本文選題：流感流行特征　切入點(diǎn)：H_3N_2　出處：《武漢科技大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【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體之一,病毒的傳播途徑廣(飛沫、氣溶膠等),速度快,并易發(fā)生變異。病毒不僅可引起人、豬、禽、犬和馬等跨物種傳播,而且人類感染嚴(yán)重時(shí)可以導(dǎo)致病毒性肺炎、多臟器功能損傷、呼吸衰竭甚至死亡。通過哨點(diǎn)醫(yī)院的監(jiān)測(cè)可以系統(tǒng)了解湖北省流感的流行趨勢(shì)特征,同時(shí)通過對(duì)季節(jié)性H_3N_2的HA基因測(cè)序了解基因變異情況,為湖北省流感防控策略的制定提供理論依據(jù)。方法1.描述性流行病學(xué)分析收集湖北省2007～2012年的流感監(jiān)測(cè)資料,Excel 2013建立數(shù)據(jù)庫,繪制流行病學(xué)圖表,SPSS17.0處理數(shù)據(jù),檢驗(yàn)標(biāo)準(zhǔn)為α≤0.05,分析流感流行的年齡分布、季節(jié)分布、流行強(qiáng)度和流行毒株型別變化等。2.分子生物學(xué)研究(1)抗原性分析方法MDCK細(xì)胞復(fù)蘇病毒,收獲的病毒液做紅細(xì)胞凝集實(shí)驗(yàn)和紅細(xì)胞凝集抑制實(shí)驗(yàn)(HI)。(2)基因分析磁珠法提取病毒核酸,RT-PCR擴(kuò)增HA基因,1%瓊脂糖凝膠電泳測(cè)定PCR產(chǎn)物,測(cè)序部分由華大基因武漢分公司完成,采用DNA Star進(jìn)行序列拼接,使用Bioedit對(duì)核苷酸與氨基酸進(jìn)行序列比對(duì)分析,糖基化位點(diǎn)用Net NGlyc1.0server分析,MEGA5.2軟件N-J法構(gòu)建進(jìn)化樹。結(jié)果(1)流感樣病例(influenza like illness,ILI)監(jiān)測(cè)特征:2007、2008年ILI%有明顯的夏季和冬季兩個(gè)流行峰,2009年呈現(xiàn)秋冬季單峰流行,2010、2011和2012年全年未呈現(xiàn)流行峰。15歲以下青少年ILI%占92%,尤其是5歲以下嬰幼兒比例占66%,是主要的感染人群。(2)流感流行特征:2007年流感以h_3n_2流行為主;2008年以h1n1流行為主;2009年以新甲型h1n1為主;2010年主要以h_3n_2流行為主;2011年主要以新h1n1流行為主;2012年以h_3n_2流行為主。2007、2008、2009、2010和2012年為典型的春冬和夏季兩個(gè)流行峰,2011年只有春冬一個(gè)流行峰。(3)h_3n_2抗原性變異分析:以a/hubei/jingzhou/0779/2012(h_3n_2)作為參照抗原,所測(cè)毒株效價(jià)與標(biāo)準(zhǔn)抗原相比,有4倍以上的差異,說明毒株間抗原性存在差異。所測(cè)毒株的核苷酸序列與同時(shí)期(2007～2012年)的標(biāo)準(zhǔn)疫苗株進(jìn)行比對(duì),有較高的同源性,為97%～99%。(4)h_3n_2病毒ha基因糖基化位點(diǎn)分析:xZ口和江岸地區(qū)毒株有9個(gè)保守的糖基化位點(diǎn),荊州和當(dāng)陽地區(qū)毒株也是有9個(gè)保守的糖基化位點(diǎn),但位置與xZ口、江岸地區(qū)都不相同,而湖北宜都則有12個(gè)糖基化位點(diǎn),數(shù)量和位置均與本研究湖北省其他地方不同。(5)h_3n_2病毒ha基因氨基酸變異分析:ha1區(qū)共有50個(gè)氨基酸位點(diǎn)發(fā)生突變,包括a區(qū)(140,142,144,145),b區(qū)(157,158,159,189,194,198),c區(qū)(53,278),d區(qū)(207)和e區(qū)(81)位點(diǎn),受體結(jié)合部位有2個(gè)位點(diǎn)發(fā)生突變,即194和225位,其中194位于抗原決定簇b區(qū),225在非抗原區(qū)。結(jié)論(1)湖北省2009年新甲型h1n1流感流行過后ili%降低,流行高峰不明顯,應(yīng)繼續(xù)全年開展監(jiān)測(cè),5歲以下嬰幼兒為主要監(jiān)測(cè)對(duì)象,是流感的易感人群,應(yīng)及時(shí)接種疫苗來增強(qiáng)保護(hù)力。(2)湖北省2007～2012年間流感流行主要為春冬和夏季兩個(gè)流行高峰,與傳統(tǒng)的流感流行季節(jié)性相吻合,流感季節(jié)以優(yōu)勢(shì)毒株流行為主,非流行季節(jié)為多種流感病毒混合發(fā)病,h_3n_2亞型流感病毒每隔一年就作為優(yōu)勢(shì)毒株引起流感流行,應(yīng)密切關(guān)注它的流行趨勢(shì)。(3)湖北省6年間h_3n_2亞型流感發(fā)生了抗原性變異,結(jié)合進(jìn)化樹的結(jié)果分析,毒株核苷酸序列與同時(shí)期疫苗株比對(duì)有較高的同源性,但同一年間,毒株間基因存在一定差異。(4)湖北省不同地域h_3n_2流感病毒的糖基化位點(diǎn)在數(shù)量和位置上都有很大差異,糖基化位點(diǎn)的增加或缺失對(duì)流感的變異有影響。(5)湖北省H_3N_2亞型流感病毒氨基酸有較大變異,且抗原決定簇的突變主要集中在A、B兩區(qū)。本文通過結(jié)合湖北省2007～2012年流感病毒的流行病學(xué)和分子生物學(xué)兩方面的研究,對(duì)流感變異的趨勢(shì)有了一定的了解,為制定湖北省未來的防控措施提供理論依據(jù),同時(shí)也為中國其他地區(qū)流感流行的研究提供參考。
[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.

【學(xué)位授予單位】：武漢科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R511.7;R181.3

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