ESTABLISHMENT OF PERSISTENT HEPATITIS-C VIRUS-INFECTION AND REPLICATION IN-VITRO

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