Schematic illustration of the method used to isolate anti-E2 antibody neutralization-resistant virus.

Each IgG antibody was diluted with phosphate-buffered saline (PBS) and combined with J6/JFH-1 cell culture-generated infectious hepatitis C virus particles (HCVcc); the mixture was allowed to react at 37°C for 1 hour. The antibody-HCVcc mixture then was added to Huh7 cells seeded in a 12-well plate to permit infection of the cells with HCVcc. Three days after the infection, the cells were subcultured into a 6-well plate, and the culture supernatant was collected at 3 and 6 days after the start of subculturing. An infectious titer measurement was performed to confirm that the collected culture supernatant contained J6/JFH-1 HCVcc. The culture supernatants that contained J6/JFH-1 HCVcc were used for subsequent infection, such that the collected culture supernatant again was mixed with the antibody, and the mixture was added to uninfected Huh7 cells. These steps were repeated for a total of 8 times. The infection-inhibiting activity of the anti-E2 antibody against the virus isolated at the final round (J6/JFH-1_EM) was determined to assess the possible presence of an escape mutant. (TIF)