Isolation and molecular characterization of an enteric isolate of the genotype-Ia bovine coronavirus with notable mutations in the receptor binding domain of the spike glycoprotein

Abstract

BCoV new isolate was plaque purified, isolated, and propagated in vitro using MDBK and HRT-18. The full-length genome sequencing of this new BCoV isolate (31 Kbs) was drafted and deported in the GenBank. The genome organization is (5′-UTR-Gene-1-32kDa-HE-S-4.9 kDa-4.8 kDa-12.7 kDa-E-M-N-UTR-3′). Phylogenetic analysis based on the sequences of (the full-length genome, S, HE, and N) showed that the BCoV-13 clustered with other North American BCoV genotype I members. 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. This research enhances our understanding of BCoV genomics and contributes to improved diagnostic and control measures for BCoV infections in cattle. • 1-The primary aim of this study was to isolate and perform an in-depth molecular characterization of a newly identified enteric field isolate of the Bovine Coronavirus (BCoV). Our research was driven by the objective to identify any genetic elements within the viral genome that could serve as markers for BCoV infection in cattle. Through our rigorous experimentation, we propagated the new BCoV isolate in the MDBK cell line across multiple blind passages, with subsequent virus confirmation by immunofluorescence assay. The isolate was plaque purified and titrated using the plaque assay on the HRT-18 cell line, and we examined viral protein expression through SDS-PAGE and Western blot techniques using BCoV-specific antibodies. We also showing the electron microscopy pictures of the typical BCoV. • 2-Our findings revealed a significant increase in viral genome copy number, protein expression, and infectivity with successive cell culture passages. The full-length genome sequence of this BCoV isolate, drafted through next-generation sequencing (NGS), is approximately 31 Kb and displays the typical BCoV genome organization (5′-UTR-Gene-1-HE-S-M-E-N-UTR-3′). Phylogenetic analysis based on the full-length genome and key structural genes (S, HE, and N) placed our isolate, BCoV-13, within the Genotype I-i clade of BCoV. • Furthermore, sequence analysis identified several notable synonymous mutations within the spike glycoprotein's receptor binding domain and nine nucleotide deletions downstream of the RNA binding domain of the nucleocapsid protein. These findings suggest that further functional studies are warranted to elucidate the roles of these mutations in BCoV pathogenesis and immune evasion. • This research provides crucial insights into BCoV genomics, advancing our understanding of its molecular biology and potential genetic markers. We believe that the findings presented in this manuscript offer valuable contributions to the field, particularly in the development of improved diagnostic and control strategies for BCoV infections in cattle populations.

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