key: cord-355290-m8875kdy
authors: Meyer, Benjamin; García-Bocanegra, Ignacio; Wernery, Ulrich; Wernery, Renate; Sieberg, Andrea; Müller, Marcel A.; Drexler, Jan Felix; Drosten, Christian; Eckerle, Isabella
title: Serologic Assessment of Possibility for MERS-CoV Infection in Equids
date: 2015-01-17
journal: Emerg Infect Dis
DOI: 10.3201/eid2101.141342
sha: 
doc_id: 355290
cord_uid: m8875kdy

nan

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. endurance horses from the United Arab Emirates that were collected for routine veterinary purposes; and 861 samples from 697 horses, 82 donkeys, and 82 mules in Spain. Because the reactivity of equid serum against MERS-CoV has not been investigated, we established a 2-stage algorithm for serologic testing that did not involve the determination of reactivity cutoff values. The screening stage involved testing of all serum samples by using a previously described ELISA with the spike protein S1-domain of MERS-CoV as the test antigen (4). The ELISA was adapted for use with horse serum by exchange of the secondary antibody. All serum samples reacted with low to medium OD values (range 0.0-0.55) (Figure, panel C) . We then tested the 50 most reactive serum samples (optical density range 0.22-0.55) by using recombinant immunofluorescent and microneutralization assays (1). These assays are more specific than the ELISA assay and therefore can be used for confirmation. None of the tested serum samples showed reactivity in the recombinant immunofluorescent or microneutralization assays; this finding suggests that no previous exposure of equids to MERS-CoV has occurred in the United Arab Emirates and Spain. Identifying all potential animal reservoirs is a critical step in controlling zoonotic diseases. Molecular data suggest that horses may be highly susceptible to MERS-CoV because of their high similarity in DPP-4 amino acids at positions critical for binding of the MERS-CoV spike protein (8) . Our in vitro data confirm the susceptibility of primary horse cells, showing production not only of viral RNA but also of infectious virus progeny, which is a prerequisite for transmission. The lower replication observed in horse cells than in VeroB4 cells may be the result of a difference in the interferon competence of the cells; replication levels in horse cells are comparable to those in bat cells (6) . Although we did not find evidence for equid infections with MERS-CoV in this study, the general susceptibility on the cell culture level suggests that equids from MERS-CoV-endemic areas, such as Africa and the Arabian Peninsula, should be further investigated for possible infection with MERS-CoV.

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We thank Dagmar Hensel for excellent technical help and Matthias Lenk for horse cell lines. We also thank Victor Corman, Monika Eschbach-Bludau, Tobias Bleicker, and Sebastian Brünink for help with horse serum samples.This study was supported by the European Commission under project ANTIGONE (contract no. 278976) and the German Centre for Infection Research.