Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and Monitoring antiviral Resistant Strains
Résumé
Directly acting antivirals have contributed considerable progress to hepatitis C treatment, but they show variable activity depending on viral genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the hepatitis C virus (HCV) near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV1a, 1b, 3a and 4a, n = 16 of 22) and 45% for recombinant forms RF_2k/1b (n = 5 of 11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in 7 of 13 samples at baseline, in the NS3 (n = 3) or NS5A region (n = 4). Of these samples, the treatment of one patient included daclatasvir and that patient experienced a relapse. Viral sequences from pre- and post-treatment samples of four patients who relapsed after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proved to be successful for both genotyping and resistance-associated variant detection on several HCV types.