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Middle East respiratory syndrome coronavirus MERS CoV causes transient lower respiratory tract infection in rhesus macaques Emmie de Wit a Angela L Rasmussen b Darryl Falzarano a Trenton Bushmaker a Friederike Feldmann c Douglas L Brining c Elizabeth R Fischer d Cynthia Martellaro a Atsushi Okumura b Jean Chang b Dana Scott c Arndt G Benecke b e Michael G Katze b Heinz Feldmann a f 1 and Vincent J Munster a 1 a Laboratory of Virology c Rocky Mountain Veterinary Branch and d Microscopy Unit Research Technologies Branch Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Hamilton MT 59840 b Department of Microbiology University of Washington Seattle WA 98195 e Department of Biology Universit Pierre et Marie Curie Centre National de la Recherche Scienti c Unit Mixte de Recherche 7224 75005 Paris France and f Department of Medical Microbiology University of Manitoba Winnipeg MB Canada R3E 0J9 Edited by Tilahun D Yilma University of California Davis CA and approved August 26 2013 received for review June 6 2013 In 2012 a novel betacoronavirus designated Middle East re spiratory syndrome coronavirus or MERS CoV and associated with severe respiratory disease in humans emerged in the Arabian Peninsula Todate 108 humancaseshave been reported including cases of human to human transmission The availability of an animal disease model is essential for understanding pathogenesis anddeveloping effectivecountermeasures Upon a combination of intratracheal ocular oral and intranasal inoculation with 7 10 6 50 tissue culture infectious dose of the MERS CoV isolate HCoV EMC 2012 rhesus macaques developed a transient lower respira tory tract infection Clinical signs virus shedding virus replication in respiratory tissues gene expression and cytokine and chemo kine pro les peaked early in infection and decreased over time MERS CoV caused a multifocal mild to marked interstitial pneu monia with virus replication occurring mainly in alveolar pneumo cytes This tropism of MERS CoV for the lower respiratory tract may explain the severity of the disease observed in humans and the up to now limited human to human transmission emerging infectious disease DPP4 I n June of 2012 a novel betacoronavirus associated with severe respiratory disease in humans emerged in the Middle East 1 2 which is closely related to betacoronaviruses circulating in bats 3 4 The rst isolate of Middle East respiratory corona virus MERS CoV 5 HCoV EMC 2012 was obtained from a patient with a fatal pneumonia and acute renal failure To date 107 additional human cases have been identi ed of which 49 were fatal 6 Aside from cases in Saudi Arabia Qatar Jordan and the United Arab Emirates imported cases have been iden ti ed in the United Kingdom Germany France Tunisia and Italy 6 Although no information is available on the source or route of primary transmission of MERS CoV human to human transmission has been recorded 7 9 Clinical data on human cases of MERS CoV infection are currently sparse but it appears that this virus mainly causes severe lower respiratory tract disease occasionally accompanied by renal disease The severity of disease distinguishes MERS CoV from other coro naviruses circulating in the human population HCoV 229E HCoV OC43 HCoV NL63 and HCoV HKU1 which are gen erally associated with upper respiratory tract infections Instead MERS CoV appears to be more similar to the severe respiratory disease caused by severe acute respiratory syndrome SARS CoV In vitro studies have shown that MERS CoV replicates ef ciently in nonciliated cells in the primary human airway epithe lium 10 and in ex vivo human lung cultures MERS CoV replicated in bronchial bronchiolar and alveolar epithelial cells 11 in line with the observed respiratory disease in humans The recently de ned receptor for MERS CoV dipeptidylpeptidase 4 DPP4 is generally expressed in endothelial and epithelial cells and has been shown to be present on cultured human nonciliated bronchiolar epithelium cells 12 providing further information on the respiratory tropism of MERS CoV Animal models that recapitulate human disease are essential for understanding pathologic processes involved in disease pro gression Moreover these models are instrumental for the de velopment of prophylactic and therapeutic countermeasures We have previously shown that rhesus macaques inoculated with a high dose of MERS CoV isolate HCoV EMC 2012 developed a respiratory disease reminiscent of that observed in humans 13 To increase our understanding of the pathogenesis of MERS CoV in the absence of clinical and pathological data from human patients we present herein a more detailed analysis of the extent of virus replication the histopathological changes in the respiratory tract and changes in systemic peripheral blood mononuclear cell PBMC and local lung tissue gene expres sion of MERS CoV infected rhesus macaques Results Clinical Signs and Hematological Changes in Animals Inoculated with MERS CoV Six rhesus macaques were inoculated with 7 10 6 Signi cance The Middle East respiratory syndrome coronavirus MERS CoV is the latest emerged coronavirus causing severe respiratory disease with a high case fatality rate in humans To better understand the disease caused by MERS CoV we developed a rhesus macaque model Infection of rhesus macaques with MERS CoV resulted in the rapid development of a transient pneumonia with MERS CoV replication largely restricted to the lower respiratory tract This af nity of MERS CoV for the lungs partly explains the severity of the disease observed in humans The MERS CoV rhesus macaque model will be instrumental in developing and testing vaccine and treatment options for an emerging viral pathogen with pandemic potential Author contributions E d W H F and V J M designed research E d W A L R D F T B F F D L B E R F C M A O J C D S H F and V J M performed research E d W A L R D F E R F A O D S A G B M G K H F and V J M analyzed data and E d W A L R H F and V J M wrote the paper The authors declare no con ict of interest This Direct Submission article had a prearranged editor Data deposition The data reported in this paper have been deposited in the Gene Ex pression Omnibus GEO database www ncbi nlm nih gov geo accession no GSE44542 and are also available to the public at http viromics washington edu 1 To whom correspondence may be addressed E mail feldmannh niaid nih gov or Vincent Munster nih gov This article contains supporting information online at www pnas org lookup suppl doi 10 1073 pnas 1310744110 DCSupplemental 16598 16603 PNAS October 8 2013 vol 110 no 41 www pnas org cgi doi 10 1073 pnas 1310744110 TCID 50 50 tissue culture infectious dose of MERS CoV isolate HCoV EMC 2012 13 Clinical signs upon inoculation with HCoV EMC 2012 were transient and varied between ani mals as speci ed in Table S1 Analysis of blood samples col lected 0 1 3 and 6 d postinfection dpi indicated increased white blood cell counts in ve of six animals on 1 dpi Fig 1 Further analysis indicated that this was the result of an increase in the number of neutrophils Moreover there was a reduction in the number of lymphocytes By 3 dpi blood cell counts had returned to numbers comparable to those before inoculation Fig 1 Viral RNA in Swabs and Bronchoalveolar Lavages Nasal oropha ryngeal urogenital and rectal swabs were obtained from the animals 1 3 and 6 dpi On 1 and 3 dpi nasal swabs from all animals were positive for the presence of viral RNA by quanti tative RT PCR qRT PCR only one animal had a positive nasal swab on day 6 postinfection Fig 2A Oropharyngeal swabs obtained from CoV 3 on days 3 and 6 postinfection and obtained from CoV 6 on day 6 postinfection were positive All urogenital and rectal swabs were negative with the exception of a urogenital swab collected from CoV 2 on day 1 postinfection Bronchoalveolar lavages BAL were also performed 1 3 and 6 dpi and analyzed for the presence of viral RNA BAL obtained 1 dpi were pos itive by qRT PCR in ve of six animals 3 dpi three of six BALs were positive and 6 dpi two of three BALs contained viral RNA Fig 2B Widespread Dissemination of Virus Through the Respiratory Tract We have previously reported the presence of viral RNA and infectious virus throughout the lungs of inoculated macaques with viral load decreasing between 3 and 6 dpi 13 In addition to the different lung lobes we have previously analyzed several other tissues including tissues of the upper respiratory tract lung lesions and kidney for complete list see Materials and Methods Although there was some variation between the different macaques with regard to the presence of viral RNA we could consistently detect the virus by qRT PCR in the nasal mucosa trachea and mediastinal lymph nodes on 3 dpi Fig 3 Furthermore we could detect viral RNA in conjunctiva tonsils oronasopharynx and in the left and right bronchus Viral loads were lower in these tissues by 6 dpi and viral RNA could no longer be detected in the nasal mucosa or conjunctiva at that time Fig 3 Viral RNA could not be detected in kidney or bladder tissue samples To further prove that virus replication occurred in the lungs of MERS CoV inoculated rhesus macaques transmission electron microscopy was performed on lung lesion samples collected 6 dpi At this time point coronavirus particles could be detected in alveolar pneumocytes Fig 4 Moreover qRT PCR analysis of RNA extracted from the right lower lung lobe and lung lesions obtained 3 and 6 dpi indicated the transcription of subgenomic mRNA ORF5 as well as ORF1 RNA in samples collected from all animals and at both time points Fig S1 another indicator of active virus replication in the lower respiratory tract Pathology of MERS CoV in the Respiratory Tract Upon necropsy of animals 3 and 6 dpi the areas of the individual lung lobes dis playing lesions were scored Lung lobes revealed varying degrees of gross lesions ranging from 0 to 75 Fig S2 Histological lesions in MERS CoV infected macaques were limited to the lungs On day 3 postinfection lesions were characterized as multifocal mild Fig 5A to marked Fig 5B interstitial pneu monia The pneumonia was characterized by thickening of al veolar septae by edema uid and brin and small to moderate numbers of macrophages and fewer neutrophils Alveoli contained moderate numbers of pulmonary macrophages and neutrophils many of these alveolar macrophages formed multinucleate syncytia Fig 5D One animal euthanized 3 dpi also displayed lesions Fig 1 Hematological changes in rhesus macaques inoculated with MERS CoV Total number of white blood cells Left neutrophils Center and lym phocytes Right were determined in blood samples obtained from animals 0 1 3 and 6 dpi Of note CoV 2 CoV 4 and CoV 6 were euthanized 3 dpi and thus no samples are available for 6 dpi Each line represents one animal CoV 1 CoV 2 CoV 3 CoV 4 CoV 5 CoV 6 Fig 2 Virus shedding in rhesus macaques inoculated with MERS CoV Nasal swabs A and BAL uid B were collected 1 3 and 6 dpi Of note CoV 2 CoV 4 and CoV 6 were euthanized 3 dpi and thus no samples are available for 6 dpi RNA was extracted and viral load was determined as TCID 50 equivalents by qRT PCR TCID 50 equivalents were extrapolated from stan dard curves generated by adding dilutions of RNA extracted from a HCoV EMC 2012 stock with known virus titer in parallel to each run Animal num bers are indicated on the x axis Black bars indicate viral load at 1 dpi white bars indicate viral load at 3 dpi and gray bars indicate viral load at 6 dpi de Wit et al PNAS October 8 2013 vol 110 no 41 16599 MICROBIO L OGY similar to those described as bronchiolitis obliterans organizing pneumonia Fig 5C This organizing pneumonia consisted of aggregations of brin Fig S3 macrophages and sloughed pul monary epithelium that ll and occlude small airways an early lesion of bronchiolitis obliterans organizing pneumonia that has yet to develop the characteristic dense collagenous stroma andmarkedin ltration of broblasts that would occur if the lesion was given time to mature Perivascular in ltrates of in ammatory cells were observed multifocally within and adjacent to affected areas of the lung Animals necropsied at 6 dpi dem onstrated moderate Fig 6A tomarked Fig 6B changes in the lungs with type II pneumocyte hyperplasia Fig 6C abundant alveolar edema and brin with formation of hyaline membranes Fig 6D MERS CoV Replicates in Alveolar Pneumocytes At 3 dpi in situ hybridization ISH demonstrated viral RNA multifocally in the lungs of all monkeys The signal was found in spindle to polyg onal cells along alveolar septae Fig 5E This location and the cellular morphology suggested that virus replication occurs mainly in the pulmonary epithelium Viral RNA was not detec ted in endothelial cells Immunohistochemistry IHC yielded similar results for the presence of viral antigen Fig 5F Addi tionally there were rare round mononuclear cells and stellate cells within the cortex of the mediastinal lymph nodes that were positive for both viral RNA by ISH and viral antigen by IHC These cells were morphologically consistent with cells of the macrophage or dendritic cell lineage Viral RNA and viral an tigen could still be detected at 6 dpi Fig 6 E and F To con rm that virus replication occurred mainly in alveolar pneumocytes uorescent IHC was performed using anticytokeratin and anticoronavirus antibodies Viral antigen was predomi nantly foundinalveolarpneumocyteson 3 as wellas 6 dpi Fig S4 A and B This distribution of viral antigen in type I and type II pneumocytes corresponded with the distribution of the receptor for MERS CoV DPP4 Fig S4 C and D as indicated by IHC performed on the lungs of infected animals Analysis of Gene Expression in Lungs and PBMC To assess genes that may play a signi cant role in disease pathology we performed a microarray analysis on lung tissue and PBMC A Welch s t test P 0 01 fold change 2 was used to compare pro les of signi cant differentially expressed genes DEG from the right lower lung lobe to observable gross lesions relative to uninfected controls The right lower lobe was selected as qPCR demon strated that viral genomes were present in those samples allowing us to compare sites of histopathologic injury to the background transcriptional pro les of sites exposed to virus On day 3 we observed a total of 173 DEG Fig 7A and Table S2 although the expression was highly variable between animals and sample sites This variability is likely multifactorial driven by differences between individual animals such as levels of virus in the lung and at lesion sites distribution of virus receptors lung sizes age and distribution and quantity of susceptible cells Using ingenuity pathway analysis IPA we determined that the majority of DEG were associated with antiviral immunity in ammation and chemotaxis Notably genes such as IL 6 che mokine C X C ligand 1 CXCL1 and matrix metalloproteinase 9 MMP9 associated with proin ammatory processes and re cruitment of in ammatory cells were signi cantly up regulated in the lesions compared with the right lower lung lobes By day 6 we identi ed only 37 DEG distinguishing right lower lung lobes from lesions Fig 7B and Table S3 and these were not grouped in any heavily enriched functional categories Additionally by 6 dpi the lung lesions showed a higher magnitude of gene ex pression compared with the right lower lung lobe samples This nding may re ect the resolving clinical status of the animals at day 6 postinfection and indicate that substantial differences in gene expression occur at sites of virus induced injury rather than throughout the lung We sought to determine if MERS CoV induced gene ex pression dynamics can be observed in PBMC and lung tissue and lesion samples from infected macaques and change over time Using singular value decomposition coupled multidimensional scaling we identi ed changes in gene expression occurring in parallel in both organs Fig S5 In PBMC a signi cant response to MERS CoV infection is observed only on day 1 postinfection In lungs a signi cant global response was found only 3 dpi Fig S5 By 6 dpi the gene expression dynamics in both organs return close to an uninfected or baseline state Furthermore functional analysis with IPA reveals that MERS CoV infection in macaques leads to very rapid innate immune activation 14 through pattern recognition receptors Leukocyte activation is evident in blood and lungs as well as evidence of a robust but self limiting in ammatory response Importantly innate immune Fig 3 Viral load in respiratory tissues of rhesus macaques inoculated with MERS CoV Rhesus macaques were euthanized on day 3 black bars and day 6 white bars postinfection and tissue samples were collected RNA was extracted and viral load was determined as TCID 50 equivalents by qRT PCR TCID 50 equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a HCoV EMC 2012 stock with known virus titer in parallel to each run Geometric mean viral loads were calcu lated error bars represent SD R right L left LN lymph node Fig 4 Virus replication in the lungs of MERS CoV inoculated rhesus mac aques Transmission electron microscopy was performed on lung lesion samples collected from MERS CoV inoculated macaques at 6 dpi Virus particles arrows are visible in type I pneumocytes Left and Right Scale bar Right 250 nm Inset 50 nm B basement membrane E endothelial cell P pneumocytes R red blood cell 16600 www pnas org cgi doi 10 1073 pnas 1310744110 de Wit et al activation occurs very rapidly after infection and is subsequently also rapidly controlled indicating quick resolution Cytokine and Chemokine Pro les in MERS CoV Infected Animals Plasma levels of 23 cytokines and chemokines were determined in serum samples obtained at 0 1 3 and 6 dpi Transient dif ferences were only observed for IL 1RA IL 2 IL 6 IL 8 IL 12 IL 13 IL 15 IFN and monocyte chemotactic protein 1 MCP 1 AT 1 dpi serum levels of IL 1RA IL 2 IL 13 IL 15 INF and MCP 1 were signi cantly higher than at 0 dpi and all levels returned to baseline by 6 dpi Fig S6 Although the serum level of IL 6 was higher and serum levels of IL 8 and IL 12 were lower at 1 dpi these differences were not statistically signi cant Discussion The emergence of MERS CoV in 2012 was the second in troduction in 10 y of a novel coronavirus causing severe re spiratory disease into the human population Both the severity of the respiratory disease and the genetic similarity to betacor onaviruses isolated from bats are reminiscent of the emergence of SARS CoV in 2003 which resulted in 8 422 human cases with 916 deaths in over 30 countries 15 To understand the disease caused by MERS CoV in humans we developed a nonhuman primate model which recapitulates the respiratory disease ob served in mild or moderate human cases but not the occasionally occurring kidney disease In this rhesus macaque model virus shedding as indicated by qRT PCR occurred predominantly via the nose and to a limited extent the throat In nasal swabs and BAL viral loads were highest day 1 postinfection and decreased over time However at 6 dpi two of three animals were still shedding virus from the re spiratory tract Although MERS CoV was detected in the upper respiratory tract and the lymphoid tissue draining the lungs replication of MERS CoV was most prominent in the lower respiratory tract MERS CoV replicated predominantly in type I and II pneumocytes in the alveoli These two cell types form the main component of the architecture of the alveolar space around the terminal bronchioles This predominant replication of MERS CoV in alveoli may explain the limited amount of virus shedding observed in our rhesus macaque model In ad dition the fact that human to human transmission so far seems limited to a few family clusters in Saudi Arabia 6 the United Kingdom 7 and Tunisia and nosocomial transmission in Jordan Saudi Arabia 6 and France 9 might be expla