Figure 6

HCV extracellular and intracellular infectivity titers and RNA genome copies in Huh7 and PLC cells. Huh7 and PLC cells were infected with JFH-1 HCVcc at an MOI of 1.0 FFU/cell. Culture supernatant and cells were collected at the indicated times p.i. (A) Extracellular infectivity titers, expressed as FFU/ml, were determined on naïve Huh7-1 cells. The dashed line represents the lower limit of detection of the assay (10¹ FFU/ml). (B) For extracellular RNA analysis, culture supernatant samples were treated with S7 Micrococcal nuclease prior to RNA purification. Two μg of total murine carrier RNA was added to all samples for normalization. HCV RNA values were determined by RT-qPCR and are displayed as HCV copies/μg total carrier RNA (means ± SD for triplicate samples). The dashed line represents the background of the assay (10¹ HCV RNA copies/μg carrier RNA). (C-D) For intracellular infectivity titer and RNA analysis, cells were pelleted, re-suspended in complete 10% cDMEM and lysed via multiple freeze/thaws. (C) Intracellular infectivity titers, expressed as FFU/ml, were determined on naïve Huh7-1 cells. The dashed line represents the lower limit of detection of the assay (10¹ FFU/ml). (D) Intracellular HCV RNA copies were determined after S7 Micrococcal nuclease treatment by RT-qPCR and are displayed as HCV copies/μg total carrier RNA (means ± SD for triplicate samples). The dashed line represents the background of the assay (10¹ HCV RNA copies/μg carrier RNA).