First linear B-cell epitope identified on the nucleocapsid protein of bovine coronavirus

Bovine coronavirus (BCoV) is an important pathogen that can cause diarrhea in calves, respiratory disease, and winter dysentery in adult dairy cows. BCoV nucleocapsid protein (N) is highly conserved structural proteins that stimulate a strong immune response in the host, inducing both humoral and cellular immune responses, and are key targets for early and rapid diagnosis of coronaviruses and vaccine development. Therefore, the identification and screening of epitopes on N proteins is essential for the development of sensitive and specific diagnostic methods. In this study, BALB/c mice were immunized with soluble recombinant BCoV N protein expressed with a prokaryotic expression system, and two N-specific monoclonal antibodies (mAbs): 2F9 and 7H6 were prepared that recognize the same linear B-cell epitope with the smallest fragment located at ³⁸⁰YQQQDG³⁸⁵. Homology analysis of the amino acid sequences of the corresponding regions of nine typical BCoV strains from the different areas showed that the epitopes were high conservation. Homology analysis of the corresponding amino acid sequences of other animal-derived coronaviruses within the β coronavirus genus revealed that the homology of this epitope across different species was remarkably low. Intriguingly, it exhibited 100 % identity exclusively with human coronavirus OC43 and canine coronavirus CRCoV/BJ - 221. Such a finding offers crucial insights into viral evolution, transmission dynamics, and strategies for disease prevention and control, thereby highlighting its significance in advancing our understanding of coronaviruses. These findings fill the gaps in the study of BCoV N protein epitopes and promote the understanding of BCoV N protein structure, which is valuable for developing diagnostic methods for detecting BCoV and exploring the biological functions of BCoV N proteins.