摘要：
目的 分析甲型肝炎减毒活疫苗H2株(hepatitis A virus H2 live attenuated vaccine strain)毒种H2M20K7(K7)经连续传代后病毒基因的遗传稳定性.方法 将H2株毒种K7用细胞工厂在人二倍体细胞KMB17中进行连续传代后,收获不同代次的病毒,提取病毒RNA,用二代高通量深度测序方法,测定不同代次病毒(K7,K10,Kll,K13,K15,K18)的全基因序列,对比分析病毒的基因变异率,同时对病毒的感染性滴度进行检测和对比.结果 H2M20K7主代毒种经连续传代后,不同代次病毒的全基因序列总体变异率较低,非同义突变率小于0.58％,基因结构稳定;在5'NCR非编码区域的基因突变率＜0.1％;在VP1/2 A、2C毒力位点区域突变率小于0.01％;不同代次病毒的感染性滴度维持在7.76～ 8.50 lgCCID50/ml之间,P值为0.981差异无统计学意义.结论 H2M20K7主代毒种、工作毒种、疫苗批病毒及经过连续传代后的各代次病毒基因结构稳定;在5'NCR非编码区未发生明显突变,毒力相关位点VP1/2 A、2B、2C等关键位点的基因均为减毒特征,变异率极低,未引起病毒毒力特征改变.病毒感染性稳定且符合要求,具有较好的遗传稳定性.%Objective To investigate the genetic stability of virus seed H2M20K7 (K7) of live attenuated Hepatitis A virus H2 strain (HAV,H2 strain) for production of hepatitis A (Live) vaccine,lyophilized after continuous passages.Methods The virus seed K7 of H2 strain was proliferated and passaged in KMB17 cells in cell factories.Viruses of different passages were harvested after continuous passages.Virus RNA was extracted and the complete genomes of different virus passages (K7,K10,K11,K13,K15,K18) were sequenced by using next-generation deep sequencing.The mutation rates of different passages were compared.The infectivity titers of different virus passages of H2 strain were tested by ELISA.Results The mutation rates of complete genomes of different passages were low after continuous passages of master virus seed.The structure of gene was stable and non-synonymous mutation rate was lower than 0.57％.The mutation rate of 5'non-coding regions was lower than 0.1％.There was no significant mutation in VP1/2 A and 2C virulence site.The infectious titers of H2 strains of different passages were within 7.76-8.50 lgCCID50/ml.No statistically significant difference was found in this study.Conclusions The gene structure of the master virus seed,working seed and different passages of H2M20K7 after subculture was stable and the mutation rate was low.No significant mutation was found in 5'non-coding regions,and the critical virulence sites such as VP1/2 A,2B and 2C showed attenuated characteristics with low mutation rate.Virulence of the virus did not changed.The H2 strain maintained stable viral infectivity and genetic stability and comply with the requirements as virus seed for vaccine manufacturing.
