Sequence Comparison and Analysis of HA Gene of Four H9N2 Avian Influenza Virus Isolates

4个H9N2亚型禽流感病毒毒株HA基因序列比较与分析(摘要)(英文)

[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 （H9N2; WD98 for short）, A/Chicken/Hebei/ZD/04 （H9N2; ZD04 for short））, A/Chicken/Beijing/MY/06 （H9N2; MY06 for short） ）, and A/Chicken/Beijing/PG/08 （H9N2; PG08 for short）） were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains.

[目的]测定H9N2AIV4个毒株血凝素基因序列,并进行比较分析,从分子生物学角度了解各毒株间的差异和变异规律,进而了解该亚型禽流感的分布及流行规律。[方法]参照H9N2亚型禽流感HA基因序列设计1对引物,对禽流感A/Chicken/Hebei/WD/98(H9N2)、A/Chiken/Hebei/ZD/04(H9N2)、A/Chicken/Beijing/MY/06(H9N2)和A/Chicken/Beijing/PG/08(H9N2)4个毒株HA基因进行扩增、克隆和测序,并将这4个毒株的HA基因序列分别与GenBank登录的10个H9N2亚型禽流感毒株(序列号分别为AF156376,AF156377,AF156378,AF461517,AF461519,AF461521,AF461530,AY364228,AY862606,D90305)进行比较分析,并绘制进化树。[结果]4个毒株HA基因(ORF)全长1683bp,编码561个氨基酸;4个毒株之间的核苷酸和氨基酸同源性分别为92.5%～95.6%和95.2%～96.8%,其中MY株和ZD株HA基因同源性和氨基酸同源性均为最高;WD株、MY株、PG株和ZD株与其他10个毒株的核苷酸和氨基酸序列同源性分别为82.6%～95.1%、83.0%～99.0%、82.7%～95.5%和81.30%～95.7%、86.6%～96.3%、86.6%～97.9%、87.0%～97.1%、86.9%～97.3%,遗传进化树分析表明,H9N2的HA基因的进化树有2个大的分支,分为2个种系:欧亚种系和北美种系。欧亚种系又分为3个群系,分别以A/Quail/HongKong/G1/97(AF156378),A/Duck/HongKong/Y439/97(AF156377)和A/Duck/HongKong/Y280/97(AF156376)为代表株,将这4个分离株与国内一些参考株、韩国代表株(AY862606)以及上述欧亚种系代表株的HA基因序列进行比较。结果表明,MY株、WD株、PG株和ZD株均属于欧亚种系,并且都属于A/Duck/HongKong/Y280/97群系,与国内参考株的亲缘关系都较近,而MY株与A/Chicken/Shandong/2/99(AF461521)亲缘关系最近。PG株与A/Chicken/Liaoning/2/00(AF461519)的亲缘关系最近,氨基酸同源性达到97%。ZD株与A/Chicken/Beijing/1/97(AF461530)的亲缘关系最近,核苷酸和氨基酸的同源性都是最高的,分别达95.7%、97.3%。韩国株(AY862606)则属于A/Duck/HongKong/Y439/97(AF156377)群系。总体来看,这4个分离株与国内参考株亲缘关系较近,而与韩国株(AF156377)较远。4个分离株除WD株的HA基因裂解位点为PSRSSR/GLF,MY、PG和ZD株的HA基因裂解位点均为PARSSR/GLF,WD株裂解位点附近的A变为S,也就是由丙氨酸变为丝氨酸,S也是非碱性氨基酸,因此,致病力没有变化,这4个分离株均为低致病性禽流感病毒。HA基因的几个受体位点分别为109aa,161aa,163aa,191aa,198aa,202aa,203aa,通过比较除了PG株的198aa为丙氨酸(Ala),其他3个都为缬氨酸(Vla)。编码198aa的3个碱基由GTG突变为GCG,其他参考株的198aa变异性也较大,A/Duck/HongKong/Y439/97(AF156377)和A/Quail/HongKong/G1/97(AF156378)的198aa都是谷氨酸(Glu),还有的国内株为T。这4个分离株的其他几个受体结合位点的氨基酸都是一样的,然而,通过对10个参考株的比较发现,191aa也容易发生变异,有的变为组氨酸(His)。202aa和203aa在所有的毒株中都比较保守。[结论]HA基因在不同毒株间具有很高的同源性。但有很多因素能够影响其HA基因的变异,从而影响其致病力,这与地域因素有很大的相关性,因此,应加强地域间的防控。