Titration of HCV Viral Stock

Viral stock was titrated as previously described (3 (link)). Briefly, 12 × 10³/well Huh7.5 were seeded on a 10-mm-diameter glass coverslip placed in a 24-well plate. A 10-fold serially diluted viral stock was added to Huh7.5 cells in a medium that was changed after 6 h. At 72 h postinfection, immunostaining against the HCV core protein was performed. Briefly, cells were washed five times with PBS and fixed with ice-cold 100% methanol at -20°C for 20 min. Cells were washed for five times with PBS; blocked for 1 h with PBS, 5% normal goat serum (BioGenex, Fremont, CA, USA), and 2% BSA; and incubated with mouse anti-HCV Core (clone [C7-50], Abcam, Cambridge, UK) diluted in blocking buffer 1:500 overnight at 4°C in a humidified chamber. Cells were washed with PBS and incubated for 1 h at room temperature with secondary antibody goat anti-mouse IgG-Alexa Fluor-568 (Invitrogen). Stained cells sections were mounted using SlowFade Gold Antifade reagent (Invitrogen) including DAPI for the nuclear counterstaining and stored in the dark. Images were acquired by a confocal microscope (TCS SP5 II, Leica). The number of foci formed at the highest dilution was used to calculate the virus titer, which was expressed as the number of focus-forming units per milliliter of supernatant (FFU/ml). The titers of our JFH1 viral stock were usually in the range of 10⁴ to 10⁶ FFU/ml.