Kurosaki M, Enomoto N, Marumo F, Sato C
Second Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Japan.
Virology. 1994 Nov 15;205(1):161-9. doi: 10.1006/viro.1994.1631.
It has been shown that hepatitis C virus (HCV) populations in vivo are composed of different but highly homologous HCV genomes (quasispecies) as shown in the hypervariable region (HVR) that exists in the N-terminal of the envelope 2 gene of HCV, and that the predominant sequence of the HVR of HCV genomes changes rapidly over time. To further investigate genetic backgrounds of the change in the HVR of HCV genomes, 45 plasma samples serially obtained from nine patients with chronic hepatitis C were studied using population-based analyses. Total RNA was recovered and the envelope gene containing the HVR was amplified by the reverse transcription and nested polymerase chain reaction. The amplified cDNA was examined by the single strand conformation polymorphism (SSCP) analysis. Furthermore, 43 HCV sequences, separated by the SSCP analysis from three patients were determined by the dideoxy chain termination method, and the phylogenetic analysis was performed using the neighbor joining method. The SSCP analysis demonstrated that HCV population within each individual were composed of 1 to 6 quasispecies. These quasispecies populations in vivo changed sequentially in eight of nine patients. Gradual selections of coexisting quasispecies were observed over 6- to 18-month periods in three patients, whereas complete replacements of previous quasispecies by new quasispecies were repeatedly observed over few-month intervals in five patients. The phylogenetic analysis on these quasispecies revealed the continuous accumulation of mutations in two patients and discontinuous appearance of evolutionarily distant quasispecies in one patient. These results indicate that HCV genomes in vivo form quasispecies populations, and that these quasispecies populations change during the natural course of chronic infection. Genetic mechanisms underlining the change of the HVR of HCV genome appear to be either continuous accumulation of mutations or selective overgrowth of preexisting minor variants from the large spectrum of quasispecies populations.
研究表明，丙型肝炎病毒（HCV）在体内的群体由不同但高度同源的HCV基因组（准种）组成，如HCV包膜2基因N端存在的高变区（HVR）所示，并且HCV基因组HVR的优势序列随时间迅速变化。为了进一步研究HCV基因组HVR变化的遗传背景，使用群体分析方法对9例慢性丙型肝炎患者连续采集的45份血浆样本进行了研究。提取总RNA，通过逆转录和巢式聚合酶链反应扩增包含HVR的包膜基因。扩增的cDNA通过单链构象多态性（SSCP）分析进行检测。此外，通过SSCP分析从3例患者中分离出43条HCV序列，采用双脱氧链终止法进行测定，并使用邻接法进行系统发育分析。SSCP分析表明，每个个体内的HCV群体由1至6个准种组成。这9例患者中有8例体内的这些准种群体依次发生变化。在3例患者中，观察到共存准种在6至18个月期间逐渐被选择，而在5例患者中，每隔几个月就反复观察到先前的准种被新的准种完全取代。对这些准种的系统发育分析显示，2例患者中存在突变的持续积累，1例患者中出现进化距离较远的准种的不连续出现。这些结果表明，HCV基因组在体内形成准种群体，并且这些准种群体在慢性感染的自然过程中发生变化。HCV基因组HVR变化的潜在遗传机制似乎是突变的持续积累或从大量准种群体中预先存在的次要变异体的选择性过度生长。
