人禽流感H_5N_1毒株NS基因特征和进化

Characteristics and evolutions in NS genes of human avian H_5N_1 strains

目的:通过对人禽流感H5N1毒株NS基因序列的变异分析,揭示毒株NS基因特征与进化。方法:检测广东地区人禽流感H5N1毒株NS基因核苷酸序列,同时检索全球人禽流感H5N1毒株NS基因序列,采用DNAStar5.0软件对检索的人禽流感H5N1毒株NS基因核苷酸序列进行比对和分析;并结合流行病学资料对变异毒株进行进化速度分析。结果:根据对NS1基因和NS2基因核苷酸序列进行同源性比较,发现分成两个系列:1997～1998年人禽流感毒株为一个系列(G1),2003～2006年毒株为另一个系列(G2);其中,2003～2006年毒株NS1基因和NS2基因中,2004～2006年越南、泰国毒株为第1亚组(G2a),2005～2006年印尼毒株为第2亚组(G2b),2006年中国大陆毒株和2003年香港毒株为第3亚组(G2c)、2006年中西亚、北非毒株为第4亚组(G2d)。NS1基因74个氨基酸位点置换,占32.2%(74/230);其中,中国大陆2006年2株毒株(ZJ-16-06、GD-01-06)的NS1基因有3个位点有改变:A086T、F201Y和P215L。NS2基因共有31个氨基酸发生置换,置换率为25.6%(31/121);中国大陆2006年2株毒株(ZJ-16-06、GD-01-06)的NS1基因有3个位点有改变,包括E/K036G、S044T和L058F。NS1基因Ks值为10.1×10-6～33.4×10-6Nt/d,Ka值为11.6×10-6～17.6×10-6Nt/d;显示NS1基因受到机体免疫压力较大,检验发现基因进化存在明显选择性压力存在,而GD-01-06的NS1基因受到选择性压力较小;与NS1基因比较,NS2基因的同义突变和错义突变速度均降低,但尤以错义突变速度降低明显(Ks值为15.5×10-6～25.5×10-6Nt/d,Ka值为7.39×10-6～10.1×10-6Nt/d)。2003～2006年毒株NS1基因丢失第80～84位氨基酸序列(TIASV),引起氨基酸结构改变;而糖基化位点未改变。结论:目前NS1基因和NS2基因进化分成两组;NS1基因除自发置换进化较快外,受到明显机体免疫机制压力,第80～84位氨基酸TIASV丢失可能对致病性发生影响。2006年中国大陆毒株NS1基因和NS2基因均有3个氨基酸与其它毒株有别,显示中国大陆人禽流感H5N1毒株NS基因正在向另一方向进化。人禽流感H5N1毒株NS基因在自然界变异非常频繁,不断变异将影响H5N1毒株在人-人传播能力和引发大流行。

Objective：To reveal characteristics and revolutions of NS genes of human avian H5N1 influenza strains by analysis of sequences of NS gene. Methods ：The NS gene of human avian H5N1 influenza in Guangdong was sequenced and the global NS genes of strains were searched out from internet ; They were analyzed by DNAStar 5. 0 by means of studying the revolutionary speeds combining with the epidemiological data. Results：According to the homologous comparison of NS1 and NS2 nuecletides,the NS genes were classified into two groups ： the strains from Hong Kong during 1997 - 1998 （ G1 ） and the strains during 2003 - 2006 （G2） ; Where the NS genes in strains from Vietnam and Thailand during 2004 -2005 were the first subtype （G2a）, the NS genes in strains from Indonesia during 2005 -2006 the second subtype（G2b） ,the NS genes in strains from China mainland and Hang Kong during 2003 -2006 the third subtype（G2c） and the NS genes in strains from Turkey, Iraq, Azerbaijan, Egypt in 2006 the 42. There were 74 substitutions of amino acids in NS1 in all strains, with a ratio of 32.2 % （74/230） ; Where there were substitutions of three amino acids （ A086T, F201Y and P215L） in ZJ-16-06 and GD-01-06. There were 31 substitutions of amino acids in NS2 in all strains,with a ratio of 25.6% （31/121） ; There were substitutions of three amino acids （E/K036 G, S044T and L058 F） in ZJ-16-06 and GD-01-06 as well. In the synonymous variation,Kss in NS1 was 10. 11×10^-6 - 33.38×10^-6 Nt/d and Kas in NS1 was 11.55 ×10^-6 - 17. 58×10^-6 Nt/d. There existed obviously the immunological pressure and obviously selective pressure by biological test on NS, s in the most strains but not obviously selective pressure on NS1 in GD-01-06. In comparison with NS1 genes, mutation in NS2 gene decreased, especially in the replacement substition （Kss were between 15.5× 10^-6 - 25.5 ×10^-6 Nt/d, Kas were between 7.39×10^-6 - 10. 1×10^-6 Nt/d）. There was a deletion of AAs0_s4（TIASV）in strains NS1 gene during 2003 -2006 but no variation of glycosylated domain. Conclusion： NS （NS1 and NS2 ） genes are classified into two groups and they not only evolved rapidly but also are born a pressure by the immunological pressure. A deletion of AAs0 -s4 TIASV in NS1 during 2003 - 2006 may result in variation of the viral pathogenesis. It is shown that the evolution on NS genes in China mainland strains is going forward other direction as there are three domain substitutions of amino acid respectively in NS1 and NS2 from other strains. NS genes of human avian H5N1 influenza strains are varied frequently in nucleotide sequence, which might have an influence on the probability of human-human transmission and initiate a new pandemic of human avian H5N1 influenza.