Mouse adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice Kun Li a Christine L Wohlford Lenane a Rudragouda Channappanavar b Jung Eun Park c James T Earnest c Thomas B Bair d Amber M Bates e Kim A Brogden e Heather A Flaherty f Tom Gallagher c David K Meyerholz g Stanley Perlman a b and Paul B McCray Jr a b 1 a Department of Pediatrics Pappajohn Biomedical Institute University of Iowa Iowa City IA 52242 b Department of Microbiology University of Iowa Iowa City IA 52242 c Department of Microbiology and Immunology Loyola University Chicago Maywood IL 60153 d Iowa Institute of Human Genetics University of Iowa Iowa City IA 52242 e College of Dentistry University of Iowa Iowa City IA 52242 f Department of Veterinary Pathology Iowa State University Ames IA 50011 and g Department of Pathology University of Iowa Iowa City IA 52242 Edited by Diane E Griffin Johns Hopkins Bloomberg School of Public Health Baltimore MD and approved March 2 2017 received for review November 21 2016 The Middle East respiratory syndrome MERS emerged in Saudi Arabia in 2012 caused by a zoonotically transmitted coronavirus CoV Over 1 900 cases have been reported to date with 36 fatality rate Lack of autopsies from MERS cases has hindered un derstanding of MERS CoV pathogenesis A small animal model that develops progressive pulmonary manifestations when infected with MERS CoV would advance the field As mice are restricted to infec tion at the level of DPP4 the MERS CoV receptor we generated mice with humanized exons 10 12 of the mouse Dpp4 locus Upon inocu lation with MERS CoV human DPP4knockin KI mice supported virus replication in the lungs but developed no illness After 30 serial pas sages through the lungs of KI mice a mouse adapted virus emerged MERS MA that grew in lungs to over 100 times higher titers than the starting virus A plaque purified MERS MA clone caused weight loss and fatal infection Virus antigen was observed in airway epithelia pneumocytes and macrophages Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane for mation associated with accumulation of activated inflammatory monocyte macrophages and neutrophils in the lungs Relative to the parental MERS CoV MERS MA viruses contained 13 22 mutations including several within the spike S glycoprotein gene S protein mutations sensitized viruses to entry activating serine proteases and conferred more rapid entry kinetics Recombinant MERS MA bear ing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice The hDPP4 KI mouse and the MERS MA provide tools to investigate disease causes and develop new therapies emerging pathogen interferon virus pathogenesis spike protein CD26 M iddle East respiratory syndrome MERS was recognized as a significant illness on the Saudi Arabian peninsula in mid 2012 and the causative agent was rapidly identified as a novel coronavirus CoV 1 MERS has a high mortality 36 asso ciated with severe lung disease that can progress to acute respiratory distress syndrome ARDS Since its emergence the World Health Organization WHO has been notified of 1 905 laboratory confirmed cases of MERS CoV infection in 27 countries result ing in at least 677 related deaths WHO website www who int emergencies mers cov en February 24 2017 MERS CoV sim ilar to the severe acute respiratory syndrome SARS CoV that caused an epidemic in 2003 has been a serious cause for global concern due to its high fatality rate Of note the MERS CoV has a genomic structure similar to that of other CoVs and contains a 30 kb positive strand RNA genome that encodes for 11 ORFs that are ordered 5 to 3 ORF1a ORF1b spike S ORF3 ORF4a b ORF5 envelope E membrane M nucleocapsid N and ORF8b 1 2 ORF1a and 1b are predicated to encode non structural proteins The subgenomic mRNAs of MERS CoV encode five unique accessory proteins designated 3 4a b 5 and 8b which are found only in the virus lineage that includes HKU4 and HKU5 1 Like other CoVs the long genomic mRNA of MERS CoV encodes replicase polyproteins that are further processed into multiple nonstructural proteins In contrast conserved S E M and N structural proteins are trans lated from separate subgenomic mRNAs Epidemiologic studies have established that MERS is zoonotic in origin with evidence for a closely related virus in dromedary camels on the Arabian Peninsula and throughout Africa 3 5 Spread from camels to people is documented 6 as is person to person spread among health care workers in hospital settings 7 The transmission of MERS CoV from person to person is inefficient but this could change with virus evolution 8 9 and if the numbers of so called superspreaders increased 10 The MERS CoV has also been detected in people with mild influenza like illnesses those with a dengue like illness and in those without obvious dis easesignsorsymptoms 11 14 Although human to human or zoonotic spread of MERS has not reached epidemic or pandemic levels its potential to spread between persons was demonstrated in healthcare settings in the Middle East 7 and by an outbreak in South Korea caused by a single infected individual 15 A lack of autopsy studies from MERS fatalities has hindered understanding of MERS CoV pathogenesis 16 None have been reported from Saudi Arabia or South Korea To better understand the pathogenesis of MERS CoV lung disease and aid the devel opment of vaccines and therapies we engineered a mouse model ofMERS Dipeptidylpeptidase4 DPP4 CD26 atypeII transmembrane ectopeptidase is the receptor for the MERS CoV 17 and the key amino acids responsible for binding the virus Significance Middle East respiratory syndrome caused by a zoonotically transmitted coronavirus MERS CoV has a high mortality 36 Because of limited autopsy data on tissues from MERS fatalities a small animal model can provide an important tool to better understand the disease We humanized the mouse locus of the virus receptor DPP4 preserving native DPP4 expression After inoculating hDPP4 knockin mice with MERS CoV there was virus replication without disease We then generated a mouse adapted MERS CoV by serial passage in hDPP4 knockin mice The resultant virus causes fatal lung disease that includes diffuse alveolar damage and immune dysregulation Here we characterize the pathologic features of the model and elucidate key aspects of the immunopathology and factors contributing to virulence Author contributions K L C L W L R C T G S P and P B M designed research 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 and D K M performed research 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 and P B M analyzed data and K L T G S P and P B M wrote the paper The authors declare no conflict of interest This article is a PNAS Direct Submission 1 To whom correspondence should be addressed Email paul mccray uiowa edu This article contains supporting information online at www pnas org lookup suppl doi 10 1073 pnas 1619109114 DCSupplemental www pnas org cgi doi 10 1073 pnas 1619109114 PNAS Early Edition 1of10 MEDI CAL SCIENC ES PNAS PLUS spike glycoprotein have been identified 18 We replaced mouse Dpp4 exons 10 12 encoding the residues required for virus bind ing with human exons 10 12 In vivo serial passage of the human MERS CoV Erasmus Medical Center EMC 2012 in the human DPP4 receptor knockin KI mice yielded a mouse adapted virus MERS MA that causes a fatal pulmonary disease phenotype as sociated with diffuse alveolar damage and a severe inflammatory response including an increased number of CD11b Ly6C hi inflam matory monocyte macrophages IMMs Sequencing of plaque purified MERS MA clones revealed several mutations that emerged with in vivo passage including a cluster in the spike S glycoprotein coding sequence A recombinant MERS CoV with these S protein mutations was more virulent in DPP4 KI mice than parental MERS CoV The human DPP4 KI mouse infected with MERS MA revealed several important disease correlates attributed to the virus and its cell entry and to the host and its immunological response Results Generation of a Mouse with Knockin of Human DPP4 Exons 10 12 Whereas human DPP4 has 26 exons the residues encoded by exons 10 12 comprise the critical MERS CoV receptor 18 19 We and others showed previously that changing the exon 10 12 codons from mouse to human conferred high affinity binding of the MERS S protein 20 23 Using a plasmid mediated gene targeting strategy SI Appendix Fig S1 a congenic C57BL 6 mouse was developed by replacing mouse Dpp4 exons 10 12 with the corre sponding human DPP4 codons hDPP4 KI The targeted founders were expanded by breeding and offspring characterized by geno typing for the presence of human DPP4 sequence as described in Materials and Methods For the studies reported we used mice homozygous for hDPP4 To identify the MERS CoV receptor dis tribution in the mouse lung we localized DPP4 in uninfected hDPP4 KImice AsshowninFig 1A airway epithelial cells had mild to moderate DPP4 immunoreactivity In the alveolus DPP4 immunostaining was most often seen in alveolar macrophages with less common immunostaining of alveolar epithelia and the pleural surface Fig 1 B and C This pattern of DPP4 expression shares similarities to the protein distribution in human lung 24 25 and identifies cell types that may support virus replication Infection of Human DPP4 Knockin Mice and Development of a Mouse Adapted MERS CoV Following intranasal i n infection with 10 4 to 10 6 pfu MERS CoV EMC 2012 Vero 81 cell passaged hereafter termed EMC Vero virus replicated in the lung titers at 1 d p i 10 5 6 pfu g tissue but mice lost no weight and there was no mortality Fig 2 A and B We next serially passaged EMC Vero in KI mice as described in Materials and Methods Tissue homoge nates were saved after each in vivo passage After 21 passages we tested the passaged virus in KI mice and noted progressive weight loss and 50 mortality SI Appendix Fig S2 After 30 in vivo passages P30 a 10 4 pfu virus inoculum caused progressive weight loss and 80 mortality Fig 2 A and B We termed this P30 virus mouse adapted MA MERS CoV or MERS MA Plaque Purified MERS MA Causes Fatal Lung Disease in DPP4 KI Mice Because CoV like all RNA viruses exist as quasispecies we fur ther purified MERS MA plaques Six of nine plaque purified isolates were evaluated in KI mice at a 10 5 pfu inoculum and each caused fatal disease SI Appendix Fig S2 Serial passage of coronaviruses may result in gene deletions 26 and we observed that deletions had occurred in most of our plaques Sequencing of the ORF3 and 4a 4b genes revealed that plaque 6 virions included a population without deletions in these regions Dataset S1 Therefore we selected virus from plaque 6 as a representative MERS MA To ensure complete clonal isolation we performed two more rounds of plaque purification Fig 2 C F shows that KI mice infected with a single triple plaque purified virus termed clone 6 1 2 exhibited dose dependent fatal disease We compared the outcomes of male Fig 2 C and D andfemale Fig 2E and F mice to several inocula of MERS MA clone 6 1 2 and observed similar outcomes An inoculum of 5 10 3 pfu was lethal in both sexes LungvirustitersinKImiceinfectedwithclone6 1 2were 1 2 log higher compared with those challenged with the EMC Vero parental virus from 1 to 5 days postinoculation d p i Fig 3A Virus was readily detected in serum of clone 6 1 2 infected mice butrarelyinmiceinfectedwithEMC Vero Fig 3B No virus was detected in the brain or other organs We also examined viral RNA distribution by PCR and identified signal in lung at 1 and 3 d p i Fig 3C A low level virus genomic RNA signal was also detected in the liver at 1 d p i only Of note the EMC Vero virus yielded more progeny than clone 6 1 2 virus in human cells Fig 3D suggesting that mouse adaptation did not enhance infection in human cells We compared the tissue histology of KI mice infected with EMC Vero or MERS MA clone 6 1 2 At 1 and 3 d p i we noted similar mild inflammatory cell infiltrates in perivascular regions and alveolar septa of mice infected with both viruses By 4 and 5 d p i lung tissue from mice infected with EMC Vero virus was notable for multifocal mononuclear cell infiltrates Fig 4A arrowheads whereas such infiltrates were mild and uncommon in the clone 6 1 2 infected animals In contrast tissues from clone 6 1 2 infected mice showed multifocal hyaline membranes Fig 4 A and B black arrows edema Fig 4B black asterisks and necrotic debris Fig 4B Inset red arrowhead consistent with diffuse alveolar damage Associated with these findings in clone 6 1 2 infected animals we observed Evans blue dye retention in lung tissue at 4 and 5 d p i indicative of increased vascular permeability Fig 4B whiteaster isks Morphometric analysis of lung tissue lesions showed that clone 6 1 2 infected lungs had significantly reduced cellular infil trates and increased hyaline membranes at 4 and 5 d p i Fig 4C We localized virus N protein in infected lung tissue at 3 d p i In EMC Vero infected mice virus antigen staining was patchy and featured immunostaining of alveolar macrophages and less com mon staining of alveolar and airway epithelia Fig 5 Middle In contrast clone 6 1 2 infected micehadmuchmorewidespread N protein staining of alveolar macrophages and alveolar epithelia ABC Fig 1 DPP4 immunostaining in uninfected hDPP4 KI mouse lung A DPP4 was observed as mild to moderate cytoplasmic staining of airway epithelia Positive staining was also noted in alveolar macrophages arrow B Mild to moderate macrophage immunostaining were commonly seen arrows Middle as was weak staining of alveolar epithelia Inset C The pleural surface arrows had multifocal DPP4 immunostaining Results shown are representative of findings from four hDPP4 KI mice Magnification 400 2of10 www pnas org cgi doi 10 1073 pnas 1619109114 Li et al and scattered airway epithelia Fig 5 Right consistent with the clinical and virological results Immune Response to MERS MA Studies of patients with MERS CoV 27 29 infected animal models 30 33 andculturedcells 34 37 indicate that delayed IFN signaling and dysregulated cytokine and chemokine production may contribute to disease severity We measured mRNA abundance for several antiviral and proin flammatory cytokines and chemokines and IFN stimulated genes ISGs in lung tissue from KI mice infected with EMC Vero and clone 6 1 2 at 1 and 3 d p i using qRT PCR As shown in SI Ap pendix Fig S3 the induction of several host defense genes was similar for both viruses Among the transcripts expressed in greater abundance in response to clone 6 1 2 were the type I and III in terferons and IL 6 IL 10 CCL2 CXCL10 and MDA5 at 3 d p i SI Appendix Fig S3 We also profiled the abundance of selected cytokine and chemokine proteins in serum and lung tissue ho mogenates at 3 d p i SI Appendix Fig S4 Notable differences between clone 6 1 2 and EMC Vero included greater elevations in CCL2 CXCL2 IL 6 CXCL10 and G CSF Overall the more virulent clone 6 1 2 elicited delayed but ultimately greater types I and III IFN transcript responses and more robust cytokine che mokine responses than the parental virus Analysis of peripheral blood smears collected at 4 d p i revealed that MERS MA clone 6 1 2 infected mice had a greater percentage of neutrophils and fewer lymphocytes than EMC Vero infected mice SI Appendix Fig S5A Both EMC Vero and clone 6 1 2 infected mice exhibited an increase in reactive lymphocytes SI Appendix Fig S5A consistent with response to infection Periph eral blood from clone 6 1 2 infected mice also had morphologic evidence of monocyte activation a reduced erythrocyte density and reduced polychromatophils immature RBCs consistent with anemia of inflammation SI Appendix Fig S5B and C These results provide further evidence of a systemic illness in clone 6 1 2 infected animals 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 determine whether the enhanced morbidity and mortality of hDPP4 KI mice to MERS MA clone 6 1 2 infection correlated with increased inflammatory cell recruitment we analyzed the percent ages and total numbers of innate immune cell populations in the lungs at 4 d p i Whereas the absolute numbers of CD45 leuko cytes were lower in clone 6 1 2 challenged mice percentages and total numbers of CD11b Ly6C hi IMMs were significantly higher in clone 6 1 2 infected mice compared with EMC Vero infected or uninfected control mice Fig 6 A and B In contrast neutrophil numbers were variable and not statistically different between groups Fig 6 A and B Staining for activation markers CD69 and CD80 revealed that IMMs and neutrophils were highly activated in clone 6 1 2 infected mice and less so in EMC Vero infected mice Fig 6 C and D IMM and neutrophil activation was significantly lower in uninfected control mice compared with the two groups of infected mice Fig 6 C and D We next enumerated NK T and B cell numbers in the lungs of the different groups and observed higher numbers in EMC Vero infected mice compared with clone 6 1 2 infected or uninfected mice Fig 6E In summary whereas substantially more mononuclear cells were detected in EMC Vero compared with MERS MA clone 6 1 2 infected lungs the infitrates were composed mainly of lymphocytes in the former group Con versely more IMMs and fewer lymphocytes were detected in the lungs of animals infected with clone 6 1 2 compared with EMC Vero virus Overall the increased accumulation of activated monocyte macrophages and neutrophils and decreased numbers of T and B cells in the lungs of MERS MA clone 6 1 2 challenged mice Log 10 PFU ml 1345 0 1 2 3 4 5 LOD Days p i EMC Vero P0 MERS MA 6 1 2 Log 10 PFU gm T i ssue 1345 0 1 2 3 4 5 6 7 8 9 LOD Days p i AB Lung Serum C Lung Heart Brain Liver Kidney Spleen Small Intestine 1 0 1 2 3 4 5 6 7 Log 10 RNA copy numbers normalized to GAPDH 1 dpi 3 dpi D Calu 3 Fig 3 Tissue virus titers and viral genomic RNA distribution Human DPP4 KI mice infected with 10 4 pfu i n inoculum of indicated MERS CoV A and B Lung and serum titers at indicated times C ORF1a quantitative RT PCR performed on indicated tissues from EMC Vero and MERS MA clone 6 1 2 infected mice at 1 and 3 d p i Limit of detection LOD for qRT PCR assay is 100 copies Mean SE n 3 D Calu 3 cells were infected with indicated MERS CoV at MOI 0 1 and virus titers at 1 d p i were determined by plaque assay n 6 representative of two replicate experiments Statistical signifi cance was assessed by Student s t test P 0 0001 02468101214 60 70 80 90 100 110 Initial W e ight Days p i 02468101214 60 70 80 90 100 110 Days p i Initial W e ight Days p i Percent survival 02468101214 0 20 40 60 80 100 5000 pfu i n 500 pfu i n 50 pfu i n Days p i Percent survival 0 2 4 6 8 10 12 14 0 20 40 60 80 100 5000 pfu i n 500 pfu i n 50 pfu i n Male Female A C EF D B Fig 2 Outcomes in hDPP4 KI mice infected with EMC Vero or MERS MA Weight loss A and survival B in KI mice infected i n with 10 4 pfu EMC Vero or passage 30 P30 MERS MA EMC Vero infected mice had 100 survival Human DPP4 KI mice infected with P30 MERS MA lost significant weight and most died Results summarize two replicate experiments n 10 EMC Vero n 11 MERS MA mixed male and female mice C F MERS MA clone 6 1 2 causes dose dependent lethal lung disease in male and female hDPP4 KI mice C and D Weight curve and survival results for male hDPP4 KI mice that received 5 000 50 pfu MERS MA clone 6 1 2 via i n inoculation For 5 000 pfu n 8 500 pfu n 12 and 50 pfu n 5 E and F Weight curve and survival results for female hDPP4 KI mice that received 5 000 50 pfu MERS MA clone 6 1 2 via i n inoculation For 5 000 pfu n 8 500 pfu n 9 and 50 pfu n 5 A C and E Data represent mean SE Li et al PNAS Early Edition 3of10 MEDI CAL SCIENC ES PNAS PLUS suggest an overt and injurious innate inflammatory response and a suboptimal adaptive immune response Id