甲型H1N1流行性感冒病毒血凝素基因变异状况及序列分析

目的 了解2009年度甲型H1N1流行性感冒(流感)病毒的检测情况和血凝素(HA)基因变异情况.方法 选择国家级流感监测哨点医院以及暴发疫情的疫点,采集流感样病例的鼻咽拭子标本,通过实时(RT)-PCR进行病毒分型及甲型H1N1流感病毒检测,对阳性标本采用狗肾细胞(MDCK)进行病原分离,采用红细胞凝集试验测定病毒效价,用血凝抑制实验进行型别鉴定,通过RT-PCR扩增毒株HA1片段的基因并进行序列测定,利用生物信息学技术进行序列分析.结果 共检测咽拭子样本996份,其中核酸检测阳性病例包括甲型H1N1 337份,季节性H1N1亚型1份,季节性H3N2亚型67份,B型12份,流感核酸检测阳性率为41.87％,其中甲型流感核酸检测阳性率为33.84％.分离出甲型H1N1病毒36株,选择18株.测序成功的10株甲型H1N1流感病毒在多个氨基酸位点发生变异,与疫苗株A/California/07/2009(H1N1)比较,有6个位点发生突变,其中1个位点位于抗原决定簇的B区.结论 2009年度分离到的流感病毒株中以甲型H1N1为绝对的优势毒株,毒株的血凝素基因与世界卫生组织(WHO)提供的疫苗株相比有变异,与疫苗株相比,抗原决定簇B区有改变,但关键位点第222位没有变化.

Haemagglutinin gene mutations and sequences of influenza viruses A (H1N1)

Objective To understand the detections of influenza A (H1N1) in 2009,and haemagglutinin (HA) gene mutations and the comparisons with standard strains.Methods The nasopharyngeal swabs from patients with influenza-like illness (ILI) in National Influenza Sentinel Surveillance Hospital and the outbreak epidemic area were collected.The virus typing and A (H1N1) viruses were tested by real time-polymerase chain reaction (RT-PCR).Then the pathogens were isolated with MDCK cells,the virus titer was determined with hemagglutination test and the virus typing was identified with hemagglutination inhibition test (HA1).The RT-PCR products of HA1 gene of virulent strains were sequenced and then analyzed through bioinformatics.Results A total of 996 pharyngeal swab specimens were tested,and nucleic acid positive cases included 337 A (H1N1) subtype,1 seasonal A (H1N1) subtype,67 A (H3N2) subtype,and 12 B type.The positive rate of nucleic acid detection of influenza was 41.87％ and that of A (H1N1) was 33.84％.Thirty-six influenza A (H1N1) virus strains were isolated,and 10 of them were successfully sequenced and several amino acid mutations were identified.There were 6 amino acid mutations found compared with vaccine strain A/California/07/2009 (H1N1),and 1 site was in area B of epitope.Conclusions A (H1N1) is absolute predominant among isolated strains in 2009.HA gene of virulent strains is mutated compared with vaccine strain provided by World Health Organization,which shows that the area B of epitope changes,while the key amino acid position 222 doesn't change.