A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

Detection of Systemic Canine Kobuvirus Infection in Peripheral Tissues and the Central Nervous System of a Fox Infected with Canine Distemper Virus

Canine kobuvirus (CaKV) is a globally distributed pathogen of dogs and is predominantly associated with infection of the gastrointestinal tract. However, an etiological link to enteric disease has not been established since CaKV has been identified in both asymptomatic dogs and animals with diarrheic symptoms. In this study, an extraintestinal CaKV infection was detected by next-generation sequencing in a fox (Vulpes vulpes) in Germany concomitant with a canine distemper virus (canine morbillivirus; CDV) co-infection. Phylogenetic analysis of the complete coding region sequence showed that this strain was most closely related to a CaKV strain detected in a dog in the United Kingdom in 2008. The tissue and cellular tropism of CaKV was characterized by the detection of viral antigens and RNA. CaKV RNA was detected by in situ hybridization in different tissues, including epithelial cells of the stomach and ependymal cells in the brain. The use of a new RT-qPCR assay for CaKV confirmed the systemic distribution of CaKV with viral RNA also detected in the lymph nodes, bladder, trachea, and brain. The detection of a CDV infection in this fox suggests that immunosuppression should be further investigated as a contributing factor to the enhanced extraintestinal spread of CaKV.

Proposed New Strain of Canine Kobuvirus from Fecal Samples of Brazilian Domestic Dogs

A proposed new strain of canine Kobuvirus was identified in fecal samples of domestic dogs from a rural community located in the municipality of Peixe-Boi, Pará, Brazil. The nucleotide identity was 92.3% similar to other representatives of the family Picornaviridae, genus Kobuvirus, and species Aichivirus A, which suggests that this is possibly a new strain within this species.