丙型肝炎病毒包膜区变异与感染慢性化关系的初步研究

Genetic variation characteristics of the envelope region of hepatitis C virus in the patients with chronic hepatitis

目的 探讨丙型肝炎病毒 (HCV)包膜区变异与其感染慢性化的关系。方法 3份HCV慢性感染者和 3份急性感染者血标本 ,采用逆转录 聚合酶链反应 (RT PCR)扩增HCV的E1区C端及E2区N端片段 (5 73bp) ,扩增产物进行克隆 ,以单链构象多态性 (SSCP)和异质性双体 (HD)分析对每份血清 30个克隆的E1 E2区准种 (quasispecies)进行筛选 ,挑选每份标本HCV的优势株与劣势株序列进行测定 ,分析非同义替换碱基数与同义替换碱基数比率 (dN dS ,间接反映选择压力 )和推导的氨基酸序列。结果 HCV慢性感染者病毒准种的复杂性和E2区dN dS明显高于急性感染者。HCV慢性感染者的E2区氨基酸替换率 (8.4 6 % )比急性感染者 (1.0 2 % )更高 ,而两者的E1区氨基酸替换率(分别为 2 .74 %和 1.0 9% )均较低。尽管HVR1变异程度更高 ,但仍存在高度保守的氨基酸位点。结论 HCV持续性感染与准种复杂性增高和宿主对HVR1的免疫选择有关。

Objective To study the association of the envelope region variation of hepatitis C virus (HCV) with the chronicity of HCV infection. Methods Acute phase plasma samples from three injection drug users who acquired HCV infecton during six month follow up and three patients with chronic hepatitis C were obtained. A 573 bp fragment containing the 5' half of E1 and 3' half of E2 were amplified by nested reverse transcription polymerase chain reaction (RT PCR). For each,30 cloned cDNAs were examined by a method that combined heteroduplex (HD) analysis and a single stranded conformational polymorphisim (SSCP) assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns for sequencing. The ratio of nonsynonymous to synonymous substitution ( dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns and glycosylation signals. Results Quasispecies complexity and E2 dN/dS ratio were higher in those with chronic hepatitis, in whom a trend toward more numbers of nonsynonymous mutations was detected at the E2. 1.02% (1.33/130) and 8.46% (11/130) amino acids within the E2 region mutated in chronic hepatitis and acute hepatitis, whereas within the E1 region 2.74% and 1.09% amino acids replaced among chronic and acute hepatitis,respectively. Some consistent amino acids were detected, although the hypervariable region 1 (HVR1) had a higher variability in subjects with chronic infection. Conclusion HCV persistence is associated with a complex quasispecies and host immune selection to HVR1.