流感减毒活疫苗中 A 型流感病毒株生产用工作毒种遗传稳定性分析

Analysis of the genetic stability of the master virus seeds for live attenuated influenza vaccine produc-tion

目的：分析流感减毒活疫苗的生产用工作毒种A／17／California／2009／38（H1N1）、A／17／Perth／09／87（H3N2）的遗传稳定性。方法将流感减毒活疫苗生产用工作毒种接种鸡胚尿囊腔进行传代，扩增第2、3、5、10代毒株的全基因组进行测序分析，其中每个代次的6个内部基因PB2、PB1、PA、NP、M、NS测序结果分别与冷适应供体毒株进行比对分析，血凝素（ HA）和神经氨酸酶（ NA）基因的测序结果分别与世界卫生组织（ WHO）推荐的适用于北半球的2011—2012年流行毒株进行比对分析，从而判断疫苗生产用工作种子毒株的基因遗传稳定性。结果扩增至10代次后，H1N1疫苗毒株基因总突变率为0．035％，H3N2疫苗毒株基因总突变率为0．022％，突变位点均未发生在冷适应性（ ca）、温度敏感性（ ts）和减毒性（ att）表型的决定位点。结论生产用工作毒种具有良好的基因遗传稳定性，扩增至第10代次的疫苗毒株同原始种子代次的同源性大于99％，工作种子批毒种符合2010年版《中华人民共和国药典》的要求。

Objective To analyze the genetic stability of master virus seed lots of live attenuated influenza vaccineA/17/California/2009/38（H1N1）andA/17/Perth/09/87（H3N2）strains.Methods The master virus seed lots were inoculated into chicken eggs for subculture.The complete genome of the 2nd, 3rd, 5th and 10th generations of viruses were amplified and sequenced.The genes encoding hemagglu-tinin （ HA） and neuraminidase （ NA） were compared with those of the WHO recommended circulating wild-type virus strains used for vaccine production in northern hemisphere during 2011-2012 influenza season.Six internal genes （PB2, PB1, PA, NP, M and NS） of each virus generation were compared with their master donor virus strain （A/Leningrad/134/17/57） for the evaluation of the genetic stability.Results The muta-tion rates of H1N1 and H3N2 strains after 10 passages were 0.035%and 0.022%, respectively.No muta-tions were found at the critical sites for controling thecold adapted （ ca） , temperature sensitive （ ts） and at-tenuated （ att） phenotypes.Conclusion The live attenuated influenza vaccine strains possessed high genet-ic stability as their tenth generations still shared 99% of homology with the original seed lots.All of the working virus seed lots met the requirements of Pharmacopoeia of the People′s Republic of China （ 2010 edition） .