Fig. 2

Characterization of TPF1-4B and sAH-N3.5 virus particles. a, b Concentrated culture medium collected from TPF1-4B (a) and sAH-N3.5 (b). RNA-transfected cells were fractionated using a 10–60 % stepwise sucrose density gradient. HCV RNA and core Ag levels were measured by qRT-PCR and sandwich EIA, respectively. Culture media after treatment with 0.2 % NP-40 prior to centrifugation were also analyzed (a, b lower panels). c Comparison of the infectivity of the culture medium from TPF1-4B RNA- and sAH-N3.5 RNA-transfected Huh7 cells. Culture media at 72 h after transfection were used to inoculate naïve Huh7 cells, and HCV core Ag levels were determined at 24, 48, 72 and 96 h after infection. d Time course of infectious TPF1-4B virus released into the culture medium of RNA-transfected Huh7 cells, as measured by FFA. The insert shows the HCV core Ag levels (red) detected by indirect immunofluorescence in cells infected with TPF1-4B viral particles at 72 h after transfection. The nuclei were counterstained with DAPI (blue). Assays (D and E) were conducted two independent experiments, and results represent the mean of three independent experiments ± standard deviations