Intracellular and extracellular HCV RNA produced by flJFH-1 mouse replicon clones. (A) Northern Blot analysis for negative-strand HCV (top panel) and cellular GAPDH (bottom panel) was performed on total RNA isolated from MMH1-1 replicon clones (lanes 1–4), Huh-7 replicon clones (lanes 5–9), and non-transfected (NT) Huh-7 cells (lane 10). (B) HCV infectivity titer in the culture medium of the same MMH and Huh-7 clones was determined by incubating serial dilutions on naïve Huh-7 cells and performing immunofluorescence for HCV 3 days post-inoculation, as previously described . The titer is expressed as NS5A-positive focus-forming units (ffu) per ml of medium. (C) RT-QPCR detection of extracellular HCV RNA following sucrose density gradient centrifugation.