H3N2亚型人流感病毒神经氨酸酶基因特性研究

Analysis of the genetic characteristic of the neuraminidase of human influenza virus subtype H3N2

目的了解H3N2亚型人流感病毒神经氨酸酶(NA)基因变异规律方法采用狗肾传代细胞法对流感病毒进行分离培养,提取病毒RNA,逆转录-聚合酶链式反应,扩增NA基因后进行核苷酸序列测定。测序结果与WHO全球流感疫苗株序列进行氨基酸序列同源比对,并采用MEGA 4.0构建系统进化树,计算遗传距离。结果 NA氨基酸序列的系统进化树表明,南宁市2007年H3N2亚型人流感病毒株被分为4个分枝,其中大部分病毒株位于分枝Ⅰ和分枝Ⅱ中。氨基酸序列比对表明,NA没有发生氨基酸的缺失与插入,总体上比较保守,NA的酶活性中心、二硫键和糖基化位点基本保持不变。NA不同抗原决定簇变异频率不同,L370S变异出现在多数病毒株中。非结构功能位点也发生了不同程度的变异情况,其中K249E变异是导致分枝Ⅱ出现的主要因素。结论 NA基因的这些特性为流感的防控以及NA抑制剂药物的应用提供了一定的参考依据。

Objective To understand the law of heredity and variation of the neuraminidase（NA） of human influenza virus subtype H3N2.Methods Influenza viruses isolated were cultured in MDCK cells.Viral RNAs were extracted and amplified by using RT-PCR.The amplified products were purified and sequenced.Multiple alignment using Clustal was performed on the NA protein sequences together with that of H3N2 vaccine strains recommended by WHO.Genetic distance and phylogenetic tree of these strains were obtained by using MEGA 4.0.Results The consensus phylogenetic tree constructed using Neighbor-Joining method showed that the strains were divided into four clades and most strains were located in Clade Ⅰ and Ⅱ.The result of multiple alignment of NA protein sequences showed that the amino acid deletion and insertion events rarely happened in NA.The NA protein sequences were conserved to some degree,especially the neuraminidase active sites,the disulphide bonds and the glycosylation sites.The distinct mutation frequencies were observed in the different antigenic determinants of NA,and L370S mutation occurred nearly in all H3N2 strains.The mutation K249E resulted in the formation of CladeⅡin the phylogenetic tree Conclusion Our analysis provides some useful information for human influenza prevention and the application of NA inhibitor drugs.