Primary hepatocyte culture supports hepatitis C virus replication: a model for infection-associated hepatocarcinogenesis.
Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections.


CONCLUSIONS
Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease.
Department of Biochemistry and Molecular Biology, George Washington University School of Medicine, Washington, DC 20037, USA.
Krishna
K
Banaudha
Jan M
JM
Orenstein
Tamara
T
Korolnek
Georges C
GC
St Laurent
Takaji
T
Wakita
Ajit
A
Kumar
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D003470:Culture Media; D016679:Genome, Viral; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D006801:Humans; D016898:Interferon-alpha; D008113:Liver Neoplasms / Q000821:virology; D012367:RNA, Viral / Q000096:biosynthesis*; D051381:Rats; D057074:Virus Release; D014779:Virus Replication
D003470:Culture Media; D016898:Interferon-alpha; D012367:RNA, Viral