PURIFICATION OF HEPATITIS C VIRUS PARTICLES AND ANALYSIS OF ITS STRUCTURAL PROTEINS

HCV病毒表征的最大障碍是无法在培养物中维持足够的复制。HCV存在于血液循环中的两种实体中，其中一种具有高密度（1.15-1.21g/cm^3），并与γ -球蛋白有关；另一株密度低（1.06 ~ 1.12g/cm^3），表现为游离病毒粒子，与感染性滴度相似。从环流中恢复的HCV浮力密度估计为1.09-1.11g/cm^3。在被感染者血浆的电子显微镜下可以看到直径为55-60nm的病毒颗粒。通过使用Nonidet P-40和Tween 80等清洁剂剥离包膜，获得直径为33 nm（峰值为1.24-1.25g/cm^3）的颗粒，并且能够与从持续感染者血清中产生的针对模拟HCV核心蛋白和IgG的寡肽的单克隆抗体结合。特异性采用兔抗分离特异性E2/高变区寡肽多克隆抗体或鼠抗HCVcore单克隆抗体偶联胶体金颗粒确定。直接免疫金电镜研究证实，HCV病毒粒子为直径55 ~ 60nm的球形颗粒，内核为33nm。由于低密度脂蛋白和免疫球蛋白的结合以及低病毒滴度，HCV在循环中的密度异质性阻碍了从HCV rna阳性血浆中大规模制备自由病毒粒子，但我们平均每网格可以观察到数十个HCV颗粒。积累的知识和技术对最近发现的人类肝炎病毒GB病毒C/ G型肝炎病毒的理化分析也很有用。

The largest obstacle to the viral characterization of HCV was the inability to sustain sufficient replication in the culture. HCV existed in two entities in the circulation, one of which had a high density (1.15-1.21g/cm^3) and was associated with gamma-globulins ; the other had a low density (1.06-1.12g/cm^3), appeared to be free virions and paralleled the infectivity titer. A buoyant density of HCV recovered from the circulation was estimated to be 1.09-1.11g/cm^3. Electron microscopy of plasma from infected individuals visualized viral particles with a diameter of 55-60nm. By peeling off the envelope with detergent such as Nonidet P-40 and Tween 80, particles were obtained, which had a diameter of 33 nm (peaked at 1.24-1.25g/cm^3) and an abilty to bind with monoclonal antibodies raised against oligopeptides mimicking the HCV core protein and IgG fractions from sera of persistently infected individuals. Specificity was determined by using rabbit polyclonal antibodies raised against isolate-specific E2/hypervariable region oligopeptides or mouse monoclonal antibodies directed to HCVcore which had been coupled with colloidal gold particles. This direct immunogold electron microscopic study confirmed that HCV virions are 55 to 60 nm diameter spherical particles and have 33nm inner core.Density heterogeneities of HCV in the circulation due to the binding of low density lipoproteins and immunoglobulins as well as low virus titers hampered the large-scale preparation of free-virions from HCV RNA-positive plasmas, but we could observe tens of HCV particles per grids in average. Accumulated knowledge and techniques were also useful for the physicochemical analysis of a recently identified human hepatitis virus, GB virus C/hepatitis G virus.