Serologic Assessment of Possibility for MERS-CoV Infection in Equids

To the Editor: In 2012, a novel coronavirus termed Middle East respiratory syndrome coronavirus (MERSCoV) emerged on the Arabian Peninsula; the virus has been responsible for >800 human cases. Recently, evidence of MERS-CoV infection in dromedaries was obtained from the Canary Islands, the Arabian Peninsula, and Africa (1– 3). Viral sequences from dromedaries and from humans infected with MERS-CoV were highly similar, suggesting a prominent role of dromedaries as an animal reservoir of the virus (4). However, the serologic assessment of other animal species has been incomplete. Investigations of domestic animal species have been restricted to goats, sheep, and cattle (3) and a limited study of horses (n = 3) (5). No evidence of recent infection was found in either study. Whereas most known CoVs have a highly restricted host range in vitro and in vivo, MERS-CoV has been found to infect a broad range of cell cultures derived from Old and New World camelids as well as humans, primates, bats, pigs, and goats (6). MERS-CoV uses the receptor dipeptidyl-peptidase-4 (DPP-4) to enter its host cell (7). Sequence comparison between the receptor-binding domain of the MERS-CoV spike protein and several mammalian DPP-4 sequences showed a higher percentage identity in the amino acid residues critical for virus entry between human and horse DPP-4 than between human and dromedary DPP-4 (8). It has been shown that MERS-CoV can use horse DPP-4 expressed on nonsusceptible cells (9), but no data are available on susceptibility of primary horse cells. Therefore, members of the family Equidae, which include domestic horses, donkeys, and mules, might be susceptible to MERS-CoV infection. According to the Food and Agricultural Organization of the United Nations (http://faostat. fao.org), >800,000 equids (horses, mules, and donkeys) are present on the Arabian Peninsula, but their role as putative MERS-CoV animal reservoirs has not been investigated. Therefore, we assessed in vitro susceptibility of primary horse cells to MERS-CoV infection and searched for serologic evidence of infection with MERS-CoV in equids originating from Spain and the United Arab Emirates. Primary cells derived from the kidney of 2 horses (termed PN-R and PFN-R) and an interferon-deficient primate cell line (VeroB4) were infected with MERS-CoV at a multiplicity of infection of 0.5 PFUs. Virus replication was quantified by real-time reverse transcription PCR (MERS-CoV upE assay) (10) and by plaque assay in Vero cells to confirm the production of infectious virus particles. Both cell lines showed clear cytopathic effects, an increase of viral RNA, and production of infectious virus progeny (Figure, panels A, B). To investigate equids for signs of infection with MERSCoV, we collected 1,053 serum samples from MERS-CoV– endemic and –nonendemic areas: 192 samples from adult