2009甲型H1N1流感病毒神经氨酸酶参照序列的建立及特征分析

Establishment of Neuramidinase Reference Sequence and Analysis of Influenza A(H1N1) Virus in 2009

目的建立2009甲型H1N1流感病毒神经氨酸酶(NA)区的参照序列并分析其特征。方法通过GenBank获得100株来自2009甲型H1N1流感病毒NA区全基因序列,利用Pri mer软件进行序列比对,采取相同碱基或氨基酸位置相同碱基或氨基酸最多者作为参照碱基或氨基酸,最终获得参照序列,同时分析所有入选病毒株的基因及氨基酸变异情况;将参照序列与1918年(1918E)、1934年(1934E)流行株,2008年(2008V)、2009年(2009V)疫苗株以及欧亚猪H1N1流感病毒流行株(SWINE)和人禽流感H5N1流行株(H5N1)序列进行比对,分析其基因变异、二硫键和活性中心分布、糖基化位点、抗原表位等特征。结果建立了2009甲型H1N1流感病毒NA区参照序列,与参照株比较,所有分离株的核苷酸及氨基酸同源性均>99.00%,核苷酸变异≥5株的位点有6个,氨基酸变异≥5株的位点有2个;该参照序列与相关病毒株核苷酸同源性分别99.79%(2009V)、77.66%(2008V)、78.01%(1934E)、83.19%(1918E)、92.13%(SWINE)、81.91%(H5N1),氨基酸同源性分别为99.57%(2009V)、80.81%(2008V)、80.81%(1934E)、87.42%(1918E)、92.32%(SWINE)、84.22%(H5N1);二硫键和活性中心分布高度保守;2009C与2009V和1918E病毒株糖基化位点类似,与其他病毒株差异较大;抗原表位与2009V完全相同,而与2008V相比,已知存在的表位分别为3?1(B细胞表位)、2:1(Th细胞表位)和7:3(CTL细胞表位)。结论该参照序列可以作为2009甲型H1N1流感病毒NA区参照序列,成为分析基因变异或氨基酸变异的标准。

OBJECTIVE To establish the consensus sequence of the neuraminidase（NA） gene of A/H1N1/2009 influenza virus and analyze some of its most important characteristics.METHODS A total of 100 different NA gene sequences of A/H1N1/2009 influenza virus were collected from GenBank,and software Primer was used to compare different sequences and the final reference sequence was created by adopting the most frequently-occurred nucleotide or amino acid in each position.We compared our established reference sequence with those of selected strains at both the nucleotide and amino acid levels in terms of nucleotide or amino acid variation,disulfide bonds,distribution of active sites,glycosylation sites,and antigenic epitopes.These strains included：1918 epidemic strain（1918E）,1934 epidemic strain（1934E）,vaccine strains of 2008（2008V）,2009（2009V）,European pandemic swine-origin H1N1 influenza virus（SWINE） and avian influenza A（H5N1） in humans.RESULTS The reference sequences of NA gene of A/H1N1/2009 influenza virus were established.The homology between the reference strain（should be reference sequence） and all isolates was greater than 99% at both the nucleotide and amino acid levels.Six sites where at least 5 nucleotide variations occurred and two sites where at least 5 amino acid variations occurred in different strains were identified.The homology between our reference sequence and 2009V,2008V,1934E,1918E,SWINE and H5N1 at the nucleotide level was 99.79%,77.66%,78.01%,83.19%,92.13%,and 81.91%,respectively.At the amino acid level,the homology was 99.57%,80.81%,80.81%,87.42%,92.32%,and 84.22% respectively.However,disulfide bonds and the distribution of active sites were highly conserved.The distribution of glycosylation sites in 2009C was similar to that of the 2009V and 1918E strains,but significantly different from that of other strains.The predicted antigenic epitopes of 2009 were identical to those of the 2009V strain,but when compared to the 2008V strain,more antigenic epitopes were identified for our 2009C reference sequence： 3 and 1 respectively for B-cell epitope;2 and 1 respectively for the cell epitope;and 7and 3 respectively for CTL cell epitope.CONCLUSION This reference sequence can serve as the NA gene reference sequence for A/H1N1/2009 influenza virus and the standard for analyzing genetic or amino acid variations.