FIG 5.

Live-cell monitoring of HCV cell-to-cell transmission. (A) Schematic diagram of HCV cell-to-cell transmission system. Huh7.5.1-VISI-GFP cells electroporated with the HCV genome were used as donor cells; Huh7.5.1-VISI-mCherry cells were used as recipient cells. Donor cells were mixed at a 1:30 ratio with recipient cells. To neutralize cell-free transmission, HCV-neutralizing antibody AR3A or control antibody (HCV anti-E2 monoclonal antibody) was added to cocultures. After incubation for 96 h, images were taken under a fluorescence microscope. (B to D) Huh7.5.1-VISI-mCherry cells were cocultured with Huh7.5.1-VISI-GFP cells electroporated with Jc1 RNA in the presence of neutralizing (AR3A) or control (control) antibody or without antibody (Mock). (B) Numbers of HCV-positive donor cells (Huh7.5.1-VISI-GFP cells) or HCV-positive recipient cells (Huh7.5.1-VISI-mCherry cells) per focus after coculture for 96 h were counted. In the scatter plot, green triangles represent number of HCV-positive donor cells per focus; red dots represent number of HCV-positive recipient cells per focus; horizontal lines represent the median for 40 randomly selected foci. Statistical significance was determined by Student's t test. (C) HCV cell-to-cell transmission phenomena were observed by fluorescence microscopy after coculture for 72 h. The green fluorescence represents HCV-positive donor cells, and the red fluorescence represents HCV-positive recipient cells. The foci indicated by yellow arrows indicate virus spreading by cell-to-cell transmission with centered donor cells; the foci indicated by white arrows indicate virus spreading by cell-free transmission without donor cells. (D) Infectivity of supernatants from cocultures was determined by TCID₅₀ titration on Huh7.5.1 cells. Means and standard deviations from three independent assays are shown (ND, not detected).