Establishment of persistent hepatitis C virus infection and replication in vitro.
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.
Seipp
S
S
Department of Internal Medicine, University of Heidelberg, Germany. Stefanie_Seipp@krzmail.krz.uni-heidelberg.de
Mueller
H M
HM
Pfaff
E
E
Stremmel
W
W
Theilmann
L
L
Goeser
T
T
D000368:Aged; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis* / Q000096:biosynthesis; D013552:Swine; D013997:Time Factors
D012367:RNA, Viral