Abstract: In order to fully understand the molecular characteristics of the major structural protein genes and non-structural protein genes at the 3' end of the genomes of Canine coronavirus (CCoV) strains JS1706 and JS1712, eight sets of primers were designed for RT-PCR assays. PCR amplification products were sequenced and assembled, an 8.7 kb genome fragment was obtained. The genome organization and its encoded proteins were 5'-S-3abc-E-M-N-7ab-3' in order. The 8.7 kb genomic nucleotide sequences of CCoV strains JS1706 and JS1712 were compared with the same region nucleotide sequences of the reference strain of Alphacoronavirus. The results showed that the two isolates had the highest nucleotide identity with the reference strain of CCoV type Ⅱ (83.4%-93.1%). The nucleotide identities were followed by 87.1%-87.9% with the FCoV type Ⅱ reference strain, 86.1%-86.8% with the TGEV reference strain, 72.0%-72.1% with the CCoV type Ⅰ reference strain and 67.5%-69.9% with the FCoV type Ⅰ reference strain. The amino acid similarity of S, E, M and N protein genes of JS1706 and JS1712 strains with the same genus Coronavirus reference strains were 46.4%-95.2%, 75.6%-100%, 82.8%-99.2% and 78.5%-99.7%, respectively. These results indicated that the S gene within the same genus of Coronavirus was highly divergent, and the E, M, and N genes were relatively conservative. According to the genetic similarity comparison of 8.7 kb nucleotide sequence and S protein amino acid sequence, JS1706 and JS1712 strains had the highest similarity with the prototype pantropic strain CB/05, which was 93.0%-93.1% and 94.8%-95.2%, respectively. Amino acid sequences alignment of other structural proteins, including E, M and N, also showed highly similarity with CB/05 strain, which were 97.6%-100%, 92.4%-93.1% and 97.9%, respectively. Further analysis of S protein gene showed that JS1706 and JS1712 had some unique amino acids at the N terminal of S protein, and there was no obvious S1/S2 proteolytic cleavage site (RRARR), but there was a S2' cleavage motif (KRKYRS) at the 958-963 amino acid position. Phylogenetic analysis based on the amino acids of S protein showed that CCoV strains JS1706 and JS1712 clustered into a branch with CCoV subtype Ⅱa reference strains and FCoV type Ⅱ reference strains. The structure and size of ORF3abc and ORF7 genes encoding non-structural protein of JS1706 and JS1712 strains were similar to those of classical vaccine strains INSAVC-1, without obvious insertion, deletion and frameshift mutations. This study would contribute to understand the molecular characteristics of CCoV epidemic strains in China and laid a foundation for subsequent molecular epidemiological investigation, diagnostic reagents development and vaccine research.

Key words: Canine coronavirus; 3'-terminal; genome; phylogenetic analysis