Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro.
Primary human hepatocytes were used to develop a culture model for in vitro propagation of hepatitis C virus (HCV). Production of positive- strand full-length viral RNA in cells and culture supernatants was monitored by PCR methods targeting three regions of the viral genome: the 5' NCR, the 3' X-tail and the envelope glycoprotein E2. De novo synthesis of negative-strand RNA was also demonstrated. Evidence for a gradual increase in viral components over a 3 month period was obtained by two quantitative assays: one for evaluation of genomic titre (quantitative PCR) and one for detection of the core antigen. Production of infectious viral particles was indicated by passage of infection to naive hepatocyte cultures. Reproducibility of the experiments was assessed using cultures from three liver donors and eleven sera. Neither the genotype, nor the genomic titre, nor the anti-HCV antibody content, were reliable predictive factors of serum infectivity, while the liver donor appeared to play a role in the establishment of HCV replication. Quasispecies present in hepatocyte cultures established from three different liver donors were analysed by sequencing hypervariable region 1 of the E2 protein. In all three cases, the complexity of viral quasispecies decreased after in vitro infection, but the major sequences recovered were different. These data strongly suggest that human primary hepatocytes are a valuable model for study of persistent and complete HCV replication in vitro and for identification of the factors (viral and/or cellular) associated with successful infection.
Rumin
Sylvie
S
INSERM U271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France1.
Berthillon
Pascale
P
INSERM U271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France1.
Tanaka
Eiji
E
Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan2.
Kiyosawa
Kendo
K
Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan2.
Trabaud
Mary-Anne
MA
INSERM U271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France1.
Bizollon
Thierry
T
Service d'Hépato-Gastroent érologie and Département de Chirurgie, Hô tel Dieu, 1 place de l'Hôpital, 69288 Lyon Cedex 02, France3.
Gouillat
Christian
C
Service d'Hépato-Gastroent érologie and Département de Chirurgie, Hô tel Dieu, 1 place de l'Hôpital, 69288 Lyon Cedex 02, France3.
Gripon
Philippe
P
INSERM U522, Hôpital Pontchaillou, 35033 Rennes Cedex, France4.
Guguen-Guillouzo
Christiane
C
INSERM U522, Hôpital Pontchaillou, 35033 Rennes Cedex, France4.
Inchauspé
Geneviève
G
INSERM U271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France1.
Trépo
Christian
C
INSERM U271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France1.
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000821:virology*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D014779:Virus Replication
D012367:RNA, Viral