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

BackgroundThe 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.ObjectivesThe present study aimed to establish simple and effective purification methods for HCV.MethodsThe infectious clone of the HCV genome (JFH-1) was transfected to the human hepatoma cell line (Huh7.5.1) and cultured in Dulbecco’smodifiedeagle medium/nutrient mixture F-12. The infectivity of JFH-1 culture was determined by reverse transcriptionquantitative 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.ResultsThe infectious titer of JFH-1 transfected Huh7.5.1 in the serum-free culture medium was 4.5104 FFU/mL, and HCV ribonucleic acid load was 3.946106 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.ConclusionsThe 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.