Isolation and molecular characterization of an enteric isolate of the Genotype-I Bovine coronavirus with notable mutations in the receptor binding domain of the spike glycoprotein and deletion downstream the RNA binding domain of the nucleocapsid protein

Abstract

AbstractBovine coronavirus (BCoV) continues to be a significant threat to cattle populations despite the implementation of vaccination programs. The continuous circulation of BCoV highlights the necessity for ongoing genomic surveillance to understand better the virus’s evolution and its impact on cattle health. The main goal of this study was to do isolation and perform a comprehensive molecular characterization of a new enteric field isolate of the BCoV. To identify any genetic elements in the sequences of this BCoV isolate that could act as genetic markers for BCoV infection in cattle. To achieve these objectives, the newly identified BCoV isolate was propagated on the MDBK cell line for several subsequent blind passages. The immunofluorescence assay verified confirmation of the virus propagation. We plaque purified this isolate and titrated it by plaque assay using the HRT-18 cell line. We examined the viral protein expression using the SDS-PAGE followed by the Western blot using the BCoV/S and BCoV/N and BCoV/S antibodies. Our results show a substantial increase in the viral genome copy number, protein expression, and virus infectivity of this BCoV isolate with the increase in cell culture passages. The full-length genome sequence of this isolate using the NGS was drafted. The vial genome is 31 Kb in length. The viral genome has the typical BCoV organization (5’-UTR-Gene- 1- HE-S-M-E-N-UTR-3’). Our phylogenetic analysis based on the nucleotide sequences of the (full-length genome, S, HE, and N) showed that the BCoV-13 clustered with other members of the BCoV (genotype I-i). The sequence analysis shows several synonymous mutations among various domains of the S glycoprotein, especially the receptor binding domain. We found nine notable nucleotide deletions immediately downstream of the RNA binding domain of the nucleocapsid gene. Further gene function studies are encouraged to study the function of these mutations on the BCoV molecular pathogenesis and immune regulation/evasion. This research enhances our understanding of BCoV genomics and contributes to improved diagnostic and control measures for BCoV infections in cattle populations.