First Molecular Insights into the Presence of Canine Kobuvirus in Ecuadorian Dogs Through the Standardization of a Sensitive SYBR Green RT-qPCR Assay

Abstract

Canine Kobuvirus (CaKoV) has been identified as an agent involved in gastrointestinal diseases among dogs worldwide, with a particular impact on young individuals. This study reports the first molecular detection of CaKoV in Ecuadorian dogs with gastroenteritis, using a sensitive SYBR Green-based RT-qPCR assay. The assay demonstrated high sensitivity, with detection limits approaching a single copy of genetic material (1 copy/μL), with a standard efficiency curve of 100.6% and a correlation coefficient of 0.997, facilitating accurate CaKoV detection even at a minimal number of gene copies; it was also highly specific for CaKoV genome amplification, as no amplification was shown for other canine enteric viruses [Canine Parvovirus (CPV-2), Canine Astrovirus (CaAstV), Canine Coronavirus (CCoV), and Canine Distemper Virus (CDV)], and demonstrated strong reproducibility across different runs. A total of 250 fecal samples were used to validate the assay and detect the presence of CaKoV, with 91 samples testing positive for CaKoV, confirming the virus’ presence across multiple provinces in Ecuador, with Pichincha reporting the highest number of positive samples. Phylogenetic analysis of the partial 3D gene sequence exhibited a nucleotide identity ≥ 90% with sequences of CaKoV strains from different countries around the world. Co-infections with other enteric viruses such as CPV-2, CCoV, and CaAstV were observed in 61.3% of CaKoV-positive samples from dogs with gastroenteritis, with triple co-infections (CPV-2, CaAstV, and CaKoV) being the most frequently detected combination in the study. The present study showed that CaKoV is circulating in domestic dogs affected with gastroenteric disease and in apparently healthy dogs. This work establishes CaKoV as a possible contributor to canine gastroenteritis in Ecuador, in addition to the typical viruses such as CPV-2 and CCoV; moreover, this study illustrates a molecular assay that is both rapid and reliable for the diagnosis of CaKoV.