中国地区H3N2亚型人流感病毒NS1基因分子进化研究
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摘要
甲型H3N2流感病毒是威胁人类健康的重要病原体之一,中国地区是世界上流感变异株策源地之一,NS1基因是流感病毒主要的毒力因子之一,对其变异规律进行深入的分析研究在流感的防治工作中具有重要的参考意义。本研究借助近年来发展起来的初步的数据挖掘技术和进化树分析方法,从生物信息学的角度对流感病毒H3N2/NS1基因的变异规律进行了系统研究,主要包括以下内容:
1、聚类分析对序列的变异规律进行初步分析
应用两步聚类法进行了NS1序列的类别分析,结果显示全部H3N2/NS1序列可以被清楚的分为4类,各类在宿主间的分布特征清楚的反映了不同宿主序列间亲缘关系的远近,而时间分布上则反映了H3N2/NS1基因在人群中不断传播和变异的过程。
2、序列的进化树分析
分析结果显示,进化树结构和聚类结果基本一致,相应结果不仅详细刻画了数十年来H3N2流感病毒NS1基因变异的基本规律,还解释了两个关键问题:猪宿主在人流感病毒变异中起基因混合器的作用;与全球其余地区相比,中国南部地区在人H3N2亚型流感病毒的NS1变异中显示出早期策源地,但并不是后期主要的变异株起源地。
3、正向选择位点鉴定
运用正向选择模型分析了所有H3N2/NS1序列信息。结果表明,人流感病毒H3N2/NS1基因所承受的选择压力是逐渐增大的,并且在90年代年后期达到高峰。在被筛选出的10个正向位点中,绝大多数都具有适应性进化的重要意义。
本研究在回答流行病学问题的同时,还试探性地建立遗传序列变异规律研究的方法体系,分析结果显示,这一系列方法能够很好的满足分子流行病学中相应分析的需求,值得进一步改进和应用。
Influenza A virus remains an important pathogen which threatens health of human in long time,China is the main resources of the new variants,and NS1 gene of influenza is the determinant virulence factor. Deeply understanding the evolution and variation rule of NS 1 gene has very important meaning for prevention of influenza.With the help of latest developed data mining technique and phylogenetic analysis method, evolution rule of H3N2/NS1 gene were explored from bioinformatics point of view,the research detail were as follows: 1、Two-step cluster analysis
Two-step cluster was applied to explore the basic rule of sequence evolution.The result shows that all sequence could be split into 4 clusters, host distribution between clusters reflect the inherit distance of different host strain,while time distribution of clusters shows the variation history of H3N2/NS1 gene in human being.
2、Phylogenetic tree analysis
Phylogenetic tree analysis was applied to study evolution rule of H3N2/NS1 gene in detail.The results were consistent with that of the cluster analysis.Beside provide evolution history detail of NS1 sequence, two importance questions were also answered:First,swine mainly play a role of exchange recipient for old variation strains.Second,as to variation of human influenza virus,China is a early stage for NS1 drift,it should not be concerned as origin of new strain all the time.
3、Test for positive selected sites
Based on the trunk of tree structure,codon-substitution model was applied to search positive selected sites.Results shows that immunization pressure on H3N2/NS1 sequences is not constant in different period, likelihood test results indicate that pressure increased with time,and reach the climax after 1990s.Ten amino acids were identified as positive selected sites,while most of them were associated with the adaptive evolution for influenza virus.
While answer the question of epidemiology,the research set up the primary method for sequence evolution analysis.Our result indicates the serious methods used in this paper could satisfy the need of related analysis;it is worthwhile to be applied in similar research.
1、聚类分析对序列的变异规律进行初步分析
应用两步聚类法进行了NS1序列的类别分析,结果显示全部H3N2/NS1序列可以被清楚的分为4类,各类在宿主间的分布特征清楚的反映了不同宿主序列间亲缘关系的远近,而时间分布上则反映了H3N2/NS1基因在人群中不断传播和变异的过程。
2、序列的进化树分析
分析结果显示,进化树结构和聚类结果基本一致,相应结果不仅详细刻画了数十年来H3N2流感病毒NS1基因变异的基本规律,还解释了两个关键问题:猪宿主在人流感病毒变异中起基因混合器的作用;与全球其余地区相比,中国南部地区在人H3N2亚型流感病毒的NS1变异中显示出早期策源地,但并不是后期主要的变异株起源地。
3、正向选择位点鉴定
运用正向选择模型分析了所有H3N2/NS1序列信息。结果表明,人流感病毒H3N2/NS1基因所承受的选择压力是逐渐增大的,并且在90年代年后期达到高峰。在被筛选出的10个正向位点中,绝大多数都具有适应性进化的重要意义。
本研究在回答流行病学问题的同时,还试探性地建立遗传序列变异规律研究的方法体系,分析结果显示,这一系列方法能够很好的满足分子流行病学中相应分析的需求,值得进一步改进和应用。
Influenza A virus remains an important pathogen which threatens health of human in long time,China is the main resources of the new variants,and NS1 gene of influenza is the determinant virulence factor. Deeply understanding the evolution and variation rule of NS 1 gene has very important meaning for prevention of influenza.With the help of latest developed data mining technique and phylogenetic analysis method, evolution rule of H3N2/NS1 gene were explored from bioinformatics point of view,the research detail were as follows: 1、Two-step cluster analysis
Two-step cluster was applied to explore the basic rule of sequence evolution.The result shows that all sequence could be split into 4 clusters, host distribution between clusters reflect the inherit distance of different host strain,while time distribution of clusters shows the variation history of H3N2/NS1 gene in human being.
2、Phylogenetic tree analysis
Phylogenetic tree analysis was applied to study evolution rule of H3N2/NS1 gene in detail.The results were consistent with that of the cluster analysis.Beside provide evolution history detail of NS1 sequence, two importance questions were also answered:First,swine mainly play a role of exchange recipient for old variation strains.Second,as to variation of human influenza virus,China is a early stage for NS1 drift,it should not be concerned as origin of new strain all the time.
3、Test for positive selected sites
Based on the trunk of tree structure,codon-substitution model was applied to search positive selected sites.Results shows that immunization pressure on H3N2/NS1 sequences is not constant in different period, likelihood test results indicate that pressure increased with time,and reach the climax after 1990s.Ten amino acids were identified as positive selected sites,while most of them were associated with the adaptive evolution for influenza virus.
While answer the question of epidemiology,the research set up the primary method for sequence evolution analysis.Our result indicates the serious methods used in this paper could satisfy the need of related analysis;it is worthwhile to be applied in similar research.
引文
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4. Ducatez MF,Webster RG,Webby RJ.Animal influenza epidemiology[J].Vaccine,2008,(26 Suppl 4):D67-69.
5. Rao BL.Epidemiology and control of influenza[J].Natl Med J India,2003,(16):143-149.
6. van Nimwegen E.Epidemiology.Influenza escapes immunity along neutral networks[J].Science,2006,(314):1884-1886.
7. Uyeki TM.Global epidemiology of human infections with highly pathogenic avian influenza A(H5N1)viruses[J].Respirology,2008,(13 Suppl 1):S2-9.
8. Mc DJ.The epidemiology and control of influenza[J].Practitioner,1957,(179):651-658.
9. Angelava NA,Angelava AV.[Epidemiology,clinical picture, prevention and treatment of Avian influenza][J].Georgian Med News,200669-76.
10.中国疾病预防和控制中心.中国流感大流行准备计划与应急预案[EB/OL].http://www.pandemicflu.ac.cn/n3520543/n3521085/20669.html,2005-09.
11.Zhang JH,Xu H,Zhang Y,Zhao X,Guo JF,Lan Y,Shu YL.[Evolutionary characterization of HA1 of influenza H1N1hemagglutinin gene surveyed in 1981-2005 in China][J].Bing Du Xue Bao,2007,(23):350-355.
12.Yu H,Zhang GH,Hua RH,Zhang Q,Liu TQ,Liao M,Tong GZ.Isolation and genetic analysis of human origin H1N1 and H3N2influenza viruses from pigs in China[J].Biochem Biophys Res Commun,2007,(356):91-96.
13.Laver G,Garman E.Virology.The origin and control of pandemic influenza[J].Science,2001,(293):1776-1777.
14.Golubev DB.[The origin of pandemic strains of the influenza A virus][J].Vopr Virusol,1992,(37):130-135.
15.Sauter C.Distinction of influenza viruses of different host cell origin[J].Experientia,1989,(45):594-596.
16.Zhirnov OP.The host origin of influenza A viruses can be assessed by the intracellular cleavage of the viral nucleocapsid protein.Brief report[J].Arch Virol,1988,(99):277-284.
17.Hayashida H,Toh H,Kikuno R,Miyata T.Evolution of influenza virus genes[J].MolBiol Evol,1985,(2):289-303.
18. Zaraket H,Saito R,Sato I,Suzuki Y,Li D,Dapat C,Caperig-Dapat I,Oguma T,Sasaki A,Suzuki H.Molecular evolution of human influenza A viruses in a local area during eight influenza epidemics from 2000 to 2007[J].Arch Virol,2009,(154):285-295.
19. Lamb RA,Choppin PW,Chanock RM,Lai CJ.Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome[J].Proc Natl Acad Sci USA,1980,(77):1857-1861.
20. Bornholdt ZA,Prasad BV.X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus[J].Nature,2008,(456):985-988.
21. Hale BG,Randall RE,Ortin J,Jackson D.The multifunctional NS 1 protein of influenza A viruses[J].J Gen Virol,2008,(89):2359-2376.
22. Twu KY,Noah DL,Rao P,Kuo RL,Krug RM.The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target[J].J Virol,2006,(80):3957-3965.
23. Cheng A,Wong SM,Yuan YA.Structural basis for dsRNA recognition by NS 1 protein of influenza A virus[J].Cell Res,2008.
24. Feigenblum D,Schneider RJ.Modification of eukaryotic initiation factor 4F during infection by influenza virus[J].J Virol,1993,(67):3027-3035.
25. Hayman A,Comely S,Lackenby A,Hartgroves LC,Goodbourn S,McCauley JW,Barclay WS.NS1 proteins of avian influenza A viruses can act as antagonists of the human alpha/beta interferon response[J].J Virol,2007,(81):2318-2327.
26. Burgui I,Aragon T,Ortin J,Nieto A.PABPl and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes[J].J Gen Virol,2003,(84):3263-3274.
27. Enami K,Sato TA,Nakada S,Enami M.Influenza virus NS1 protein stimulates translation of the Ml protein[J].J Virol,1994,(68):1432-1437.
28. Li Z,Jiang Y,Jiao P,Wang A,Zhao F,Tian G,Wang X,Yu K,Bu Z,Chen H.The NS1 gene contributes to the virulence of H5N1 avian influenza viruses[J].J Virol,2006,(80):11115-11123.
29. Jackson D,Hossain MJ,Hickman D,Perez DR,Lamb RA.A new influenza virus virulence determinant:the NS1 protein four C-terminal residues modulate pathogenicity[J].Proc Natl Acad Sci USA,2008,(105):4381-4386.
30. Zhu Q,Yang H,Chen W,Cao W,Zhong G,Jiao P,Deng G,Yu K,Yang C,Bu Z,et al.A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens[J].J Virol,2008,(82):220-228.
31. Li Y,Anderson DH,Liu Q,Zhou Y Mechanism of influenza A virus NS1 protein interaction with the p85beta,but not the p85alpha,subunit of phosphatidylinositol 3-kinase(PI3K)and up-regulation of PI3K activity[J].J Biol Chem,2008,(283):23397-23409.
32. Jiao P,Tian G,Li Y,Deng G,Jiang Y,Liu C,Liu W,Bu Z,Kawaoka Y,Chen H.A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice[J].J Virol,2008,(82):1146-1154.
33. Hale BG,Knebel A,Botting CH,Galloway CS,Precious BL,Jackson D,Elliott RM,Randall RE.CDK/ERK-mediated phosphorylation of the human influenza A virus NS1 protein at threonine-215[J].Virology,2009,(383):6-11,
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