犬冠状病毒JS1706和JS1712毒株基因组3'端分子特性分析

为了全面了解犬冠状病毒（CCoV）分离毒株JS1706和JS1712基因组3'端主要结构蛋白基因和非结构蛋白基因的分子特征，本研究设计了8组引物进行RT-PCR扩增，产物经测序和拼接后，获得了约8.7 kb基因组片段，该基因组结构及其编码蛋白顺序为5'-S-3abc-E-M-N-7ab-3'。对CCoV JS1706、JS1712株8.7 kb基因组核苷酸序列与α冠状病毒属参考毒株的相同区域核苷酸序列进行比对，结果表明，2个分离株与CCoV Ⅱ型参考毒株相似性最高（83.4%~93.1%），其次为FCoV Ⅱ型参考毒株（87.1%~87.9%）、TGEV参考毒株（86.1%~86.8%）、CCoV Ⅰ型参考毒株（72.0%~72.1%）和FCoV Ⅰ型参考毒株（67.5%~69.9%）。JS1706、JS1712毒株与同属冠状病毒参考株的结构蛋白S、E、M和N蛋白氨基酸相似性分别为46.4%~95.2%、75.6%~100%、82.8%~99.2%和78.5%~99.7%。说明同属内冠状病毒的S基因变异度大，E、M、N基因相对保守。根据基因组3'端8.7 kb核苷酸序列和S蛋白氨基酸序列相似性比对结果，JS1706和JS1712毒株均与泛嗜型原型株CB/05相似性最高，分别为93.0%~93.1%、94.8%~95.2%，其他结构蛋白包括E、M和N氨基酸序列比对也发现与CB/05株的相似性较高，分别为97.6%~100%、92.4%~93.1%和97.9%。S蛋白氨基酸序列的进一步分析表明，JS1706和JS1712毒株的S蛋白N端有一些特有氨基酸，S蛋白氨基酸序列中没有明显的S1/S2蛋白酶切位点（RRARR），但在958—963位氨基酸有S2'裂解位点特征基序（KRKYRS）。基于S蛋白氨基酸序列构建的系统发育进化树分析显示，CCoV JS1706和JS1712株与CCoV Ⅱa亚型参考毒株和FCoV Ⅱ型参考毒株聚集形成一个分枝。CCoV JS1706和JS1712株非结构蛋白的编码基因ORF3abc和ORF7，其结构、大小与经典疫苗株INSAVC-1相似，无明显插入、缺失和移码突变。本研究有助于深入了解国内CCoV流行毒株的分子特性，为后续分子流行病学调查、诊断试剂和疫苗研发奠定了基础。

Molecular Characterization of 3'-terminal Genome of Canine Coronavirus Strains JS1706 and JS1712

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.