Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system.
Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.
Ito
T
T
Second Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan. tito@med.showa-u.ac.jp
Yasui
K
K
Mukaigawa
J
J
Katsume
A
A
Kohara
M
M
Mitamura
K
K
D002459:Cell Fusion; D016174:Hepacivirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006526:Hepatitis C / Q000175:diagnosis* / Q000378:metabolism; D022781:Hepatocytes / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000032:analysis; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D012367:RNA, Viral