The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 μg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 2¹⁷, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Dairy cows are economically significant ruminants in China, and the dairy industry is closely linked to food safety and the agricultural economy. However, various factors such as pathogenic microorganisms often lead to frequent diseases in dairy cows. Furthermore, as potential hosts for diverse viruses, dairy cows can harbor zoonotic pathogens, which pose a threat to public health. The Shaanxi–Gansu–Ningxia region boasts abundant natural resources and extensive pastures. It is a major animal husbandry base in Northwest China, and dairy farming plays a significant role in the local economy. However, research on dairy cow virus diversity in this region remains limited; epidemic prevention and control capabilities are constrained, and the risk of disease outbreaks is elevated. In this study, 790 dairy cow samples were collected from 13 large-scale farms and free-range households in the Shaanxi–Gansu–Ningxia region from 2021 to 2023. Sample types consisted of nasal and anal swabs. Six viral metagenomic libraries were constructed and analyzed using high-throughput sequencing and bioinformatics methods, leading to the identification of 51 viral families. These comprised 16 positive-sense single-stranded RNA virus families, one Retroviridae family, four double-stranded RNA virus families, 21 double-stranded DNA virus families, and nine single-stranded DNA virus families. Among these, RNA viruses were represented by families such as Astroviridae, Coronaviridae, Caliciviridae, Picornaviridae, and Picobirnaviridae; DNA viruses were primarily detected in Circoviridae, Papillomaviridae, Genomoviridae, and Smacoviridae. Alpha diversity analysis revealed no significant differences in viral diversity and abundance among the three regions (p > 0.05); however, significant differences were observed in the read counts and proportions of RNA and DNA viruses across the provinces. Phylogenetic analysis further indicated that viruses carried by dairy cows exhibit considerable genetic diversity and pose potential cross-species transmission risks. This study established a reference database for the dairy cow virome in the Shaanxi–Gansu–Ningxia region, elucidated the phylogenetic relationships of key viruses, and provided a scientific basis for future monitoring and prevention of dairy cow viruses.