甲型H1N1流感死亡病例三株病毒分离株血凝素基因测序分析

Sequence analysis of the hemagglutinin gene of isolates viruses from 3 novel influenza A （ H1N1 ） deaths

目的 对长沙市3株甲型H1N1流感死亡病例病毒分离株HA基因的来源和变异情况进行研究.方法 对长沙市的3例甲型H1N1流感死亡病例的鼻/咽拭子标本进行RT-PCR检测和流感病毒分离,利用WHO推荐的测序引物和CEQTM8000 Genetic Analysis System对3株甲型H1N1流感死亡病例病毒株[A/湖南开福/SWL4142/2009（H1N1）,A/湖南长沙/SWL4346/2009（H1 N1）,A/湖南芙蓉/SWL4224/2009（H1N1）]HA基因进行测序,测序方法为末端标记循环法（Dye Terminator Cycle sequencing）,测序结果提交至GenBank,并用ClustalX和Mega4.1软件对测序结果进行氨基酸比对分析和构建进化树.结果 A/湖南开福/SWL4142/2009（H1N1）,A/湖南长沙/SWL4346/2009（H1N1）和A/湖南芙蓉/SWL4224/2009（H1N1）3株病毒株HA基因序列分别与甲型H1 N1流感病毒A/NewYork/3502/2009（H1N1）,A/Shanghai/71T/2009（H1N1）和A/Chita/01/2009（H1N1）分离株高度同源,同源性为99%,与国内甲型H1N1流感病毒株A/Sichuan/1/2009（H1N1）核苷酸同源性在99.5%以上 进化树分析显示包括3株病毒株在内的甲型H1N1流感病毒与A/Swine/Indiana/P12439/00亲缘关系近,但与人季节性A流感病毒（H1N1）、禽流感亲缘关系较远.与A/Swine/Indiana/P12439/00 HA基因比较发现：3株病毒株HA基因分别有28、30、27个氨基酸发生变异,但3株病毒株在21个重要抗原位点中只有一个R53K氨基酸替换 对全球364条甲型H1N1流感病毒HA基因序列进行多重比对发现有119个非保守氨基酸位点,其中5个位于重要抗原位点.结论 3株病毒株和其他甲型H1N1流感病毒HA基因可能由北美猪流感病毒变异而来 3株病毒株HA基因与国内外甲型H1N1流感病毒高度同源,同全球绝大多数甲型H1N1流感病毒一样处于稳定状态.

Objective To understand the origin and variation of the hemagglutinin gene of isolates viruses from 3 novel influenza A（ H1N1 ） deaths in Changsha （ A/Hunan Kaifu/SWL4142/2009 （ H1N1 ） , A/Hunan Changsha/SWL4346/2009 （ H1 N1 ） and A/Hunan Furong/SWL4224/2009（ H1N1 ））. Methods The nasopharyngeal swab specimens from the 3 novel influenza A（ H1N1 ） deaths in Changsha were tested by RT-PCR and influenza viruses were isolated simultaneously. With the sequencing primers recommended by World Health Organization （WHO）, the HA gene of sequences of 3 novel influenza A（ H1N1 ） deaths were tested by CEQTM 8000 Genetic Analysis System, through dye terminator cycle sequencing. The sequencing results were submitted to GenBank, then the results were analyzed for amino acid alignment and phylogenetic tree analysis with ClustalX and Mega4.1 software. Results All the nucleotide homologies of HA gene sequences in A/Hunan Kaifu/SWL4142/2009 （ H1N1 ）, A/Hunan Changsha/SWL4346/2009 （ H1N1 ） and A/Hunan Furong/SWL4224/2009（ H1N1 ） are 99% as compared with the novel influenza A（ H1N1 ） virus strains of A/NewYork/3502/2009 （ H1N1 ）, A/Shanghai/71T/2009 （ H1N1 ） and A/Chita/01/2009 （ H1N1 ）The nucleotide homology of the 3 HA gene sequences are more than 99. 5% the same compared with the novel influenza A（ H1N1 ） virus strain （ A/Sichuan/1/2009（ H1N1 ） ） in China. Phylogenetic tree analysis reveals that 2009 novel influenza A（H1N1 ） viruses including 3 HA gene sequences of A/Hunan Kaifu/SWL4142/2009 （ H1 N1 ）, A/Hunan Changsha/SWL4346/2009 （ H1N1 ）, A/Hunan Furong/SWL4224/2009（ H1N1 ） had a close evolutionary relationship with the swine H1 virus isolates in North America （ A/Swine/Indiana/P12439/00）, but a distant evolutionary relationship with those human seasonal A（ H1 N1 ） influenza virus and avian. After comparing with genes of A/Swine/Indiana/P12439/00, we found that the HA gene sequences of the 3 viruses isolated had 28,30 and 27 amino acids with mutation respectively, but only one （R53K） amino acids mutation at 21 important antigenic sites in the 3 viruses isolated. Multiple alignment of 364 HA genes sequences of novel influenza A （ H1N1 ） viruses in the world showed they had 119 nonconserved amino acids, 5 non-conserved position at important antigenic sites. Conclusions The HA gene sequences from 3 viruses isolated in this study and other influenza A （ H1N1 ） viruses might originate from swine A（ H1N1 ） in North America by variation. The 3 HA gene sequences of viruses isolated have high homology as compared with the novel influenza A （ H1N1 ） virus strains worldwide, and the 3 HA gene sequences of viruses isolated are in stable condition as the vast majority of novel influenza A（ H1N1 ） virus strains in the world.