我国部分地区1996～2008年传染性支气管炎病毒分离株膜蛋白基因的遗传变异与系统进化分析

GENETIC VARIATION AND PHYLOGENETIC ANALYSIS OF M GENES OF INFECTIOUS BRONCHITIS VIRUSES ISOLATED IN CHINA FROM 1996 TO 2008

1996-2008年从我国不同地区分离30株传染性支气管炎病毒（Infectious Bronchitis Viruses，IBV）野毒株的M基因，采用RT-PCR方法克隆测定所分离的野毒株和澳大利亚T株的M基因序列，利用生物信息学软件与GenBank中公布的部分国内外IBV毒株的M基因序列进行比较分析，研究我国IBV的分子流行学特点和分子遗传变异规律。结果发现所测毒株M基因具有4种不同长度的开放阅读框：669bp、672bp、678bp和681bp，分别编码222、223、225和226个氨基酸的多肽，这些长度的差异是由5′端的核苷酸插入或缺失造成的。30个IBV分离株间的同源性在89．5％～100％之间。以疫苗株H120氨基酸位置为参照，在被比较的73株IBVM蛋白中发现62个位点存在变异，其中以2～5、10～16、44～46、217～222等4个区域氨基酸取代率较高。系统进化分析显示，被比较的73个IBV毒株分为5个进化群，我国的IBV分属于其中的4个群，其中第一群和第四群与我国所使用的疫苗病毒株相距较远。同时发现部分近年的分离株与10多年前分离株具有很近遗传进化关系。从M基因看，在我国出现了多种基因型IBV共存的现象，分离株与疫苗株的遗传差异提示我们需要对疫苗的选用做出重新评估。

The M genes of 30 infectious bronchitis virus （IBV） strains isolated in China from 1996 to 2008 and Australian strain T were cloned and sequenced by RT-PCR to investigate the molecular epidemiology and genetic variation. Their sequences were also compared with those published in GenBank using bioinformatics softwares. Results showed that the nucleotide sequences of M gene had four different lengths： 669 bp （T strain）, 672 bp （J and ZJ981isolates）, 678 bp （rest strains） and 681 bp （W and JH051 isolates）, which encoded four different sizes of polypeptide of 222, 223, 225 and 226 amino acid residues, respectively. The nucleotide difference in length resulted from insertion or deletion at the 5＇ terminus of M gene. The nucleotide similarities of M genes of 30 Chinese isolates were 89. 5% - 100%. When the H120 strain was used as a reference, 62 mutation sites were identified among a total of 73 IBV strains include in this study. Four regions at nucleotide positions of 2 - 5, 10 - 16,44 - 46 and 217 - 222 showed a higher frequency of substitution than other regions. Five dusters could be found among 73 strains by the phylogenetic analysis and the Chinese strains belonged to four clusters. Clusters Ⅰ and Ⅳ strains showed a large distance from the IBV vaccine strains currently used in China. Some of the strains recently isolated showed a close relatedness with those isolated 10 years ago. In conclusion, the present data have indicated that different genotypes of IBV strains are circulating in China. The distinct genetic diversity between the vaccine strains and the field IBV isolates implies a need to reassess the vaccine strains widely used in China.