Current Hepatitis C treatment guidelines recommend pegylated interferon (peglFN) with ribavirin (RBV) for patients with detectable plasma HCV RNA and liver histology showing fibrosis. Unfortunately, approximately 50% of treated patients do not achieve a sustained virologic response (SVR), especially those infected with HCV genotype 1 strains. The viral factors that mediate peglFN+RBV resistance remain to be identified. Genetic changes within the HCV nonstructural 5A (NS5A) and the nonstructural 5B (NS5B) genes have been associated with IFN and RBV failure, respectively. There are antivirals under development that target the HCV protease and polymerase enzymes but drug resistant strains have developed to these agents as well. The HCV genes that encode for these biologic targets are >5 Kb; thus, extensive sequencing is required to perform the important investigations into the genetic determinants of drug resistance. We have developed a novel HCV microarray that can determine the sequence of the HCV genotype la NS3, NS5A and NS5B genes in a single hybridization assay. We have validated this assay with HCV genotype la clones, sequencing >30 Kb with approximately 99% accuracy. In this study, we will 1) compare the abilities of a HCV microarray assay and standard sequencing methods to detect genetic changes in the NS3, NS5A and NS5B genes of clinical HCV genotype la strains. With this assay we will 2) test the hypothesis that peglFN+RBV therapy drives HCV RNA sequence selection within the NS5A and NS5B genes. We will perform investigations into the genetic determinants of peglFN and RBV resistance by determining the sequence changes in the NS3, NS5A and NS5B genes in HCV genotype la strains from 3 distinct patient populations: a. HCV genotype la infected patients who do not received peglFN+RBV b. Baseline HCV la samples from patients who had a SVR to peglFN + RBV. c. Pre and post samples from HCV la infected patients who are non-responders to peglFN+RBV. If successful this project will create a tool that can rapidly and accurately provide the sequence of HCV genes that encode drug targets and facilitate the investigations into the genetic determinants of HCV drug resistance.
