甲型肝炎病毒Js-4株经人二倍体细胞传代后的核苷酸序列分析

目的分析甲型肝炎病毒(Hepatitis A virus,HAV)Js-4株经人二倍体细胞MRC-5传代后的核苷酸序列变异情况。方法将HAV Js-4株经Vero细胞适应14代获得的原始株Js-4-V14经人二倍体细胞MRC-5于35℃连续适应培养15代,取其中8个适应株(Js-4-M2、Js-4-M3、Js-4-M4、Js-4-M5、Js-4-M10、Js-4-M12、Js-4-M14、Js-4-M15)及原始株,提取病毒基因组RNA,通过RT-PCR扩增病毒全基因组,采用生物信息学软件DNAstar Lasergene 7进行序列分析。结果 8个适应株全基因组与Js-4-V14株的核苷酸序列同源性在99.8%～100.0%之间;第5代适应株在5′-NCR(101～200 bp)发生较大变异,表现为105～157 bp缺失,之后该突变稳定遗传;第10、12、14、15代适应株在2A(3 012～3 210 bp)区段3 122位点发生点突变;8个适应株和Js-4-V14毒株与野毒株HM175为同一基因亚型,均为IB型;9个毒株的主要抗原位点与野生株HM175完全一致。结论 HAV Js-4-V14株在MRC-5细胞传代适应过程中,主要抗原位点未发生突变,其与细胞适应性和病毒增殖有关的基因在传代早期已经变异,传代后期这些变异保持相对稳定。

Nucleotide sequencing of hepatitis A virus Js-4 strain subcultured in human diploid cells

Objective To analyze the variation of nucleotide sequence of hepatitis A virus(HAV) Js-4 strain after subculture in human diploid MRC-5 cells.Methods HAV Js-4 strain was subcultured in Vero cells for 14 passages,and the obtained Js-4-V14 strain was inoculated to MRC-5 cells and further subcultured at 35 ℃ for 15 passages.Genomic RNAs were extracted from Js-4-V14 strain as well as eight adapted strains,i.e.Js-4-M2,Js-4-M3,Js-4-M4,Js-4-M5,Js-4-M10,Js-4-M12,Js-4-M14 and Js-4-M15,with which the whole genomes were amplified by RT-PCR and sequenced by bioinformatics software DNAstar Lasergene 7.Results The homologies of whole genomes of eight adapted strains to those of strain Js-4-V14 were 99.8% ～ 100.0%.However,obvious variation was observed in 5′-NCR(101 ～ 200 bp) of strain Js-4-M5,expressed as deletion of 105 ～ 157 bp,which showed genetic stability.Point mutations were observed at site 3 122 of 2A region(3 012 ～ 3 210 bp) of Js-4-M10,Js-4-M12,Js-4-M14 and Js-4-M15 strains.The eight adapted strains,Js4-V14 strain and wild strain HM175 were of subgenotype IB,of which the major antigenic sites were completely in agreement.Conclusion There was no mutation in the major antigen site of HAV Js-4-V14 strain during subculture in MRC-5 cells,while the mutations of genes related to cell adaptability and virus multiplication appeared at early stage and were relatively stable at late stage of subculture.