Naturally Occurring Resistance Mutations to New Antivirals in the Hepatitis C Virus NS5b Polymerase in Treatment-Naďve Patients

The current treatment for hepatitis C virus (HCV) chronic infection consists of peglyated interferon alpha plus ribavirin. This treatment is unspecific, generates important side effects and is only effective in 50% of patients. New treatments based in specific inhibitors aimed at targeting virus-specific enzymes, such as the HCV NS3 protease and the NS5b RNA dependent RNA polymerase (RdRp), are entering the clinical field. Previous studies have identified emergence of viral resistance in vitro for this compounds, but few have provided information regarding natural variation of polymerases between or within infected individuals. We collected viral isolates from different patients with chronic HCV infection from reference hospitals in Valencia, Spain. Natural variability and the presence of majority mutants with resistance or compensatory mutations to NS5B RdRp nucleosidic (NI) and non-nucleosidic (NNI) inhibitors was determined through direct Sanger sequencing of the HCV NS5B gene; whereas minority mutants were determined by cloning and sequencing >20 individual clones. Signature polymorphism patterns were found in the direct Sanger sequence of the polymerase at various sites, when compared to the reference isolate HCV-J; probably as a consecuence of the high error rate of viral replication of this RNA genome. In addition, five frequent polymorphisms at amino acid sites 71, 338, 426, 523, 556 were observed as minority or majority mutations within the 21 clonal NS5b sequences from patient CL14; at frequencies of 0.0476, 0.9545, 0.476, 0.0476, and 0.9545, indicating potential resistance to NNI such as thiopenes, dihydropyrones, benzothiazidines and benzofurans; and also to cyclosporine analogues. This data indicates that natural polymorphism and minority resistant mutants do exist in HCV NS5B polymerases isolated form treatment-naïve individuals. Analyses such as the one used here will assist in determining the likelihood of HCV resistance upon the administration of new NS5B polymerase inhibitors and will aid in the development of assays for genotypic resistance testing.