Wilson J J, Polyak S J, Day T D, Gretch D R
Viral Hepatitis Laboratory, Department of Laboratory Medicine, University of Washington Medical Center, Seattle 98195, USA.
J Gen Virol. 1995 Jul;76 ( Pt 7):1763-71. doi: 10.1099/0022-1317-76-7-1763.
In infected humans, hepatitis C virus (HCV) exists as a quasispecies typically characterized by multiple nucleotide substitutions within the second envelope gene hypervariable region 1 (HVR1). In the current study, we used heteroduplex gel shift analysis (GSA) of HVR1 sequences amplified directly from patients' sera to define two patterns of HCV quasispecies: (i) simple quasispecies, which gave a mostly homogeneous gel shift profile with a single predominant band and (ii) complex quasispecies, which gave a gel shift profile with multiple bands. Recombinant HVR1 libraries were generated from two patients with complex HCV quasispecies (cases 1 and 2) and two patients with simple HCV quasispecies (cases 3 and 4), and 129 individual clones were analysed by either GSA, nucleotide sequencing or both techniques. In case 1 we identified a highly complex HCV quasispecies with 11 distinct HVR1 variants differing by 1-51 nucleotide changes. We found a general but not absolute correlation between GSA pattern and the number or position of nucleotide changes within HVR1. In case 2, the complex HCV quasispecies consisted of three distinct major variants; GSA of individual HVR1 clones allowed us to reconstruct the complex quasispecies pattern in vitro. In case 3, the simple quasispecies comprised 66% homogeneous clones and 33% unique minor variants differing by 1-3 nucleotides from the consensus sequence. In case 4, the simple quasispecies was 84% homogeneous, but six unique major shift variants were identified among 31 clones by GSA. In summary, HCV quasispecies can be characterized based on GSA profiles following direct PCR amplification of HVR1 sequences from patients' serum; the GSA profiles approximate the clonal population of HCV as determined by clonal analysis. GSA of HVR1 clones showed a strong correlation with nucleotide sequencing.
在受感染的人类中，丙型肝炎病毒（HCV）以准种形式存在，其典型特征是在第二个包膜基因高变区1（HVR1）内存在多个核苷酸替换。在本研究中，我们对直接从患者血清中扩增的HVR1序列进行异源双链凝胶迁移分析（GSA），以确定HCV准种的两种模式：（i）简单准种，其凝胶迁移图谱大多为均匀图谱，有一条主要条带；（ii）复杂准种，其凝胶迁移图谱有多个条带。从两名患有复杂HCV准种的患者（病例1和2）和两名患有简单HCV准种的患者（病例3和4）中构建重组HVR1文库，并通过GSA、核苷酸测序或两种技术对129个单个克隆进行分析。在病例1中，我们鉴定出一种高度复杂的HCV准种，有11个不同的HVR1变体，核苷酸变化为1 - 51个。我们发现GSA模式与HVR1内核苷酸变化的数量或位置之间存在一般但非绝对的相关性。在病例2中，复杂HCV准种由三个不同的主要变体组成；对单个HVR1克隆进行GSA使我们能够在体外重建复杂准种模式。在病例3中，简单准种包含66%的同源克隆和33%与共有序列相差1 - 3个核苷酸的独特次要变体。在病例4中，简单准种的同源性为84%，但通过GSA在31个克隆中鉴定出6个独特的主要迁移变体。总之，在从患者血清中直接PCR扩增HVR1序列后，可根据GSA图谱对HCV准种进行特征描述；GSA图谱近似于通过克隆分析确定的HCV克隆群体。对HVR1克隆进行GSA显示与核苷酸测序有很强的相关性。
