2007～2012年湖北省流感流行病调查和H3N2病毒HA基因分析

发布时间：2018-03-13 19:01

本文选题：流感流行特征　切入点：H_3N_2　出处：《武汉科技大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的流感病毒是引起急性呼吸道感染性疾病的主要病原体之一,病毒的传播途径广(飞沫、气溶胶等),速度快,并易发生变异。病毒不仅可引起人、猪、禽、犬和马等跨物种传播,而且人类感染严重时可以导致病毒性肺炎、多脏器功能损伤、呼吸衰竭甚至死亡。通过哨点医院的监测可以系统了解湖北省流感的流行趋势特征,同时通过对季节性H_3N_2的HA基因测序了解基因变异情况,为湖北省流感防控策略的制定提供理论依据。方法1.描述性流行病学分析收集湖北省2007～2012年的流感监测资料,Excel 2013建立数据库,绘制流行病学图表,SPSS17.0处理数据,检验标准为α≤0.05,分析流感流行的年龄分布、季节分布、流行强度和流行毒株型别变化等。2.分子生物学研究(1)抗原性分析方法MDCK细胞复苏病毒,收获的病毒液做红细胞凝集实验和红细胞凝集抑制实验(HI)。(2)基因分析磁珠法提取病毒核酸,RT-PCR扩增HA基因,1%琼脂糖凝胶电泳测定PCR产物,测序部分由华大基因武汉分公司完成,采用DNA Star进行序列拼接,使用Bioedit对核苷酸与氨基酸进行序列比对分析,糖基化位点用Net NGlyc1.0server分析,MEGA5.2软件N-J法构建进化树。结果(1)流感样病例(influenza like illness,ILI)监测特征:2007、2008年ILI%有明显的夏季和冬季两个流行峰,2009年呈现秋冬季单峰流行,2010、2011和2012年全年未呈现流行峰。15岁以下青少年ILI%占92%,尤其是5岁以下婴幼儿比例占66%,是主要的感染人群。(2)流感流行特征:2007年流感以h_3n_2流行为主;2008年以h1n1流行为主;2009年以新甲型h1n1为主;2010年主要以h_3n_2流行为主;2011年主要以新h1n1流行为主;2012年以h_3n_2流行为主。2007、2008、2009、2010和2012年为典型的春冬和夏季两个流行峰,2011年只有春冬一个流行峰。(3)h_3n_2抗原性变异分析:以a/hubei/jingzhou/0779/2012(h_3n_2)作为参照抗原,所测毒株效价与标准抗原相比,有4倍以上的差异,说明毒株间抗原性存在差异。所测毒株的核苷酸序列与同时期(2007～2012年)的标准疫苗株进行比对,有较高的同源性,为97%～99%。(4)h_3n_2病毒ha基因糖基化位点分析:xZ口和江岸地区毒株有9个保守的糖基化位点,荆州和当阳地区毒株也是有9个保守的糖基化位点,但位置与xZ口、江岸地区都不相同,而湖北宜都则有12个糖基化位点,数量和位置均与本研究湖北省其他地方不同。(5)h_3n_2病毒ha基因氨基酸变异分析:ha1区共有50个氨基酸位点发生突变,包括a区(140,142,144,145),b区(157,158,159,189,194,198),c区(53,278),d区(207)和e区(81)位点,受体结合部位有2个位点发生突变,即194和225位,其中194位于抗原决定簇b区,225在非抗原区。结论(1)湖北省2009年新甲型h1n1流感流行过后ili%降低,流行高峰不明显,应继续全年开展监测,5岁以下婴幼儿为主要监测对象,是流感的易感人群,应及时接种疫苗来增强保护力。(2)湖北省2007～2012年间流感流行主要为春冬和夏季两个流行高峰,与传统的流感流行季节性相吻合,流感季节以优势毒株流行为主,非流行季节为多种流感病毒混合发病,h_3n_2亚型流感病毒每隔一年就作为优势毒株引起流感流行,应密切关注它的流行趋势。(3)湖北省6年间h_3n_2亚型流感发生了抗原性变异,结合进化树的结果分析,毒株核苷酸序列与同时期疫苗株比对有较高的同源性,但同一年间,毒株间基因存在一定差异。(4)湖北省不同地域h_3n_2流感病毒的糖基化位点在数量和位置上都有很大差异,糖基化位点的增加或缺失对流感的变异有影响。(5)湖北省H_3N_2亚型流感病毒氨基酸有较大变异,且抗原决定簇的突变主要集中在A、B两区。本文通过结合湖北省2007～2012年流感病毒的流行病学和分子生物学两方面的研究,对流感变异的趋势有了一定的了解,为制定湖北省未来的防控措施提供理论依据,同时也为中国其他地区流感流行的研究提供参考。
[Abstract]:The purpose of the influenza virus is one of the major pathogens of acute lower respiratory tract infection, virus propagation (Guangzhou aerosol droplets, etc.), fast, and easy to mutate. The virus can cause not only human, pig, bird, dog and horse cross species transmission, and human infection can lead to serious viral pneumonia, multiple organ damage, respiratory failure and even death. The sentinel hospitals can understand the trends feature of influenza in Hubei Province, at the same time through the HA gene sequencing of H_3N_2 gene to understand the seasonal variation, and provide a theoretical basis for the development of Hubei province flu prevention and control strategy. Methods 1. descriptive epidemiological analysis of influenza surveillance data collection Hubei province for 2007~2012 years, Excel 2013 database, drawing the chart data in SPSS17.0, epidemiology, inspection standards for alpha = 0.05, the age distribution of the popular sense of flow analysis, Seasonal distribution of molecular biology of.2. type strains and epidemic intensity changes (1) antigenicity analysis method of MDCK cell recovery virus, the virus was harvested for hemagglutination test and hemagglutination inhibition assay (HI). (2) gene analysis method to extract bead virus nucleic acid amplification of HA gene, RT-PCR, determination of PCR products 1% agarose gel electrophoresis and sequencing in part by BGI Wuhan branch, using DNA Star sequences, Bioedit of nucleotide and amino acid sequences of Net, analysis of NGlyc1.0server glycosylation sites, the phylogenetic tree of MEGA5.2 software N-J method. Results (1) influenza like illness (influenza like illness, ILI) monitoring feature: 20072008 years ILI% has obvious in summer and winter of two peaks, 2009 autumn winter peak epidemic in 20102011 and 2012 full year did not show peaks of.15 years of age. ILI% years accounted for 92%, especially in infants under 5 years of age accounted for 66%, is the main infected population. (2) the epidemic characteristics of influenza h_3n_2 influenza epidemic: 2007 to 2008; in 2009 with a new H1N1 epidemic; influenza A H1N1; in 2010, mainly in the h_3n_2 epidemic; 2011 in New popular H1N1; 2012 the popular h_3n_2.2007200820092010 and 2012 mainly for the typical winter and spring and summer two peaks, 2011 spring and winter a popular peak. (3) analysis of h_3n_2 antigenic variation: a/hubei/jingzhou/0779/2012 (h_3n_2) as reference antigen, measured strain titer and standard antigen compared to a difference of more than 4 times, indicating the presence of strains of antigenic differences. Nucleotide sequence of measured strains with the same period (2007~2012 years) of the vaccine strain were compared, had high homology, 97% ~ 99%. (4) h_3n_2 virus HA gene glycosylation Analysis: strain xZ port and Jiangan District, there are 9 conserved glycosylation sites, strains of Jingzhou and Dangyang area also has 9 conserved glycosylation sites, but the position of the mouth and xZ, Jiangan District, Hubei and Yidu are not the same, there are 12 glycosylation sites, number and location are different with this research in other areas of Hubei province. (5) analysis of h_3n_2 virus HA gene amino acid mutation: HA1 district has a total of 50 missense mutations, including a (140142144145), B (157158159189194198), C (53278), D (207) and E (81) receptor binding sites. Part 2 mutations, namely 194 and 225, of which 194 is located in the antigenic determinant of B region, 225 in non antigenic region. Conclusion (1) in Hubei Province in 2009 the new H1N1 influenza epidemic after ili% decreased, the epidemic peak is not obvious, should continue to carry out the monitoring of the infant under the age of 5, as the main monitoring the object is the flu The susceptible population, should be timely vaccination to enhance the protection force. (2) Hubei province 2007~2012 years influenza epidemic mainly in spring and winter and summer two peaks, consistent with the traditional flu season, the flu season to the dominant strains prevalent in non epidemic season for influenza virus mixed disease, h_3n_2 type A influenza virus every year as the dominant strain caused by influenza, should pay close attention to trends in it. (3) the antigenic variation of Hubei Province during 6 years of h_3n_2 influenza, analysis and phylogenetic tree analysis has high homology with the same period of vaccine strain than strain nucleotide sequence, but the same there are some differences between strains, gene. (4) glycosylation sites in different regions of Hubei province h_3n_2 influenza viruses have great differences in the number and position, increase or lack of variation of glycosylation sites of influenza. Ring. (5) there is considerable variation in Hubei province H_3N_2 subtype influenza virus mutation and epitope amino acids, mainly concentrated in A, B district two. Based on two aspects of epidemiology and molecular biology of influenza virus in Hubei province for 2007~2012 years, have a certain understanding of the trend of flu, provide the theoretical basis for the prevention and control of Hubei Province in the future, but also provide reference for the study of influenza China in other areas.

【学位授予单位】：武汉科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R511.7;R181.3

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