key: cord-0993676-2pnqpljt
authors: Munster, Vincent J.; Adney, Danielle R.; van Doremalen, Neeltje; Brown, Vienna R.; Miazgowicz, Kerri L.; Milne-Price, Shauna; Bushmaker, Trenton; Rosenke, Rebecca; Scott, Dana; Hawkinson, Ann; de Wit, Emmie; Schountz, Tony; Bowen, Richard A.
title: Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis)
date: 2016-02-22
journal: Sci Rep
DOI: 10.1038/srep21878
sha: 577c6a13f9ef70e9756890fc66e98f537c01ac0a
doc_id: 993676
cord_uid: 2pnqpljt

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the zoonotic potential of Betacoronaviruses. Investigations into the origin of MERS-CoV have focused on two potential reservoirs: bats and camels. Here, we investigated the role of bats as a potential reservoir for MERS-CoV. In vitro, the MERS-CoV spike glycoprotein interacted with Jamaican fruit bat (Artibeus jamaicensis) dipeptidyl peptidase 4 (DPP4) receptor and MERS-CoV replicated efficiently in Jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for MERS-CoV. To shed light on the intrinsic host-virus relationship, we inoculated 10 Jamaican fruit bats with MERS-CoV. Although all bats showed evidence of infection, none of the bats showed clinical signs of disease. Virus shedding was detected in the respiratory and intestinal tract for up to 9 days. MERS-CoV replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. Analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. Our results indicate that MERS-CoV maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for MERS-CoV.

Scientific RepoRts | 6:21878 | DOI: 10 .1038/srep21878

Arab Emirates and Egypt 23, [28] [29] [30] . MERS-CoV sequences and virus isolates obtained from dromedary camels in Qatar and the Kingdom of Saudi Arabia showed high sequence identity with those obtained from epidemiologically linked human cases 28, 31 . Together, these data suggest that rather than direct zoonotic transmission from a bat reservoir, dromedary camels are involved as the primary reservoir host for MERS-CoV. Phylogenetic and epidemiological data suggest that rather than a single introduction in the human population, MERS-CoV appears to continue to be transmitted by multiple independent spillover events from dromedary camels 32, 33 .

After the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, the targeted focus on reservoir studies in bats has resulted in a vast increase of our knowledge on the genetic diversity of bat coronaviruses. Despite the increase in genetic data on coronavirus diversity in their natural reservoirs, only very limited data are available on the impact of these viruses on the reservoir host and controlled infection experiments with coronaviruses in their reservoir hosts have not been performed. To understand the drivers of MERS-CoV emergence, a more comprehensive understanding of the interaction between the virus and its natural and intermediate reservoir hosts is needed. Here we present data on the first experimental infection of bats with MERS-CoV to model the infection kinetics in a coronavirus host species, the Jamaican fruit bat (Artibeus jamaicensis).

Artibeus jamaicensis DPP4 receptor and cell susceptibility. The MERS-CoV receptor DPP4 is the main host restriction factor 34 ; therefore, we first studied the interaction between MERS-CoV and Jamaican fruit bat DPP4. The pAJ-DPP4 plasmid expressing the DPP4 coding sequence of Jamaican fruit bat under control of a CMV promoter was transfected into BHK cells, which are not permissive to MERS-CoV 34 . The expression of AJ-DPP4 in transfected cells was confirmed by flow cytometry, showing the presence of bat DPP4 on the surface of transfected BHK cells by determining the increase over untransfected cells ( Figure S1 ). Transient expression of bat DPP4 in BHK cells supported MERS-CoV replication, whereas transient expression of hamster DPP4 in BHK cells did not (Fig. 1A) . Subsequently, the replication kinetics of MERS-CoV were compared in LLC-MK2 cells (Macaca mulatta) and Jamaican fruit bat primary kidney cells. MERS-CoV replicated efficiently to high titers in both cell lines (Fig. 1B) , indicating that there is no restriction at the receptor or cellular level for MERS-CoV replication in Jamaican fruit bat cells.

Clinical signs in bats inoculated with MERS-CoV. Ten adult Jamaican fruit bats were inoculated via the intranasal and intraperitoneal routes with 10 5 TCID 50 of MERS-CoV strain EMC/2012; 2 mock inoculated Jamaican fruit bats, housed in a separate cage, were used as controls, the mock inoculated animals were inoculated with tissue culture medium via the same routes and volumes. The bats were observed at least once daily for signs of disease. Bodyweight and temperature were measured throughout the experiment for a maximum of 28 days post inoculation (dpi) for bat 9 and 10 (MERS-CoV inoculated) and bat 11 and 12 (mock inoculated controls). None of the bats showed signs of disease, weight loss or increased body temperature throughout the experiment ( Figure S2 ).

To examine MERS-CoV shedding in inoculated bats, oral and rectal swabs were collected for the duration of the experiment. MERS-CoV shedding was first detected on 1 dpi, as indicated by the presence of viral RNA in throat and rectal swabs and continued for a maximum duration of 9 days. All animals, except bat 10, shed MERS-CoV from the respiratory tract ( Fig. 2A) ; all bats except 4 and 10, shed MERS-CoV from the intestinal tract (Fig. 2B ). Viral loads in swabs collected from the respiratory tract were higher than viral loads in swabs from the intestinal tract.

Tissues collected at the sequential necropsy dates of 2, 4, 7, 14 and 28 dpi were analyzed for the presence of viral RNA, infectious virus, and evaluated by histopathology and immunohistochemistry. MERS-CoV viral RNA was detected in various tissues of all inoculated bats, except bat 8 on 14 dpi and bat 9 on 28 dpi. The highest viral loads were detected in the lower respiratory tract (Fig. 3) . MERS-CoV viral RNA was detected at 2 dpi in trachea, lung, liver, spleen, bladder and nasal turbinates; at 4 dpi in lung, spleen, duodenum, colon, bladder, turbinates and brain; at 7 dpi in lung, liver, turbinates and brain; at 14 dpi in heart, lung, liver, spleen and duodenum. No MERS-CoV viral RNA was detected at 28 dpi (Fig. 3) . In additon, MERS-CoV viral RNA was detected in blood on 2 and 4 dpi, in bats 1-3, indicative of viremia ( Figure S3 ). MERS-CoV mRNA was detected in tissues of bats 1 to 7, confirming MERS-CoV replication on the transcriptional level (Table S2) .

Infectious MERS-CoV was isolated from the lungs of bats 1 (2 dpi) and 6 (7 dpi), the bladder and nasal turbinates of bat 7 (14 dpi), and the duodenum of bat 10 (28 dpi), indicating active virus replication, mainly in the respiratory tract.

Only two of ten bats (bat 3 and bat 5) exhibited histopathology associated with MERS-CoV infection, which was mild. All MERS-CoV associated lesions were detected in the respiratory tract of the infected bats (Fig. 4 , Table S1 ). Bat 3 and bat 4 (4 dpi) displayed a mild acute rhinitis, but MERS-CoV replication was not detected by immunohistochemistry (Table S1 and S2). Bat 3 and 5 displayed a multifocal interstitial pneumonia that was characterized by minimal alveolar interstitial thickening by small numbers of macrophages and neutrophils (Fig. 4 , Table S1 ). The adjacent alveolar spaces contained small numbers of alveolar macrophages. MERS-CoV antigen and RNA was detected by immunothroughout the lungs of bat 1 (2 dpi), but no associated pulmonary pathology was detected (Fig. 4 , Table S2 ). Cytokeratin and anti-MERS-CoV co-staining demonstrated MERS-CoV antigen in type I pneumocytes of the lungs of bat 1 (Fig. 5 ).

Scientific RepoRts | 6:21878 | DOI: 10.1038/srep21878

Innate immune response to MERS-CoV. MERS-CoV was most consistently detected in the lower respiratory tract of the bats. The Mx1, ISG56 and RANTES gene expression in the lungs of Jamaican fruit bats was analyzed as an indicator of the induction of an innate immune response to MERS-CoV infection. A 6-fold increase in expression of Mx1was observed in the lungs of the infected Jamaican fruit bats at 2 dpi. A statistically significant upregulation of Mx1 gene expression was detected when comparing the lungs of bats collected on 2 dpi and 28 dpi (two-tailed unpaired t-tests, p < 0.034). The maximum ISG56 expression of 7.4-fold occurred at 2 dpi. Statistically significant differences were observed between the 2 dpi and 7 dpi, 14 dpi and 28 dpi animals (two-tailed unpaired t-tests, p < 0.035, p < 0.0178 and p < 0.0192 respectively. In addition significant differences were observed between the 7 dpi and the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0009 and p < 0.0085). The RANTES expression at its peak at 2 dpi was increased 22.5 fold. Statistically significant differences were observed between the 2 versus the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0147 and p < 0.0136), the 4 versus the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0092 and p < 0.0075), and the 7 dpi versus the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0390 and p < 0.0366) (Fig. 6 ).

Antibody response to MERS-CoV. Sera were collected prior to inoculation and at the scheduled necropsy dates. Each of the bats was seronegative for MERS-CoV prior to inoculation. Only bat 7 developed a MERS-CoV specific antibody response, both by ELISA and virus neutralization assay. The sera obtained from bat 7 had a neutralizing titer of 320 at 14 days post inoculation.

The high sequence similarity of MERS-CoV to coronavirus sequences detected in bats suggests that MERS-CoV or its immediate ancestor originated in bats 35 . Direct contact between bats and humans is uncommon, and a domestic or peridomestic intermediate species often plays a role in the emergence of zoonotic viruses from natural reservoirs to humans [36] [37] [38] [39] . Similar to the emergence of SARS-CoV in 2002 from the masked palm civet (Paguma larvata) as an intermediate host 40 , the dromedary camel appears to have initially served as the intermediate host for MERS-CoV 41 . Several aspects of the emergence of MERS-CoV are currently still unknown, including the role of the natural reservoir and the relationship between the natural and intermediate reservoirs. with their ability to support efficient replication of MERS-CoV, the availability of an annotated transcriptome 45 , and the relative easy housing and husbandry practices of Jamaican fruit bats suggest that this bat species can become an important model system to investigate the relationship between coronaviruses and their bat hosts 46 . Although the Jamaican fruit bat is not the direct ancestral reservoir for MERS-CoV, as it is a new world bat species, generalized responses towards viruses of bat-origin rather than a direct host-pathogen relationships can be modelled.

The ability of MERS-CoV to use DPP4 of multiple species as a receptor, including DPP4 of human, dromedary camel, and bat origin 34, 47 , suggests that no prior adaptation was needed on the DPP4 receptor level for cross-species and zoonotic transmission to occur. With batCoV-HKU4, a closely related coronavirus, it was shown that replication in human cells required two mutations in the spike protein 48 . These amino acid residues, which are conserved in MERS-CoV, results in the activation of the batCoV-HKU4 spike protein by human cellular proteases. This suggests that batCoV-HKU4 needs these residues for replication in humans. Interestingly, our data show that MERS-CoV replicates efficiently in Jamaican fruit bat cells, suggesting that the MERS-CoV spike can efficiently be processed by Jamaican fruit bat cellular proteases and that there is no host restriction on the post-translational modification level of the MERS-CoV spike in dromedary camels, humans and bats.

Bat coronaviruses have been primarily detected in fecal samples in field studies suggesting that these viruses have a intestinal tract tropism 9,19,43 . MERS-CoV was able to replicate to higher titers in the respiratory tract in comparison with the intestinal tract of the Jamaican fruit bats. The tissue tropism of MERS-CoV in Jamaican fruit bats is comparable to the respiratory tract tropism observed in dromedary camels and humans 49, 50 . This might suggest that MERS-CoV, upon cross-species transmission from bats into dromedary camels evolved from a gastrointestinal tract virus into a respiratory tract virus, similar to influenza A viruses 51 .

The ability for MERS-CoV to antagonize the innate immune response appears to correlate with its pathogenic potential in humans. MERS-CoV and related batCoV-HKU4 can inhibit innate immune signaling in a variety of human cell lines in vitro via the ORF4b-encoded accessory proteins 52 Lungs of Jamaican fruit bat 5 were stained with α -cytokeratin as an epithelial marker (purple) and with a polyclonal α -coronavirus antibody (brown-red) to demonstrate that viral antigen was located along the basement membrane of alveolar pneumocytes of bat 1 at 2 dpi (indicated by black arrows). Original magnification: 40× .

with 2% fetal calf serum (Hyclone, Logan), 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 μ g/ml streptomycin (Gibco). Vero E6, LLC-MK2, BHK and Jamaican fruit bat primary kidney cells were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal calf serum, 50 U/ml penicillin and 50 μ g/ml of streptomycin.

Sequencing and cloning of the Jamaican fruit bat DPP4 sequence. Total RNA was extracted from primary kidney cells using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized using random hexamer primers and SuperScript III Reverse Transcriptase (Applied Biosystems). DPP4 was then amplified using iProof High-Fidelity DNA Polymerase (BioRad) and primers DPP4UnvF1 and DPP4UnvR12 (primer sequences are available upon request). The obtained DPP4 gene sequence was synthesized in expression plasmid pcDNA3.1(+ ) 

cate with MERS-CoV with a multiplicity of infection (MOI) of 0.01 (cell lines) or 1 (transfected cell lines) 50% tissue culture infectious dose (TCID 50 ) per cell. One hour after inoculation, cells were washed once with DMEM and culture medium replaced. Supernatants were sampled at 0, 24, 48 and 72 h after inoculation. MERS-CoV was titrated by end-point titration in quadruplicate in Vero E6 cells cultured in DMEM supplemented with 2% fetal calf serum, 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 μ g/ml streptomycin. Cells were inoculated with ten-fold serial dilutions of virus, and scored for cytopathic effect 5 days later. The TCID 50 was calculated by the method of Spearman-Karber.

Animal experiments. Twelve captive-bred Jamaican fruit bats were used for this work 46, 57 . Ten bats were inoculated with 10 5 TCID 50 EMC/2012 via a combination of intranasal (25 μ l each nostril) and intraperitoneal (100 μ l) routes. Two mock inoculated bats were included as controls for histopathology and gene expression analyses. Mock inoculated bats were inoculated with standard tissue culture media via the same routes and volumes. Bats were injected with an IPTT-300 temperature transponder (BMDS) to monitor body temperature daily. Animals were weighed daily and observed for signs of disease. Oropharyngeal and rectal swabs were obtained on 1, 2, 3, 4, 5, 6, 7, 9 and 11 dpi and analyzed for the presence of viral RNA. On 2, 4, 7, 14 and 28 days post inoculation (dpi), two bats were euthanized and trachea, heart, lung, liver, spleen, kidney, duodenum, colon, bladder, nasal turbinates and brain were collected for virological and histopathological analysis.

Histopathology. Histopathology was performed on bat tissues. After fixation for at least 7 days in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (H & E) staining. Immunohistochemistry was performed using a MERS-CoV EMC/2012 polyclonal rabbit antibody at a 1:1000 dilution and in situ hybridization was performed using probes directed against the MERS-CoV EMC/2012 N gene as described previously 62 . RNA extraction. RNA was extracted from swab samples using the QiaAmp Viral RNA kit (Qiagen). RNA was eluted in 60 μ l. Tissues (30 mg) were homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen). RNA was eluted in 50 μ l.

Quantitative PCR. For detection of viral RNA in samples, 5 μ l RNA was used in a one-step real-time RT-PCR upE assay 63 using the Rotor-Gene TM probe kit (Qiagen) according to the manufacturer instructions. In each run, standard dilutions of a titered MERS-CoV stock were run in parallel, to calculate TCID 50 equivalents in the samples. For the detection of viral mRNA, 5 μ l RNA was used in a one-step real-time RT-PCR using the MERS-CoV M mRNA assay in the Rotor-Gene TM probe kit 64 . Artibeus jamaicensis orthomyxovirus resistance gene 1 (Mx1) gene expression was determined by qRT-PCR using Mx1, ISG56 and RANTES specific primers (derived from transcriptome sequencing 45 ). The fold-change of each gene was calculated by normalizing the change in CT (cycle threshold) value of Mx1 (Δ CT) to the CT values for hypoxanthine phosphoribosyltransferase (HPRT) as an internal reference gene for each sample and comparing this to the CT values of mock inoculated bats 11 and 12 (2 ∧ (−Δ Δ CT). Mx1 specific primers: 5′ -CCAGACCTGACCCTGATAGA-3′ , 5′ -TGGATGTACTTCCTGAATGAGTTG-3′ and 5′ -FAM-ATCTAGTGTCCGATGTCAGCTGGC-IABkFQ-3′ . ISG56 specific primers: 5′ -GCTGTCTATCGTCTGAATGGG-3′ , 5′ -TTCTTGTCCGATGTCCTGAAG-3′ and 5′ -HEX-CGATGAGGC/Zen/ATTTTGTCTGCAAACCC-IABkFQ-3′ . RANTES specific primers: 5′ -AGTTGTCCTAATCACCCGAAAG-3′ , 5′ -CAGAGTGTTGATGTAGTCCCG-3′ and 5′ -FAM-TGTGCCGA C/Zen/CCGGAGAAGAAAT-IABkFQ-3′ . HPRT specific primers: 5′ -AGATGGTGAAGGTCGCAAG-3′ , 5′ -CCTGAAGTATTCATTATAGTCAAGGG-3′ and 5′ -FAM-ACTTTGTTGGATTTGAAATTCCAG ACAAGTTTG-BHQ1.

Virus isolation. Tissue samples were homogenized in a TissueLyzer II (Qiagen) after addition of 1ml DMEM.

Homogenates were centrifuged to pellet cellular debris and subsequently inoculated onto VeroE6 and LLC-MK2 cells. After 1hr adsorption, cells were washed once with DMEM and media was replaced.

ELISA. Antibody responses were measured in an enzyme-linked immunosorbent assay (ELISA) using hCoV-EMC/2012 as described previously 65 . Briefly, EMC/2012 containing cell culture supernatant was used to coat immuno 96 microwell maxisorp plates (NUNC) at 4 °C overnight and diluted serum samples were added. Bound antibodies were detected using a secondary protein A/G conjugated with horseradish peroxidase (HRP; Pierce). Sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera. Sera obtained from rabbits immunized with EMC/2012 were used as a positive control.

Virus Neutralization Assay. Two-fold serial dilutions of heat-inactivated sera were prepared in a 96 microwell tissue culture plate and 100 TCID 50 of MERS-CoV was added and incubated for 1 hour at 37 °C. After incubation the virus-sera mixture was transferred to a 96 microwell tissue culture plate with a 95% confluent Scientific RepoRts | 6:21878 | DOI: 10.1038/srep21878 monolayer of VeroE6 cells. The virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, that still inhibited EMC/2012 virus replication.

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The authors would like to thank Drs. Bart