甲型H1N1流感病毒基因特征及耐药性研究

Study of the genetic characteristics and drug resistance of the H1N1 influenza A virus

目的研究甲型H1N1流感病毒的基因特征和变化规律,为流感疫苗评价提供依据。方法选取2009年6月-2013年4月本院分离出的15株甲型H1N1病毒分离株,提取病毒RNA进行反转录。采用PCR方法扩增15株甲型H1N1流感病毒HA和NA全基因并进行测序分析。利用软件Bioedit和MEGA软件对序列进行拼接,绘制种系发生树,采用邻位相临法构建进化树。结果 15株甲型H1N1流感病毒均为低致病性病毒,对神经氨酸酶抑制剂敏感,对金刚烷胺类呈耐药性。实验毒株HA基因序列与国内和国外毒株同源性为99.1%-99.7%。该地区流感毒株抗原变异程度较大,其中毒株HA发生14个氨基酸点位改变,分别为：A215E,D127E,E235K,E374K,G170E,H138R,L161I,I321V,L191I,P83S,R45K,S185T,S203T和V234I。其中第83位和第321位与国内代表株相同,但是与国外代表株不同。NA发生15个氨基酸点位改变,分别为A20V,N44S,V83M,V106I,E128G,H144Y,I188T,V241I,S247N,N248D,S334N,N369K,N449K,D451G和G454S。其中所有毒株均发生V106I和S247N的变化。结论通过对HA和NA基因序列对比分析,该地区流感疫苗对当地居民有保护作用,但是毒株与疫苗株间发生相对抗原漂移,需要进一步观察毒株变异情况。

Objective To study the genetic characteristics and variation of the H1N1 influenza A virus in order to provide a basis for assessment of flu vaccines. Methods Fifteen strains of the H1N1 influenza A virus were isolated in hospitals from June 2009 to April 2013.RNA was extracted and reverse transcribed.The HA and NA genes from 15 strains of the H1N1 influenza A virus were sequenced. Results The 15 strains of the H1N1 influenza A virus were mildly pathogenic and they were sensitive to neuraminidase inhibitors but resistant to amantadine.The sequence of the HA gene of the tested strains was highly similar to that of domestic and foreign strains,with a similarity of 99.1%-99.7%.Influenza virus antigens varied considerably,with modification of amino acids at 14 positions in HA：A215E,D127 E,E235K,E374 K,G170E,H138 R,L161I,I321 V,L191I,P83 S,R45K,S185 T,S203T,and V234 I.The amino acids at positions 83 and 349were the same as those in typical domestic strains but differed from those in typical foreign strains.There was modification of amino acids at 15 positions in NA：A20V,N44 S,V83M,V106 I,E128G,H144 Y,I188T,V241 I,S247N,N248 D,S334N,N369 K,N449K,D451 G,and G454 S.The mutations V106 Iand S247N were evident in all of the strains. Conclusion Comparison of the HA and NA gene sequences indicated that vaccines are protecting people in this region,but there is an antigenic drift between the wild strains and vaccine strains.Mutations in these strains need to be observed more closely in the future.