Isolation of MERS Coronavirus from a Dromedary Camel, Qatar, 2014

We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels.

MERS-CoV (99.5% identity) isolated from camels at a different location in Qatar and in Egypt (9,11). Comparison of spike protein amino acid sequences from various human and camel isolates showed that this protein is highly conserved between this camel virus and other human isolates (online Technical Appendix Table, wwwnc.cdc.gov/EID/ article/20/8/14-0663-Techapp1.pdf).
In addition, most amino acid residues critical for receptor binding (14) are identical in human and camel isolates, except for L506F in England/Qatar_1_2012. The biologic relevance of this mutation has not been investigated. The presence of arginine at position 1020 in the camel virus isolate might indicate that selective pressure at this site has probably not taken place as previously postulated. The fact that a MERS-CoV from a camel is highly similar to that from a human patient who probably became infected >1 year earlier in the same region suggests that this virus is maintained within camel populations and further supports the hypothesis that MERS-CoV can be transmitted from camels to humans.
To test for the presence of infectious virus, we titrated the swab sample on Vero cells (ATCC no. CCL-81). After 48 hours, we observed cytopathic changes in cells (320 50% tissue culture infectious dose/mL). After isolation, the passage-3 virus stock was used for all subsequent experiments.
To check for adaptive mutations obtained during cell culture, we used 454 deep-sequencing technology (Roche, Indianapolis, IN, USA) to analyze the full-genome sequence as described elsewhere (3). A total of 57,655 sequence reads were obtained, of which 17,056 were specific for MERS-CoV, revealing ≈99.77% of the virus genome. Genome coverage ranged from 1 to 2,082 reads at single nucleotide positions. Gaps or regions with coverage of <4 reads were confirmed by Sanger sequencing. When the genome of the passaged virus was aligned with the genome of the initial clinical isolate, we did not observe any mutations acquired during passaging.
To further functionally characterize this virus isolate, we subsequently inoculated human hepatoma (Huh-7) cells with MERS-CoV camel/Qatar_2_2014. After 2 days, virus-induced cytopathic effects were observed in the inoculated cell cultures (online Technical Appendix Figure). In addition, a strong increase in virus titer was measured in the cell supernatant ( Figure 2 These data demonstrate that the MERS-CoV obtained from a dromedary camel is able to replicate in human cells and uses DPP4 as entry receptor, similar to MERS-CoV isolates obtained from human patients (15).

Conclusions
We isolated MERS-CoV from the nasal cavity of 1 dromedary camel and demonstrated its infectiousness. Further studies are needed to test whether camels infected at a young age are more likely than adult dromedary camels to excrete infectious virus, possibly because of the MERS-CoV seronegative status of the younger camels. In addition, our results add to recent findings that MERS-CoVs from camels and humans are nearly identical (9-11). As might be expected from the high level of conservation in the critical interacting amino acids in the receptor-binding domain of the camel and human MERS-CoV isolates (online Technical Appendix Table), we show that camel MERS-CoV can infect human Huh-7 cells by using the same entry receptor as the human MERS-CoV isolates (15). Collectively, combined with the observation that the sequence of this virus was most closely related to that of a virus from a human patient who acquired MERS-CoV in Qatar a year earlier, these data support the hypothesis that dromedary camels are a reservoir for MERS-CoV and can transmit the infection to humans. However, whether exposure of humans to camels directly can lead to human infection cannot be concluded from our data. We are not aware of a connection between the camel population sampled in this study and the patient infected with MERS-CoV England/Qatar 1. Future epidemiologic studies are needed to investigate whether contact with camels or camel products constitutes a risk factor for MERS-CoV infection.

Acknowledgments
We are grateful to the Joint Supreme Council of Health and Animal Resources Department of Ministry of Environment field investigation team for exceptional research assistance; to the Doha Camel slaughterhouse veterinarians, staff, and workers for their help; and to the Supreme Council of Health Administration for funding this study through a grant from the Health Promotion Department and the Communicable Disease Control Department routine budget. We also thank Thomas P.Samuel, Redentor Cuizon, Ronald R. Manaor, Khalid Yousif, and Farid Abdoudia for help with collecting samples.

Consensus P V S G N D H Y R A I A A L D W E M E A S Q A R A A A Q T C E
a Amino acid residues in the Middle East respiratory syndrome (MERS-CoV) spike protein that differ from the EMC/2012 isolate are displayed in red.
b Critical amino acid in binding to DPP4 receptor, displayed in green.