Abstract
Aichivirus D (AiV-D) is a newly emerging diarrhea-associated pathogen in cattle and sheep, and comprised two officially recognized genotypes (AiV-D1 and AiV-D2) and two potential novel genotypes (AiV-D3 and AiV-D4). This study aimed to establish a fluorescent RT-PCR assay for detecting AiV-D and to investigate its prevalence in yak diarrheic samples. A SYBR Green fluorescent RT-PCR assay was successfully established by targeting the conserved region of viral 3D genes, enabling detection of all known AiV-D genotypes. The established assay was specific for AiV-D, exhibiting no cross-reactivity with other common diarrhea-causing pathogens in ruminants, with coefficients of variation for reproducibility ranging from 0.23% to 2.16% and detection limits for the positive plasmids of AiV-D1 to AiV-D4 at 2.21, 4.78, 4.10, and 5.94 copies/μL, respectively. Of the 166 diarrheic samples collected from calf yaks across nine farms in Sichuan province, China, between May and August 2023, 22.3% (37/166) tested positive for AiV-D, suggesting its substantial prevalence within the sampled region. Furthermore, seven complete 3D gene sequences were cloned from positive samples, and subsequent phylogenetic analysis revealed distinct evolutionary patterns among these isolates. In conclusion, this newly established assay represents a reliable diagnostic tool for AiV-D detection, while our findings provide significant insights into the epidemiological distribution and evolutionary dynamics of AiV-D in the yak population.
