山东省H3N2亚型流感病毒的分子流行病学研究

发布时间：2018-07-03 17:27

本文选题：H3N2 + 流感病毒　；参考：《山东大学》2017年硕士论文

【摘要】：[背景]流行性感冒病毒共分为甲、乙、丙、丁型,其中季节性H3N2亚型流感在人群中普遍流行。HA蛋白和NA蛋白是H3N2流感病毒表面主要的两种抗原,经常频繁地发生变异,对发生流感感染和造成流行具有重要意义,其中HA蛋白的遗传变异进化发生得最频繁。HA蛋白的抗原位点、受体结合部位及关键的糖基化位点如果发生氨基酸替换,病毒的致病性和感染的宿主种类就会相应发生改变。NA蛋白又作为药物治疗的重要靶点,酶活性中心的关键位点发生变异会导致耐药性。因此研究H3N2亚型流感病毒的HA基因和NA基因在流感流行中的基因进化规律和分子变异特点具有重要意义。[目的]了解2014年4月至2015年3月山东省流感病毒病原学监测的描述性流行病学分布情况,以及在H3N2亚型流感病毒的流行过程中HA基因和NA基因的分子流行病学分布特点,为预测流感发展趋势及预防控制流行提供基础理论依据。[方法]本研究首先对山东省流感监测数据库进行流感病毒病原学监测情况的基本描述,然后选择山东省疾病预防控制中心流感实验室于2014年4月至2015年3月期间分离保存的H3N2亚型流感毒株102株,并对其进行复苏,最后共95株H3N2亚型流感病毒毒株进行RT-PCR扩增和基因测序。利用DN AStar软件的EditSeq模块编辑序列及MegAlign模块分析同源性;多序列比对采用Molecular Evolutionary Genetics Analysis 5.0(MEGA 5.0)软件的ClustalW程序;系统发生树的构建使用MEGA 5.0软件Neighbor-Joining(NJ)法。[结果]1.山东省于2014年4月至2015年3月共收集了21276份流感样病例鼻咽拭子标本,描述分析发现2014年10月至2015年3月为流感流行高峰期,感染人群主要为0～15岁人群,以5岁以下比例最高;共分离出2371株流感病毒细胞株,其中A(H3N2)流感病毒占比例最高(1665株),并且2014年11至2015年1月是H3N2亚型流感的流行高峰期。2.山东省所分离的H3N2亚型流感病毒的HA和NA基因序列的同源性分析结果分别为98.0～100%和97.7～100%。HA基因系统发生树分析显示,大多数山东省分离的H3N2流感病毒的HA基因聚集成一簇,与A/Switzerland/9715293/2013(H3N2)的亲缘关系最近,与A/Texas/50/2012(H3N2)的亲缘关系较远。NA基因系统发生树分析提示,大多数山东省分离的H3N2流感病毒NA基因聚集分布在一个大分支,与 A/Switzerland/9715293/2013(H3N2)的遗传距离最短,与 A/Texas/50/2012(H3N2)的遗传距离最长,均未发现明显的地域和时间特征。3.HA蛋白共52个位点发生氨基酸替换,其中抗原决定簇(HA1蛋白)位点发生了 17个,裂解位点序列并没有发生变异,丢失糖基化位点NES134-136和NSS156-158,同时新增糖基化位点NYT170-172。NA蛋白有51个位点发生氨基酸替换,酶活性中心位点118(D-N)、371(V-I)发生变异,催化位点156(V-I)、425(T-N)发生变异,5个抗原位点发生变异,丢失糖基化位点NWS77-79和NDS320 322,同时新增糖基化位点NIT84-86和NAS236-238。[结论]1.2014年4月至2015年3月山东省流感病毒流行的优势毒株是H3N2亚型流感病毒,其流行高峰时间为冬春季(2014年11月、2014年12月、2015年1月)。2.2014年4月至2015年3月山东省分离的H3N2亚型流感流行株的HA基因和NA基因的同源性较高,但与当年疫苗株的遗传距离稍远,与下一年的疫苗株遗传距离较近;HA蛋白和NA蛋白均有部分抗原决定位点出现氨基酸替换现象,HA基因普遍可能发生了抗原性漂移,WHO推荐的2014～2015年疫苗株对H3N2亚型流感病毒保护效果可能较差,而2015～2016年的疫苗株可能对其具有一定保护效果。3.部分H3N2亚型分离毒株的HA基因和NA基因的糖基化位点也发生了变异,不同功能的基因片段发生糖基化位点的变异可能产生不同的影响。4.所有毒株的NA蛋白的酶活性中心的NA抑制剂抑制位点未发现变异,所以推测病毒均对奥司他韦敏感。
[Abstract]:[background] influenza viruses are divided into a, B, C, and B, among which seasonal H3N2 subtype influenza pandemic.HA and NA are the main two antigens on the surface of the H3N2 influenza virus, often frequent variation, which is of great significance for the occurrence of influenza infection and epidemic, in which the genetic variation and evolution of the HA protein are evolved. The antigen site of the most frequent.HA protein, the receptor binding site and the key glycosylation site if the amino acid substitution occurs, the virus pathogenicity and the host species of the infection will change the.NA protein as an important target for the drug treatment. It is of great significance to investigate the gene evolution and molecular variation of the H3N2 subtype influenza virus HA gene and NA gene in influenza epidemic. [Objective] to understand the descriptive epidemiological distribution of influenza virus pathogens in Shandong province from April 2014 to March 2015, and the HA gene and N in the epidemic process of H3N2 subtype influenza virus. The distribution characteristics of the molecular epidemiology of A gene provide basic theoretical basis for predicting the trend of influenza development and prevention and control. [Methods] the basic description of influenza virus pathogen monitoring in Shandong province was first described in this study, and then the Influenza Laboratory in Shandong province was selected in April 2014. During the period of March 2015, 102 strains of H3N2 subtype influenza strain were separated and recovered. In the end, 95 strains of H3N2 subtype influenza virus strains were amplified and sequenced by RT-PCR. The EditSeq module editing sequence and MegAlign module of DN AStar software were used to analyze the homology, and Molecular Evolutionary Genetics Ana was used in the multi sequence column ratio. ClustalW program of lysis 5 (MEGA 5) software; construction of phylogenetic tree using MEGA 5 software Neighbor-Joining (NJ). [results]1. Shandong province collected 21276 cases of influenza like nasopharyngeal swab specimens from April 2014 to March 2015, describing the peak of influenza epidemic from October 2014 to March 2015 and the main infection population The highest proportion of 0~15 year olds under 5 years old, 2371 strains of influenza virus cells were isolated, among which A (H3N2) influenza virus accounted for the highest proportion (1665 strains), and from 11 to January 2015 2014 was the homology analysis of HA and NA gene sequences of H3N2 subtype of H3N2 subtype virus isolated from.2. Shandong province. The analysis of 98 ~ 100% and 97.7 ~ 100%.HA gene phylogenetic tree showed that most of the HA genes isolated from the H3N2 influenza virus in Shandong province were integrated into one cluster, closest to A/Switzerland/9715293/2013 (H3N2), and the relative relationship with A/Texas/50/2012 (H3N2) was far away from the genetic tree analysis of the.NA gene system, most of the Shandong Province The isolated H3N2 influenza virus NA gene is distributed in a large branch, with the shortest genetic distance to A/Switzerland/9715293/2013 (H3N2), the longest genetic distance from A/Texas/50/2012 (H3N2), and no obvious geographical and temporal characteristics of the 52 loci of the.3.HA protein. The antigen determinant (HA1 protein) site occurs. 17, there was no variation in the sequence of lysis sites, the loss of glycosylation sites NES134-136 and NSS156-158, and the new glycosylation site NYT170-172.NA protein had 51 amino acid substitutions, the enzyme activity center site 118 (D-N), 371 (V-I) mutation, the catalytic site 156 (V-I), 425 (T-N) mutation, 5 in situ variation, The loss of glycosylation sites NWS77-79 and NDS320 322, and the addition of glycosylation sites NIT84-86 and NAS236-238.[concluded that the dominant strain of influenza virus epidemic in Shandong province from April to March 2015]1.2014 was H3N2 subtype influenza virus. Its peak time was winter and spring (November 2014, December 2014, January 2015).2.2014 April to 3 months of 2015. The HA and NA genes of H3N2 subtype influenza strains isolated from East Province have high homology, but the genetic distance from the vaccine strain is a little far away, and the genetic distance of the vaccine strain is close to the next year. The HA protein and NA protein all have amino acid replacement at some antigen determination points, and the HA gene may have the antigenicity drift, WHO recommends. The protective effect of the 2014~2015 year vaccine strain on the H3N2 subtype influenza virus may be poor, and the 2015~2016 year vaccine strain may have some protective effect on it, the HA gene and the glycosylation site of the NA gene are also mutated, and the mutation of the glycosylation site of the different functional base fragments may produce the mutation. There was no variation in the inhibitory sites of the NA inhibitors of the NA active protein of all.4. strains, so we speculated that the virus was sensitive to oseltamivir Wei Min.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R511.7;R181.3

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