南宁分离株A(H3N2)流感病毒神经氨酸酶遗传变异及蛋白结构分析

Analysis of the characteristics of NA gene and the structure of its protein in the influenza virus A subtype H3N2 in Nanning

目的应用生物信息学方法分析南宁市2007-2012年A(H3N2)亚型流感病毒分离株神经氨酸酶NA的遗传变异规律、二硫键、酶活性中心位点、抗原决定簇位点、糖基化位点及蛋白结构变化情况。方法通过RT-PCR扩增H3N2病毒NA基因并测序,运用生物软件对所测序列进行拼接和同源比对;通过构建系统进化树分析进化规律,通过同源建模分析蛋白结构的变化。结果系统进化树将40株NA基因序列分为4个类群,呈多侧支流行;毒株的二硫键高度保守,酶活性中心位点224和位点276出现氨基酸的替换,部分毒株NA抗原决定簇328、332、336、434氨基酸位点发生替换;所有毒株200、329和402糖基化位点全部丢失,部分毒株在367位点增加一个糖基化位点,在234位点丢失一个糖基化位点;HA晶体结构分析氨基酸突变位点主要发生在β折叠部位和无规则卷曲部位。结论南宁分离株A(H3N2)亚型流感病毒变异活跃,抗原决定簇位点、酶活性中心位点、糖基化位点均出现氨基酸的替换。因此应继续加强对南宁市的流感监测,及时了解当地流感的流行趋势和病毒的抗原变异情况,为全国的流感预防、控制、预测及流感疫苗株的开发提供理论依据。

Objectives To use bioinformatics to analyze genetic variation in the neuraminidase （NA） gene and variations in disulfide bonds, enzyme active sites, antigenic determinant sites, glycosylation sites, and protein structures in the influenza A virus subtype H3N2 in Nanning from 2007 to 2012. Methods The NA gene was amplified with RT-PCR and sequenced. Sequencing results were assembled and their homology was compared by constructing a phylogenic tree and analyzing evolutionary patterns. Changes in protein structures were analyzed by modeling homology. Results Accord- ing to the phylogenetic tree, sequences of the NA gene from 40 strains of the influenza virus were divided into four groups that were predominantly located on numerous branches. All of the strains had disulfide bonds that were highly conserved. All of the strains displayed mutations in enzyme active sites at amino acids 224 and 276. Some of the strains had antigenic determinant sites displayed mutations at amino acids 328, 332, 336, and 434. All of the strains lacked mutations in glycosylation sites at amino acids 200, 329, and 402. Some strains added a glycosylation site at amino acid 367 and lost a glyco sylation site at amino acid 234. Mutations at amino acid sites occurred mainly at sheets and random coils according to a nalysis of the crystal structure of NA. Conclusion Strains of the influenza A virus subtype H3N2 in Nanning actively varied, and the subtype had amino acid substitutions at antigenic determinant sites, enzyme active sites, and glycosylation sites. Influenza surveillance in Nanning should be enhanced to promptly discern trends in influenza prevalence and viral antigenic variation in order to provide a theoretical basis for flu prevention, control, and forecasting and in order to develop strains for influenza vaccines.