HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels

J Antimicrob Chemother. 2016 Mar;71(3):739-50. doi: 10.1093/jac/dkv403. Epub 2015 Dec 17.


Objectives: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen.

Methods: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays.

Results: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11).

Conclusions: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Drug Resistance, Viral*
  • Genotype
  • Genotyping Techniques / methods*
  • Hepacivirus / classification*
  • Hepacivirus / drug effects*
  • Hepacivirus / genetics
  • Hepacivirus / isolation & purification
  • Hepatitis C / virology*
  • Humans
  • Mutation*
  • RNA, Viral / genetics
  • Retrospective Studies
  • Sequence Analysis, DNA
  • Viral Nonstructural Proteins / genetics*


  • NS3 protein, hepatitis C virus
  • RNA, Viral
  • Viral Nonstructural Proteins