Epidemiological survey and genetic diversity of bovine coronavirus in Northeast China

In 2020, to trace the prevalence and evolution of bovine coronavirus (BCoV) in China, a total of 1383 samples (1016 fecal samples and 367 nasal swab samples) were collected from 1016 cattle exhibiting diarrhea symptoms on dairy farms and beef cattle farms in Heilongjiang Province, Northeast China. All samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) detection of the BCoV N gene, followed by an analysis of its epidemiology and genetic evolution. The results indicated that of the 1016 diarrhea-affected cattle, 15.45% (157/1016) were positive for BCoV, in which positive rates of the fecal and nasal swab samples were 12.20% (124/1016) and 21.53% (79/367), respectively. Of the 367 cattle whose nasal swab samples were collected, the BCoV positive rate of the corresponding fecal samples was 15.26% (56/367). BCoV infection was significantly associated with age, farming pattern, cattle type, farm latitude, sample type, and clinical symptom (p < 0.05). Of the 203 BCoV-positive samples, 20 spike (S) genes were successfully sequenced. The 20 identified BCoV strains shared nucleotide homologies of 97.7-100.0%, and their N-terminal domain of S1 subunit (S1-NTD: residues 15-298) differed genetically from the reference strains of South Korea and Europe. The 20 identified BCoV strains were clustered in the Asia-North America group (GII group) in the global strain-based phylogenetic tree and formed three clades in the Chinese strain-based phylogenetic tree. The HLJ/HH-10/2020 strain was clustered into the Europe group (GI group) in the S1-NTD-based phylogenetic tree, exhibiting N146/I, D148/G, and L154/F mutations that affect the S protein structure. Of the identified BCoV strains, one potential recombination event occurred between the HLJ/HH-20/2020 and HLJ/HH-10/2020 strains, which led to the generation of the recombinant BCV-AKS-01 strain. A selective pressure analysis on the S protein revealed one positively selected site (Asn509) among the 20 identified BCoV strains located inside the putative receptor binding domain (residues 326-540). These data provide a greater understanding of the epidemiology and evolution of BCoV in China.