2016 A mouse model for MERS coronavirus-induced acute respiratory distress syndrome

2016·3.71 MB·English

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PUBLISHED:28NOVEMBER2016|ARTICLENUMBER:16226|DOI:10.1038/NMICROBIOL.2016.226
A mouse model for MERS coronavirus-induced
acute respiratory distress syndrome
Adam S. Cockrell1*, Boyd L. Yount1, Trevor Scobey1, Kara Jensen1, Madeline Douglas1, Anne Beall1,
Xian-Chun Tang2,3, Wayne A. Marasco2,3, Mark T. Heise4,5*† and Ralph S. Baric1,4*†
Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute
respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate
of ∼36%.Limited clinical studies indicate that humans infected with MERS-CoVexhibit pathology consistent with thelate
stages ofARDS, which is reminiscentof thedisease observed inpatients infectedwith severe acute respiratorysyndrome
coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal
models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant
to MERS-CoV. Therefore, we used CRISPR–Cas9 gene editing to modify the mouse genome to encode two amino acids
(positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible
to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a
mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS,
including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and
pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV
neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-
inducedARDS.
T
heseverityofrespiratoryillnesscausedbyMiddleEastrespir- MERS-CoVfailstoreplicateintraditionalsmall-animalmodels
atory syndrome coronavirus (MERS-CoV), its pandemic (mouse,hamster,guinea-pig andferret)due totheinabilityofthe
potentialthroughhuman-to-humanrespiratorytransmission receptor-bindingdomainintheMERS-CoVspikeproteintointer-
and a dearth of effective treatments necessitate the development act with the respective DPP4 receptor7–10. In addition to acting as
of new MERS-CoV therapies and vaccines. Effective vaccine and theMERS-CoVreceptor,DPP4regulatesT-cellactivation,cytokine
therapeutic development require preclinical animal models that functionandtrans-endothelialmigrationtositesofinflammation11.
resemble the pathogenesis of human MERS-CoV infection. Therefore,overexpressionofDPP4mayresultinimmunedysregu-
Additionally, these models should: (1) include a measure of mor- lation.Effectivemodelswouldthereforeideallypromotefunctional
tality associated with severe respiratory disease; (2) not be con- MERS-CoV/DPP4 interactions, with minimal perturbations of
founded by neurological complications due to high viral loads in innate DPP4 expression, signalling activity or tissue distribution.
the brain; (3) exhibit sustained, high-level virus replication within Classicalstrategiestoovercomereceptorincompatibilitiestogener-
thelungsofinfectedanimals;(4)exhibitlungpathologyassociated ate susceptible mice have relied on generalized or tissue-specific
withhumanacuterespiratorydistresssyndrome(ARDS);(5)main- transgenic overexpression approaches to drive expression of
tain innate expression of the MERS-CoV host receptor, dipeptidyl the human receptor (hDPP4) in the mouse12–15. Although
peptidase 4 (DPP4), to prevent perturbation of immunological MERS-CoVcanelicitrespiratorydiseaseinhDPP4overexpression
homeostasis; (6) be genetically tractable to study host genes that models, these models exhibit afatalcentral nervous system (CNS)
regulate responses to MERS-CoV vaccines and therapeutics; and andsystemicmulti-organdisease12–14,probablyduetonon-specific
(7)exhibitreproducibility. overexpressionofthereceptorthroughouttheanimal,whichcom-
Conventional non-human primate (NHP) models have been plicatesthe studyof MERS-CoV-induced respiratory pathogenesis
established for MERS-CoV in both the rhesus macaque and inthesemodels.
common marmoset1–4. NHPs are instrumental for the preclinical In this study, we used our knowledge of which determinants
development of therapeutics; however, these models are cost- allow mouse DPP4to act as afunctional MERS-CoV receptor7 by
prohibitive for initial screening of large numbers of vaccine and usingCRISPR–Cas9(clusteredregularlyinterspacedshortpalindro-
therapeutic candidates, challenging to work with for routine mic repeats and CRISPR-associated gene 9) genome editing tech-
pathogenesis studies, limited in availability and typically require nology to insert codons that match the human sequence at
high virus challenge doses into multiple sites. Furthermore, two positions 288 and 330 in the mouse Dpp4 gene. This strategy
recentstudieshave contradictedtheinitial studiesinNHPs,which resulted in a mouse that is permissive for MERS-CoV infection,
may complicate use of the rhesus macaque5 or the common while maximally preserving the species-specific interaction
marmoset6modelforroutinevaccineortherapeutictesting. networks critical for DPP4 immune function. Generation of mice
1DepartmentofEpidemiology,UniversityofNorthCarolina-ChapelHill,ChapelHill,NorthCarolina27599,USA.2DepartmentofCancerImmunologyand
AIDS,Dana-FarberCancerInstitute,HarvardMedicalSchool,Boston,Massachusetts02215,USA.3DepartmentofMedicine,HarvardMedicalSchool,
Boston,Massachusetts02115,USA.4DepartmentsofMicrobiologyandImmunology,UniversityofNorthCarolina-ChapelHill,ChapelHill,North
Carolina27599,USA.5DepartmentsofGenetics,UniversityofNorthCarolina-ChapelHill,ChapelHill,NorthCarolina27599,USA.†Theseauthorsjointly
supervisedthiswork.*e-mail:[email protected];[email protected];[email protected]
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a b c 288/330+/+ 288/330+/− C57BL/6J WT
Exon 10 A288 Exon 11 T330 WT + −
Exon 10 A288 Exon 11 T330 6J +/0 +/0
/ 3 3 Lung
C57BL/6J WT BL /3 /3
7 8 8
5 8 8
Exon 10 288L Exon 11 330R C 2 2
Exon 10 A288 Exon 11 T330
288/330+/− Brain
Exon 10 288L Exon 11 330R
Exon 10 288L Exon 11 330R
288/330+/+
Kidney
d
1 × 109 288/330+/+ abc
288/330+/− def
e) 1 × 108 a d b e C57BL/6J WT
ng tissu 11 ×× 110067 c f e 288/330+/+ 288/330+/− C57BL/6J WT
u
g l
er 1 × 105 H&E
p
−1ml 1 × 104
u.
p.f. 1 × 103
e (
Titr 1 × 102 LOD IHC
1 × 101
1 × 100
EMC/2012 Camel MERS icMERS MERS-0
Figure1|ACRISPR–Cas9geneticallyengineeredmousemodelforMERS-CoVreplication.a,C57BL/6Jmiceweregeneticallyengineeredusing
CRISPR–Cas9genomiceditingtoencode288Land330RinmDPP4ononechromosome(heterozygous,288/330+/−)oronbothchromosomes
(homozygous,288/330+/+).b,NorthernblotofmDPP4mRNAexpression.c,Immunohistochemistry(IHC)ofmDPP4proteininthelungs,brainandkidneys
ofindividualC57BL/6Jwild-type(WT),288/330+/−and288/330+/+mice.d,ViraltitresforMERS-CoVat3dayspost-infectionfromC57BL/6JWT,
288/330+/−and288/330+/+(alln=4)miceinfectedwith5×105plaque-formingunits(p.f.u.)oftheindicatedviruses.Bargraphsshowmeans+s.d.
Student’st-testwasusedtocalculateP<0.05forcomparisonsofMERS-0witheachvirusin288/330+/+mice(a–congraph)and288/330+/−mice
(d–fongraph).e,Pathologyof288/330+/+,288/330+/−andC57BL/6JWTmiceatday3afterinfectionwithMERS-0.Lungtissuesectionswerestainedto
examinethepathologybyhaematoxylinandeosinstaining(H&E),orwerestainedbyIHCtodetectnucleocapsidproteinfromMERS-0infection.IHCand
H&Epathologyimagesarerepresentativeofatleastthreesamples.Scalebars(c,e),1mm.
carrying a chimaeric mouse DPP4 (mDPP4) molecule patterns in the lungs, kidneys and brains of 288/330+/+ and 288/
(A288L/T330R), combined with a mouse-adapted strain of MERS- 330+/− mice reflected those observed in C57BL/6J wild-type mice
CoV, allowed us to generate a mouse model that resembles severe (Fig. 1b,c; Supplementary Fig. 2). DPP4 is central to the
MERS-CoV-induced respiratory disease without bystander neuro- maintenance ofglucosehomeostasisinmammals16.Blood glucose
logical disease. In parallel, we demonstrated that thismodel system levels were within the normal range observed in C57BL/6J wild-
canbeusedforthedevelopmentandtestingofMERS-CoVvaccines type mice, supporting the hypothesis that biological mDPP4
andtherapeutics. functions were not altered in the 288/330+/+ and 288/330+/− mice
(Supplementary Fig. 2). Moreover, basal CD4+ T-cell expression
Results of interleukin-2, tumour-necrosis factor-α, interferon-γ, CD69,
A CRISPR–Cas9-generated mouse model for MERS-CoV CD25 and mDPP4 (CD26) from the 288/330+/+ and 288/330+/−
infection. We have demonstrated previously that the introduction lineswascomparabletothelevelsobservedinC57BL/6Jwild-type
of two amino acids that match the human sequence at positions mice (Supplementary Fig. 3). Notwithstanding functional T-cell
288 and 330 in the mDPP4 receptor can support MERS-CoV assessment, these results suggested that minimal alteration of the
docking,entryandreplicationincellculture7.Thesedeterminants 288 and 330 alleles does not alter basal T-cell activation status.
are located within exons 10 and 11 of mDPP4 on chromosome 2 Overall expression levels, expression patterns, biological function
(Fig. 1a and Supplementary Fig. 1). Therefore, we used and the immunological profiles of mDPP4 were comparable to
CRISPR–Cas9 genome editing to introduce these determinants those of C57BL/6J wild-type mice following site-specific
(A288L and T330R) into the mDPP4 receptor (Fig. 1a and modificationofthe288and330alleles.
Supplementary Table 1). Two lines of C57BL/6J-derived mice The288/330+/+and288/330+/−micesupportedefficientinfection
were generated that were either homozygous (288/330+/+) or andreplicationofthehumanMERS-CoVstrainHCoV-EMC/2012,
heterozygous (288/330+/−) for the chimaeric mDPP4 alleles the camel MERS strain Dromedary/Al-Hasa-KFU-HKU13/2013
(Fig. 1a). The 288/330+/+ homozygous mice encoded the 288L and a recombinant virus derived from a molecular infectious
and 330Rchanges on both chromosomes, therebyexpressing only clone(icMERS)inthelungs,butthesevirusstrainscouldnotrepli-
mDPP4withbothchanges(Fig.1a).The288/330+/−heterozygous cate inC57BL/6J wild-typemicethat retainedthe original murine
mice encoded the 288L and 330R changes on one chromosome A288 and T330 alleles (Fig. 1d). In parallel, a MERS-CoV tissue-
and the C57BL/6J wild-type amino acids, A288 and T330, on the culture-adaptedvariant,derivedbyinfectionofNIH/3T3cellsecto-
other chromosome, thereby expressing both mutated and wild- picallyexpressing the chimaeric mDPP4 (A288L/T330R) receptor,
type mDPP4 (Fig. 1a). The innate mDPP4 expression levels and was found to encode a three amino acid insertion (RMR) and a
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a 120 b 110
105
100
%) 100
80 y 0 ( 95
Survival (%) 4600 ht relative to da 898005
g
Wei 75 288/330+/+, MERS-15
288/330+/−, MERS-15
20 288/330+/+, MERS-15 70 288/330+/+, MERS-0
288/330+/−, MERS-15 C57BL/6J WT, MERS-15
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection
c 1 × 109
Day 3
1 × 108 Day 6
d i ii
e) 1 × 107
u
s
s
g ti 1 × 106
n
u
er g l 1 × 105
p
−1ml 1 × 104
u. iii iv
p.f. 1 × 103
e (
Titr 1 × 102 LOD
< LOD
1 × 101
1 × 100
MERS-15 MERS-0 MERS-15 MERS-15
288/330+/+ 288/330+/+ 288/330+/− C57BL/6J WT
Figure2|Mouse-adaptedMERS-CoVcausesfataldiseasein288/330+/+mice.Micewereinoculatedintranasallywith5×106p.f.u.a,Mortalityof
288/330+/−(n=10)and288/330+/+(n=16)micewasmonitoreddailyuptoday6p.i.Datashowthepercentageofsurvivingmice.b,Mouseweights
weremeasureddailyuptoday6p.i.for288/330+/+miceinfectedwithMERS-15(n=16)orMERS-0(n=10),288/330+/−miceinfectedwithMERS-15(n=10)
andC57BL/6Jwild-type(WT)miceinfectedwithMERS-15(n=7).Dataaredailymeansofthepercentageweightrelativetoday0±s.d.c,Virallungtitresfor
MERS-CoVweredeterminedatday3p.i.(288/330+/−+MERS-15,n=4;288/330+/++MERS-15,n=5;288/330+/++MERS-0,n=5;C57BL/6JWT+MERS-
15,n=4)andday6p.i.(288/330+/−+MERS-15,n=4;288/330+/++MERS-15,n=4;288/330+/++MERS-0,n=5;C57BL/6JWT+MERS-15,n=3).Thelimit
ofdetection(LOD)isindicated.Barsaremeans+s.d.d,Immunohistochemistryoflungsectionsforanti-MERSnucleocapsidat3daysp.i.Resultsshow
288/330+/+mice+MERS-15(i),288/330+/−mice+MERS-15(ii),288/330+/+mice+MERS-0(iii)andC57BL/6JWTmice+MERS-15(iv).IHCimagesare
representativeofatleastthreesamples.Scalebar(d),1mm.
singleaminoacidchange(S885L)intheS2regionofthespikegene. MouseadaptationofMERS-CoVinducessevereARDS-likedisease.
Thisrecombinantlyderivedvirus,MERS-0,encodingtheRMRand The recombinantly derived MERS-0 virus was mouse adapted by
S885L S2 mutations, demonstrated significantly enhanced replica- serial passage for 15roundsthrough the lungs in 288/330+/−mice
tion both in cell culture (Supplementary Fig. 4) and in the lungs at 3-day intervals, resulting in the MERS-15 strain. Infection of
of288/330+/+and288/330+/−mice(Fig.1d;P<0.05).Despiterepli- 288/330+/+ mice via the intranasal route with MERS-15 resulted
catingtosignificantlyhighervirustitresinvivothantheotheriso- in ∼70% mortality (genuine mortality, rather than mice meeting
lates, MERS-0 exhibited no evidence of severe clinical disease the typical 20% weight loss cut-off associated with humane
symptoms (Supplementary Fig. 4). Lung histology demonstrated euthanasia criteria), while 100% of infected 288/330+/− mice
that nucleocapsid antigen from MERS-0 (Fig. 1e), and from the survived (Fig. 2a). However, both lines exhibited 20–25% weight
other strains (not shown), was readily detected in the lungs of lossbyday6p.i.,incontrasttoMERS-0-infected288/330+/+mice
infectedmicebyimmunohistochemistry,butinfectedlungsexhib- or MERS-15-infected C57BL/6J wild-type mice, which exhibited
itedonlymoderatesignsofrespiratorypathologyandinflammation. no weight loss (Fig. 2b). Significantly higher levels of MERS-15
These results demonstrated that we had developed a MERS-CoV replication were detectable in the lungs of both 288/330+/+ and
modelthatcouldsupporthighlevelsofvirusreplicationuptoday 288/330+/− mice at days 3 and 6 p.i., while MERS-0 was mostly
3 post-infection (p.i.), but that further in vivo adaptation was clearedfromthelungs byday6p.i.(Fig. 2c,d). SimilartoMER-0,
required to achieve the respiratory symptoms characteristic of MERS-15 did not replicate in C57BL/6 wild-type mice.
MERS-CoVinfectioninhumans. Importantly, the observed decreases in survival and weight loss
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a 50 b 0.8
MERS-15, 288/330+/+ MERS-15, 288/330+/+
MERS-15, 288/330+/− 0.7 MERS-15, 288/330+/−
MERS-0, 288/330+/+ MERS-0, 288/330+/+
** MERS-15, C57BL/6J WT 0.6 MERS-15, C57BL/6J WT
**
Penh(log)10 5 ** ** ** −1 (ml s)EF50 000...453 * ** ** **
0.2 ** **
** ** ** 0.1 * ** *
**
**
0.5 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection
c d
i ii i ii iii vii
iii iv iv v vi viii
Figure3|LungfunctioninMERS-15-infectedmice.a,b,Respiratoryfunctionwasmonitoredinlivemiceuptoday6p.i.usingwhole-bodyplethysmography
tomeasurePenh(a)andEF (b)in288/330+/+miceinfectedwithMERS-15(n=9)orMERS-0(n=3),288/330+/−miceinfectedwithMERS-15(n=4)
50
andC57BL/6Jwild-type(WT)miceinfectedwithMERS-15(n=3).Dataaredailymeans±s.d.Student’st-testwasusedtocompare288/330+/+mice
infectedwithMERS-15andMERS-0(*P<0.05;**P<0.01).c,Pathologyoflungsfrominfectedmiceatday3p.i.demonstratingsevereinflammationfor
288/330+/+(i)and288/330+/−(ii)miceinfectedwithMERS-15,andmoderateinflammationfor288/330+/+miceinfectedwithMERS-0(iii)andC57BL/6J
WTmiceinfectedwithMERS-15(iv).d,Pathologyatday6p.i.infectedinmice.In288/330+/+miceinfectedwithMERS-15,therewassevereinflammation
andoedemainthelargeairways(i)andalveoli(ii),andhyalinemembraneformation(iii).The288/330+/−miceinfectedwithMERS-15exhibitedsevere
inflammationthroughouttheparenchyma(iv),hyalinemembraneformation(v)andperivascularcuffing(vi).The288/330+/+miceinfectedwithMERS-0(vii)
andC57BL/6JWTmiceinfectedwithMERS-15(viii)exhibitedmild-to-moderateinflammation.Sampleswerestainedwithhaematoxylinandeosinandare
representativeofatleastthreesamples.Scalebars(c,d),1mm.
induced by MERS-15 were not confounded by neurological mice infected with MERS-15 (Fig. 3a,b), demonstrating that
complications from brain infection, as plaque assays for MERS-15 elicited severe respiratory distress in mice carrying the
replication-competent virus and PCR with reverse transcription chimaericDPP4receptor.Thiswasfurthersupportedbyourobser-
(RT-PCR) at days 3 and 6 p.i. were negative (Supplementary vation of severe haemorrhage in the lungs of both 288/330+/+ and
Fig. 5). Moreover, quantitative RT-PCR on the same samples 288/330+/− mice infected with MERS-15 at days 3 and 6
demonstrated an increase of >106 detectable viral transcripts in (Supplementary Fig.6),inflammatoryinfiltratesbyday3 (Fig. 3c)
infected lungs compared with similarly infected C57BL/6J mice, and respiratory pathology associated with severe acute respiratory
with no detectable viral transcripts in the brains of these mice distress, including hyaline membrane formation, intra-alveolar
(SupplementaryFig.5c). oedema, perivascular cuffing and severe inflammation, at day
Althoughmortalityandweightlossprovideimportantmeasures 6 p.i. (Fig. 3d). Quantitative comparison of the lung pathology in
of MERS-CoV-induced disease, these parameters do not directly 288/330+/+miceinfectedwithMERS-15andMERS-0demonstrated
assess the impact of virus replication on respiratory function. that MERS-15 induced significant levels of pathology commensu-
Therefore, to directly assess the impact of MERS-15 infection on rate with ARDS by day 6 p.i. (Supplementary Fig. 7). Although
respiratory function in 288/330+/+ and 288/330+/− mice, we wedidnotconductanexhaustiveassessmentofallextrapulmonary
measuredrespiratoryfunctionusingunrestrainedplethysmography, tissuesinthe288/330+/+mice,wecouldnotdetectvirusreplication
as demonstrated previously for respiratory pathogenesis in mouse in the brain, even at doses of 5×106plaque-forming units (p.f.u.)
models of severe acute respiratory syndrome (SARS) and influ- up to day 6 when the humane euthanasia end points were
enza17. MERS-15 elicited severe lung disease as quantified by reached, and the pathology observed in our model was consistent
enhanced pause (Penh), a unitless measure that reflects airway with the severe respiratory pathology associated with fatal ARDS
obstruction/restrictionduetodebrisintheairway,andbymidtidal intheonlypublishedcasestudyofahumanMERS-CoVinfection18.
expiratoryflow(EF ),whichrepresentstheflowrateatwhich50%
50
ofthetidalvolumehasbeenexpelledinasinglebreath17.MERS-15 IdentificationofMERS-CoV-adaptedmutationsassociatedwith
infection led to significant increases in both Penh and EF in severe respiratory disease. We anticipated that the MERS-CoV
288/330+/+ and 288/330+/− mice up to day 6 p.i. compared5w0ith genome would acquire mutations due to immunological pressure
288/330+/+ mice infected with MERS-0 and C57BL/6J wild-type and/orenhanced virusfitness during mouseadaptation. Twoviral
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a 120 b 110
105
100
%) 100
80 y 0 ( 95
%) da
al ( 60 e to 90
v v
Survi 40 ght relati 8805 MERS-15 C1, 288/330+/+
MERS-15 C1, 288/330+/+ Wei 75 MERS-15 C2, 288/330+/+
20 MERS-0, 288/330+/+
MERS-15 C2, 288/330+/+ 70 MERS-15 C2, C57BL/6J WT
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection
c
1 × 109
Day 3
1 × 108 Day 6
sue) 1 × 107 d i ii iii
s
ng ti 1 × 106
u
g l
er 1 × 105
p
−1ml 1 × 104
p.f.u. 1 × 103 iv v vi
Titre ( 1 × 102 < LOD LOD
1 × 101
1 × 100
MERS-15 C1 MERS-15 C2 MERS-0 MERS-15 C2
288/330+/+ 288/330+/+ 288/330+/+ C57BL/6J WT
Figure4|Clonalisolatesofmouse-adaptedMERS-CoVexhibitsevererespiratorydisease.Micewereinoculatedintranasallywith5×106p.f.u.a,The
mortalityof288/330+/+miceinfectedwithMERS-15C1(n=7)orMERS-15C2(n=11)wasmonitoreddailyuptoday6p.i.Datareflectthepercentageof
survivingmice.b,Mouseweightsweremonitoreddailyfor288/330+/+miceinfectedwithMERS-15C1(n=7),MERS-15C2(n=11)orMERS-0(n=6),and
C57BL/6Jwild-type(WT)miceinfectedwithMERS-15C2(n=6).Dataaredailymeans±s.d.c,Virallungtitresweredeterminedatday3p.i.(288/330+/++
MERS-15C1,n=4;288/330+/++MERS-15C2,n=3;288/330+/++MERS-0,n=3;C57BL/6JWT+MERS-15C2,n=3)andday6p.i.(288/330+/++
MERS-15C1,n=4;288/330+/++MERS-15C2,n=6;288/330+/++MERS-0,n=3;C57BL/6JWT+MERS-15C2,n=3).Thelimitofdetection(LOD)is
indicated.Barsaremeans+s.d.d,IHCoflungsectionsat3daysp.i.from288/330+/+miceinfectedwithMERS-15C1(i)orMERS-15C2(ii)andstainedfor
nucleopcapsid.Thepathologyofthelungsfrom288/330+/+miceinfectedwithMERS-15C2wasassessedbyhaematoxylinandeosinstaining(H&E)atday
6p.i.anddemonstratedsevereinflammation(iii),oedema(iv),hyalinemembraneformation(v)andperivascularcuffing(vi).AllH&Eimagesare
representativeofatleastthreesamples.Scalebar(d),1mm.
clones, MERS-15 clone 1 (MERS-15 C1) and MERS-15 clone 2 orf4b into orf5 (Supplementary Fig. 9). Moreover, 5′ rapid
(MERS-15 C2), were isolated by plaque purification from the amplification of cDNA ends revealed that the nucleotide at
MERS-15 heterogeneous virus population. MERS-15 C2 infection position2ofthe5′untranslatedregionwasdeletedinbothclones
resulted in increased mortality of the mice (Fig. 4a) and (Supplementary Fig. 9). Generation of an infectious clone
significantly increased haemorrhage up to day 6 p.i. compared harbouring all of the MERS-15 C2 mutations (icMERSma1)
with MERS-15 C1 (Supplementary Fig. 8), whereas both clonal demonstrated that the disease could be reproduced with an
isolates caused 25–30% weight loss (Fig. 4b) and high levels of infectiousdoseof5×106p.f.u.(SupplementaryFig.10).Decreasing
virus replication in the mice up to day 6 p.i., when humane the dose by 10-fold to 5×105p.f.u. resulted in weight loss that
euthanasia end points were reached (Fig. 4c). The lung pathology paralleledthatobservedwith5×106p.f.u.;however,the5×105p.f.u.
elicitedbyMERS-15C2resembledthatobtainedwiththeprimary dose exhibited a slight decrease in mortality up to day 7 p.i.
MERS-15 virus (Fig. 4d), demonstrating similar pathologies (Supplementary Fig. 10). Although additional studies will be
including oedema, hyaline membrane formation and perivascular needed, comparative genomic analysis of the two clones, C1 and
cuffing. Sequencing of the entire MERS-15 C2 genome revealed a C2, indicated that nsP2 and Orf5 may have significant roles in
set of unique missense mutations, acquired during in vivo determiningdiseaseoutcome.Therefore,theMERS-15C2derived
passaging, in the genes encoding the non-structural proteins, virus, combined with the 288/330+/+ mouse line, represent useful
nsP2, nsP6 and nsP8, and a large deletion in orf4b that may be tools for studying MERS-CoV pathogenesis or assessing
responsible for the enhanced disease observed with MERS-15 therapeuticcountermeasuresagainstMERS-CoV-inducedARDS.
(Supplementary Fig. 9). Sequencing of MERS-15 C1 revealed
some differences that may influence the capacity of the virus to Human monoclonal antibody 3B11 protects from MERS-CoV-
elicit the increased mortality observed with MERS-15 C2, namely elicited severe respiratory disease. Humanmonoclonalantibodies
mutations in nsP2 and an expanded deletion that extended from providearobuststrategyforthetreatmentofnewlyemergedviruses
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a 120 b
105
100 100
%)
80 y 0 ( 95
%) o da 90
urvival ( 60 elative t 85
S 40 ht r 80
g
Wei 75
20 3B11 3B11
70
Isotype control Isotype control
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection
c 1 × 109 d
Day 3 3B11
16
1 × 108 Day 6 Isotype control
e) 1 × 107 14 **
u
s
g tis 1 × 106 12
n
u
−1ml per g l 11 ×× 110045 Penh 108
u.
p.f. 1 × 103 6 *
e (
Titr 1 × 102 LOD 4
< LOD
1 × 101 2
** *
1 × 100 0
Isotype control 3B11 0 3 6
Day post-infection
Figure5|Humanmonoclonalantibody3B11protectsmicefromsevererespiratorydisease.The288/330+/+micewereintraperitoneallyadministered
250µgof3B11humanmonoclonalantibodyorisotypecontrolantibody12hbeforechallengewith5×106p.f.u.MERS-15C2.a,b,Mortality(a)andmouse
weight(b)weremonitoreddailyformicereceiving3B11(n=12)oranisotypecontrol(n=12)uptoday6p.i.Datashowthepercentageofsurviving
mice(a)ordailymeanweight±s.d.(b).c,Virallungtitresweredeterminedatday3p.i.(n=6forboth3B11-andisotypecontrol-treatedmice)andday6p.i.
(n=6for3B11-treatedmice;n=3forisotypecontrol-treatedmice).Thelimitofdetection(LOD)isindicated.Barsaremeans+s.d.d,Lungfunctionwas
assessedbyPenhat0,3and6daysp.i.formicereceiving3B11(n=6atdays0,3and6)orisotypecontrol(n=6atdays0and3;n=3atday6).Dataare
means+s.d.Student’st-testwasusedtocomparemicereceiving3B11withthosereceivingtheisotypecontrolatday3andday6p.i.(*P<0.05;**P<0.01).
in humans. 3B11 is a human monoclonal antibody that targetsthe particles (VRPs) expressing MERS-CoV spike protein (spike–VRP)
receptor-binding domain of the MERS-CoV spike protein19, and is ormockvaccinatedwithVRPsexpressinggreenfluorescentprotein
effective in NHPs5. Asthe MERS-15 C2 adapted virus acquired no (GFP–VRP), boosted at 4 weeks postprime and challenged with
receptor-binding domain mutations (Supplementary Fig. 9), we MERS-15 C2 at 4 weeks postboost. All mice receiving spike–VRP
reasoned that 3B11 should protect 288/330+/+ mice from MER-15 survivedandexhibitednoweightlossfollowingchallengecompared
C2 challenge. Pretreating mice for 12h with 3B11 provided 100% with GFP–VRP mock-vaccinated animals (Fig. 6a,b). Spike–VRP
protection against MERS-15 C2 challenge (Fig. 5a,b). Moreover, vaccination significantly reduced MERS-15 C2 replication in the
3B11treatmentreducedviralloads inthelungs of infectedmiceto lungs of infected mice as shown by both plaque titre (Fig. 6c) and
undetectable levels (Fig. 5c and Supplementary Fig. 11) and viralantigenstaining(SupplementaryFig.14),whilealsoprotecting
protectedfromlossofrespiratoryfunction(Fig.5d)(Supplementary against severe respiratory disease, as assessed by measurement of
Fig. 11), pulmonary haemorrhage (Supplementary Fig. 11) and Penh(Fig.6d)andEF (SupplementaryFig.13),lunghaemorrhage
50
severe pathological changes (Supplementary Fig. 12). In contrast, (SupplementaryFig.13)andpathologicalindicationsofsevereacute
pretreatment with isotype control antibody provided no protective respiratory disease (Supplementary Fig. 14). Neutralization of
effect. Therefore, these data convincingly demonstrated that our MERS-15 C2 with prechallenge serum from spike–VRP-vaccinated
preclinicalmouse modelof severe respiratorydisease and mortality mice validated the presence of high-titre neutralizing antibodies in
canserveasaplatformforassessingMERS-CoVtherapeutics. the serum of vaccinated 288/330+/+ mice (Supplementary Fig. 13).
Our data demonstrated that the spike–VRP vaccine provoked an
Spike protein vaccines derived from Venezuelan equine adaptiveimmuneresponsecapableofprotectingmicefromalethal
encephalitisvirusrepliconparticlesprotectfromlethalinfection. challenge with MERS-CoV, thereby extending the utility of our
To examine vaccine efficacyinthe 288/330+/+MERS-15C2 model, preclinical mouse model of severe respiratory disease to include
micewere vaccinated with Venezuelan equine encephalitis replicon vaccineevaluation.
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a 120 b
110
100 105
%) 100
%) 80 ay 0 ( 95
Survival ( 4600 ht relative to d 898005
g
Wei 75
20
Mock vaccinated (GFP−VRP) Mock vaccinated (GFP−VRP)
70
Vaccinated (spike−VRP) Vaccinated (spike−VRP)
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection
c 1 × 109 d
Day 3 12 Mock vaccinated (GFP−VRP)
1 × 108 Day 6 ** Vaccinated (spike−VRP)
ue) 1 × 107 10
s
s
g ti 1 × 106
un 8 *
−1ml per g l 11 ×× 110045 Penh 6
u.
p.f. 1 × 103 4 **
e (
Titr 1 × 102 < LOD LOD 2
1 × 101 *
1 × 100 0
GFP−VRP Spike−VRP 0 3 6
Day post-infection
Figure6|Vaccinationof288/330+/+micewithaVRPdeliveringMERS-CoVspikeproteinprotectsmicefromchallengewithMERS-CoV.Thevaccination
protocolisdescribedinMethods.a,b,Aftera5×106p.f.u.challenge,mortality(a)andmouseweight(b)weremonitoreddailyformicereceivingGFP–VRP
(n=19)orspike–VRP(n=19)vaccineuptoday6p.i.Datareflectthepercentagesurvival(a)ordailymeanweight±s.d.(b).c,Virallungtitreswere
determinedonday3p.i.(n=7forspike–VRPandGFP–VRP)andday6p.i.(n=12forspike–VRP;n=6forGFP–VRP).Thelimitofdetection(LOD)is
indicated.Barsaremeans+s.d.d,LungfunctionwasassessedbyPenhat0,3and6daysp.i.formicereceivingGFP–VRP(n=12atdays0and3p.i.;n=6
atday6p.i.)orspike–VRP(n=12atalldaysp.i.).Datarepresentmeans+s.d.Student’st-testwasusedtocomparemicereceivingGFP–VRPwiththose
receivingspike–VRPatdays3and6p.i.(*P<0.05;**P<0.01).
Discussion reportedhistopathologythatincludeddiffusealveolardamagewith
The MERS-CoV preclinical mouse model described here demon- denudingofbronchiolarepithelium,hyalinemembraneformation,
stratesforthefirsttimethattheCRISPR–Cas9systemcanbeused type II pneumocyte hyperplasia and oedema18. Furthermore,
togeneticallyeditanon-permissivehostreceptortogenerateasus- MERS-CoV antigen staining localized the virus to pneumocytes
ceptiblemodelforanemerginginfectiouspathogen.The288/330+/+ and syncytial cells18. Infection of type I and II pneumocytes can
MERS-CoVmousemodelresemblesthesevere,andoftenfatal,res- lead to cell death, as observed in autopsies of patients who have
piratory distress syndrome observed in humans, and can be pre- died from severe respiratory infections with influenza virus and
vented through treatment with the 3B11 neutralizing monoclonal SARS-CoV21,22.Moreover,pneumocytecelldeathhasbeenproposed
antibody19 or a VRP-based vaccine directed against the tocausedecreasedrespiratoryfunction,asmeasuredbywhole-body
MERS-CoV spike protein15,20. Coupled with an inability to elicit plethysmographyinamousemodelofinfluenza23.Commensurate
escapemutants,3B11 hasalso beentested inNHPs5, makingitan withthesepreviousstudies,ourMERS-CoVmousemodeldemon-
excellentcandidatefordownstreamhumanstudies.However,exist- stratedwidespreadinfectionofpneumocytesandpathologyconsist-
ingNHPmodelsrelyonquantitativeRT-PCR,ratherthanmeasures entwithdiffusealveolardamageandsevererespiratorydisease.This
ofinfectiousvirus,toquantifyviralloads,andthesemodelsdonot was corroborated by decreased pulmonary function in the
reproducibly result in the severe respiratory disease or mortality MERS-CoV model, as measured by plethysmography, which may
observed in human MERS patients1–4. Therefore, the results from be associated with widespread infection and possibly the death of
our model complement the NHP 3B11 studies by directly demon- pneumocytes and airway epithelial cells. Therefore, this model
strating that 3B11 reduces levels of infectious MERS-CoV in the system provides the field with the opportunity to investigate the
lungs, while preventing severe virus-induced respiratory pathology, mechanisms that lead to MERS-CoV-induced pathology and
lossofrespiratoryfunctionandmortality. severerespiratorydisease,intheabsenceofanyCNScomplications.
In fatal cases of human MERS-CoV infections, individuals OtherMERS-CoVmousemodelshaveusedthemoretraditional
exhibitsevererespiratorydistressrequiringmechanicalventilation, methodofexpressingthefull-lengthhumanDPP4receptortofacili-
andtheonlypublishedhumanautopsyfromaMERS-CoVfatality tateMERS-CoVinfection12–15,24.Thesemodelsexhibitedinfection/
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replication in the lungs following intranasal administration at low modelstoevaluatetherapeuticcountermeasures,therebynecessitat-
viraldoses(102–105p.f.u.),whichinsomecasesresultedinpathol- ingvirusadaptationtohostimmunitytoachieveeffectivemodels.It
ogyindicativeofpneumonia-likedisease12–14.Onelimitationofthe is critical that the GOF regulatory structure does not impede the
288/330+/+ MERS-CoV model described here was the use of high development of robust animal models of human disease, which
viral loads to achieve severe, and often fatal, respiratory disease. areessentialforprotectingthepublichealth.
Further adaptation may allow the use of lower infectious doses
thatwouldproduceamodelofmilddiseasewithsubsequentrecov- Methods
eryatlatertimepoints,ashasbeendescribedwithSARS-CoV25.In Viruses,cellsandplaqueassays.AllvirusstockswerepreparedonVeroCCL81
thiscontext,itisinterestingthatthedeletionofMERS-CoVORF4b, cells(ATCC).CCL81cellsweremaintainedroutinelyinDulbecco’smodifiedEagle’s
medium(Gibco)supplementedwith10%fetalbovineserum(FBS;Sigma)and
a phosphodiesterase and antagonist of RNase L activity in human
1×antibiotic/antimycotic(Gibco).AllviruseswereharvestedinOpti-MEMmedium
cells, appeared less critical foreliciting severe disease in rodents26, (Gibco)supplementedwith3%FBS,1×antibiotic/antimycotic,1×non-essential
suggesting possible species-specific modes of action in vivo. aminoacids(Gibco)and1×1mMsodiumpyruvate(Gibco).Thewild-typeHCoV-
Deletions in some SARS-CoV interferon antagonist genes and EMC/2012strainofMERS-CoVwasusedatpassage10(originallyprovidedbyBart
accessoryopen reading frames have also yielded subtle changes in Haagmans(ErasmusMedicalCenterRotterdam,TheNetherlands)atpassage8),
icMERSwasgeneratedpreviouslybyScobeyetal.33,MERScamelstrainDromedary/
overallvirulenceinvivo27,28.However,itisimportanttopointout
Al-Hasa-KFU-HKU13/2013wasusedatpassage5andwasprovidedatpassage4by
that a balance must be achieved between the virulence of mouse- MalikPeiris(UniversityofHongKong,China)andMERS-0wasgeneratedinthe
adapted virus, which enhances the model’s capacity to replicate laboratoryofR.S.B.asdescribedbelow.Virustitresweredeterminedbyplaque
humandiseasephenotypes,andthenumberofmutationsrequired assaysonVeroCCL81cells33.AllvirusesweremaintainedunderBiosafetyLevel
tosignificantlyreducethedoselethalto50%ofanimalstested. (BSL)3conditionswithredundantfansandpersonnelpoweredair-purifying
respirators,scrubs,Tyveksuits,Tyvekapronsanddoublelayersofgloves.
Conventional models using constitutive overexpression of the AnicMERSvirustaggedwithtomatoredfluorescentprotein33waspassagedfor
hDPP4 MERS-CoVreceptor havedemonstrated widespreadinfec- tenroundsonNIH/3T3cells(ATCC)thatweregeneratedtostablyoverexpressthe
tion of extrapulmonary tissues including brain, kidney, liver, mDPP4receptorcontainingA288LandT330R.ThemDPP4A288L/T330R
spleen and heart12–14, and two of these studies indicated that the expressioncassetteusedtogeneratethecelllinehasbeendescribedpreviously7.
mice exhibited multi-organ failure13,14. Furthermore, high viral Sequencingofthepassagedvirusidentifiedaninsertionofthreeaminoacids(RMR)
afteraminoacid884inthespikeproteinandanS885Lchange.Thesechangeswere
loadsweredetectedinthebrainsofmiceinthetransgenichDPP4
subclonedbackintotheMERS-CoVinfectiousclonebyoverlap-PCRoftheF
overexpression models12–14. Importantly, Li et al.13 concluded that fragmentwiththefollowingprimers:5′-GGTTTCCAGAAGTGTGAGCAATTA
“mortalitycorrelatedwithbraininfection,suggestingthatinfection CTGCGCG-3′,5′-GCAGGCCTCTGCAGTCGACGGGCCCGGGATCCAA
ofthisorganwasmostimportantforthehighmortalityobservedin TGCC-3′,5′-CCTGTTTCTATATCTACTGGCAGTCGTAGAATGCGGCTTGCA
K18-hDPP4mice”.Whiletheselethalmodelshavevalueforvaccine CGTAGTGCTATTGAGGATTTGC-3′and5′-GCAAATCCTCAATAGCACTA
CGTGCAAGCCGCATTCTACGACTGCCAGTAGATATAGAAACAGG-3′.TheF
andimmunotherapeutictesting29,small-moleculeinhibitorsthatare
fragmentisonepartofaseven-plasmidsystem(A,B,C,D1,D2,EandF)that
effective in the lung may be limited in their efficacy due to an permitspartitioningoftheentiregenometogenerateinfectiousMERS-CoV,as
inability to cross the blood–brain barrier30. Similar neurological describedpreviously33.ThePCRproductencodingtheinsertionwassubclonedback
complicationshavebeenobservedinmodelsystemsforSARSthat intotheFfragmentusingtherestrictionenzymesMscIandBamHIandvalidatedby
used overexpression, or tissue-specific constitutive promoters, to sequencing.RecombinantMERS-0viruswasusedfortheinvivopassage
experiments,whichwereGOFstudiesreviewedandapprovedbytheNational
express human angiotensin 1-converting enzyme 2 (ACE2) in
InstitutesofHealth(NIH).VeroCCL81andNIH/3T3cellswereoriginallyreceived
mice31. While additional human pathology studies are needed to fromtheATCC,whichindicatesthatcelllinesareauthenticandconfirmsthatcell
determinetheextentofextrapulmonarysitesofMERS-CoVreplica- linesaremycoplasmafree.Noneoftheworkingcelllinestockswasauthenticatedor
tionandtheirimpactonMERS-CoVdisease,itisclearthatrespir- testedformycoplasmarecently,althoughtheoriginalseedstocksusedtocreatethe
workingstocksarefreefromcontamination.Additionally,bothcelllineshavebeen
atory replication and pathology is an important aspect of human
authenticatedbymorphologicalandcytopathologicalevaluation.Furthermore,Vero
MERS-CoV disease. Therefore, an important attribute of the CCL81cellswereconfirmedforDPP4overexpressionbythecapacitytobeinfected
288/330+/+ model isthat the lackof detectable virus replication in andtoreplicateMERS-CoV.
the CNS means this model can be used to study MERS-CoV-
induced pulmonary disease without the confounding effects of GenerationofmicewithmDPP4modifiedatpositions288(exon10)and
deathduetoCNSinfection. 330(exon11).Theallelesencodingaminoacids288and330areshownin
SupplementaryFig.1.GenomicengineeringofthesealleleswiththeCRISPR–Cas9
Recently,adebatehasemergedaroundthesafetyofperforming
genomeeditingsystemwasperformedattheUniversityofNorthCarolinaatChapel
gain-of-function (GOF) studies with highly pathogenic viruses Hill(UNC-CH)AnimalModelsCoreFacility.ThemessengerRNA(mRNA)
(such as MERS-CoV, SARS-CoV, influenza virus H5N1) (http:// encodingCas9endonucleaseandguideRNAs(gRNAs)werebasedonthesystem
www.gryphonscientific.com/gain-of-function/). As demonstrated establishedbyMalietal.34andpreparedasdescribedbelow.ThegRNAs
here, GOF studies were absolutely necessary to develop a mouse (SupplementaryTable1)weregeneratedbyaninvitrotranscriptionreactionusinga
model that reflects the ARDS pathology observed previously in T7HighYieldRNASynthesiskit(NEB),where1µgDraI-linearizedtemplateDNA
wasusedina20µlreactionfollowingthekitguidelinesforshortRNAtranscripts.
humans infected with respiratory pathogens. Importantly, the Thereactionwasincubatedat37°Covernight,followedbyDNaseI(RNase-free)
GOF studies performed here yielded MERS-CoV strains that digestionfor15minat37°C.ThegRNAswerethenpurifiedusinganRNeasy
reflect the complexity of clinical isolates identified recently in column(Qiagen)followingtheguidelinesforshortRNApurification.Cappedand
humans, where deletions were identified in ORF3 and ORF4A32. polyadenylatedCas9mRNAwaspreparedbyaninvitrotranscriptionreactionusing
anmMESSAGEmMACHINET7ULTRAkit(LifeTechnologies).CappedmRNA
Moreover,theseGOFstudieshaveallowedustoidentifymutations
wasgeneratedwith1µghCas9-T7linearizedplasmidDNAina20µlreaction
inMERS-CoVproteinsthatmayinfluencehowMERS-CoVinter- containing1×NTP/ARCASolution,1×T7reactionbufferand2µlT7enzyme.The
acts with, and possibly circumvents, host immune responses. reactionwasincubatedat37°Cfor1h,followedbyadditionof1µlTURBODNase
Nevertheless,futurestudiestoevaluatehostfactorsthatcontribute anddigestionat37°Cfor15min.Toaddapoly(A)tailtothecappedmRNA,the
20µlreactionmixfromstep1wasmixedwithnuclease-freewater(36µl),5×E-PAP
toMERS-CoVdiseasewillbeconstrainedinthismodel,aswellas
buffer(20µl),25mMMnCl (10µl),ATPsolution(10µl)andE-PAP(4µl)toa
inhDPP4expressionmodels,bythefactthatthemicemustbeback- finalreactionvolumeof100µ2l.Thereactionwasincubatedat37°Cfor30–45min.
crossed to mouse lines harbouring modified endogenous genes, ThecappedandpolyadenylatedRNAwaspurifiedbylithiumchlorideprecipitation
such as knock-out mice. This limitation may be overcome andresuspendedinmicroinjectionbuffer(5mMTris/HClbuffer,0.1mMEDTA,
through additional GOF studies that facilitate MERS-CoV adap- pH7.5).
FertilizedzygoteswerecollectedfromC57BL/6Jfemalesthathadbeen
tation to the innate mDPP4 receptor molecule. The continued
superovulatedandmatedtoC57BL/6Jmales.Pronuclearmicroinjectionwas
threats from novel emerging pathogens, such as Zika virus, will performedwith50ngµl−1Cas9mRNA,50ngµl−1gRNADpp4-g59B,25ngµl−1
demand the rapid development of physiologically relevant animal gRNADpp4-g88Band50ngµl−1eachofDpp4-A288L-BandDpp4-T330R-B
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donoroligonucleotides(SupplementaryTable1).Injectedembryoswereimplanted day0toestablishabaselineandagainatdays3and6p.i.Theisolationand
intopseudopregnantrecipients,andtheresultingpupswerescreenedforalleles productionofthe3B11andF10antibodieshasbeendescribedpreviously19.
encodingchanges(SupplementaryFig.1)atpositions288and330.Mutationsat
the288positionweredetectedbyamplifyingbiopsyDNAsampleswiththe Spike–VRPvaccinestudyandvirusneutralizationassay.TheMERS-CoVgene
followingprimers:Dpp4-E10ScF1:5′-GATTCTGAGCAAGCAAACACGC-3′, encodingthespikeproteinandGFPgenewerepackagedintoVRPsgeneratedwith
andDpp4-E10ScR1:5′-CCACAAGGTATCCCACAGAGACG-3′.The helperconstructsfromtheV3526attenuatedstrainofVenezuelanequine
752bpPCRproductwassequencedwithprimerDpp4-E10-SqR1: encephalitisvirus,underBSL2conditions,asdescribedpreviously20.Micewere
5′-CAAGAACCACACCAATGGAAAGTC-3′.Mutationsatthe330positionwere administeredaprimaryvaccinationof10µlbyfootpadinjectionofspike–VRP
detectedbyamplifyingbiopsyDNAsampleswiththefollowingprimers: (1×105p.f.u.)orcontrolGFP–VRP(1×105p.f.u.).After28days,themicewere
Dpp4-E11ScF1:5′-AAGTGCTGGGATTATAGGTGGTCAC-3′,andDpp4-E11ScR1: boostedwiththesamedoseoftheirrespectiveVRPstrain.At28dayspostboost,all
5′-GTGTTTACATTCTAAGTTGGGTTTCTGC-3′.The767bpPCRproductwas vaccinatedmicewerechallengedwith5×106p.f.u.MERS-15C2.Micewere
sequencedwithprimerDpp4-E11-SqF1:5′-GCATGTTATCCACTGTGCCATCTC-3′. monitoreddailyforweightandsurvival,andkilledatdays3and6p.i.tocollect
Fiveof66liveanimalsproducedshowedevidenceofboththe288and330modifications. lungsforhistology,assessmentofviraltitrebyplaqueassayandevaluationoflung
FounderswithbothexpectedmodificationswerebackcrossedtoC57BL/6Jmiceto haemorrhaging.Respiratoryfunctionwasmeasuredatday0toestablishabaseline
identifyanimalswiththe288and330modificationsincis.F animalswithboththe288 andthenagainatdays3and6p.i.
1
and330modificationswerethenintercrossedtoproducehomozygousbreederpairsfor ThepresenceofMERS-15C2-specificserumantibodieswasassessedbyaplaque
colonyenrichmentanddownstreamstudies. reductionneutralizationtitreassay.Prechallengeserumsampleswerecollectedat
25dayspostboostwiththespike–VRPvaccineortheGFP–VRPcontrol.Virus
Mouseinfections.GeneticallyengineeredmicewithamodifiedmDPP4receptor neutralizationassayswereperformedasdescribedpreviously35.Thepercentageof
werehousedandbredinaccordancewithguidelinesestablishedbytheDepartment plaquereductionwascalculatedas:1− (numberofplaqueswithspike–VRPor
ofLaboratoryAnimalMedicineatUNC-CH.Asthe288/330+/+and288/330+/−mice GFP–VRPserum/numberofplaqueswithserumfromnaïve288/330+/+mice)×100.
arenovelmouselinesdevelopedinthelaboratoryofR.S.B.,experimentsutilized
availablemaleandfemalemicethatrangedfrom12to20weeksofage.Basedon Respiratoryfunction.Respiratoryfunctionwasmeasuredforindividualmice,atthe
availabilityatthetimeofeachexperiment,experimentalandcontrolanimalswere indicatedtimepoints,asdemonstratedpreviouslyforSARS-CoVandinfluenzaA
age-andsex-matched.Noblindingwasusedinanyanimalexperiments,and virus17.Briefly,individualmicewereacclimatedfor30mininindividual
animalswerenotrandomized.Samplesizesweredeterminedfrompreliminarydata plethysmographychambers(BuxcoSystems)andeachbreathwasthenquantified
thatwouldyieldstatisticallysignificantdifferences.Mousestudieswereexecuted overa5minperiod.Micewereroutinelyrandomizedintodifferentchambersto
underanimalBSL3conditionsasdescribedpreviously35.Beforeviralinfection,mice avoidmeasurementbiasesthatcouldresultbetweenchambers.DataforPenhand
wereanesthetizedbyadministering50µlketamine/xylazinemixture EF wereanalysedasdescribedpreviouslybyourgroup17.
50
intraperitoneallyandtheninfectedintranasallywith50µlvirussolutioncontaining
5×106p.f.u.Incompleteinfectionsduetobubblingofinoculumfromthenasal Histology.Lung,brainandkidneysampleswereplacedin10%phosphate-buffered
cavity,theinabilitytoinhaletheentiredoseorinoculumgoingintothemouthwere formalinfor>7daysat4°Cforfixation.Fixedtissuesampleswerethenremoved
noted,andthesemicewereconsideredfailuresandwereexcluded,asdescribed fromtheBSL3,placedintocassettesforembeddinginparaffinandsubmittedtothe
previously35.Followingsedationandinfection,miceweremonitoreddailyforweight LinebergerComprehensiveCancerCenterAnimalHistopathologyCorefor
lossandsurvival,aswellasforsignsthattheanimalsweremoribund(including processing,sectioningandstaining.Tissuesections(5µm)werestainedwith
labouredbreathing,lackofmovementandlackofgrooming).Micethatreached20% haematoxylinandeosin,andMERS-CoVnucleocapsidantigenwasdetectedusing
weightlosswereplacedunderexceptionandmonitoredatleasttwicedaily.Micethat mouseanti-MERSnucleocapsidserumata1:250dilution.Thenucleocapsid
approached30%weightlosswereeuthanizedimmediately.Micedeemedmoribund antiserumwasgeneratedinthelaboratoryofR.S.B.usingMERS-CoVnucleocapsid–
wereeuthanizedatthediscretionoftheresearcher.Micewereeuthanizedwithan VRPparticlesinBALB/cmiceasdescribedpreviously20.Antigenwasvisualized
isofluraneoverdosefollowedbyasecondarythoracotomy,atvarioustimepoints,to using3,3′-diaminobenzidinestaining.AnOlympusDP71cameraattachedtoan
collectlungtissues.Intheabsenceofathoracotomy,cervicaldislocationwasusedas OlympusBX41microscopewasusedtocaptureimageswith×10and×40objectives.
asecondaryeuthanasiamethod.AllareapprovedmethodsoftheInstitutional Histopathologywasscored,blindedtoinfectionandanimalstatus,forairway
AnimalCareandUseCommittee(IACUC)attheUNC-CH. disease,vasculardisease,parenchymalpneumonia,diffusealveolardamage,
eosinophilsandimmunohistochemistryonascaleof0–3(0,none;1,mild;2,
Ethicsstatement.Mousestudieswerecarriedoutinaccordancewiththe moderate;3,severe).
recommendationsforthecareanduseofanimalsbytheOfficeofLaboratory
AnimalWelfareatNIH.IACUCatUNC-CHapprovedtheanimalstudies Flowcytometryanalysis.Wholeperipheralbloodfromeachmousestrainwas
performedhere(protocol,IACUC13-272),usingaweightlosscut-offpointof collectedinEDTAandperipheralbloodmononuclearcellswerepurifiedusing
∼30%forhumaneeuthanasia.SyntheticallyreconstructedMERS-CoVswere density-gradientcentrifugationoverFicoll-PaquePLUS(GEHealthcareLife
approvedbytheUNC-CHInstitutionalBiosafetyCommittee,whichalsoconsidered Sciences).Mouselungtissueswerefirstdissociatedmechanicallyusingscalpelblades
GOFresearchconcernsbeforeexecutionoftheseexperiments. andthendigestedfurtherusingtypeAcollagenase(WorthingtonBiochemical)in
mediumpreparedwithDNaseI(1mgml−1)for1hinashakingincubatorat37°C.
Analysisofserumglucoselevels.Allbloodglucosemeasurementsweretaken Digestedlungsuspensionswerefiltered,centrifugedandtreatedwithACKlysis
followinga6hfast.Bloodglucosewasmeasuredbytailclipsamplingusingan buffer(Gibco).Single-cellsuspensionsfrombloodandlungtissuewereresuspended
AlphaTRAK2glucometer(AbbottLaboratories)36.Thissystemisdesignedforuse at1×107cellsml−1inRPMI1640mediumsupplementedwith10%heat-inactivated
inlaboratorymice,withanormalrangeof111–205mgdl−1(6.1–11.38mmoll−1) FBSandantibiotic/antimycoticcocktail(allfromGibco),andwereused
bloodglucoseandadetectionlimitof20–750mgdl−1(1.1–41.62mmoll−1) immediatelyforflowcytometry.Single-cellsuspensionswereplatedin200µlper
bloodglucose. wellon96-well,round-bottomedplatesandstainedforflowcytometryusingan
IntracellularFixationandPermeabilizationBufferSet(eBioscience)accordingtothe
AdaptationofMERS-0inhumanizedmice.TherecombinantvirusMERS-0was manufacturer’sprotocolandusingantibodiesspecificforCD3(clone145-2C11;BD
passedthroughthelungsof288/330+/−miceevery3daysfor15passagestoobtaina Biosciences),CD4(cloneGK1.5;BDBiosciences),CD8(clone53-6.7;eBioscience),
MERS-CoVthatwasadaptedtocauserespiratorydiseaseinmice(MERS-15).At CD25(clonePC61;BDBiosciences),CD26/DPP4(cloneH194-112;Biolegend),
eachpassage,thelungswerehomogenizedand50µllunghomogenatewasusedfor CD69(cloneH1.2F3;eBioscience),interferon-γ(cloneXMG1.2;BDBiosciences),
intranasalinfectionofanaïve288/330+/−mouse.TheMERS-15mouse-adapted tumour-necrosisfactor-α(cloneMP6-XT22;eBioscience)andinterleukin-2(clone
viruswasassessedinmicebysurvival,weightloss,lungtitre,haemorrhaging, JES6-5H4;eBioscience).CellswerealsotreatedwithafixableLIVE/DEAD
respiratoryfunctionandhistopathologyindicativeofdiffusealveolardamageand discriminator(Invitrogen).Stainedcellswerefixedfor20minin1%
ARDS.ClonalisolatesoftheMERS-15viruswereobtainedbyplaquepurification paraformaldehyde,resuspendedinPBSandstoredat4°Cbeforeacquisitionwithin
andamplificationinVeroCCL81cells,andweresequencedtodeterminethe 24h.Cellpopulationswerefirstgatedtoexclude(1)debris,(2)doubleteventsand
mutationsacquiredduringmouseadaptationofthevirus.Allsequencingwas (3)deadcellsbeforephenotypicandfunctionalgating.Allsampleswerestainedin
performedattheUNC-CHGenomeAnalysisFacility.MERS-15C2reproducedthe parallelwithcontrolsoffluorescence–1.Aminimumof100,000live,singletevents
severerespiratorydiseaseobservedwithMERS-15;therefore,allsubsequent wereacquiredpersample,anddatawereanalysedusingFlowJosoftware(TreeStar).
experimentswereperformedwiththeplaque-purifiedMERS-15C2.
NorthernblotanalysisandRT-PCR.Northernblotanalysiswasperformedfor
Humanmonoclonalantibody3B11protectionstudy.The288/330+/+micewere detectionoffull-lengthmDPP4inthelungsof288/330+/+,288/330+/−andC57BL/6J
prophylacticallyadministered250µgofhumanmonoclonalantibody3B11or wild-typemice.Poly(A)RNAwasisolatedtoeliminateribosomalRNA(rRNA;
isotypecontrolantibodyF10byintraperitonealinjection1912hbeforeinfectionwith Qiagen).EquivalentamountsofRNAwereresolvedon0.8%agarosegelsand
MERS-15C2.Miceweremonitoreddailyforweightandsurvival,andkilledatdays3 transferredtonitrocellulosemembrane.Abiotinylatedprobe(5′-biotin-gATg
and6p.i.tocollectthelungsforhistology,forviraltitreassessmentbyplaqueassay (biotin-dT)gCTggTgAgCTgTgCTgCTAgCgATCCCgTggTCTTCATCC-3′)was
andforevaluationoflunghaemorrhaging.Respiratoryfunctionwasmeasuredat usedtodetectmDPP4.
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ARTICLES
NATURE MICROBIOLOGY
DOI:10.1038/NMICROBIOL.2016.226
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Received27April2016;accepted14October2016;
nuclearimportblockenhanceshosttranscriptioninhumanlungcells.J.Virol.
published28November2016 87,3885–3902(2013).
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