First detection and genetic characterization of canine Kobuvirus in domestic dogs in Thailand

Background Canine Kobuvirus (CaKoV) has been detected both in healthy and diarrheic dogs and in asymptomatic wild carnivores. In this study, we conducted a survey of CaKoV at small animal hospitals in Bangkok and vicinity of Thailand during September 2016 to September 2018. Results Three hundred and seven rectal swab samples were collected from healthy dogs (n = 55) and dogs with gastroenteritis symptoms (n = 252). Of 307 swab samples tested by using one-step RT-PCR specific to 3D gene, we found CaKoV positivity at 17.59% (54/307). CaKoVs could be detected in both sick (19.44%) and healthy (9.09%) animals. In relation to age group, CaKoV could be frequently detected in younger dogs (25.45%). Our result showed no seasonal pattern of CaKoV infection in domestic dogs. In this study, we characterized CaKoVs by whole genome sequencing (n = 4) or 3D and VP1 gene sequencing (n = 8). Genetic and phylogenetic analyses showed that whole genomes of Thai CaKoVs were closely related to Chinese CaKoVs with highest 99.5% amino acid identity suggesting possible origin of CaKoVs in Thailand. Conclusions In conclusion, this study was the first to report the detection and genetic characteristics of CaKoVs in domestic dogs in Thailand. CaKoVs could be detected in both sick and healthy dogs. The virus is frequently detected in younger dogs. Thai CaKoVs were genetically closely related and grouped with Chinese CaKoVs. Our result raises the concerns to vet practitioners that diarrhea in dogs due to canine Kobuvirus infection should not be ignored. Electronic supplementary material The online version of this article (10.1186/s12917-019-1994-6) contains supplementary material, which is available to authorized users.

a survey of canine Kobuvirus in domestic dogs at small animal hospitals in 5 provinces of Thailand. The survey was conducted under the Chulalongkorn University's animal use and care protocol # 1731074. The result of this study provided the first detection and genetic characterization of CaKoV isolated from domestic dogs in Thailand.

Canine Kobuviruses in domestic dogs in Thailand
During September 2016 to September 2018, we conducted a survey of viral enteric diseases in domestic dogs in small animal hospitals in 5 provinces of Thailand (Bangkok, Nakhon Ratchasima, Ratchaburi, Suphanburi, and Tak). We tested 307 rectal swab samples for CaKoV by using one-step RT-PCR specific to 3D gene. Based on a two-year survey, we found CaKoV positivity at 17.59% (54/307). CaKoVs could be detected in both sick (19.44% (49/252)) and healthy (9.09% (5/55)) animals. Our result showed no seasonal pattern of CaKoV infection in dogs (Figs. 1 and 2). In relation to age group, CaKoV could be frequently detected in younger dogs at 25.45% (42/165) (Additional file 2: Table S2). The coinfections of CaKoV with other enteric viral pathogens were observed including CaKoV/Canine parvovirus/Canine Coronavirus (n = 6), CaKoV/Canine parvovirus (n = 20) and CaKoV/Canine Coronavirus (n = 2). In this study, 12 CaKoVs were selected and characterized by whole genome sequencing (n = 4) or 3D and VP1 gene sequencing (n = 8). The viruses were selected to represent epidemiological and demographic data such as age, date of isolation and breed. In this study, nucleotide sequences of the CaKoV were submitted to the GenBank database under the accession numbers MK201776 -MK201795 (Table 1).

Phylogeny of the Thai canine Kobuviruses
Phylogenetic analysis of whole genome of CaKoVs showed that the Thai CaKoVs were closely related to each other and clustered with Aichivirus A. The cluster Aichivirus A contains Kobuviruses from dogs, cats, rodents, bats and human. While Aichivirus B and C contain Kobuviruses from cattle and pigs, respectively. Based on whole genome sequence, Thai CaKoVs were closely related to Chinese CaKoVs sub-cluster but in separated sub-cluster from the viruses from the US, UK, Brazil and Tanzania (Fig. 3). Phylogenetic analysis of 3D and VP1 of Thai CaKoVs and reference CaKoVs from various animal species were also performed. Similarly, 3D gene of Thai CaKoVs were grouped together with Chinese CaKoVs (G1 sub-cluster) but separated from the viruses in sub-clusters G2 as well as G3 (Fig. 4). Phylogenetic analysis of VP1 gene, the viruses can be clustered into 2 major subgroups, US/EU/Africa subgroup and China/Thailand subgroup (Fig. 5).

Genetic analysis of the Thai canine Kobuviruses
We compared the nucleotide and deduced amino acid sequences of Thai CaKoVs against those of reference viruses from the US, UK, Italy, China, and Korea (Tables 2 and 3  most variable region of VP1 is position 201-243, especially proline rich region. Putative proline rich region at VP1-228-240 (P 228 XPPPPXPPXPXP 240 ) was also observed in Thai CaKoVs as well as reference viruses (Table 4). In this study, unique amino acids were found in Thai and Chinese CaKoVs at the position, 65 V, 67D, 119L, 138T, 150P, 151M, 153D, 201S, 204Q, 205Q, 201Q, 213T and 241E ( Table 4). Analysis of predicted amino acid cleavage sits of whole genome were conserved among Thai CaKoVs (Table 5).

Discussions
Canine Kobuvirus (CaKoV) is an emerging pathogen in Thailand. To the best of our knowledge, the CaKoV was described in Asia in retrospective study in Korea in 2011 and have been reported in Japan, China and Australia, respectively [2,15,17,21]. However, the CaKoV have   never been reported in the country or South East Asia region. In this study, during the 2 year-survey program, we found CaKoV positivity at 17.59% in both sick (19.44%) and healthy (9.09%) animals. Compare to other studies, CaKoV % positivity in this study was lower than those in China (54%) and Korea (32.2%) [14,22]. Our result showed that the CaKoV could be frequently detected in younger dogs at 27% which consistence with previous reports [15]. Similar to other previous studies, co-infections with other enteric viral pathogens were observed such as CaKoV/Canine parvovirus and CaKoV/ Canine Coronavirus [12,14,15]. Moreover, CaKoVs were detected in both diarrheic and non-diarrheic dogs which consistent with other studies [2,15]. Our result supported that this virus may not be the only cause of enteric disease in dogs. Nevertheless, the CaKoV infection have still been identified in symptomatic dogs without other enteric pathogen infections [12]. Our observation supported that the role of CaKoV as a primary pathogen of acute gastroenteritis remain unclear.
In this study, the genome size of 4 Thai CaKoVs is 7, 530 bp with one ORF encoding 2,444 amino acids of a putative polyprotein, which comparable to previous reports. Genome organization of CaKoV includes leader protein (L), structural proteins (VP0, VP3, VP1), nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D). Phylogenetic analyses showed that the Thai CaKoVs were closely related to each other and clustered with Aichivirus A. It is noted that Thai CaKoVs were closely related to Chinese CaKoVs sub-cluster but in separated sub-cluster from the viruses from the US, UK, Brazil and Tanzania (Fig. 3). Phylogenetic analyses of 3D gene showed similar result which Thai CaKoVs were grouped together with Chinese CaKoVs (G1 sub-cluster). This observation regarding to the sub-clusters of CaKoVs was in agreement with the previous study [23]. On the other hand, based on VP1 gene, the viruses can be clustered into 2 major subgroups, US/EU/Africa subgroup and China/Thailand subgroup which similar to the previous reports [16,22] (Figs. 4 and 5).
Genetic analyses of Thai CaKoVs showed that whole genome of 4 Thai CaKoVs posed highest nucleotide similarity to Chinese CaKoVs including SMCD-59 and CH-1. This observation supported phylogenetic analysis that Thai CaKoVs were closely related to Chinese CaKoVs sub-cluster but in separated sub-cluster from   (100) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) (100) 100 (100) 97.1 (100) 99.4 (100) 100 (100) 100 (100) 100 (100) (100) 96 (100) the viruses from the US, UK, Brazil and Tanzania. Of all viral genes, the VP1 gene was the most diverse gene among Thai CaKoVs and other reference CaKoVs. Similar observation was also reported in previous study that VP1 protein is the most variable capsid protein [24]. It is noted that the putative proline rich region at VP1-228-240 (P 228 XPPPPXPPXPXP 240 ) was observed both in Thai CaKoVs and reference viruses. Previous studies indicated that proline rich region may associate with enteric receptor binding of the viruses [14,24]. It is noted that Thai CaKoVs posed unique PPP (VP1; 228-240), which also observed most reference viruses from China, Korea, Japan, US, UK suggesting unique characteristic. These unique amino acids were not observed in the CaKoV from the Australia (CE9), Brazil (BRA/26) and Tanzania (TZ/75, TZ82) [16,20]. However, the association of these unique amino acids and viral pathogenesis is still need to be further investigated. Based on genetic analysis, unique amino acids at the position, 65 V, 67D, 119L, 138 T, 150P, 151M, 153D, 201S, 204Q, 205Q, 201Q, 213 T and 241E were observed. These unique amino acids of China/ Thailand sub-cluster could be benefit for the detection of virus origin or diagnostic purpose in the future. Similar to previous study, analysis of predicted amino acid cleavage sits of whole genome were conserved among CaKoVs except one variation at 776/777 (VP3/VP1) which unique in wild carnivores [16].

Conclusions
In conclusion, this study is the first to report of canine Kobuvirus in dogs in Thailand. CaKoVs were mostly detected in clinical dogs of young age. However, the viruses

Sample collection
Sample collection was conducted in domestic dogs at small animal hospitals in Bangkok and vicinity of Thailand During September 2016 to September 2018. 307 rectal swab samples were collected from healthy dogs (n = 55) and dogs with gastroenteritis symptoms (n = 252) including vomiting, watery diarrhea, hemorrhagic diarrhea and dehydration. The swab samples were collected from dogs of young age (< 1 year) (n = 165), adult (1-5 years) (n = 98) and older (> 5 years) (n = 44). The animal demographic data including age, sex, breed, and vaccination history were also recorded. The ethics was conducted under the Chulalongkorn University's animal use and care protocol # 1731074. The consent to participate of the owners of the animals used in this study was obtained in writing.

Canine Kobuvirus (CaKoV) detection
All 307 samples were subjected to canine Kobuvirus identification by one step RT-PCR using primers specific to 3D gene of CaKoV [21]. First, RNA extraction was performed using the QIAsymphony DSP Viral/Pathogen mini kit (Qiagen, Hilden, Germany) following manufacturer's instructions. To detect CaKoV, RNA samples were screened for 3D gene of CaKoV by using one step RT-PCR assay.

Phylogenetic and genetic analyses of canine Kobuviruses
The phylogenetic and genetic analyses were performed by comparing nucleotide sequences of Thai CaKoVs with those of Kobuvirus available from the GenBank database. The reference nucleotide sequences of CaKoVs were retrived based on their different geographic locations, host species and date of isolation. Phylogenetic analysis of CaKoV was performed by using MEGA v.6.0 (Tempe, AZ, USA) [29] with neighbor-joining method with Kimura 2-parameter with 1,000 bootstrap replicates and Beast program with Bayesian Markov chain Monte Carlo (BMCMC) with 10,000,000 generations and an average standard deviation of split frequencies < 0.05 [30]. For genetic analysis, the nucleotide sequences and deduced amino acids of CaKoV were aligned and compared using MegAlign software v.5.03 (DNASTAR Inc.; Wisconsin, USA). Pairwise comparison of nucleotides and amino acids of Thai CaKoV and those of reference CaKoVs were conducted. The variable and unique amino acids related to receptor binding of the viruses and host preferences of CaKoVs were monitored.

Additional files
Additional file 1: