Two New Purification Methods of Hepatitis C Virus Particles from Serum-Free Culture System

Abstract

Background: The traditional ultracentrifugation purification method of hepatitis C virus (HCV) particles requires special equipment, limiting its wide application. Therefore, more effective and convenient methods for HCV are needed. Objectives: The present study aimed to establish simple and effective purification methods for HCV. Methods: The infectious clone of the HCV genome (JFH-1) was transfected to the human hepatoma cell line (Huh7.5.1) and cultured in Dulbecco’s modified eagle medium/nutrient mixture F-12. The infectivity of JFH-1 culture was determined by reverse transcription-quantitative polymerase chain reaction and immunofluorescence. After concentration by centrifugal filter devices, HCV particles were purified by heparin-affinity chromatography and magnetic separation technique. The purified viruses were detected by the western blot and immune-electron microscopy. Results: The infectious titer of JFH-1 transfected Huh7.5.1 in the serum-free culture medium was 4.5 × 104 FFU/mL, and HCV ribonucleic acid load was 3.946 × 106 IU/mL in 30 days of cell culture post-transfection. After purification by heparin-affinity chromatography or magnetic separation method, viral particles were visualized with spherical morphology and an average diameter of 55 nm assessed by electron microscopy. The viruses were confirmed by the western blot and immune-electron microscopy with specific antibodies to HCV. Conclusions: The heparin-affinity chromatography and magnetic separation methods were established for the purification of HCV, which were simple and efficient methods for the stable purification of HCV particles on a large scale.

The Role of Cytosolic Lipid Droplets in Hepatitis C Virus Replication, Assembly, and Release

“…The HCV assembly and release steps are the least characterized, mostly because of unauthentic HCV species that replicate efficiently in cell culture to enable microscopic or biochemical characterizations. The subcellular HCV-induced membranous structures and interacting organelles such as cLDs are below the resolution limit of most common high-resolution microscopic systems and attempts to visualize HCV particles with electron microscopy had yet to produce conclusive results [161,162]. However, biochemical and genetic methods demonstrated tight coupling of the HCV assembly and releases and cLD morphogenic and metabolic dynamics (Figure 5).…”