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

HCV病毒特征的最大障碍是不能在培养物中维持足够的复制。HCV存在于两个循环实体中，其中一个具有高密度（1.15-1.21g/cm ^3），并与γ-球蛋白相关;另一个具有低密度（1.06-1.12g/cm ^3），似乎是游离病毒粒子，并降低了感染性滴度。从循环中回收的HCV的浮力密度估计为1.09-1.11g/cm ^3。来自感染个体的血浆的电子显微镜观察到直径为55- 60 nm的病毒颗粒。通过用去污剂如Nonidet P-40和Tween 80剥离包膜，获得直径为33 nm（峰值为1.24-1.25g/cm ^3）的颗粒，并且能够与针对模拟HCV核心蛋白的寡肽和来自持续感染个体血清的IgG组分的单克隆抗体结合。通过使用针对分离株特异性E2/高变区寡肽的兔多克隆抗体或针对已与胶体金颗粒偶联的HCV核心的小鼠单克隆抗体来确定特异性。直接免疫金电镜研究证实，HCV病毒粒子为直径为55 ~ 60 nm的球形粒子，内核为33 nm，由于低密度脂蛋白和免疫球蛋白的结合以及病毒滴度低，HCV在循环中的密度不均匀性阻碍了从HCV RNA阳性血浆中大规模制备游离病毒粒子，但我们平均每个网格可以观察到数十个HCV粒子。积累的知识和技术也是有用的理化分析最近确定的人肝炎病毒，GB病毒C/庚型肝炎病毒。

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.