key: cord-0885380-fbkkb4l7 authors: Kim, J. H.; Jang, J.‐H.; Yoon, S.‐W.; Noh, J. Y.; Ahn, M.‐J.; Kim, Y.; Jeong, D. G.; Kim, H. K. title: Detection of bovine coronavirus in nasal swab of non‐captive wild water deer, Korea date: 2018-03-05 journal: Transbound Emerg Dis DOI: 10.1111/tbed.12847 sha: 934600fa1078e96c0a50499f189409e7a17caaa7 doc_id: 885380 cord_uid: fbkkb4l7 Bovine coronavirus (BCoV) is a causative agent of respiratory and enteric diseases in cattle and calves. BCoV infection was also evident in captive wild ruminants. Recently, water deer are recognized as the most common wildlife to approach farmhouses and livestock barns in Korea. Therefore, we investigated 77 nasal swab samples from non‐captive wild water deer (Hydropotes inermis) between November 2016 and September 2017 and identified three samples positive for coronavirus, indicating potential for respiratory shedding. The full genomic sequences of the water deer coronavirus were closely related to BCoV (>98%). Therefore, effective biosecurity system in bovine farms would be necessary to prevent contact between farm ruminants and free‐ranging wild water deer. Bovine coronavirus (BCoV) is a causative agent of respiratory and enteric diseases in calves and adult cattle. It belongs to the virus species Betacoronavirus 1 of the lineage A of the genus Betacoronavirus (Bidokhti et al., 2013) . BCoV has relatively recent common ancestors with human coronavirus OC43 (HCoV-OC43) and porcine haemagglutinating encephalomyelitis virus (PHEV) (Vijgen et al., 2006) . Due to their genetic closeness, the interspecies transmission potential of BCoV should not be ignored. In fact, human enteric coronavirus 4408 strain (HECV-4408) was first isolated from a child's diarrhoeic stool and was genetically and antigenically more closely related to BCoV than HCoV-OC43 (Zhang, Herbst, Kousoulas, & Storz, 1994) . In the late 1970s, bovine-like CoVs were first detected in other ruminants by electron microscope (EM) and they have since been detected in the faecal samples of wild ruminants such as sambar deer (Cervus unicolor), waterbuck (Kobus ellipsiprymnus), sable antelope (Hippotragus niger), white-tailed deer (Odocoileus virginianus), wisent (Bison bonasus), Himalayan tahr (Hemitragus jemlahicus), sitatunga (Tragelaphus spekii), nyala (Tragelaphus angasii) and giraffe (Giraffa camelopardalis) (Alekseev et al., 2008; Chung, Kim, Bae, Lee, & Oem, 2011; Hasoksuz et al., 2007) . As no specific genetic markers were identified to discriminate the ruminant bovine-like CoVs from BCoVs (Alekseev et al., 2008) , interspecies transmission of BCoVs may be common among the ruminants. However, the majority of cases were found in the captive wild ruminants rather than non-captive ruminants and most of them were detected in the faecal samples. In Korea, no evidence of BCoV infection in water deer (Hydropotes inermis) was reported between 2010 *These authors contributed equally to this work and 2012 (Kim et al., 2014) . Therefore, we investigated coronaviruses in water deer in Korea, using 77 nasal swabs collected between November 2016 and September 2017. In this study, 77 nasal swab samples from water deer (Hydropotes inermis) were obtained from Chungnam Wild Animal Rescue Center, between November 2016 and September 2017. The water deer in this study were rescued wildlife, and the nasal swabs were immediately collected as soon as they were delivered by wildlife veterinarians. The nasal swab samples were placed into universal viral transport medium (BD Diagnostics, USA) and transported to the laboratory for analysis. The genome of water deer coronavirus was further sequenced by RT-PCR using primers that were designed based on the genome of a related BCoV, strain Quebec (AF220295). Subsequently, 3ʹ and 5ʹ end sequences were obtained by rapid amplification of cDNA end (RACE) sequencing based on PCR using the adapteroligo dTVN primer from Cosmo Genetech Co., Ltd. The obtained partial viral genome was assembled and manually annotated by (Table 1) . Phylogenetic analysis, based on 256 bases of the partial RDRP gene, showed they were closely related to BCoV and HECV-4408 (Figure 1a) . The nucleotide identities of the viruses with BCoV and HECV-4408 were 99.2% and 100%, respectively. The fully sequenced water deer coronavirus W17-18 genome (GenBank Accession No. MG518518) was 31,034 bp (37.1% G+C content); the overall gene content and arrangement in the strain were almost identical to those from its related BCoVs from captive wild ruminants in the GenBank database. Moreover, the OrthoANI value analysis suggested that the water deer coronavirus W17-18 genome was very similar (>98%) to those BCoVs from bovine and wild animals and closely related to HCoV-OC43 strains (>95%) and PHEV (>93%). The full genome sequence-based phylogenetic analysis by maximum likelihood method revealed that the virus was closely related to BCoV, bovine-like coronavirus and HECV-4408 ( Figure 1b) . Distinctively, the N2 protein (208 amino acids), which was solely found in the BCoVs, was detected in the sequenced genome, whereas the predicted 4.8-kDa non-structural protein in water deer coronavirus strain W17-18 was elongated due to a 13-nucleotide deletion compared to other BCoVs ( Figure 2) . Therefore, the phylogenetic analysis based on the 4.8 kDa-or 4.8 kDa-like non-structural protein amino acid sequences of other reference viruses infers that water deer coronavirus W17-18 has created a unique clade within the BCoVs (Figure 1c) . Collectively, the newly found water deer coronavirus in this study was closely related to BCoV showing minor unique genetic characteristic in the 4.8 kDa-like non-structural protein. As the water deer coronavirus was detected from nasal swabs during the winter season, it could be assumed that the water deer coronaviruses might also cause respiratory disease and circulate during the winter season like other BCoVs. The majority of BCoV cases previously detected in wild ruminants have been in captive animals. In this study, BCoV was detected in non-captive wild water deer, which may provide further evidence that BCoV could be transmitted between freely ranging wild ruminants and farm ruminants. These findings may attract public attention for BCoV transmission between wild water deer and farms during winter season. In addition, BCoVlike human coronavirus (HECV-4408) was demonstrated to be able to infect gnotobiotic calves and provide cross-protection against BCoV infection (Han, Cheon, Zhang, & Saif, 2006) , and interspecies transmission of BCoV in turkey poults was also demonstrated under experimental condition (Ismail, Cho, Ward, Saif, & Saif, 2001 ). Therefore, interspecies transmission ecology of BCoV among livestock, wild animals and humans should be further investigated to control the virus. We conclude that the water deer coronavirus is a new strain of BCoV, rather than a new species. This is the first report of BCoV detection in non-captive wild water deer and presents a possible respiratory infection risk to water deer in Korea. Water deer (H. inermis) have recently been recognized as the most common wildlife to approach farmhouses and livestock barns in Korea (Kim et al., 2014) . BCoV transmission between water deer and livestock farms should not be overlooked. Therefore, effective biosecurity systems in bovine farms should be investigated to prevent interspecies transmission of BCoV between farm ruminants and wild water deer. This work was supported by grants from the KRIBB Initiative pro- Bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences Evolutionary dynamics of bovine coronaviruses: Natural selection pattern of the spike gene implies adaptive evolution of the strains Detection and characterization of bovine-like coronaviruses from four species of zoo ruminants Cross-protection against a human enteric coronavirus and a virulent bovine enteric coronavirus in gnotobiotic calves Biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe Experimental bovine coronavirus in turkey poults and young chickens Pathogens in water deer (Hydropotes inermis) in South Korea Identification of a novel coronavirus in bats MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution Evolutionary history of the closely related group 2 coronaviruses: Porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus OC43 Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child