key: cord-303289-qoukiqr7
authors: Hemida, M. G.; Chu, D. K. W.; Perera, R. A. P. M.; Ko, R. L. W.; So, R. T. Y.; Ng, B. C. Y.; Chan, S. M. S.; Chu, S.; Alnaeem, A. A.; Alhammadi, M. A.; Webby, R. J.; Poon, L. L. M.; Balasuriya, U. B. R.; Peiris, M.
title: Coronavirus infections in horses in Saudi Arabia and Oman
date: 2017-03-13
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12630
sha: 
doc_id: 303289
cord_uid: qoukiqr7

Equine coronaviruses (ECoV) are the only coronavirus known to infect horses. So far, data on ECoV infection in horses remain limited to the USA, France and Japan and its geographic distribution is not well understood. We carried out RT‐PCR on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in Saudi Arabia and Oman. We document evidence of infection with ECoV and HKU23 coronavirus by RT‐PCR. There was no conclusive evidence of Middle East respiratory syndrome coronavirus infection in horses. Serological data suggest that lineage A betacoronavirus infections are commonly infecting horses in Saudi Arabia and Oman but antibody cross‐reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections.

infect or cause disease in horses (Balasuriya, 2013) . It belongs to the genus Betacoronavirus lineage A, as does human coronavirus OC43, bovine coronavirus and porcine hemagglutinating encephalomyelitis virus. Equine coronavirus was first isolated from faeces of a diarrhoeic foal in 1999 (ECoV-NC99) in North Carolina, USA (Guy, Breslin, Breuhaus, Vivrette, & Smith, 2000) , and was initially believed to only affect foals. Since 2010, there have been several reports of ECoV-associated respiratory and enteric infections in adult horses in Japan, Europe and the United States, but its global distribution is still poorly defined (Kooijman, Mapes, & Pusterla, 2016; Miszczak et al., 2014; Oue, Morita, Kondo, & Nemoto, 2013; Pusterla, Holzenkaempfer, Mapes, & Kass, 2015) .

Middle East respiratory syndrome coronavirus (MERS-CoV) is a betacoronavirus within lineage C first identified in humans in 2012 and continues to pose a threat to global health (WHO, 2016 May 27) . The evidence points to dromedary camels as a natural host and a major source for human MERS-CoV infections (Alexandersen, Kobinger, Soule, & Wernery, 2014; Chu et al., 2015; Gutierrez, Tejedor-Junco, Gonzalez, Lattwein, & Renneker, 2015; Memish et al., 2014; Perera et al., 2013; Reusken et al., 2014) . There is no convincing evidence of MERS-CoV infections in other domestic livestock species so far (Adney et al., 2016; Alexandersen et al., 2014; Hemida et al., 2013; Meyer et al., 2015; Perera et al., 2013) .

Sequence similarity comparisons of dipeptidyl peptidase-4 (DPP4

[CD26]), the functional receptor for MERS-CoV, have revealed that equine DPP4 is phylogentically closely related to human DPP4 and the binding affinity of MERS-CoV spike S1 domain for equine DPP4 is similar to that of human and camel DPP4, raising the possibility that MERS-CoV infections may occur among horses (Barlan et al., 2014) . Serological studies of horses originating from Spain and the United Arab Emirates gave negative results for MERS-CoV (Alexandersen et al., 2014; Meyer et al., 2015) , but it is still relevant to investigate for evidence of MERS-CoV in horses in areas endemic for MERS-CoV.

We have tested for coronavirus infections in apparently healthy horses in Saudi Arabia and Oman by testing nasal and rectal swabs by RT-PCR assays for MERS-coronavirus and using a pan-coronavirus RT-PCR with potential to detect all coronaviruses, hitherto known and unknown. Following the detection of ECoV and HKU23 coronavirus (HKU23) by the pan-coronavirus RT-PCR, we tested the swab specimens with specific RT-PCR assays for ECoV and HKU23 and tested the sera with serological assays to detect ECoV, bovine CoV (BCoV) (closely related to HKU23), as well as MERS-CoV. (Guy et al., 2000; Zhang et al., 2007) and the bovine coronavirus (ATCC BRCV-OK-0514-2).

Viral RNA was extracted from nasal and rectal swabs using the QIAamp viral RNA minikit (Qiagen, Hilden, Germany) following the manufacturer's instructions. RNA extracts were tested for evidence of conserved coronavirus nucleic acid genetic sequences using previously reported RT-PCR assays (Chu et al., 2014) , RTqPCR assay for MERS-CoV upE gene (Corman et al., 2012) , RTqPCR assay for ECoV (Miszczak et al., 2014) , and a RTqPCR assay for HKU23 reported below. RNA was reverse transcribed in a 20 ll reaction mixture containing 19 first-strand buffer, 5 mM DTT, 0.5 mM deoxynucleotide triphosphates (dNTP), 2.5 ng/ll random hexamers and 200 units of SuperScript III (Life Technologies).

The pan-coronavirus nested PCR targeted the RNA-dependent RNA polymerase (RdRp) gene of coronaviruses developed by us as previously described (Chu et al., 2014) . Using the cDNA synthesized as described above, first round PCR was carried out using forward primer 5 0 -GGKTGGGAYTAYCCKAARTG-3 0 (position 15,287 of ECoV strain NC99; GenBank accession number EF446615) and reverse pri- of ECoV strain NC99) and 5 ll of cDNA as template for the reaction. PCR products with expected size of 658 bp were purified for DNA sequencing using the forward and reverse primers.

We developed a two-step real-time quantitative RT-PCR (RT-qPCR) assay for the detecting HKU23 targeting N gene of the virus.

Virus RNA was reverse transcribed as detailed above. Real-time PCR The details of all RT-PCR and real-time PCR assays used in this study are summarized in Table S1 .

The primers used for sequencing were those used to generate the PCR products. DNA amplicons were purified and sequenced in both forward and reverse directions with the PCR primers using BigDye 

All sera were tested using previously validated MERS-CoV spike pseudoparticle neutralization test (ppNT) (Hemida et al., 2014; Perera et al., 2013) , and the positive sera were confirmed using microneutralization (MN) test and plaque reduction neutralization tests (PRNT) using live MERS-CoV strain EMC in a Biosafety Level 3 containment laboratory (Park et al., 2015) . Selected sera were tested for antibodies to ECoV and BCoVs . The North American ECoV-T A B L E 1 Geographic location of specimens collected and animal management practices BCoV is ubiquitous in cattle worldwide and is genetically and antigenically related with HKU23, a coronavirus endemic in dromedaries (Woo et al., 2014 (Woo et al., , 2016 . Antibody to BCoV was tested using MN assay as described previously (Park et al., 2015; Perera et al., 2013) . Serology for BCoV and ECoV was carried out with culture medium without foetal calf serum. Known positive and negative control sera were included in all serology assays. (Woo et al., 2014) . (Table 4 ). In the confirmatory PRNT which is considered the "gold standard" serological test for MERS-CoV, horse serum 9-2 reduced plaque numbers by 90% (PRNT 90 ) up to a titre of 40. This serum was negative in ECoV and BCoV MN assays (Table 4 ). This serum was from a racing horse stable in Al-Qassem and may represent a rare example of transmission of MERS-CoV to horses or cross-reaction with another yet undocumented coronavirus infecting horses. This horse was not known to frequently come into contact with camels. Three of the other sera (serum ID nos 5.35; 5.41; 2.12) positive in the MERS-CoV ppNT assay had detectable

ECoV MN antibody and one of these also had detectable BCoV MN antibody (Table 4 ). Thus, these may well represent evidence of cross-reactive responses.

T A B L E 5 Cross-neutralization titres (denoted as reciprocal titres) for Middle East respiratory coronavirus (MERS-CoV), bovine coronavirus (BCoV) and equine coronavirus (ECoV) in hyperimmune or naturally infected sera known to be positive for different coronaviruses NR460pig antiserum to porcine respiratory coronavirus 1,200 a <20 <20 <20 <20

NR2518 -Guina pig antisera feline infectious peritonitis virus 2,000 a <20 <20 <20 <20

NR458pig antisera for porcine transmissible gastroenteritis virus 1,400 a <20 <20 <20 <20 ECoV-negative sera 2 (horse, Hong Kong) Not relevant <20 <20 <20 <20

BCoV-positive camel serum #740293 e Not relevant <20 ND 640 80

BCoV-positive camel serum #467468 e Not relevant <20 ND 80 <20

C99-10 -BCoV antisera from guinea pig 20,480 b <20 <20 320 <20

Bcov antisera from germ free bovine calf 580 bneutralisation titre <20 <20 1,280 80

NR456 -BCoV antisera from gnotobiotic calf 10,000 a <20 <20 80 <20

NRC772 -Rabbit antisera SARS S protein high titre 640 <20 <20 <20 <20

Mouse hepatitis virus(JHM strain) hyperimmunized mouse dam 1 1,778 cneutralisation titre <20 <20 <20 <20

Mouse hepatitis virus(JHM strain) hyperimmunized mouse dam 2 363 cneutralisation titre <20 <20 <20 <20

Mouse hepatitis virus(A59 strain)-infected mice 1,000 cneutralization titre <20 <20 <20 <20

NRC774antisera for SARS coronavirus sero titre <10 <20 <20 <20 <20

NRC769 -Rabbit antisera to SARS S protein sero titre <10 <20 <20 <20 <20

Gamma coronavirus NR2515-Guina pig antiserum to infectious bronchitis virus 50,000 a <20 <20 <20 <20 7-3 <20 <20

7-4 <20 20 7-1 <20 <20

7-2 <20 <20

Al-Qassem 9 (Racing) 9-A3 20 <20

9-A4 <20 40 9-A5 <20 <20

9-B1 <20 <20

9-C1 20 20 9-C2 80 640 9-E1 <20 <20

9-E2 <20 <20

Qateef 10 (Farming) 10-13 <20 80

10-14 <20 20

10-15 <20 80

10-16 20 160 11 (Farming) 11-8 80 40

11-9 <20 320 11-11 <20 <20

11-12 <20 <20

11-13 <20 80

(Continues)

The extent of the serological cross-reactivity of MERS-CoV, ECoV and BCoV was systematically investigated. We did not have an isolate of HKU23 virus available for serological testing and BCoV was used as a virus that is genetically closely related to HKU23 with pairwise amino acid similarity of 94.1% in the spike protein gene, the determinant of antigenic cross-reaction in neutralization tests (Woo et al., 2014) . Horse sera obtained from the USA reported to be strongly reactive by ELISA (optical density ranging from 1.458 to 1.939) for ECoV showed high titre (>1,280) for ECoV in MN assay as expected, but also had high antibody titres (ranging from 160 to 1,280) in BCoV MN tests (Table 5) . As these horse sera were from horses in the "field," they may well have been exposed to multiple coronaviruses. Thus, these results may reflect cross-reactivity or coinfection of US horses with ECoV-and BCoV-like viruses. Two of these sera showed low titres (20) in the MERS-CoV ppNT assay but were negative in the more stringent MERS-CoV MN assay. As MERS-CoV is not circulating in the USA, these results suggest crossreactivity between MERS-CoV and ECoV or related CoVs may occur, albeit at low titre. Similarly, a BCoV immune serum from an experimentally infected gnotobiotic calf showed detectable, but 16-fold reduced antibody titre with ECoV but no cross-reaction with MERS-CoV. Spike proteins of equine coronavirus and bovine coronavirus have an overall amino acid similarity of 80.8% and 73.1% similarity of the S1 region which contains the receptor-binding domain and neutralization epitopes. This probably explains the large difference in neutralization titres observed in Table 5 . A dromedary serum from Kazakhstan, a region known to be free of MERS-CoV activity (Miguel et al., 2016) , had detectable antibody to BCoV (probably reflecting HKU23 or BCoV infection) and also to ECoV. We cannot conclude whether this reflects cross-reactivity or infection with BCoV, HKU23 and/or ECoV. None of the beta coronavirus immune sera to SARS-CoV or mouse hepatitis virus, nor immune sera to alpha or gamma coronaviruses gave cross-reactions in BCoV or ECoV MN assays (Table 5) . As had been previously reported, none of these dromedary sera cross-reacted with MERS-CoV (Hemida et al., 2014) .

Fifty-four horse sera from Saudi Arabia and Oman were selected to represent different collection locations, excluding those five sera previously reported to be MERS-CoV ppNT positive (Table 6 ). These sera were seronegative for MERS-CoV (as expected); 40 (74%) of them had detectable MN antibody to ECoV (titres ranging from <20 to 640; median 40), and 18 (33.3%) had MN antibodies to BCoV (titres ranging from <20 to 160; median <20). The reciprocal geometric mean antibody titres (assigning sera with titres <20 the value of 10 for computational purposes) for ECoV and BCoV were 44 and 25.5, respectively. There were twenty-four sera with undetectable antibody to BCoV with detectable ECoV antibody titres ranging from 20 to 640. The titres to ECoV were higher than or equal to titres to BCoV, with two exceptions where the titre to BCoV (20) was marginally higher than that with ECoV (Table 6 ). These results are compatible with the RT-PCR detection of ECoV in rectal swabs in horses and probably suggest that ECoV or a closely related virus is commonly infecting horses in many regions of the Arabian Peninsula.

Two of the five horses in whom ECoV RNA was detected by RTqPCR had low ECoV MN antibody titres (20) while the other three had titres ranging from 160 to 640 (Table 6 ). This may reflect virus shedding persists into convalescence or that re-infection of previously seropositive horses can occur.

The RTqPCR detection of two HKU23-like viruses indicates that HKU23 which is common in dromedaries in the region is also infecting horses. In the absence of an HKU23 isolate for use in neutralization tests and given the potential serological cross-reactivity between these three viruses, it is difficult to conclude how commonly equines are infected with HKU23. The antibody titres to BCoV, which is genetically closely related to HKU23 in the spike protein gene, may suggest that BCoV-or HKU23-like virus infection of horses does occur in Saudi Arabia. MERS-CoV did not result in virus replication or seroconversion (Adney et al., 2016) .

In conclusion, the serological data suggest that lineage A betacoronaviruses are commonly infecting horses in Saudi Arabia and Oman but antibody cross-reactions between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. RT-PCR detection of ECoV and HKU23 in equine swabs confirms the circulation of these two viruses in horses in Saudi Arabia. Cocirculation of related viruses may provide potential for recombination, a potential means of generating genetic diversity and facilitating host jumps in coronaviruses. This is the first report of HKU23 being detected in horses and the first detection of ECoV in Asia outside of Japan.

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