1990～2004年上海市甲型流感病毒基因特性的研究

Study on genetic characteristic of influenza A virus in Shanghai City between 1990 and 2004

目的研究1990～2004年上海市甲型流感病毒流行株血凝素(HA)基因的特性,探讨流感病毒基因的变异与流感流行的关系。方法采用鸡胚和MDCK细胞两种方法分离甲型流感病毒。提取病毒RNA进行逆转录-聚合酶链反应(RT-PCR)扩增产物纯化,核苷酸序列测定,并用MegAlign软件进行基因种系发生树分析。结果2000年以来上海市流行的甲3亚型与A/Sydney/5/1997(H3N2),A/Wuhan/359/1995(H3N2)相比,HA1区的核苷酸同源性为95.7%～98.6%和96.8%～98.6%。与90年代流行甲3亚型同源性为88.4%～92.8%,氨基酸的替换发生在抗原决定簇A、B、E、D区和受体结合位点(RBS)的左臂。甲1亚型与A/HongKong/1131/1998(H1N1)、A/Shanghai/7/1999(H1N1)相比,核苷酸同源性为97.8%～99.3%,与90年代流行的甲1亚型同源性为96.8%～97.8%。基因种系发生树表明近几年甲型流感病毒与90年代流行株存在基因特性不同的分支。结论2000年以来上海市H1N1亚型的抗原性未发生明显的变异,H3N2亚型流感病毒的基因发生变异使抗原性发生漂移,是近年引起局部地区流感暴发的主要因素。

Objective The study was designed to study the genetic characteristic of HA of epidemic strain for influenza A virus in Shanghai City between 1990 and 2004, and explore the relationship between the genetic variation of influenza virus and flu epidemic. Methods The two methods of chick embryo and MDCK cell were used to separate the influenza A virus.Virus RNA was extracted for purification of amplified products of Reverse Transcription Polymerase Chain Reaction （RT-PCR） and determination of nucleotide sequence, and the MegAlign software was adopted for analysis of phylogenetic tree. Results From 2000, compared with A/Sydney/5/1997 （H3N2） and A/Wuhan/359/1995 （H3N2）, the nucleotide homology in the HA1 area for H3N2 prevalent in Shanghai City was 95.7%-98.6% and 96.8%-98.6% respectively, and that compared with H3N2 prevalent in 1990s was 88.4%-92.8%, and the replacement of amino acid took place in the areas of A, B, E and D of antigenic determinant and left arm of receptor binding site （RBS）, while compared with A/HongKong/1131/1998 （H1N1） and A/Shanghai/7/1999 （H1N1）, the nucleotide homology for the H1N1 was 97.8%-99.3%, and that compared with HIN1 prevalent in 1990s was %.8%-97.8%. The phylogenetic tree showed that in recent years there existed a branch for influenza A virus of genetic characteristic different from that of epidemic strains in 1990s. Conclusion There has been no apparent variation for antigenicity of H1N1 in Shanghai City since 2000, but the gene of influenza A （H3N2） virus is mutated, and the antigenicity is so shifted, which is the main reason for local outbreak of influenza in recent years.