whereas such infiltrates were mild and uncommon in the clone
pleural surface (Fig. 1 B and C). This pattern of DPP4 expression
6.1.2-infectedanimals.Incontrast,tissuesfromclone6.1.2-infected
sharessimilaritiestotheproteindistributioninhumanlung(24,25)
miceshowedmultifocalhyalinemembranes(Fig.4AandB,black
andidentifiescelltypesthatmaysupportvirusreplication.
arrows),edema(Fig.4B,blackasterisks),andnecroticdebris(Fig.
InfectionofHumanDPP4KnockinMiceandDevelopmentofaMouse- 4B,Inset,redarrowhead)consistentwithdiffusealveolardamage.
AdaptedMERS-CoV.Followingintranasal(i.n.)infectionwith104to Associated with these findings in clone 6.1.2-infected animals, we
106pfuMERS-CoV(EMC/2012,Vero81cellpassaged,hereafter observedEvansbluedyeretentioninlungtissueat4and5d.p.i.,
termed“EMC/Vero”),virusreplicatedinthelung(titersat1d.p.i. indicativeofincreasedvascularpermeability(Fig.4B,whiteaster-
∼105–6 pfu/g tissue), but mice lost no weight and there was no isks). Morphometric analysis of lung tissue lesions showed that
mortality(Fig.2AandB).WenextseriallypassagedEMC/Veroin clone 6.1.2-infected lungs had significantly reduced cellular infil-
KI mice as described in Materials and Methods. Tissue homoge- tratesandincreasedhyalinemembranesat4and5d.p.i.(Fig.4C).
natesweresavedaftereachinvivopassage.After21passages,we We localized virus N protein in infected lung tissue at 3 d.p.i. In
testedthepassagedvirusinKImiceandnotedprogressiveweight EMC/Vero-infected mice, virus antigen staining was patchy and
loss and ∼50% mortality (SI Appendix, Fig. S2). After 30 in vivo featured immunostaining of alveolar macrophages and less com-
passages(P30),a104pfuvirusinoculumcausedprogressiveweight mon staining of alveolar and airway epithelia (Fig. 5, Middle). In
loss and ∼80% mortality (Fig. 2 A and B). We termed this contrast, clone 6.1.2-infected mice had much more widespread
P30virusmouse-adapted(MA)MERS-CoVor“MERS .” N-proteinstainingofalveolarmacrophagesandalveolarepithelia
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A B C
Fig.1. DPP4immunostaininginuninfectedhDPP4KImouselung.(A)DPP4wasobservedasmildtomoderatecytoplasmicstainingofairwayepithelia.
Positivestainingwasalsonotedinalveolarmacrophages(arrow).(B)Mildtomoderatemacrophageimmunostainingwerecommonlyseen(arrows,Middle),
aswasweakstainingofalveolarepithelia(Inset).(C)Thepleuralsurface(arrows)hadmultifocalDPP4immunostaining.Resultsshownarerepresentativeof
findingsfromfourhDPP4KImice.(Magnification:400×.)
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A B evidence of monocyte activation, a reduced erythrocyte density, P
andreducedpolychromatophils(immatureRBCs)consistentwith
anemia of inflammation (SI Appendix, Fig. S5 B and C). These
resultsprovidefurtherevidenceofasystemicillnessinclone6.1.2-
infectedanimals.
Examination of autopsy samples from patients who succumbed
to SARS and MERS (n = 1) showed substantial accumulation of
monocyte–macrophages and neutrophils in the lungs (38–41). To
C110 Male D determine whether the enhanced morbidity and mortality of
100 5000 pfu i.n.
% Initial Weight107890000 Percent survival 24680000 55000 p pfufu i. ni.n.. ialhungDcnergPsesPaa4sane-tdKd4Itiondmtf.alpialc.mien.muWtomahtMboeerrEryesRacoseSfltMlhinAreenccaalrbtouesnioteimlmu6etm.en1ut.n2,nuweimnecfbeeaeclnlrtaispolyoonzfpecuCdolraDtrthei4oel5an+ptseedlirencuwektnihtothe--
cytes were lower in clone 6.1.2-challenged mice, percentages and
600 2 4 6 8 10 12 14 00 2 4 6 8 10 12 14 totalnumbersofCD11b+Ly6ChiIMMsweresignificantlyhigherin
Days p.i. Days p.i.
clone 6.1.2-infected mice compared with EMC/Vero-infected or
E Female F
110 uninfected control mice (Fig. 6 A and B). In contrast, neutrophil
100 5000 pfu i.n.
% Initial Weight107890000 Percent survival 24680000 55000 p pfufu i. ni.n.. ngCclruoDomnu8ebp0es)6r(r.s1Fe.vi2wge-.aien6lrefeeAdcttvaheandardtiamIBbMi)lce.MeStasaannainddnidnlnegnosefstourstsoratoacipttniihsvitEalisctMiawolCnleyr/meVdaheirifrgkfoehe-rilrneysnfa(etCccttDibeveda6tt9wemdaeinecidnne
60 0 (Fig.6CandD).IMMandneutrophilactivationwassignificantly
0 2 4 D6ays p8.i. 10 12 14 0 2 4 D6ays p8.i. 10 12 14 lowerinuninfectedcontrolmicecomparedwiththetwogroupsof ES
infectedmice(Fig.6CandD).WenextenumeratedNK-,T-,and NC
Fig. 2. Outcomes in hDPP4 KI mice infected with EMC/Vero or MERSMA. B-cell numbers in the lungs of the different groups and observed CIE
Weightloss(A)andsurvival(B)inKImiceinfectedi.n.with104pfuEMC/Vero S
highernumbersinEMC/Vero-infectedmicecomparedwithclone L
oHmruompstaadsnsiaeDgdPe.PR43e0sKuI(lPtms30iscu)emMinmEfRaerScitMzeeAd.twwEMiothCre/PVp3el0ircoaM-tienERfeeSxcMpteeArdliommsetincseitgs,nhniafid=ca11n00t0(w%EMeisCgu/hVrtveiavrnoad)l., 6su.1b.s2t-ainnfteiacltleydmoorreunminofneoctneudclmeaircece(lFlsigw.e6rEe)d.eItnecstuemdminarEyM, wCh/eVreeraos MEDICA
n = 11 (MERSMA), mixed male and female mice. (C–F) MERSMA clone compared with MERSMA clone 6.1.2-infected lungs, the infitrates
6.1.2causesdose-dependentlethallungdiseaseinmaleandfemalehDPP4 were composed mainly of lymphocytes in the former group. Con-
KImice.(CandD)WeightcurveandsurvivalresultsformalehDPP4KImice versely, more IMMs and fewer lymphocytes were detected in the
that received 5,000–50 pfu MERSMA clone 6.1.2 via i.n. inoculation. (For lungs of animals infected with clone 6.1.2 compared with EMC/
5,000pfun=8;500pfun=12;and50pfun=5).(EandF)Weightcurveand Vero virus. Overall, the increased accumulation of activated
survivalresultsforfemalehDPP4KImicethatreceived5,000–50pfuMERSMA monocyte–macrophagesandneutrophilsanddecreasednumbersof
clone6.1.2viai.n.inoculation.(For5,000pfun=8;500pfun=9;and50pfu
n=5).(A,C,andE)Datarepresentmean±SE. TandBcellsinthelungsofMERSMAclone6.1.2-challengedmice
and scattered airway epithelia (Fig. 5, Right), consistent with the
A Lung B Serum
clinicalandvirologicalresults.
9 5
ue8 EMC/Vero (P0)
(icmInm2hde7emai–mcsu2auon9treke)e,idRntinheefasmeptpcoRdtrneoeNsdldeaAuaytcneotdiiamoMbInaFuElRnNmmSdMaoasiAnydg.cenSeclastolui(nfnd3otgi0rrei–bas3suno3etdf)ve,edpartyaanostldrieedcnaguintussletltiauawvtsrieireetdahdlscMceyeavtlEnoelsdrkRi(itnS3ype4-.rC–oaW3oin7nVde)- ogPFU/gm Tiss10 1234567 LOD Log PFU/ml101234 MERSMA 6L.O1D.2
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flammatory cytokines and chemokines, and IFN-stimulated genes 0 0
1 3 4 5 1 3 4 5
(ISGs) in lung tissue from KI mice infected with EMC/Vero and Days p.i. Days p.i.
clone6.1.2,at1and3d.p.i.usingqRT-PCR.AsshowninSIAp- C D Calu-3
s
pendix, Fig. S3, the induction of several host defense genes was erH 7 1 dpi
similarforbothviruses.Amongthetranscriptsexpressedingreater mbPD 6 3 dpi
at(cmbCveSyibeCrrotItuufowgLenAlkeere2dipnonen,apantneneCtsccenclXaeasdolonniCnaixnddne,tLeFIc263r6Lhei,.g1.s-de1.I6.pm2..LSp2,o-3o.eIni6aL).lksn,i.eic-(dnW1iCStet0eIEoXe,dApMCCcardpllCLoCosepot1nLl/eeaV0en2ipy,nd,eer6siroaCdx.o1nf,,iXi.ndlbi2FenCudicsGwgLtel.tue1ur-hCdurS0leetem4,Simd)aaFt.bhna.agNuedtnrOeneodtMldayyvttaapeleDgunberrralncAeelIeegla5l,daetoetnvitafifrhsatdf,sseeteu3itrIolyeeeImpdnInce.sohtpciseronei.ndieIs--. Log RNA copy nu10normalized to GA-0123451Lung Heart Brain Liver Kidney Splmeaell nIntestine
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and III IFN-transcript responses and more robust cytokine/che-
mokineresponsesthantheparentalvirus. Fig.3. TissuevirustitersandviralgenomicRNAdistribution.HumanDPP4
Analysisofperipheralbloodsmearscollectedat4d.p.i.revealed KImiceinfectedwith104pfui.n.inoculumofindicatedMERS-CoV.(AandB)
thatMERS clone6.1.2-infectedmicehadagreaterpercentage Lung and serum titers at indicated times (C) ORF1a quantitative RT-PCR
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of neutrophils and fewer lymphocytes than EMC/Vero-infected performed on indicated tissues from EMC/Vero and MERSMA clone 6.1.2-
infectedmiceat1and3d.p.i.Limitofdetection(LOD)forqRT-PCRassayis
mice (SI Appendix, Fig. S5A). Both EMC/Vero and clone 6.1.2-
∼100copies.Mean±SE;n=3.(D)Calu-3cellswereinfectedwithindicated
infected mice exhibited an increase in reactive lymphocytes (SI
MERS-CoVatMOI0.1andvirustitersat1d.p.iweredeterminedbyplaque
Appendix,Fig.S5A)consistentwithresponsetoinfection.Periph- assay.n=6,representativeoftworeplicateexperiments.Statisticalsignifi-
eral blood from clone 6.1.2-infected mice also had morphologic cancewasassessedbyStudent’sttest.****P<0.0001.
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Fig.4. Lungpathologyand scoringatdays 4–5postinfection.(A)Theuninfected grouplackedlesions.EMC/Verovirus infection wascharacterized by
multifocalmononuclearinfiltrates(arrowheads),butmononuclearinfiltratesweremildanduncommonintheclone6.1.2-infectedgroup.Rather,clone6.1.2-
infectedlungswerecharacterizedbymultifocalhyalinemembranes(A,arrows),edema,andnecroticdebristhatwasconsistentwithdiffusealveolardisease:
40x(Top)and200x(Bottom).(BandC)FurthercharacterizationofalveolardiseaseinEMC/Veroandclone6.1.2virusinfection.Grossly(B,Topimages),Evans
bluedye(administeredi.v.beforekilling)wasnotretainedinEMC/Veroinfectionbutwasretainedinclone6.1.2(whiteasterisks),indicativeofvascular
leakage.Histopathology(B,Bottomimages)ofEMC/Veroandclone6.1.2lungs.EMC/Vero-infectedlung(Left)hadperivasculartointerstitialmononuclear
infiltrates.Clone6.1.2-infectedlungexhibitededema(blackasterisks,BottomMiddle,400x),scatteredcelldeath/debris(Insetandredarrow,BottomRight),
and/orhyalinemembranes(blackarrows,600x).(C)Morphometryoflungtissuesshowedthatclone6.1.2lungshadsignificantlyreducedcellularinfiltrate
andincreasedhyalinemembranes(P=0.01andP=0.003,respectively,Mann–Whitney).n=5–6micepercondition.
suggestanovertandinjuriousinnateinflammatoryresponseanda (tissue culture passaged in Vero 81 cells), passage 21 MERS ,
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suboptimaladaptiveimmuneresponse. 9plaquesfrompassage30MERS ,and6tripleplaque-purified
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MERS clones. Each sequenced MERS genome had be-
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Identification of MERS-CoV Mutations Acquired During Mouse tween 12 and 21 nonsynonymous differences compared with the
Adaptation. To identify the changes fixed into MERS-CoV dur- publishedEMC/2012isolate(42).Thesedifferencesarenotedin
ing serial passaging, we sequenced several mouse-adapted virus SIAppendix,Fig.S5andDatasetS1,inthecontextofMERS-CoV
genomesusingtheIlluminaMiSeqplatformincluding:EMC/Vero ORFs. EMC/Vero differed from EMC/2012 at three loci: NSP3
Uninfected hDPP4 KI EMC/Vero Clone 6.1.2
Fig.5. MERS-CoVN-proteinimmunolocalizationinuninfectedKImiceormiceinfectedwithEMC/VeroorMERSMAclone6.1.2.Noantigenstainingwasobserved
inuninfectedhDPP4KImice(Left).At3d.p.i.,miceinfectedwithEMC/Verohadapatchydistributionofvirusantigen(brownstain),includingmacrophage
immunostainingalongwithuncommonepitheliaofalveoliandairways(Middle).Incontrast,clone6.1.2-infectedmicehadwidespreadinfectionwithstainingof
macrophages,alveolarepithelia,andscatteredairwaycells(Right).n=3–4micepercondition.[Magnification:100x(Top)and400x(Bottom.]
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Fig.6. Accumulationofactivatedinnateimmunecellsinthelungsofclone6.1.2-infectedhDPP4KImice.LungsharvestedfromuninfectedhDPP4KImiceor
hDPP4KImicechallengedwith1×104pfuofEMC/VeroorMERSMAclone6.1.2wereanalyzedforinfiltratingimmunecellsonday4p.i.(A)FACSplots(Left)
showpercentages of leukocytes,inflammatorymonocyte–macrophages, and neutrophilsinthe lungs ofuninfected,EMC/Vero, andclone 6.1.2 groups.
(B)Scatterplots(Right)representpercentagesandtotalnumbersofimmunecellsinthelungsofuninfected,EMC/Vero,andclone6.1.2groups.(CandD)
Histogramsandscatterplotsshowlevelsofactivationmarkers,CD69andCD80onlung-derivedIMMs(C),andneutrophils(D).(E)Bargraphswithscatterplots
showtotalnumberofNK,T,andBcellsinthelungsofuninfected,EMC/Vero,andclone6.1.2groups.Datawerederivedfromtwoindependentexperiments
withthreetosixmicepergroupperexperiment.*P<0.05,**P<0.01,***P<0.001,****P<0.0001.
(S1112F), spike (T1015N), and Orf5 (W108X). Missense muta- (see passage 30 isolates in SI Appendix, Fig. S2 and Dataset S1).
tionswerepresentinallMERS isolates,including13in1a/1b, ComparedwithEMC/Vero,6uniquemutationsemergedinthe
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7inS,1in3,1in4a,2in4b,1in5,1inM,and2inN.Wealso S-protein coding sequence of MERS viruses. Every isolate
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observed nonsense or frameshift mutations in ORF5 that arose hadmutationsintheSprotein,includingthepassage21isolate,
during early Vero cell tissue culture amplification of the original which was not fully virulent. Because variations in CoV S
EMC/2012isolate.Interestingly,mostclonesencodedvirusdeleted proteins frequently correlate with virulence (43, 44), we next
inORFs3a,3bandor4,butclone6.1.2expressedfull-length3,4a, assessedwhethertheS-proteinchangesaccountedforthegain
and 4b geneproducts(SIAppendix, Fig.S6 and Dataset S1). We in virulence described above.
observed no evidence for differences in virulence when isolates Wenotedthatthevirusthatinitiatedtheserialpassages,EMC/
containingORF3/4a/4borlackingtheirexpressionwerecompared Vero, differed from the prototype human isolate EMC/2012 at
Lietal. PNASEarlyEdition | 5of10
TherecombinantEMC/2012MERS-CoV(42)andtheMA1.0and
MA2.0 recombinants were inoculated into 12- to 18-wk male
hDPP4-KI mice to assess disease outcomes and virus titers. Fol-
lowinga104pfuinoculum,therecombinantvirusescausedamild
transientweightlosswithnomortality(Fig.8AandB).Atahigher
105 pfu inoculum, the MA1.0 and MA2.0 recombinant viruses
caused greater weight loss than the recombinant EMC/2012 virus
(42),with∼40%mortalityinMA2.0-infectedmice(Fig.8CandD).
Lungtissuetitersat1d.p.i.wereslightlyincreasedinthemicein-
fectedwiththeMA2.0Sproteinatthe105pfuinoculum(Fig.8E).
Of interest,theserecombinant virusescausedminimalweight loss
andnomortalityinyoung6-to8-wkhDPP4-KImice(SIAppendix,
Fig. S7). The replication properties of these three recombinant
virusesweresimilarinhumanCalu-3andHuh7celllines(Fig.8F),
indicatingthat,whereasmutationsarisingintheSproteinpartially
contribute to the increased virulence observed in MERS -
MA
infected mice, they did not enhance growth in human cells.
Fig.7. Adaptedmutationsinspikeproteininfluencevirusentry.(A)Linear
depictionofMERS-CoVS1(receptorbinding)andS2(fusion)domains.Amino Discussion
acidchangesinEMC/2012andmouse-adaptedMERS-CoVSproteins(MA1.0and
WhereasMERSrepresentsanongoingrisktohumanhealth,there
MA2.0)areindicatedrelativetotheparentvirusEMC/Vero.(BandC)Effectsof
hasbeenonlyonepostmortemstudyofapatientwithMERS(16),
E64dandcamostatonMERSpseudovirusentryintoLET1cells.(D)Kineticsof
MERSpseudovirusentryintoLET1cells.Errorbarsrepresentmean±SE*P< and our knowledge of human disease pathophysiologyis poor.
0.05,**P<0.01,***P<0.001comparedwithEMC/2012S. TodevelopamorerobustsmallanimalmodeltostudyMERS
pathogenesis, we generated hDPP4 knockin mice with exons
10–12replacedbythehumancodons,retaininggeneregulation
S residue 1015 (Fig. 7A). The EMC/Vero contained N1015, a by the native promoter. Although hDPP4-KI mice supported
residue reported intissue culture adapted MERS-CoV (45). We modest replication of EMC/Vero MERS-CoV in the lung, the
observed a reversion of this residue during serial passage in KI
mice,whereN1015returnedtotheTresiduefoundinEMC/2012.
On continued passage in KI mice, additional S mutations arose,
notably the P747H and S749R residues comprising part of the A 104pfuinoculum B
S-proteolytic cleavage site. To gain insights into how these S
mutationsmightcontributetovirulence,weconstructedaseriesof
SgenesreflectingEMC/Vero,EMC/2012,andthoseinMERS
MA
p30(Fig.7A).TheconstructstermedMA1.0andMA2.0featured
S-proteinmutationsrepresentativeofallthataroseduringmouse
adaptation, including those present in clone 6.1.2 (Fig. 7A). We
evaluated the effect of these S mutations on cell entry using ve-
sicularstomatitisvirus-basedMERSS pseudoviruses(46).These C D
105pfuinoculum
viruses were transduced into murine lung epithelial type I cells
(47)expressinghDPP4(48).Strikingly,wefoundthatEMC/Vero
S-directed cellentry washighlysensitive tothecysteineprotease
inhibitorE64d,whichinactivatesendosomalcathepsins,butrela-
tively resistant to the serine protease inhibitor camostat, which
inactivatescell-surfacetransmembraneproteases(Fig.7BandC).
Thesefindings indicatedthat theMA mutations, particularlythe
N1015Tchange,shiftedvirusesfromusingendosomalcathepsins, E F
overtocell-surfacetransmembraneproteases.Cell-surfacetrans-
membrane proteases “trigger” a rapid virus–cell entry (46, 49).
Consistently,theEMC/2012andMApseudovirusesenteredcells
withrapidkinetics,achieving50%ofmaximalcellentryin20min.
By contrast, theentry ofEMC/Veroviruseswererelatively slow,
with less than 40% of maximal cell entry in 45 min (Fig. 7D).
TheseresultsindicatedthatMERS-CoVspikesevolveinvivofor
utilization of cell-surface transmembrane proteases, thereby ac-
celerating cell entry and increasing productive infections in the
mouselung. Fig. 8. Influence ofadapted spikeprotein mutations oninvivovirulence.
(AandB)KImice(age12–13wk,male)received104pfuofindicatedrecombinant
S-ProteinMutationsinMERSMAContributetoVirulence.Toevaluate MERS-CoVandweremonitoreddailyforweight(A)andmortality(B)(n=
the effects of the evolved S-protein mutations on virulence, we 8pergroup,representativeoftworeplicateexperiments).(CandD)KImice
used reverse genetics to introduce MERS S-gene mutations (age12–18wk,male)received105pfuofindicatedrecombinantMERS-CoV
MA
into an infectious MERS-CoV (EMC/2012 genome) bacterial andweremonitoreddailyforweight(C)andmortality(D)(n=10pergroup,
artificialchromosome(pBAC-MERSFL)asdescribedinMaterials representativeoftworeplicateexperiments).(E)Lungvirustitersat1d.p.i.
determinedbyplaqueassayinanimalsthatreceivedthe105pfuinoculum(n=
and Methods (50, 51). Recombinant MERS-CoV were prepared
6 per group, representative of two replicate experiments). (F) Calu-3 and
bearing the S proteins from EMC/2012 (42) or mouse-adapted Huh7cellswereinfectedwith indicatedrecombinant virusesatMOI=0.1.
viruses (MA1.0, or MA2.0). The sequence-verified BACs were Progenywerecollectedat1d.p.i.andtiteredbyplaqueassay.(n=6,repre-
transfected into Huh7 cells to generate recombinant MERS- sentativeoftworeplicateexperiments).Errorbarsrepresentmean±SE*P<
CoVs,whichwerethenamplifiedinCalu3cellstoobtainstocks. 0.05,**P<0.01,***P<0.001comparedwithrMERS-CoVEMC/2012.
6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1619109114 Lietal.
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infection was largelyasymptomatic. Thirtyinvivoserial passages The most severe manifestation of MERS in humans is a lethal P
ofEMC/VeroMERS-CoVinKImiceledtoanevolvedMERS pneumonicprocesswithassociateddiffusealveolardamage(2,16).
MA
strainthatcausedlethalpneumonia.Comparedwiththeparental Autopsy results from a single individual that died from MERS
virus, MERS infection featured diffuse alveolar damage, dys- wererecentlyreported(16).Keyhistopathologicfindingsincluded
MA
regulatedinnateimmuneresponses,andthepresenceofactivated MERS-CoV antigen localization in pneumocytes, macrophages,
inflammatory monocyte–macrophages and neutrophils. Sequenc- andendothelialcellswithevidenceofdiffusealveolardamage.No
ing of MERS genomes revealed that mouse-adapted viruses evidenceofantigenstainingwasfoundinotherorgans(16).Sim-
MA
developedmutationsinseveralgenesincludingtheSprotein.The ilarly,MERS clone6.1.2infectioninhDPP4KImicecauseda
MA
virulent MERS clone 6.1.2 had 12 mutations in coding se- predominantly parenchymal lung disease associated with diffuse
MA
quences and one mutation in a noncoding region of the virus alveolardamage,acommonpathologicfeatureofARDS.
genome. Studies of recombinant MERS-CoVs bearing these Arapidand well-coordinatedinnateimmuneresponseisa first
S-proteinmutationsshowedthatthesevirusescausedincreased lineofdefenseagainstviralinfections,butdysregulatedandexces-
morbidity and mortality in adult KI mice. The hDPP4-KI sive immune responses may cause immunopathology and increase
mouse and the MERS provide tools to investigate disease morbidity and mortality (65–68). Although there is no direct evi-
MA
pathogenesis and evaluate new therapies. dence for the involvement of proinflammatory cytokines and che-
mokinesindiseasepathogenesis,correlativefindingsfrompatients
ComparisonwithOtherModelsofMERS-CoVInfection.Several ani- with MERS with severe disease suggest a role for exuberant in-
malspecieshavebeenscreenedfortheirsusceptibilitytoMERS- flammatoryresponsesinthediseaseprocess.MERS-CoVinfection
CoVinfection.Mice(52),ferrets(53),andSyrianhamsters(54)do resultsinadelayedbutelevatedantiviralandproinflammatorycy-
not support MERS-CoV replication. In contrast, the lungs of tokine and chemokine expression in dendritic cells, monocyte–
rhesusmacaques(31,55),marmosets(30),rabbits(56),andcamels macrophages,andepithelialcells(31,34,69).Additionally,studies
(57) are permissive to virus replication. Marmosets develop lung inpatientswithMERSwithvaryingdiseaseseverityshowedhigher
disease and associated mortality in response to a large inoculum virustitersandelevatedlevelsofserumproinflammatorycytokines
(30),butothersreportminimaldiseaseinmarmosets(58).Overall (IL-6andIFN-α)andchemokines(IL-8,CXCL-10,andCCL5)in CES
N
the phenotypes in other mammals studied are mild and do not individuals with severe MERS compared with those with mild to E
CI
result in severe MERS-like disease. We reported a small animal moderate disease (28, 29, 70, 71). Similarly, mice infected with S
L
mseoqdueelntolyfMweERanSd, tohteheArds-h(3D3P,P549)segnesnietirzaetdedmmouicsee (t4ra8n),sgaenndicsufobr- Ma dEeRlaSyMiAnctlhoenein6d.1u.c2tidoinspolafyIeFdNhirgehspluonngsevsir.uFsutrittehresrmasosorec,iaeteledvawtiethd EDICA
M
hDPP4usingvariouspromoters.Whentransgenicmiceexpressing proinflammatory cytokines and the accumulation of activated
hDPP4 under control of the cytokeratin 18 promoter (32) or a monocyte–macrophagesandneutrophilsinthelungsuggestapos-
ubiquitous promoter (33) were exposed to MERS-CoV, they de- sibleroleforimmunopathologicresponsesinthegreatertissuein-
veloped fatal disease, butlethality occurred after mice developed juryandfatalpneumoniaweobserved.
anoverwhelmingencephalitis.
Pascaletal.recentlyreportedmakingamousewithhumanized Viral Mutations Associated with Adaptation to the Mouse Lung.
DPP4exonsusinganalternativestrategy(60).Followinganinoc- Sequencing the genomes of MERS strains and triple plaque-
MA
ulum of 2 × 105 pfu of human MERS-CoV isolates, these mice purified clones from virulent strains identified several coding se-
supportedvirusreplication(106to107pfupergramoftissue),with quencemutations,includingchangesintheSprotein.TheS-protein
histopathologic features of mild–moderate peribronchiolar in- mutations were not within the receptor binding domain. Rather,
flammation, interstitial inflammation, and alveolar thickening they arose in proteolytic substrate sites and in subdomains that
observed at 2 and 4 d.p.i. Additional studies in this model de- controlS-protein-catalyzedvirus–cellmembranefusions.MERS
MA
monstrated that a dosage of 2.5 × 104 pfu MERS-CoV caused clone 6.1.2 and the recombinant MA1.0 and MA2.0 viruses all
∼20% weight loss, necessitating euthanasia. In these mice, de- exhibitedS-proteinmutationsassociatedwith“earlyentry”(46),a
+
pletionofCD8 Tcellsreduceddiseaseseverity,whereasmacro- characteristic of coronaviruses that preferentially use cell-surface
phagedepletionexacerbatedMERS-CoV–inducedlungpathology transmembrane proteases, such as TMPRSS2 (72, 73), for their
and disease signs (61), but whether CD8+ T-cell or macrophage virus–cellmembranefusionactivationandresultantcellentry.We
depletionwouldhavethesameeffectsinmicewithlethaldisease speculatethatthemouseadaptedvirusesareselectedonthebasis
remainstobedetermined.Incontrast,thehumanDPP4 knockin oftheirutilizationofthesetransmembraneproteases,whichmaybe
mouse infected with MERS reported here showed enhanced abundantlyavailableinthemouselung,butsparseonothersettings,
MA
virusreplication,significantlungpathology,andmortalitywithan such as Vero cell cultures. The MERS clone 6.1.2 genome
MA
inoculumaslowas5×102pfu.Thesedifferenceslikelyderivefrom contained 10 additional missense mutations outside of the the S
the array of mutations that arose following 30 in vivo serial pas- protein.BecausetheS-proteinchangesinisolationresultedinonly
sages,includingthoseintheSprotein.Whilethismanuscriptwas amodestgaininvirulence(Fig.8),itwillbeimportanttodetermine
underreview,Cockrelletal.reportedengineeringaMERSmodel whichothermutationsarealsocriticalforthepathogenicchanges
by changing two amino acids in the mouse Dpp4 locus (62). This that we observed. The mouse passage-associated mutations that
innovation, combined with in vitro and in vivo serial passage, aroseinMERS-CoVnonstructuralproteinsmayalsocontributeto
yielded a virulent mouse-adapted MERS-CoV with mutations virulence and evasion of the host immune response. Of note, at
distinctfromthoseidentifiedhere,indicatingthatthereareseveral leastoneofthem,nsp9,wasalsomutatedinmouse-adaptedSARS-
evolutionarypathwaystomousevirulence. CoV and contributes to increased virulence (74), although the
functionofthisproteinremainsunknown.
Comparison of MERS-CoV Disease in Human Lung and Human DPP4
KnockinMice.ThecelltypessusceptibletoMERS-CoVinfectionin Advantages and Limitations of the Mouse Model. The hDPP4 KI
human lung were first identified in ex vivo studies and include mouse model offers some advantages over previous small animal
nonciliatedbronchial epithelial cells(17),aswell astypeIand II modelsforMERS-CoVdisease.UnliketheAd-hDPP4–sensitized
pneumocytes, endothelia, and fibroblasts (45). Investigators using mousemodel,hDPP4KImicedonotrequireapriortransduction
human bronchial and lung tissue grown in ex vivo organ cultures process (48).In contrast totransgenicmiceexpressing hDPP4by
observedinfectionofnonciliatedbronchialepithelium,bronchiolar tissue-specific (32) or ubiquitous promoters (33, 59), hDPP4 KI
epithelial cells, alveolar epithelial cells, and endothelial cells (63) micedonotdevelopbraindisease.Themodelshouldbeusefulin
and detected morphological correlates of severe lung injury (64). studiesofdiseasepathogenesis,vaccineapplications,andevaluation
Lietal. PNASEarlyEdition | 7of10
ofsmallmoleculeinhibitorsandothertherapies.Inaddition,hDPP4 VirusTiters.Tissueswereasepticallyremovedanddissociatedin1×PBSusinga
KI mice may be useful in modeling the comorbidities associated manual homogenizer. Tissue homogenates were centrifuged and super-
withincreasedmortalityinhumans,includingaging,diabetes,and natantsremoved.SamplesweretiteredonVero81cellsasreported(48).
chronicdiseases(2).
Extraction of Total RNA and Quantitative RT-PCR. Mouse organs were ho-
Apotentialdisadvantageofourmodelisthatthelungdisease
mogenizedinTRIzolandtotalRNAisolatedusingtheDirect-zolRNAMiniPrep
that develops following infection with MERS may rely upon
MA kit (Zymo Research). A DNase treatment step was included. Two hundred
species-specific mutations arising from selective pressures in the nanogramsoftotalRNAwasusedasatemplateforfirst-strandcDNA.The
newhost.Suchselectivepressuresmaynotrecapitulateallaspects resultingcDNAwassubjectedtoamplificationofselectedgenesbyreal-time
ofthehumaninfection.Forexample,themutationsthatarosein quantitative PCR using Power SYBR Green PCR Master Mix (Applied Bio-
theSproteinduringinvivoserialpassagehavenotbeenreported systems).Averagevaluesfromduplicatesofeachgenewereusedtocalculatethe
in human disease isolates although they are in S domains that relativeabundanceoftranscriptsnormalizedtohypoxanthineguaninephos-
phoribosyltransferaseandpresentedas2-ΔCT.Theprimersusedwerepreviously
show variability among human isolates (75, 76). Our findings
reported(32).
suggestthatthesevariablehumanisolatesshouldbeevaluatedfor
distinct host-cell protease dependencies (77). We note that a
Mouse-AdaptedMERS-CoV.Todevelopamodelofseveredisease,weserially
mouse-adapted SARS-CoV (MA15) that evolved after 15 serial passagedMERS-CoV(EMC/Vero)inhDPP4-KImice.Twomiceweregiven105pfu
passagesinBALB/cmicecontainedsixnucleotidesubstitutionsin ofvirusintranasally.Twodayslater,micewerekilled,lungshomogenizedin
fivegeneproducts:ORF1a(nsp5and9),ORF1b(nsp13),andthe PBS,50μLofclarifiedlunghomogenatewasadministeredtotwohDPP4-KI
SandMproteins(74,78).Acombinationofthesegeneticchanges miceintranasally,andtheprocesswasrepeated.Tissuehomogenateswere
wasrequiredtocausevirulence.Additionalstudiesareneededto savedateachpassage.
investigatethecontributionsofindividualMERS mutationsto To obtain a single plaque of a mouse-adapted variant of MERS-CoV, a
MA monolayerofVero81cellsinsix-wellplateswasinfectedwithseriallydiluted
thelethalphenotype.
virusfor1handoverlaidbymediumcontaining1%low-melting-pointagarose.
Threedaysafterincubation,asingleplaquewaspickedusingasterilePasteur
Conclusions
pipetteandtransferredinto500μLDMEM.Theisolatedplaqueswerethen
TheemergenceofMERSin2012representsthelatestexampleof freeze thawed to release the viral particles from agarose and applied to
thezoonotictransmissionofaCoVfromanimalhoststohumans. Huh7cellstopropagateviruses.
It isavirtual certainty that other examples of zoonoticallytrans- Huh7andVero81cellsweregrowninDMEMwith10%FBSand1%penicillin/
mittedCoVinfectionswilloccurinthefuture.Theavailabilityofa streptomycin(PS).Calu-3cellsweregrowninMEMwith20%FBSand1%PS.
small animal model of MERS with a severe disease phenotype Cellsin24-wellplateswereinfectedwithindicatedMERS-CoVatamultiplicityof
providesa tool fortheevaluationofnovel vaccinestrategies,es- infection(MOI)of0.1.After1hofvirusadsorptionat37°C,thevirusinocula
wereremoved,thecellswerewashedwithPBS,andnewmediumwasadded.
peciallyindetermining theimmunopathologicalconsequencesof
At18–24h.p.i.,virusreplicationwasdeterminedbytitratinginfectiousvirus
any intervention. Additionally, future studies of the viral muta-
fromsupernatantsonVero81cells.
tionsassociatedwithmouseadaptationmayprovidenewinsights
intothevirologicaldeterminantsofseverediseasethatoccurwith Histology and Immunohistochemistry. Lungs were prepared for histological
evolutioninahost. examination and for immunohistochemistry for virus antigen and cellular
markersafterfixationin10%formalinandembeddinginparaffin.Forroutine
MaterialsandMethods histology,tissuesections(∼4μmeach)werestainedwithhematoxylinandeosin.
To detect virus N-protein antigen, sections were incubated with a mouse
AllanimalstudieswereapprovedbytheAnimalCareandUseCommitteeof
polyclonalantibody[generatedbyimmunizingmicetotheMERS-CoVNprotein
theUniversityofIowa.DetailedinformationisprovidedinSIAppendix,SI
withavirusrepliconparticles-Nvector(48)(1:3,000dilution)],incubatedwitha
MaterialsandMethods.
biotinylatedanti-mousesecondaryantibody,followedbysequentialincubation
GenerationofMicewithHumanizedExons10–12oftheMouseDpp4Gene.A withstreptavidin–horseradishperoxidaseconjugate(VectastainABCkit,Vector
Laboratories) and 3,3′-diaminobenzidine (DAB) (Vector Laboratories). Immu-
congenic C57BL/6 mouse with mouse Dpp4 exons 10–12 replaced with the
nolocalizationofDPP4inmouselungwasadaptedfrompreviouswork(24).
humanDPP4codonswasgeneratedbyTaconicBiosciences.Thischangeinexon
Briefly,primaryAbusedwasarabbitpolyclonal(1:400,PA1-8455;Pierce)and
codingsequenceallowedthemodifiedmousegeneproducttodisplayamino
thiswasfollowedbyacommercialsecondarykit(EnvisionRabbit,DAKO).DAB
acidsrequiredforvirusbinding(18–20).Thetargetingstrategywasbasedon
wasthechromogenandslideswerecounterstainedwithHarrishematoxylin.
National Center for Biotechnology Information transcripts NM_010074_3
Tissueswereevaluatedbyboardcertifiedveterinarypathologistsandscored
(mouse)andNM_001935_3(human).Thetargetingvectorwasgeneratedusing
usingapostexaminationmaskingmethod(79).Atleastfivemice(maleand
BACclonesfromthemouseC57BL/6JRPCIB-731andhumanRPCIB-753BACli-
female)wereanalyzedpertimepointandperexperimentalcondition.
braries.MousegenomicsequencefromcodonI264inexon10tocodonV340in
exon12wasreplacedwithitshumancounterpart.Positiveselectionmarkers EvansBlueDyeExtravasation.Tosedatemice,1%EvansBluein1×PBSwas
wereflankedbyFRT[neomycinresistance(NeoR)]andF3[puromycinresistance
retroorbitally instilled. Thirty minutes postinstillation, mice were killed. PBS
(PuroR)] sites and inserted into intron 9 and intron 12, respectively. The (1×) was perfused through the right ventricle, followed by 10% formalin
remainingrecombinationsiteswerelocatedinnonconservedregionsofthe perfusion.Lungswereexcisedandphotographed.
genome.SIAppendix,Fig.S1showsthegenetargetingstrategyusedtogen-
eratethehumanDPP4knockinmice.Furtherdetailsonthegenerationofthe PeripheralBloodSmears.Bloodwascollectedretroorbitallyandtransferredtoa
modelareprovidedinSIAppendix,SIMaterialsandMethods. bloodcollectiontubecontainingEDTAtopreventclotting.Bloodsmearswere
producedusingthetraditionalmanualmethod,allowedtodry,andstainedwith
InfectionofhDPP4KIMicewithMERS-CoV.TheEMC/2012strainofMERS-CoV Diff-Quik(SiemensHealthcareDiagnostics)orwithmodifiedWright’sstain.
(passage8,designatedMERS-CoV)wasprovidedbyBartHaagmansandRon
Fouchier, Erasmus Medical Center, Rotterdam, The Netherlands. Virus was CellSurfaceStainingforFlowCytometry.Micewerekilledon4d.p.i.andthe
propagatedandtiteredbyplaqueassayonVero81cells.Thistissueculture lungsperfusedviatherightventriclewith10mLPBS.Lungtissueswereminced
amplifiedviruswastermedEMC/Vero.HumanDPP4KImicewereanesthetized anddigestedinHBSSbuffercontaining2%FCS,25mMHepes,1mg/mLCol-
with ketamine/xylazine (87.5 mg/kg ketamine/12.5 mg/kg xylazine) and in- lagenaseD(Roche),and0.1mg/mLDNase(Roche)for30minatroomtem-
fectedintranasallywithMERS-CoVin50μLDMEM.Infectedmicewereex- perature.Forsurfacestaining,5×105cellswereblockedwith1μganti-CD16/
amined daily and weights recorded. The majority of infections were 32antibodyandsurfacestainedwiththeindicatedantibodiesat4°C.Cells
performed in young, 6- to 8-wk-old mice. Selected experiments were per- werethenfixedusingCytofixsolution(BDBiosciences).Forsurface/intracellular
formedinadultmiceasnotedinResultsandfigurelegends.Noninfectedlit- staining,cellswereincubatedwithfluorochrome-labeledantibodiesspecificfor
termatesservedascontrols.AllMERS-CoVworkwasconductedinaBiosafety mouse:FITCorPECy7-anti-CD45(30-F11),PEorFITCanti-Ly6G(1A8),PE/PerCp-
level3(BSL3)laboratory. Cy5.5anti-Ly6C(AL-21orHK1.4),PEorV450anti-CD11b(M1/70),APCanti-F4/80
8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1619109114 Lietal.
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(BM8),V450anti-CD11c(N418),APC-CD69(H1.2F3),APCanti-CD80(16-10A1), 2012recombinantMERS-CoVandmodifiedbyPerlmanandcolleagues(80). P
APC-CD3(145-2C11),FITCanti-CD4(RM4-5),APCanti-CD8α (53-6.7),PE-CD19 WeverifiedproperconstructionoftherecombinantcompleteBACbySanger
(1D3),PE-Cy7-NKp46(29A1.4),andPerCp-Cy5.5anti-IA/IE(M5/114.15.2).Anti- sequencing.Thefull-lengthBACcDNAclonewastransfectedintoHuh7cellsto
bodieswereprocuredfromBDBiosciencesoreBiosciences. rescue the recombinant virus. Plaque-purified MERS-CoV was amplified to
developstocks.
MERS-CoV Genome Sequencing and Assembly. Viral RNA was isolated from
producercellsupernatantsusingaQIAampViralRNAMiniKit(Qiagen)according StatisticalAnalysis.Student’sttests,ANOVAwithDunn’smultiplecompari-
tomanufacturerspecifications.ViralcDNAwasgeneratedusingSuperScriptIII sontest,orMann–Whitneyranksumtestwereusedtoanalyzedifferences
reversetranscriptaseandarandomhexamerprimingapproach.Specifically,11μL in mean values between groups unless otherwise specified. Results are
oftotalRNAwasaddedto1μLofFR26RV-N,1μLof10mMdNTPmix,4μL expressedasmean±SE.Pvaluesof≤0.05wereconsideredsignificant.
of5×FirstStrandBuffer,1μLof0.1MDTT,1μLofRNaseOUT,and1μLof
SuperScriptIIIReverseTranscriptase.Furtherdetailsoflibraryproduction,
ACKNOWLEDGMENTS.WethankLuisEnjuanesforprovidingthebacterial
sequencing,andgenomeassemblyareprovidedinSIAppendix,SIMaterials artificial chromosome (pBAC-MERSFL) and Jennifer Bartlett and Sateesh
andMethods.
Krishnamurthyfortheircarefulreviewofthemanuscript.Thisworkissup-
portedbytheNationalInstitutesofHealth(NIH)GrantP01AI060699;and
GenerationofRecombinantMERS-CoVbyReverseGenetics.Weusedreverse theCellMorphologyCoreandPathologyCore,whicharepartiallysupported
genetics to introduce the MERSMA S-protein mutations into an infectious bytheCenterforGeneTherapyforCysticFibrosis(NIHGrantP30DK-54759)
MERS-CoV bacterial artificial chromosome (pBAC-MERSFL). The BAC system andtheCysticFibrosisFoundation.P.B.M.issupportedbytheRoyJ.Carver
was developed by Enjuanes and colleagues (50, 51) to generate a EMC/ CharitableTrust.
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