Transient replication of full-length HCV-Con1 genome and variant genomes with adaptive mutations. Huh-7 cells were transfected with the wild-type Con1 genome, with a replication-deficient variant of Con1 (Con1/D318N), or with two Con1 variants that contain cell culture-adaptive mutations [Con1/5.1 (E1202G, T1280I and S2197P) and Con1/NS5A (S2197P)]. Cells were harvested and replication was monitored by Northern and Western blot. (A) Detection of HCV RNA by Northern blot. Total RNA was prepared from harvested cells, and 10 μg was analyzed by using ³²P-labeled riboprobes complementary to a sequence within the HCV NS5B and 3′ UTR (Upper) and β-Actin (Lower), respectively. As a reference, 10 μg of total RNA from control Huh-7 cells, as well as serial dilutions of in vitro transcripts of a subgenomic HCV replicon spiked with 2 μg of total RNA from naïve Huh-7 cells, were analyzed in parallel. (B) Detection of HCV protein by Western blot. Lysates, each corresponding to 5 × 10⁵ cells, were loaded on a gel, and proteins were separated by electrophoresis and blotted. The upper part of the blot was probed with an HCV NS5B-specific monoclonal antibody, whereas the bottom part was incubated with antibodies specific for β-Actin.

Intrahepatic inoculation of chimpanzee 1580 with RNA transcripts of the wild-type Con1 clone (pFK-Con1). Serum samples were tested for HCV-RNA by in-house RT-nested PCR with 5′ UTR primers and/or the Roche Monitor Test 2.0: filled rectangle, positive by RT-nested PCR and/or by Monitor; open rectangle, negative by RT-nested PCR in two independent assays; open circle, HCV Monitor titers; samples found to be below the detection limit of 600 units/ml (indicated by dashed line) are shown as not detected (ND). Seroconversion in the Abbott second-generation ELISA for anti-HCV is represented by a horizontal bar. Shaded area, serum levels of ALT. Liver Histology: necroinflammatory changes of liver biopsy samples graded as 0 (normal), 1 (mild), 2 (mild–moderate), 3 (moderate–severe), or 4 (severe).

Intrahepatic inoculation of chimpanzee 1614 with RNA transcripts of pFK-Con1/NS5A, a variant of Con1 with a S2197P mutation in NS5A. The proline at position 2197 had reverted to the wild-type sequence (serine) in viruses recovered from chimpanzee serum at week 1. See also legend to Fig. 2.