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Abstract:

Background: Resistance-associated variants (RAVs) emerge in HCV viral quasispecies at low frequency, reducing treatment response of Direct Antiviral Agents (DAAs). Next Generation Sequencing (NGS) technologies and a new analysis approach are used to disclose hot spot positions in HCV NS3 protease. We investigated presence of baseline RAVs in a cohort with a specific workflow not previous reported. Materials and methods: In 2014, serum samples from eight HCV chronic infected patients were collected. HCV RNA was measured by routine diagnostic method. HCV NS5B region was sequenced by Sanger technique. HCV genotyping was performed by phylogenetic analysis of NS5B region (100 aa) using RAxML. Ultra-deep sequencing of NS3 region (181 aa) using PGM Sequencer was performed. NS3 consensus sequence was generated by homemade script: after pre-processing and quality control of raw data using FastQC, a de novo assembly was performed using software packages VICUNA and V-FAT, to create consensus sequences. Resistance mutation positions for NS5B and NS3 sequences by Geno2pheno were analyzed. Results: All patients were DAAs-naïve, not co-infected with HIV or HBV, and infected by HCV 1b. Seven patients were experienced and only one was naïve to interferon/ribavirin. Treatment included Telaprevir (TVR) in 7 patients and Boceprevir (BOC) in one patient. Median age was 55 years (range 44-68), 6/8 were males. Surgery and/or cohabitation with HCV positive individuals were the most frequent risk factors reported by patients, followed by tattoo. At baseline, median HCV RNA was 6.32 log10 IU/ml, median AST and ALT levels were 40IU/l and 61IU/l, respectively. Overall, patients had undetectable HCV RNA at the end of therapy. V36L mutation, conferring resistance to BOC and possible resistance to TVR or Simeprevir, was detected in naïve patient’s HCV isolate at baseline. In 4/8 patients, we identified I132V mutation associated with resistance to TVR. The C316N mutation for Dasabuvir (DSV) was found in two patients’ isolates. No RAVs were found for Paritaprevir or Sofosbuvir resistance, however V338A (NS5B region) and D30E+I170V (NS3 region), not drug resistance related, were detected in all samples at baseline. Conclusions: PGM and our script homemade analysis are a powerful method to analyse viral variability and minority variants on NS3 coding region. Indeed, free software package allows to detect non-synonymous mutation, generating in silico specific Sanger consensus sequence per each sample through de novo assembly. Interestingly, five patients carried RAVs associated to TVR, suggesting a possible failure of therapy. Also, despite the fact that only a portion of the NS5B region was analysed by Sanger method, two patients’ isolates carried C316N mutation, which would have contraindicated DSV use.