Prevalence and Genomic Features of Aichivirus D in Yaks in Western China

Aichivirus D (AiV-D), a member of the genus Kobuvirus within the family Picornaviridae, is an emerging pathogen associated with diarrhea in multiple livestock species, including cattle, dairy cattle, Tibetan sheep, and yaks. In this study, a total of 280 fecal samples were collected from yaks with diarrhea in Sichuan, Gansu, and Qinghai Provinces, western China, between June 2025 and February 2026. The overall positive rate of AiV-D was 30.00% (84/280), and the farm-level positive rate was 87.5% (7/8). Four complete yak AiV-D genomes (GenBank: PZ424445-PZ424448) were obtained from the positive samples, with genome sizes ranging from 8369 to 8475 nt and GC contents ranging from 55.66 to 55.85%. Each genome contained a single open reading frame (ORF) of 7509-7518 bp, encoding a typical picornaviral polyprotein consisting of a leader protein (L), structural proteins P1 (VP0, VP3, and VP1), and nonstructural proteins P2 (2A, 2B, and 2C) and P3 (3A, 3B, 3C, and 3D). Pairwise comparisons of the ORF revealed nucleotide and amino acid identities of 82.07-99.63% and 81.96-99.56%, respectively, among the four strains. Phylogenetic analysis based on the complete genomes revealed that strain BKV/YAK/FX1/2025/CHN clustered with the bovine reference strain BKV5 (84.70% nt/88.35% aa identity), whereas the other three strains grouped with the yak reference strain BKV/Yak/SCDF77/2021/CHN (GenBank: OP776109.1) (89.80-93.07% nt 95.53-96.84% aa identity). Recombination analysis predicted a recombination event in strain BKV/YAK/SF1JK6/2025/CHN, with breakpoints located in the nonstructural protein coding region (2C-3D). Additionally, eight full-length VP0, VP3, and VP1 gene sequences were amplified from the AiV-D-positive samples, and a few co-mutational amino acid substitutions were identified in these structural proteins. Phylogenetic analysis based on the VP1 gene further divided the eight strains into two distinct clades: six strains formed an independent clade and were designated as a novel sub-genotype within AiV-D4, tentatively named AiV-D4-L1, closely related to but clearly distinguishable from the AiV-D4 reference strains reported in China, while the remaining two strains (BKV/YAK/FX1/2025/CHN and BKV/YAK/FX77/2025/CHN) clustered with the bovine reference strain and were designated as a novel genotype, AiV-D5. These results reveal genetic diversity among yak-derived AiV-D strains and suggest possible host adaptability or cross-host transmission, warranting further investigation in future studies. However, as all the samples were collected from diarrheic yaks without a healthy control group, the potential association between AiV-D and diarrhea requires further validation.