深圳市2007年流感流行特征及病毒分子变异研究

发布时间：2018-03-29 08:50

本文选题：流感病毒　切入点：流感样病例　出处：《郑州大学》2008年硕士论文

【摘要】：目的了解深圳市2007年流感流行特征,探讨流感病毒血凝素蛋白重链(HA1)区基因特性、抗原变异及其与流感流行的关系,为流感防治提供科学依据。材料与方法收集深圳市2007年流感样病例和流感样病例暴发疫情资料,用EXECL和SAS 8.0统计软件对流感样病例就诊百分比进行时间趋势、年龄分布情况统计分析,并设计流感监测预警线和平均水平线。采集流感样病例鼻咽拭子标本,同时接种狗肾传代细胞和9～11日龄鸡胚的羊膜腔和尿囊腔进行流感病毒分离培养,用红细胞凝集及凝集抑制试验实现病毒滴度及型别鉴定。采用随机整群抽样法于流感流行期前、后采集一般人群血清了解深圳人群的血清抗体水平。对所有分离病毒提取RNA,用RT-PCR扩增HA1区基因片段,产物纯化后测序;用DNASTAR软件、SIMMONIC软件和MEGA3.1软件进行核苷酸—氨基酸序列分析、同源性比较、基因进化树构建等分子变异研究,并与WHO推荐北半球疫苗株和国家代表株进行比较,揭示流感病毒HA1区基因变异、抗原性改变及其与流感流行的关系。结果 1.深圳市2007年流感流行特征 2007年深圳市流感样病例(influenza-like illness,简称ILI)就诊百分比曲线呈单峰分布,流感活动高峰主要分布在4～7月,ILI就诊百分比于第25周达到峰值11.22%,超过预警线值10.86%,较2005、2006年明显提高,且持续时间长;ILI主要集中在0～14岁年龄组儿童,占流感样病例总数的72.97%;学校流感流行高峰较早出现;深圳流感监测预警线具有一定的季节性和灵敏度,能及时发现异常值。2007年共报告流感样病例暴发疫情50起,经实验室PCR检测,35起流感病毒阳性,其中A型29起,B型6起;时间上主要分布在3～6月,占92.0%,88.0%发生于中小学校。2007年共检测流感样病例鼻咽拭子标本2831份,343份阳性,分离率为12.12%;H1N1亚型、H3N2亚型、B型流感病毒所占构成比分别为3.5%、62.68%和33.82%;其中1～6月份以H3N2亚型为主要流行株,7～12月份则以B(Yamagata)系为主要流行株。月病毒分离率1～6月份逐步升高,6月份最高为28.38%,后按月递减,与ILI就诊百分比时间变化趋势一致。结合往年资料发现主要流行株交替出现。2007年3、9月份共采集一般人群血清1700份,H3N2亚型血清抗体阳性率最高,3、9月份分别为:65.82%和72.94%,经检验具统计学意义(P＜0.05),这与2007年H3N2亚型病毒活动水平较高有关;H1N1亚型、B(Victoria)系、B(Yamagata)系毒株3、9月份抗体阳性率分别为46.98%、43.80%、43.84%、21.90%、41.18%和21.67%;除H3N2亚型外,其它三个型别流感病毒几何平均滴度偏低。 2.深圳市2007年流感病毒血凝素蛋白重链区基因变异 H3N2亚型流感病毒血凝素重链(HA1)区核苷酸同源性和基因进化树结果均显示:2007年H3N2亚型毒株分为两个分支,1～4月份部分毒株与国家代表株A/JXDH/312/2006(H3N2)在一个分支,该部分毒株占1～4月份H3N2亚型分离株的33.33%;5月份之后的毒株与2008～2009年疫苗株A/Brisbane/10/2007(H3N2)为同一分支,并在流行上占优势,该部分毒株占2007年H3N2亚型分离株的89.30%;2005～2006年毒株与2006～2007年度疫苗株A/Wisconsin/67/2005(H3N2)距离较近;A/Brisbane/10/2007(H3N2)是从A/Wisconsin/67/2005(H3N2)进化而来的抗原变异株。氨基酸位点是随着时间发展逐步变化。与参照株A/Wisconsin/67/2005(H3N2)比较发现,所有毒株发生D122N、1223V氨基酸替换,1～4月份部分毒株在抗原决定簇A区发生R142G和N144D氨基酸变异,该变异没有被保留下来,5月份之后的毒株发生G50E、K140I氨基酸替换;D122N、N144D氨基酸置换分别导致增加和丢失一个糖基化位点。受体结合位点氨基酸较保守。 H1N1亚型流感病毒HA1区核苷酸同源性和基因进化树结果一致。HA1区基因进化树结果为2007年、2006年毒株和国家代表株A/GDLH/219/2006(H1N1)在一个分支,2005年毒株分为两个分支,4月份之前分离株与A/NewCaledonia/20/1999(H1N1)为同一分支,5月份之后的分离株与A/SolomonIsland/3/2006(H1N1)为一支。氨基酸序列分析显示,2005年5月以后的大部分毒株出现以下氨基酸变异:T82K、Y94H、R146K、R209K和T267N,2006年5月份之后的毒株在抗原决定簇B区发生了A190T、H193Y和E195D氨基酸变异,同时也发生A区R146K的置换。所有毒株的糖基化和受体结合位点比较保守。 2007年B型流感病毒HA1区基因进化树分为B(Victoria)和B(Yamagata)两个谱系,其中B(Victoria)系毒株与深圳2006年分离株、本年度疫苗株B/Malaysia/2506/2006、国家代表株B/Shenzhen/155/2005同在一个分支;B(Yamagata)系毒株与国家代表株B/FJXL/54/2006距离最近,其次为2008～2009年度疫苗株B/Florida/4/2006。氨基酸位点分析显示:B(Victoria)毒株发生S134P氨基酸变异,B(Yamagata)系毒株发生R48K、P108A、L131P和D197N氨基酸替换;197-199糖基化位点变异频率较高,76.66%的B(Yamagata)系毒株发生D197N氨基酸置换导致增加一个糖基化位点。结论深圳市流感流行高峰在夏季;流感监测预警线和平均水平线具有较好的季节性和灵敏度,能及时发现异常值。低年龄组人群较易受流感病毒感染,学校流感监测值得重视。深圳每年主要流感流行株交替出现。2007年深圳市H3N2亚型流感病毒的基因特性和抗原性已发生变化,引起该型病毒较高水平的流行,流感病毒抗原变异和人群免疫水平共同影响着流感的流行;建议将分子生物学研究纳入流感监测,密切关注新型流感病毒株的出现。
[Abstract]:objective
Objective to understand the epidemiological characteristics of influenza in Shenzhen in 2007, and to explore the characteristics of influenza virus hemagglutinin protein heavy chain (HA1) region, antigen variation and its relationship with influenza epidemic, so as to provide a scientific basis for prevention and treatment of influenza.
Materials and methods
The collection of influenza like cases of influenza like cases in 2007 and the Shenzhen outbreak data, time trend by EXECL and statistical software SAS 8 influenza like cases percentage, age distribution statistical analysis, and design the warning line of influenza surveillance and the average. Influenza like cases were collected nasopharyngeal swab specimens, the influenza virus and amniotic cavity allantoic cavity inoculation MDCK cells and 9~11 day old chick embryos were cultured to achieve viral titer and type identification of red cell agglutination and agglutination inhibition test. By cluster random sampling in the influenza epidemic period before, after the acquisition of the general population to understand the serum serum antibody level in population of Shenzhen. All the isolated virus extract RNA. HA1 gene was amplified by RT-PCR, the products were sequenced after purification; using DNASTAR software, SIMMONIC software and MEGA3.1 nucleotide - amino acid sequence analysis, the same Genetic variation studies such as genetic comparison, construction of phylogenetic tree and comparison with WHO recommended northern hemisphere vaccine strains and national representative strains, reveal the genetic variation, antigenicity changes of influenza virus HA1 region and its relationship with influenza pandemic.
Result
1. influenza epidemic in Shenzhen in 2007
Shenzhen city in 2007 of influenza like illness (influenza-like illness, referred to as ILI) visiting percentage curve showed a unimodal distribution, the peak of influenza activity mainly in 4~7 months, visiting percentage of ILI at twenty-fifth weeks to reach a peak of 11.22%, exceeding the warning line value of 10.86%, compared to 20052006 years is higher, and lasted for a long time; ILI mainly concentrated in children aged 0~14 the age group, accounting for 72.97% of the influenza influenza epidemic peak appears earlier; school; Shenzhen influenza surveillance warning line has certain seasonal and sensitivity, can detect outliers in.2007 has reported an outbreak of influenza like 50, the laboratory of PCR detection, 35 influenza virus positive type A 29 B, type 6; time is mainly distributed in the 3~6 month, accounted for 92%, 88% occurred in primary and middle schools and.2007 were tested for influenza nasopharyngeal swab specimens of 2831 copies, 343 copies of the positive isolation rate was 12.12%; H1N1. Subtype, H3N2 subtype influenza virus B proportion were 3.5%, 62.68% and 33.82%; 1~6 months to subtype H3N2 was the predominant strain, 7~12 months to B (Yamagata) is the predominant strains. Virus isolation rate of 1~6 months increased gradually, the highest in June 28.38%, after the monthly rate, and ILI were the percentage of time trends consistent with former data. We found that the predominant strains appear alternately during 3,9 month.2007 were collected in the general population of 1700 serum samples, the positive rate of H3N2 subtype antibody was highest, 3,9 months were 65.82% and 72.94%, the test was statistically significant (P < 0.05), the relevant with the 2007 H3N2 virus subtype activity level is higher; H1N1 subtype, B (Victoria), B (Yamagata) 3,9 antibody positive rate in strain were respectively 46.98%, 43.80%, 43.84%, 21.90%, 41.18% and 21.67%; in addition to the H3N2 subtype, three other types of influenza virus A He Pingjun's titer is on the low side.
2. gene variation of hemagglutinin protein heavy chain region of influenza virus in Shenzhen city in 2007
H3N2 subtype influenza virus hemagglutinin heavy chain (HA1) nucleotide homology and phylogenetic tree showed that the 2007 influenza virus H3N2 is divided into two branches, 1~4 month strains and national representative strain A/JXDH/312/2006 (H3N2) in a branch, the strains accounted for 1~4 in H3N2 subtype 33.33% after May; 2008~2009 years of virulent strain and vaccine strain A/Brisbane/10/2007 (H3N2) for the same branch, and dominant in prevalence, the strains of H3N2 subtype isolates in 2007 accounted for 89.30%; 2005~2006 years in 2006~2007 strains and vaccine strain A/Wisconsin/67/2005 (H3N2) distance; A/Brisbane/10/2007 (H3N2) from A/Wisconsin/67/2005 (H3N2) antigenic variants evolved. With the gradual change of amino acid sites is time. With the development of reference strain A/Wisconsin/67/2005 (H3N2) found that the toxic strains D122N, 1223V amino 1~4 month acid replacement, R142G and N144D strains determined amino acid mutation cluster A in antigen, the mutation was not retained after May strains G50E, D122N, N144D and K140I amino acid substitutions; amino acid substitutions were increased and the loss of a glycosylation site. The receptor binding sites of amino acids is more conservative.
H1N1 subtype influenza virus HA1 nucleotide homology and phylogenetic tree of.HA1 gene phylogenetic tree results are consistent with the results for the 2007 2006, strains and national strains A/GDLH/219/2006 (H1N1) in a branch, 2005 were divided into two branches, before April isolates and A/ NewCaledonia/20/1999 (H1N1) for the same branch, May after the isolates with A/SolomonIsland/3/2006 (H1N1) is a. Amino acid sequence analysis showed that most of the strains in May 2005 after the following: T82K, Y94H, amino acid mutation of R146K, R209K and T267N, in 2006 May after the decision happened A190T cluster B strains in H193Y antigen, and E195D amino acid variation, and the occurrence of A replacement R146K. All strains of glycosylation and receptor binding sites are conservative.
The 2007 B influenza virus HA1 gene phylogenetic tree is divided into B (Victoria) and B (Yamagata) two lineages, including B (Victoria) and strain were isolated from Shenzhen in 2006 this year, the national vaccine strain B/Malaysia/2506/2006, B/Shenzhen/155/2005 strains with a branch; B (Yamagata), and the national strains B/FJXL/54/2006 strain the nearest, next the display for the 2008~2009 annual vaccine strain B/Florida/4/2006. amino acid analysis: B (Victoria) strain S134P amino acid mutation, B (Yamagata) R48K strain were P108A, L131P, and D197N amino acid substitutions; 197-199 glycosylation sites of high frequency, 76.66% B (Yamagata) D197N strain were resulted in amino acid substitution add a glycosylation site.
conclusion
Shenzhen city in the summer peak of influenza; influenza surveillance and early warning line average line has better seasonal and sensitivity, can detect outliers. The lower age group is more susceptible to influenza virus infection, influenza surveillance school is worthy of attention. The main Shenzhen annual influenza strain characteristics and antigenicity of.2007 gene appeared alternately in Shenzhen City H3N2 influenza A virus has changed, the virus caused higher levels of popularity, variation and immunity level of influenza virus antigen affect influenza; suggested molecular biology research into flu monitoring, pay close attention to the emergence of novel influenza virus.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R181.3

【参考文献】