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

Sequence alignment of N protein from seven different coronavirus strains. The highly conserved sequence is indicated by a red frame. and respiratory infections, and signs of infections vary from mild to moderate, but are severe in puppies or in combination with other pathogens (Kong, 2007) . N protein is an abundant and crucial structural protein in coronavirus particles, and has an important role in the viral immune response (Li et al., 2005) . Antigen-capture enzyme-linked immunosorbent assays (ELISAs) using monoclonal antibodies against the N protein of SARS-CoV have been validated as useful for early diagnosis of SARS (Che et al., 2004) . Thus, serological assays targeting the NP of coronaviruses are considered important to for diagnosis of viral infection. Characterization of the antigenicity of nucleocapsid proteins derived from individual coronaviruses is essential for the development of serological assays for coronaviruses. It is notable that CoVs are characterized as having high rates of mutation and recombination, which is important to the transfer of hosts and alterations of virulence. We cloned the NP coding genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) from diseased pet dogs in Beijing and followed with sequence analysis. Our data identified that CCoV-BJ70 and CRCoV-BJ202 belong to two distinctly different subgroups with close relationships with strains isolated from relatively near regions, based on the N gene phylogenetic tree. The CCoV-BJ70 clustered with other CCoVs in the Alphacoronavirus genus, including a Chinese strain isolated from deceased giant pandas, but was distinct from heterologous coronaviruses HCoV-229E and NL63. CRCoV-BJ202 was closely related to a Korean strain: CRCoV-K37, as well as HCoV-OC43. Furthermore, N protein identity between CCoVs and HCoVs also confirmed the phylogeny. Notably, the N protein identity observed between CCoV-BJ70 and HCoVs was only 25.5% to 39.9%, but was highly similar with other CCoV strains. However, CRCoV-BJ202 has a high identity of 96.7% to HCoV-OC43, suggesting that CRCoV might share the same origin with HCoV-OC43 or recombination has occurred between them. Although the phylogenic relationship and amino acid sequence similarity was observed, the potential of crossreactivity between these coronaviruses is still unclear.