Walvax-2细胞基质培养甲型肝炎病毒YN5株的遗传稳定性分析

Genetic stability of hepatitis A virus YN5 strain cultured in Walvax-2 cells

目的分析Walvax-2细胞基质培养甲型肝炎病毒(hepatitis A virus,HAV)YN5株的遗传稳定性。方法将HAV YN5株在Walvax-2细胞中于35℃连续适应培养,取P24、P27、P29、P35和P40代病毒,提取RNA,以其为模板进行RT-PCR扩增,并测序。采用生物信息学软件DNAMAN、Vector NTI、BioEdit等进行序列分析,与GenBank中登录的标准株HM175(M14707)进行同源性比较。结果 5代病毒全基因组间的核苷酸序列同源性为100%。与标准株HM175比较,5代病毒基因组5′-NCR(101～200 bp)片段的124和152位发生突变,且存在134～137 bp缺失;2A(3 012～3 210 bp)片段的3 025和3 196位发生突变,其中,3 025位点A变为T,与标准株HM175 VP1/2A(3 024～3 191 bp)的差异小于7. 5%,基因亚型与其相同,均为IB型;2C(3 986～4 960 bp)片段的4 043、4 087、4 185、4 222、4 563、4 802、4 880位点发生突变。5代病毒与标准株HM175氨基酸序列同源性为99. 4%,共有29个氨基酸发生突变,主要抗原位点与标准株HM175完全一致。结论 Walvax-2细胞基质培养HAV YN5株可保持良好的遗传稳定性,有望用于甲肝疫苗的研发及生产。

Objective To analyze the genetic stability of hepatitis A virus(HAV)YN5 strain cultured in Walvax-2 cells.Methods HAV YN5 strain was adapted to Walvax-2 cells and further subcultured at 35 ℃. Genomic RNAs were extracted from the virus of passages(P)24,27,29,35 and 40 and used as templates for amplification by RT-PCR. The PCR products were sequenced and analyzed by bioinformatics software such as DNAMAN,Vector NTI and BioEdit,of which the homologies to the standard virus strain HM175(M14707)in GenBank were evaluated. Results The homology of whole genomes from five passages of YN5 strain was 100%. However,compared with standard strain HM175,mutations at sites 124 and 152 of 5′-NCR(101 ~ 200 bp) as well as deletion of 134 ~ 137 bp were observed in the genomes of the five passages. Meanwhile,two point mutations were observed in 2 A region 3 012 ~ 3 210 bp,including the change of A to T at site 3 025,with a difference of less than 7. 5% with HM175 VP1/2 A(3 024 ~ 3 191 bp). YN5 strain was identified as subgenotype IB,which was identical to that of standard strain HM175. Mutations were also observed at sites 4 043,4 087,4 185,4 222,4 563,4 802 and 4 880 of 2 C(3 986 ~ 4 960 bp) region. All the homologies of YN5 strain of five passages to HM175 strain were 99. 4%,with mutations of 29 amino acids,while the major antigenic sites were completely consistent with those of HM175 strain. Conclusion The HAV YN5 strain cultured in Walvax-2 cells showed high genetic stability,which was expected to be used for the development and production of HAV vaccine.