Molecular epidemiology, genetic characterization and infection of canine respiratory coronavirus in dogs in Bangkok, Thailand

Abstract

Canine respiratory coronavirus (CRCoV) is one of the etiological agents of the canine infectious respiratory disease complex (CIRDC). This virus is highly contagious and has worldwide distribution. However, information regarding its genetic characteristics, diagnostic methods, and pathogenesis remains limited. This study aimed to obtain complete coding genomes of Thai CRCoV strains, and successfully identified 21 strains from two time periods (2013–2015, group A; and 2021–2022, group B). Comparative analysis of these two cohorts revealed ongoing genetic variation, while phylogenetic analysis demonstrated that Thai CRCoV strains from the two time periods shared a common ancestor but clustered into distinct lineages. Full-length genome analysis revealed that all Thai CRCoV strains possessed a nonsense mutation within a nonstructural gene located between the spike (S) and envelope (E) genes, resulting in a truncated putative nonstructural protein. Analysis of the S gene further indicated that group B strains exhibited signature nonsynonymous mutations that were not observed in group A strains, suggesting ongoing viral evolution. No evidence of recombination was detected among the Thai CRCoV strains; however, one Thai CRCoV strain was identified as a potential parental virus of a CRCoV strain reported in the United States. In addition to genomic analysis, limitations in existing diagnostic methods hinder accurate detection, particularly in samples with low viral loads. Accordingly, this study established probe-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) and nanoplate-based reverse transcription digital polymerase chain reaction (RT-dPCR) assays as versatile molecular tools for the detection and quantification of CRCoV. RT-dPCR demonstrated higher analytical sensitivity than RT-qPCR. However, RT-qPCR is more suitable for large-scale screening, whereas RT-dPCR is advantageous for detecting low-abundance targets and challenging sample types, such as rectal swabs. The developed assays were subsequently utilized to investigate the epidemiology of CRCoV in Thailand using a cohort of 267 dogs, including both animals with respiratory illness and clinically healthy dogs. The overall prevalence was 10.49%, with the majority of positive cases detected in dogs presenting with respiratory disease. Clinical samples were collected from three anatomical sites: nasal swabs (NS), oropharyngeal swabs (OS), and rectal swabs (RS). CRCoV was detected at all three sites, with the highest detection rate observed in NS and the lowest in RS. Furthermore, postmortem analysis was conducted on 86 dogs, among which only one puppy tested positive for CRCoV. High viral loads were detected in respiratory-associated organs, while the virus was also identified in extra-respiratory tissues. In situ hybridization demonstrated the presence of CRCoV in the mesenteric lymph node, a non-respiratory organ, and this finding was further supported by immunohistochemistry. These results suggest that CRCoV may disseminate beyond the respiratory tract. In summary, this study provides a comprehensive genomic characterization of CRCoV in Thailand, along with the development of versatile molecular diagnostic tools. It further elucidates the epidemiology, tissue distribution, and tissue localization of CRCoV, providing a foundation for future investigations.