FIG. 8.

Serial passage of HCV_TCP with Luc-NS3-5B (Luc-SG) or Jc1Δp7 genome on Huh7.5 and Huh7.5[CE1][E2p7NS2] packaging cells. (A) Cells were inoculated with a high dose of the respective HCV_TCP to initiate the first round of infection (1st) and cultured for 72 h before they were fixed and stained using NS5A-specific monoclonal antibodies (green). Nuclei were counterstained with DAPI (blue). Cell-free culture fluid of these cells was harvested and passed to naïve Huh7.5 cells (second round of infection) or naive packaging cells, which were again fixed and stained 72 h later. (B) Infectivity of the culture fluids harvested 72 h after the first and second infections (left) as well as the quantity of HCV RNA in the lysate (right) of the cells at this time point was determined. The solid line (arrowhead) represents the detection limit of the limiting dilution assay. (C) Absence of superinfection exclusion in Huh7.5[CE1][E2p7NS2] packaging cells. Huh7.5[CE1][E2p7NS2] cells and control cells transduced with the empty lentiviral vector (Huh7.5 pWPI) were inoculated with Luc-Jc1 reporter viruses and harvested 72 h later. Infection efficiency was quantified by determining cell-associated reporter gene activity. Mean values of quadruplicate measurements and the standard errors of the means are given. The solid line (arrowhead) represents the background level of luciferase activity measured in uninfected cells.