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.Genetic variation provides a selective advantage for RNA virus populations, promoting escape from immune selection and rapid adaptation to novel environments (8). The absence of proofreading-repair mechanisms in RNA replicases and transcriptases is thought to contribute to mutation rates in the range of 10 Ϫ3 to 10 Ϫ5 substitutions per nucleotide per round of RNA replication (32). However, two factors exert a counterbalancing pressure against excessive genetic variation. A high degree of conservation of viral genomic sequences must be maintained for interactions with specific cellular proteins, as in the case of internal ribosomal entry. Excessive error rates can also contribute to net loss of fitness by leading to "error catastrophes" that threaten the viability of populations of quasispecies present in viral swarms. While the RNA genome of hepatitis C virus (HCV) contains hypervariable regions that are thought to contribute to immune escape, little is known about their intrinsic origins or their control in the absence of immune or drug selection.The extraordinary genetic diversity of HCV is reflected in its in vivo generation of quasispecies, whic...

The high prediction rate of false-positive anti-HCV results using very low levels by the Ortho VITROS anti-HCV assay safely avoids the need for supplemental testing.

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 ⌬ 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-␣, 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.cell-based assay ͉ amantadine ͉ ribavirin I nfection with hepatitis C virus (HCV) is a leading cause of chronic liver disease throughout the world (1). Chronic infection nearly always follows acute exposure to HCV, and chronically infected persons develop cirrhosis and hepatocellular carcinoma at dramatically elevated rates (2). Considering both the failure of humoral immunity to prevent reinfection and the virus' propensity for sequence diversity (3, 4), prospects for the development of an HCV vaccine seem remote. Unfortunately, available antiviral therapies, including IFN-␣ and ribavirin (RBV), have limited effectiveness (5).The lack of tissue-culture systems permissive for HCV replication has limited the development of new treatments. Although self-replicating HCV RNA replicons may permit investigation of RNA replication inhibitors (6), this system is limited to expression of the nonstructural region of the genome and requires mutant viral sequences to replicate (7). The successful introduction of RNA transcripts from an infectious cDNA clone into chimpanzees has made possible the adaptation of clones to animal studies (8). However, the repetitive use of large animals to explore HCV biology is impractical.Although IFN directly inhibits the replication of many viruses and alters immune function (9), current evidence that it directly inhibits HCV replication is inferential. Sequence changes and protein interaction assays implicate several HCV proteins as particular targets of IFN (10, 11). Given the need to screen for antiviral compounds, we developed a system capable of recapitulating the early steps of the HCV life cycle by adapting the infectious cDNA clone with elements that permit HCV RNA replication in vivo....