Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China

Lu, Shuai; Chen, Yingzhu; Qin, Kun; Zhou, Jianfang; Lou, Yongliang; Tan, Wenjie

In this study, we expressed the N proteins of CCoV and CRCoV for serological studies. To our knowledge, the cloning and sequencing of the NP of CRCoV was first reported in China. Furthermore, we analyzed the antigenic relationship among CCoV, CRCoV and HCoVs. As shown in Figure 5 , CCoV NP reacted with antisera from HCoV-229E and NL63 belonging to Alphacoronavirus, but showed weaker antigenicity, which may be due to the limited number of conserved antigenic sites among the subgroup. No significant cross-reactivity was observed to polyclonal antisera from three other HCoVs in the Betacoronavirus genus, indicating an extremely low avidity of the respective antibody, or a distinctly far antigenic relationship between these different clades. In contrast, CRCoV NP showed cross-reactive binding to antisera from mice immunized with the N proteins of HCoV-HKU1 and OC43, respectively, to a nearly same degree, and they all resolved into several fuzzy bands, except to the anti-MERS-CoV. Interestingly, CRCoV seems to be phylogenetically distant with HCoV-229E and NL63, but showed weaker reactivity than that of HCoV-HKU1 and OC43. This might be explained by conserved NP immune epitopes shared between both alpha and beta viruses, such as the highly conserved motif FYYLGTGP. In addition, CRCoV NP reacted strongly with anti-OC43 as did anti-HKU1, which may be attributed to the high identity of the NP amino acid sequence of CRCoV to HCoV-OC43 and HKU1 respectively. However, CRCoV NP showed weaker reactivity with anti-MERS-CoV than antisera derived from four other HCoVs. This may be due to the variations in their primary sequence, which can influence the antigenic sites and lead to a variety of cross-reactivity. Furthermore, the crossreactivity generated between the two rNPs and anti-HCoVs was inconsistent with the identity of the NP sequences. This difference is possibly due to insufficient recognition by HCoVs antibodies, or different antigenic sites within the N proteins were exposed to an immune response, also indicating the potential complexity in serological diagnosis.