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目的比较新甲型H1N1流感病毒对MDCK细胞和鸡胚的敏感性差异,了解病毒分离株特征,为疫苗制备和开展实验室常规监测等奠定基础。方法用MDCK细胞和鸡胚进行流感病毒分离[1],收获病毒后提取病毒RNA,逆转录-聚合酶链式反应(RT-PCR)扩增,阳性扩增产物纯化后测序,测序结果使用Clustal和MEGA4.1软件进行比较分析,通过与NCBI数据库中不同时间和地区的流感病毒H1N1株同源比对后,绘制种系发生树。结果从334份患者呼吸道标本中,用MDCK细胞分离法分离出134株新甲型H1N1病毒,分离率为40.12%,用鸡胚分离法分离出150株新甲型H1N1病毒,分离率为44.91%;所测10株新甲型H1N1毒株HA1区的344个氨基酸,与2009年新甲型H1N1国际代表株A/California/07/2009相比,只有少数位点发生氨基酸替换,并且这些位点都不在抗原决定簇上;种系发生树显示,10株新甲型H1N1和国际代表株同源性较大,形成一簇,与A/Swine/guangxi/13/2006较为接近。结论新甲型H1N1病毒对MDCK细胞和鸡胚的敏感性相差不大;新甲型H1N1病毒未发生变异,短期内不会产生新变种。
Objective To compare the sensitivity of new influenza A (H1N1) virus to MDCK cells and chicken embryos, understand the characteristics of virus isolates, and lay a foundation for vaccine preparation and routine laboratory monitoring. Methods The virus was isolated from MDCK cells and chicken embryos [1]. The virus RNA was harvested after the virus was harvested and amplified by reverse transcription-polymerase chain reaction (RT-PCR). The positive PCR products were purified and sequenced. The sequencing results were analyzed using Clustal And MEGA4.1 software were compared and analyzed. The phylogenetic tree was drawn by homology comparison with the H1N1 strain of influenza virus at different time and place in the NCBI database. Results From 134 respiratory samples of patients, 134 new type A H1N1 viruses were isolated by MDCK cell isolation method with a separation rate of 40.12%. 150 new type A H1N1 viruses were isolated by chicken embryo isolation with a separation rate of 44.91% ; 344 amino acids of the HA1 region of 10 new type A H1N1 strains tested showed amino acid substitution only in a few sites compared with the 2009 A / California / 07/2009 new A H1N1 strain, and these sites Are not on the epitopes. The phylogenetic tree shows that the 10 new H1N1 strains are highly homologous with the international representative strains and form a cluster, which is closer to that of A / Swine / guangxi / 13/2006. Conclusion The sensitivity of the new H1N1 virus to MDCK cells and chicken embryos is similar. The new H1N1 virus does not mutate, and no new variant will be produced in a short term.