Development of CDV-specific monoclonal antibodies for differentiation of variable epitopes of nucleocapsid protein

The highly contagious canine distemper viruses (CDVs) are still a major threat to a wide range of natural susceptible hosts. The nucleocapsid (N) protein plays various roles in the virus-induced immune response. But precise mapping of epitopes and antigenic variations in N protein of CDV are still scant. In this study, two monoclonal antibodies (MAbs), designated as F8N and G3N, against the N protein of CDV were generated and characterized. The epitopes recognized by the two MAbs were mapped by truncated N protein fragments expressed in E.coli based on western blotting. The 470ESRYDTQ476 and 385GITKEEAQL393 were identified as the minimal linear epitopes recognized by F8N and G3N, respectively. The amino acid residues of the epitope (385-393aa) were highly conserved in a variety of CDV strains from the databases as well as five CDV strains in this study, indicating that MAb G3N can detect various CDV strains. However, MAb F8N was found not to react with an older CDV 851 strain of the five CDV strains due to both of two amino substitution (S471P and Y473H) in the epitope, whereas either single mutant S471P or Y473H did not eliminate the binding of F8N. Further, the variable epitopes existed in the N protein of six CDV strains resembling CDV3 in phylogenic tree by alignment with sequences from the databases. This is the first record of a precise epitope affecting antigenity of N protein of CDV. These results may facilitate future investigations into the function of NP of CDV and diagnostic methods for CDV infection.