Isolation, identification and pathogenicity analysis of BCoV epidemic strains in Xinjiang

Bovine coronavirus (BCoV) causes diarrhea in calves, winter dysentery in adult cattle, and respiratory diseases, posing a significant threat to the cattle industry. In this study, a BCoV strain was isolated from intestinal lymph node tissues of infected calves in Hami, Xinjiang, using HRT-18G cells with an optimized trypsin-HEPES synergistic culture system. Following plaque purification, the virus was confirmed by RT-PCR, indirect immunofluorescence assay, and transmission electron microscopy, and designated as BCoV-XJHM. The viral titer reached 10^8.0 TCID₅₀/mL. Whole-genome sequencing revealed that BCoV-XJHM shares 98.4-99.2% nucleotide identity with 35 representative domestic strains, clustering with the Guangxi strain (GX-NN230328, PP599028.1) in the same evolutionary subclade. Two amino acid substitutions (S81A and S149A) were observed in the N-terminal domain of the nucleocapsid (N) protein. In a BALB/c mouse model, oral inoculation of BCoV-XJHM induced significant body weight loss (P < 0.001) and mild pulmonary pathology, with viral RNA detected in lung and colon tissues. This study reports an optimized protocol for BCoV isolation in HRT-18G cells, describes two amino acid substitutions in the N protein of the BCoV-XJHM strain, and establishes a BALB/c mouse model for evaluating BCoV pathogenicity in a heterologous host.