Abstract: ObjectiveTo establish a method of hepatitis B virus(HBV)infection of HepG-2 cells by HBV positive serum in vitro.MethodsThe HepG-2 cells were treated with low temperature for the synchronization,then HBV positive serum and 4% polyethylene glycol(PEG)in serum-free mininum essential medium(MEM)were added.The HBV negative serum and culture medium were used in negative control group and blank control group.The cells were cultured with culture medium for 6 days.Then,the culture medium and cells were collected every 24 hour.HBV DNA was detected by fluorescence quantitative(FQ)-PCR,and HbsAg in the cells was detected by indirect immunofluorescent assay.ResultsIn the infection group,HBV DNA was detected in the cell culture medium and cells at the first day after infection by FQ-PCR (16.04±7.99×10³ copies/mL,8.84±3.97×10³ copies/mL).The DNA content of cells in the second day reached a peak (3.51±1.86×10⁵ copies/mL)and then declined smoothly and there was no HBV DNA deteated at the fourth day.While the virus DNA in culture supernatant gradually increased with the time with the content of 8.41±5.34×10⁵ copies/mL at the sixth day.HBsAg in the cell membrane and cytoplasm was detected by indirect immunofluorescent assay in infection group.HBV DNA and HBsAg were not detected in control groups.The infected HepG-2 cells were subcultured in the first generation of the cells.HBV DNA could be detected in culture medium and cells.A small amount of HBV DNA could be detected in cultured supernatants of the second generation but not detected in the cells.HBV DNA was not detected in the supernatant and the cells of third generation.ConclusionHepG-2 cells were infected by HBV positive serum under certain conditions and the virus could replicate in the cells for short time.