本文選題：流感病毒　切入點：流感樣病例　出處：《鄭州大學(xué)》2008年碩士論文

【摘要】：目的 了解深圳市2007年流感流行特征,探討流感病毒血凝素蛋白重鏈(HA1)區(qū)基因特性、抗原變異及其與流感流行的關(guān)系,為流感防治提供科學(xué)依據(jù)。 材料與方法 收集深圳市2007年流感樣病例和流感樣病例暴發(fā)疫情資料,用EXECL和SAS 8.0統(tǒng)計軟件對流感樣病例就診百分比進行時間趨勢、年齡分布情況統(tǒng)計分析,并設(shè)計流感監(jiān)測預(yù)警線和平均水平線。采集流感樣病例鼻咽拭子標(biāo)本,同時接種狗腎傳代細胞和9～11日齡雞胚的羊膜腔和尿囊腔進行流感病毒分離培養(yǎng),用紅細胞凝集及凝集抑制試驗實現(xiàn)病毒滴度及型別鑒定。采用隨機整群抽樣法于流感流行期前、后采集一般人群血清了解深圳人群的血清抗體水平。對所有分離病毒提取RNA,用RT-PCR擴增HA1區(qū)基因片段,產(chǎn)物純化后測序;用DNASTAR軟件、SIMMONIC軟件和MEGA3.1軟件進行核苷酸—氨基酸序列分析、同源性比較、基因進化樹構(gòu)建等分子變異研究,并與WHO推薦北半球疫苗株和國家代表株進行比較,揭示流感病毒HA1區(qū)基因變異、抗原性改變及其與流感流行的關(guān)系。 結(jié)果 1.深圳市2007年流感流行特征 2007年深圳市流感樣病例(influenza-like illness,簡稱ILI)就診百分比曲線呈單峰分布,流感活動高峰主要分布在4～7月,ILI就診百分比于第25周達到峰值11.22%,超過預(yù)警線值10.86%,較2005、2006年明顯提高,且持續(xù)時間長;ILI主要集中在0～14歲年齡組兒童,占流感樣病例總數(shù)的72.97%;學(xué)校流感流行高峰較早出現(xiàn);深圳流感監(jiān)測預(yù)警線具有一定的季節(jié)性和靈敏度,能及時發(fā)現(xiàn)異常值。2007年共報告流感樣病例暴發(fā)疫情50起,經(jīng)實驗室PCR檢測,35起流感病毒陽性,其中A型29起,B型6起;時間上主要分布在3～6月,占92.0%,88.0%發(fā)生于中小學(xué)校。2007年共檢測流感樣病例鼻咽拭子標(biāo)本2831份,343份陽性,分離率為12.12%;H1N1亞型、H3N2亞型、B型流感病毒所占構(gòu)成比分別為3.5%、62.68%和33.82%;其中1～6月份以H3N2亞型為主要流行株,7～12月份則以B(Yamagata)系為主要流行株。月病毒分離率1～6月份逐步升高,6月份最高為28.38%,后按月遞減,與ILI就診百分比時間變化趨勢一致。結(jié)合往年資料發(fā)現(xiàn)主要流行株交替出現(xiàn)。2007年3、9月份共采集一般人群血清1700份,H3N2亞型血清抗體陽性率最高,3、9月份分別為:65.82%和72.94%,經(jīng)檢驗具統(tǒng)計學(xué)意義(P＜0.05),這與2007年H3N2亞型病毒活動水平較高有關(guān);H1N1亞型、B(Victoria)系、B(Yamagata)系毒株3、9月份抗體陽性率分別為46.98%、43.80%、43.84%、21.90%、41.18%和21.67%;除H3N2亞型外,其它三個型別流感病毒幾何平均滴度偏低。 2.深圳市2007年流感病毒血凝素蛋白重鏈區(qū)基因變異 H3N2亞型流感病毒血凝素重鏈(HA1)區(qū)核苷酸同源性和基因進化樹結(jié)果均顯示:2007年H3N2亞型毒株分為兩個分支,1～4月份部分毒株與國家代表株A/JXDH/312/2006(H3N2)在一個分支,該部分毒株占1～4月份H3N2亞型分離株的33.33%;5月份之后的毒株與2008～2009年疫苗株A/Brisbane/10/2007(H3N2)為同一分支,并在流行上占優(yōu)勢,該部分毒株占2007年H3N2亞型分離株的89.30%;2005～2006年毒株與2006～2007年度疫苗株A/Wisconsin/67/2005(H3N2)距離較近;A/Brisbane/10/2007(H3N2)是從A/Wisconsin/67/2005(H3N2)進化而來的抗原變異株。氨基酸位點是隨著時間發(fā)展逐步變化。與參照株A/Wisconsin/67/2005(H3N2)比較發(fā)現(xiàn),所有毒株發(fā)生D122N、1223V氨基酸替換,1～4月份部分毒株在抗原決定簇A區(qū)發(fā)生R142G和N144D氨基酸變異,該變異沒有被保留下來,5月份之后的毒株發(fā)生G50E、K140I氨基酸替換;D122N、N144D氨基酸置換分別導(dǎo)致增加和丟失一個糖基化位點。受體結(jié)合位點氨基酸較保守。 H1N1亞型流感病毒HA1區(qū)核苷酸同源性和基因進化樹結(jié)果一致。HA1區(qū)基因進化樹結(jié)果為2007年、2006年毒株和國家代表株A/GDLH/219/2006(H1N1)在一個分支,2005年毒株分為兩個分支,4月份之前分離株與A/NewCaledonia/20/1999(H1N1)為同一分支,5月份之后的分離株與A/SolomonIsland/3/2006(H1N1)為一支。氨基酸序列分析顯示,2005年5月以后的大部分毒株出現(xiàn)以下氨基酸變異:T82K、Y94H、R146K、R209K和T267N,2006年5月份之后的毒株在抗原決定簇B區(qū)發(fā)生了A190T、H193Y和E195D氨基酸變異,同時也發(fā)生A區(qū)R146K的置換。所有毒株的糖基化和受體結(jié)合位點比較保守。 2007年B型流感病毒HA1區(qū)基因進化樹分為B(Victoria)和B(Yamagata)兩個譜系,其中B(Victoria)系毒株與深圳2006年分離株、本年度疫苗株B/Malaysia/2506/2006、國家代表株B/Shenzhen/155/2005同在一個分支;B(Yamagata)系毒株與國家代表株B/FJXL/54/2006距離最近,其次為2008～2009年度疫苗株B/Florida/4/2006。氨基酸位點分析顯示:B(Victoria)毒株發(fā)生S134P氨基酸變異,B(Yamagata)系毒株發(fā)生R48K、P108A、L131P和D197N氨基酸替換;197-199糖基化位點變異頻率較高,76.66%的B(Yamagata)系毒株發(fā)生D197N氨基酸置換導(dǎo)致增加一個糖基化位點。 結(jié)論 深圳市流感流行高峰在夏季;流感監(jiān)測預(yù)警線和平均水平線具有較好的季節(jié)性和靈敏度,能及時發(fā)現(xiàn)異常值。低年齡組人群較易受流感病毒感染,學(xué)校流感監(jiān)測值得重視。深圳每年主要流感流行株交替出現(xiàn)。2007年深圳市H3N2亞型流感病毒的基因特性和抗原性已發(fā)生變化,引起該型病毒較高水平的流行,流感病毒抗原變異和人群免疫水平共同影響著流感的流行;建議將分子生物學(xué)研究納入流感監(jiān)測,密切關(guān)注新型流感病毒株的出現(xiàn)。
[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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R181.3

【參考文獻】

相關(guān)期刊論文 前10條

1 王勇,陳淑霞,薛穎,董杰,金奇,侯云德;中國歷年H_3N_2亞型人流行性感冒病毒血凝素基因的序列測定及分析[J];病毒學(xué)報;2002年02期

2 王勇,薛穎,陳淑霞,金奇,侯云德;H_3N_2亞型人流行性感冒病毒HA_1的蛋白序列同源性比較、變異規(guī)律及結(jié)構(gòu)與功能的分析[J];病毒學(xué)報;2002年04期

3 劉傳楠,朱洪浩,何浩,涂毛妹,彭毓清,張慶枝;1995～1999年武漢地區(qū)流感監(jiān)測結(jié)果分析[J];湖北預(yù)防醫(yī)學(xué)雜志;2000年06期

4 袁方玉,葉國軍,潘南勝,陳麗,霍細香;2001～2003年湖北省流行性感冒監(jiān)測結(jié)果分析[J];公共衛(wèi)生與預(yù)防醫(yī)學(xué);2004年06期

5 潘南勝;劉傳楠;陳麗;朱洪浩;葉國軍;林新明;袁方玉;霍細香;;湖北省2001~2004年流感病毒分離情況分析[J];公共衛(wèi)生與預(yù)防醫(yī)學(xué);2006年01期

6 程錦泉;張順祥;蔣麗娟;武南;;從深圳首例人禽流感疫情應(yīng)對看突發(fā)公共衛(wèi)生事件的大眾溝通[J];中國公共衛(wèi)生管理;2007年03期

7 楊春,余佳;甲型流感病毒抗原變異的機理[J];國外醫(yī)學(xué).病毒學(xué)分冊;1999年03期

8 倪漢忠,鄢心革,張欣,柯昌文,彭國文,周慧瓊;2000—2003年廣東省流感監(jiān)測結(jié)果分析[J];海峽預(yù)防醫(yī)學(xué)雜志;2005年04期

9 趙群;徐聞;秦明芳;白優(yōu)昌;寧德明;羅春蕊;蔣立;尹建雯;伏曉慶;;2002～2004年云南省流感監(jiān)測結(jié)果分析[J];疾病監(jiān)測;2006年02期

10 李玉青,汪寧;流感的分子流行病學(xué)研究進展[J];疾病控制雜志;2001年01期

，

本文編號：1680407