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Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin. Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days. Infectivity of released virions in supernatants after long-term culturing (day 30-80) was shown by successful infection of fresh cells. In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months. These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.

Background/aims: Recently, the hepatitis GB-C virus (GBV-C) has been identified as another virus potentially causing chronic hepatitis. Although high rates of coinfection are emerging in drug addicts with chronic hepatitis C virus infection, no detailed data on clinical presentation are available. Therefore, co-infection was sought in hepatitis C virus patients to determine the impact of GB-C virus on clinical presentation., Methods: GBV-C was determined by nested reverse transcriptase-polymerase chain reaction in serum of 70 HIV negative intravenous drug abusers with chronic hepatitis C. Biochemical, histological and virological parameters were compared between patients with or without GBV-C coinfection., Results: Hepatitis C virus and GBV-C coinfection was found in 18 of 70 (25.7%) patients. Cases with coinfection were younger and had shorter duration of disease (31.4+/-6.2 vs. 35.3+/-7.3 (p=0.09) and 9.9+/-6.8 vs. 12.9+/-7.7 (p=0.17) years) than those without coinfection. Neither hepatitis C virus genotype distribution and HCV RNA levels nor serum liver function tests, titers of immunoglobulins or autoantibodies differed between the two groups. Histologically, chronic active hepatitis (16.7 vs. 46.4%, p=0.07), fibrosis (8.3% vs. 21.4%, p=0.3), and cirrhosis (0% vs. 8.2%, p=0.31) were less prevalent in coinfected patients. After interferon treatment, 5/6 coinfected and 11/19 patients with hepatitis C virus infection alone had cleared HCV RNA and 4/6 lost GBV-C RNA from serum. The two patients with GBV-C/HCV infection who persistently cleared hepatitis C virus but not GBV-C from serum had normal transaminases during follow-up despite persistence of GBV-C., Conclusions: Coinfection of chronic hepatitis C patients with GBV-C does not lead to a significant change in clinical presentation, severity of liver disease, hepatitis C viremia, or response to interferon treatment.