Ana M. Contreras

Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System

ABSTRACT High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid “error catastrophe.” Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5′ untranslated region (5′ UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3′ cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5′ UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.

Hepatitis C virus replication is directly inhibited by IFN-α in a full-length binary expression system

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The absence of culture systems permissive for HCV replication has presented a major bottleneck to antiviral development. We sought to recapitulate the early steps in the life cycle of HCV by means of DNA-based expression of viral genomic sequences. Here we report expression of replicating HCV RNA by using a, to our knowledge, novel binary expression system in which cells were transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis Δ ribozyme to control 3′ cleavage, and infected with vaccinia-T7 polymerase. HCV genomic and replicative strand synthesis, in addition to protein synthesis, was detectable and depended on full-length HCV sequences. Moreover, the system was capable of generating HCV RNA quasispecies, consistent with the action of the low-fidelity HCV NS5B RNA polymerase. IFN-α, but not ribavirin, directly inhibited the viral replicative cycle in these cells, identifying the virus itself and not solely the immune system as a direct target of IFN action. The availability of a cell-based test for viral replication will facilitate screening of inhibitory compounds, analysis of IFN-resistance mechanisms, and analysis of virus–host cell interactions.

Very low hepatitis C antibody levels predict false-positive results and avoid supplemental testing.

BACKGROUND: False‐positive results for hepatitis C virus antibody (anti‐HCV) occur with unacceptable frequency in low‐prevalence populations. The purpose of the study was to determine whether signal‐to‐cutoff (S/CO) ratios of anti‐HCV assay–reactive samples could be used to discriminate false‐positive from true‐positive anti‐HCV results and avoid the need for supplemental testing.

High antibody level: an accurate serologic marker of viremia in asymptomatic people with hepatitis C infection.

BACKGROUND: The screening and diagnosis of hepatitis C virus (HCV) infection is initiated by testing for antibody to HCV (anti‐HCV). A positive anti‐HCV test in blood donors represents ongoing infection in only a variable proportion of individuals. Because a high anti‐HCV level has been associated with viremia, a study was conducted to determine whether a high antibody level is an accurate serologic marker for viremia in asymptomatic anti‐HCV–positive persons.

Hepatitis C antibody intraassay correlation: is retest in duplicate necessary?

BACKGROUND: The hepatitis C virus antibody (anti‐HCV) can be identified with third‐generation immunoassays. The purpose of this study was to define the correlation or agreement between first and second reactive results of anti‐HCV microparticle‐based enzyme immunoassay (MEIA) and of chemiluminescence assays (ChLIAs) in blood donors, to determine whether repeat testing is necessary.