北京市1998-2004年婴幼儿A3型流感病毒分离株HA1基因序列分析

Sequence analysis of the HA1 regions of hemagglutinin genes of influenza viruses(H3N2) isolated from children in Beijing from 1998-2004

目的 了解1998-2004年北京地区婴幼儿中流行的A3（H3N2）亚型流感病毒血凝素基因HA1区的基因变异特点。方法 提取该期间采集的呼吸道标本中H3N2亚型流感病毒的RNA，经逆转录多聚酶链反应扩增得到HA1区基因片段。通过目的基因的克隆、测序或PCR产物直接测序，进行序列分析。结果 19982004年问北京地区婴幼儿中流行的H3N2亚型流感病毒血凝素HA1区基因均为987bp，编码一个含329个氨基酸的蛋白质。这些H3N2亚型流感病毒血凝素HA1区的核苷酸和氨基酸同源性分别在95．5％～100．0％和93．0％～100．0％之间，分离年份越近同源性越高。在HA1区有7个糖基化位点非常保守，分别位于8、22、38、63、126、165和285位氨基酸。与1997年以前的毒株相比，1997年以后的毒株均在122和133位增加了一个糖基化位点。自1999年以后的毒株均在144位增加了一个糖基化位点。每年流行的毒株都较前一年的毒株出现了位于不同抗原决定簇或者受体结合位点的氨基酸替换。进化分析显示，每年分离的毒株均与以前的毒株出现了不同程度的变化，不断出现新的进化分支。结论 1998—2004年间北京地区婴幼儿中流行的H3N2亚型流感病毒持续不断地发生着点突变，导致抗原性不断发生漂移。加强监测和密切关注其变异动向对防控流感流行有极其重要的意义。

Objective To characterize the HA1 regions of hemagglutinin gene of influenza viruses （H3N2） isolated from children in Beijing from 1998 - 2004. Methods The HA1 regions of hemagglutinin gene were amplified by RT-PCR from the viruses isolated and identified as A3（H3N2） from clinical samples collected from infants and children during the peak seasons of influenza between 1998 and 2004. PCR products were sequenced or cloned into T-A vector and were analyzed after being sequenced. Results The HA1 regions of hemagglutinin genes amplified from those isolates were 987 bp in length, encoding a protein of 329 amino acids in length. The identities of nucleotides and amino adds among these H3N2 isolates in Beijing and vaccines strains from 1998 - 2004 were 95.5 % - 100.0 % and 93.0 %-100.0 % , respectively. The homology of the HA1 regions were related to the date of virus isolation, meaning the homology was higher among those strains isolated in nearer dates than others. Seven potential N-linked glyeosylation sites in the HA1 regions located at amino acid positions 8, 22, 38, 63, 126, 165 and 285 were conserved in all the viruses analyzed. Two sites at 122 and 133 were inserted in those virus isolated after 1997, and another site at 144 appeared in those isolated after 1999. More amino acid substitutions located in the five putative antigenic sites or receptor binding sites were found more in the isolates than the isolates from previous year. Phylogenetic analysis showed new branches appeared continuously during 1998 -2004. The strains isolated during winter in 2004 belonged to different branches, suggesting the appearance of new variants. Conclusion Amino acid substitutions continuously occurred in the HA1 regions of hemagglutinin genes in influenza virus（H3N2） isolated from children in Beijing from 1998- 2004, which might have resulted in antigenic drift and led to the appearance of new variants.