Enomoto N, Kurosaki M, Tanaka Y, Marumo F, Sato C
Second Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Japan.
J Gen Virol. 1994 Jun;75 ( Pt 6):1361-9. doi: 10.1099/0022-1317-75-6-1361.
Hepatitis C virus (HCV) populations in vivo consist of heterogeneous mixtures of genetically different but closely related variants defined as a 'quasispecies'. The longitudinal fluctuation of HCV quasispecies populations in chronic hepatitis C has not been elucidated. Serial plasma samples were obtained from four patients with chronic hepatitis C (two patients without any treatment and two patients treated with interferon), and cDNA fragments containing the 5'-terminal region of the E2 gene of HCV were amplified from plasma RNA using PCR. Since conventional cloning of PCR products detects only a small part of the entire population, PCR products of each sample were separated by electrophoresis using single-strand conformation polymorphism (SSCP) analysis, which can distinguish DNA fragments of the same size as different electrophoretic bands depending on their sequence-specific conformation. Separated DNA fragments were recovered from SSCP bands in gels and their nucleotide sequences determined. SSCP electrophoresis separated PCR products into bands with different mobility. Sequence analysis of these bands confirmed that HCV populations in each patient are composed of quasispecies with different E2-hypervariable regions (HVR), which are known to contain antibody epitopes. Different patterns of variation in the HVR of quasispecies were observed in individual patients with different clinical features over time during chronic infection. Following interferon treatment, some quasispecies disappeared during the treatment and reappeared after the end of the treatment, whereas other quasispecies in the same patient remained during the treatment suggesting that the sensitivity to interferon is different among quasispecies.
丙型肝炎病毒（HCV）在体内的群体由基因不同但密切相关的变体组成的异质混合物构成，这些变体被定义为“准种”。慢性丙型肝炎中HCV准种群体的纵向波动尚未阐明。从四名慢性丙型肝炎患者（两名未接受任何治疗的患者和两名接受干扰素治疗的患者）获取系列血浆样本，使用聚合酶链反应（PCR）从血浆RNA中扩增出包含HCV E2基因5'-末端区域的cDNA片段。由于常规的PCR产物克隆仅检测到整个群体的一小部分，因此使用单链构象多态性（SSCP）分析通过电泳分离每个样本的PCR产物，SSCP分析可根据DNA片段的序列特异性构象将相同大小的DNA片段区分为不同的电泳条带。从凝胶中的SSCP条带回收分离的DNA片段并测定其核苷酸序列。SSCP电泳将PCR产物分离为具有不同迁移率的条带。对这些条带的序列分析证实，每位患者体内的HCV群体由具有不同E2高变区（HVR）的准种组成，已知这些高变区含有抗体表位。在慢性感染期间，随着时间推移，在具有不同临床特征的个体患者中观察到准种HVR的不同变异模式。干扰素治疗后，一些准种在治疗期间消失并在治疗结束后重新出现，而同一患者体内的其他准种在治疗期间持续存在，这表明准种对干扰素的敏感性不同。
