Summary auto-generated
Researchers successfully demonstrated that primary cultures of adult human hepatocytes from uninfected donors could be infected with hepatitis C virus (HCV) in vitro. Using 33 serum samples from HCV-infected patients with varying viral loads and genotypes, they inoculated hepatocyte cultures and monitored infection using a strand-specific reverse transcription PCR assay. The assay distinguished between positive-strand RNA (present in the inoculum) and negative-strand RNA (evidence of viral replication). Positive-strand HCV RNA was detected in cells infected with most serum samples, while negative-strand RNA appeared in cells infected with 10 serum samples, peaking at days 3-5 post-infection before declining by day 14. At day 5, viral RNA showed approximately 15-fold amplification compared to the inoculum. Infection was reproducible across different hepatocyte cultures when using the same serum sample, indicating no inter-individual variability in hepatocyte susceptibility. This primary human hepatocyte culture system retained differentiated metabolic characteristics and represents a valuable model for studying early HCV replication steps and evaluating antiviral compounds.
Key findings
- Adult human hepatocytes in primary culture can sustain HCV infection and support RNA replication, as evidenced by detection of both positive and negative-strand RNA
- Negative-strand HCV RNA appeared by day 1 post-infection with maximum levels at days 3-5, indicating active viral replication
- Viral RNA showed at least 15-fold amplification by day 5 post-infection compared to the inoculum
- Infection reproducibility across different hepatocyte cultures with the same serum sample demonstrates no inter-individual variability in hepatocyte susceptibility
- High viral load in the inoculum was necessary but not sufficient for productive infection, suggesting other viral or cellular factors influence infectivity
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Abstract
In vitro infection of adult normal human hepatocytes in primary culture has been performed for investigating the replication cycle of hepatitis C virus (HCV) in differentiated cells. Hepatocytes were prepared from liver tissue resected from donors who tested negative for HCV, and inoculation was performed 3 days after plating with 33 HCV serum samples of different virus load and genotype. The presence of intracellular HCV RNA, detected by a strand-specific rTth RT-PCR assay, was used as evidence of infection. A kinetics analysis of HCV replication revealed that intracellular negative-strand RNA appeared at day 1 post-infection with a maximum level at days 3 and 5, followed by a decrease until day 14. At day 5, we estimated that the copy level of viral RNA was amplified at least 15-fold in infected cells. The level of intracellular HCV RNA in response to different serum samples was reproducible from one hepatocyte culture to another, suggesting that there is no inter-individual variability in the susceptibility of hepatocytes to HCV infection. These findings indicate that adult human hepatocytes in primary culture retain their susceptibility to in vitro HCV infection and support HCV RNA replication. This model should represent a valuable tool for the study of initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.