浙江省近年甲型H1N1流感病毒神经氨酸酶基因序列分析

Sequence analysis of neuraminidase gene of influenza A( H1N1) pdm09 viruses in Zhejiang

目的分析浙江省2009—2013年初甲型H1N1流感病毒神经氨酸酶基因（NA）序列进化特征。方法提取浙江省2009—2013年初29株甲型H1N1流感病毒基因组RNA，反转录一聚合酶链反应（RT—PCR）扩增NA基因，测序并拼接出ORF。以浙江省不同年份与地域15份代表性毒株NA序列和GenBank数据库中选取的22株2009—2012年国内外甲型H1N1流感病毒NA基因序列，采用MEGA5．1软件对其进行序列比对并构建种系发生树。结果扩增并测序获得29株甲型N1N1流感病毒的NA基因ORF的全长序列，与国内外甲型H1N1流感病毒NA序列比对后显示序列的同源性较高，浙江省毒株与北美早期甲型H1N1流感病毒典型代表株A／California／04／2009（H1N1）的同源性为98．20％-99．50％，其中采集于2012年末和2013年初的4份病毒NA与A／California／04／2009（H1N1）的同源性为98．63％～99．14％。在1株采集于2010年1月的甲型H1N1流感病毒NA的基因序列中发现可导致奥司他韦耐药的H275Y突变基因型。结论虽然浙江省后期的甲型H1N1流感病毒神经氨酸酶基因累积了更多的变异，但所有毒株之间基因同源性仍然较高，所有毒株的神经氨酸酶基因同源性达到98．20％及以上，序列分析结果证实1份毒株携带奥司他韦耐药基因型突变。

Objective To understand the evolutionary characteristics of neuraminidase gene of influenza A （ H1 N1 ） pdm09 virus isolated in Zhejiang province from 2009 to early 2013. Methods Viral RNA was extracted from 29 strains of influenza A （ H1N1 ） pdm09 virus. Neuraminidase gene was amplified by RT-PCR and the ORF sequence was assembled together by using sequencing data. Neuraminidase genes from 22 representative domestic and overseas strains during 2009 -2012 were downloaded from GenBank database for analysis. All the sequences were used for the construction of phylogenetic tree by using MEGA 5.1 software. Results The 1410 bp ORF sequences of neuraminidase gene were obtained by analyzing sequencing results of the 29 strains. After alignment, high homology was observed among all the NA genes of the strains isolated all over the world. The homology between strains isolated in Zhejiang during 2009 -2012 and the representative strain of A/California/04/2009 （H1N1） from North America was as high as 98. 20% -99.50%. The homology between the 4 strains isolated in Zhejiang in late 2012 and early 2013 and A/Califoruia/04/2009 （H1 N1 ） strain was 98.63% -99. 14%. A H275Y mutation in neuraminidase protein which could confer resistance to oseltamivir was observed in an A （H1N1） pdm09 strain isolated in January 2010 through nucleic acid sequence analysis. Conclusion Although more mutations were accumulated in NA genes in the strains isolated in Zhejiang in late epidemic stage, high homology of 〉 98.20% was observed in all the NA genes. A H275Y mutation in neuraminidase protein which could confer resistance to oseltamivir was observed.