Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro

Abstract

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.

Fig. 1.

HCVcc is infectious in chimpanzees. (A) HCV RNA viral load (open circles) was measured in the weekly serum or plasma of each animal (see Methods) and expressed as World Health Organization IU per ml. Serum levels of ALT (dashed line) and AST (gray line), measured at each time point, are expressed as IU/liter. (B) HCV-specific antibody levels were quantitated by a standard HCV-diagnostic ELISA (see Methods) and expressed as optical density (OD) units. The cutoff for a positive reaction was 0.4 OD. (C) Liver biopsies were taken at 0, 1, 2, 3, and 4 months postinfection. The relative levels of CD3ε, IFN-γ, and OAS1 mRNAs, quantitated and normalized to the levels at month 0 (see Methods), are expressed as the fold induction.

Fig. 2.

HCVcc is infectious in chimeric uPA-SCID mice. Viral loads (open circles) and levels of human albumin (dashed line) were quantitated (see Methods) in the plasma from mice infected with HCV-J4 (A), HCVcc strain FL-J6/JFH (B and C), or plasma from mouse C (D). Mice A and B were killed at 5 weeks postinfection, and mouse C was killed at 14 weeks postinfection. The limit of HCV RNA detection is indicated by a horizontal bar.

Fig. 3.

HCV grown in vivo has a lower buoyant density than HCV grown in vitro. The relative viral RNA levels (open circles) and buoyant density (filled circles) were plotted for each fraction. Gradients were loaded with HCVcc (A), plasma from chimpanzee 4x0483 at 3 weeks postinfection (B), and virus recovered from chimpanzee 4x0483 week 3, then passaged in cell culture for 9 days (C). Fraction 1 corresponds to the top of each gradient.