title: HCV NS3 quasispecies in liver and plasma and dynamics of telaprevir-resistant variants in breakthrough patients assessed by UDPS: A case study.
abstract: BACKGROUND
  The impact of pre-existing variants in hepatitis C virus (HCV) quasispecies, carrying resistance-associated mutations (RAMs), on the outcome of treatment with direct acting antiviral agents (DAA) is debated and it is complicated by the lack of knowledge of quasispecies distribution between the viral reservoir (liver) and the circulating compartment.
  
  
  OBJECTIVE
  To evaluate NS3 protease heterogeneity and presence of RAMs on baseline plasma and liver biopsy samples. Plasma dynamics were also analyzed during therapy and after its suspension. Study design Ultra-deep pyrosequencing (UDPS) was performed in two HCV genotype 1a patients who received telaprevir (TVR)-based therapy and developed treatment failure due to TVR-resistance.
  
  
  RESULTS
  In both patients the baseline diversity of NS3 quasispecies in plasma was higher than in liver (183.6×10(-4) vs 47.8×10(-4) and 246.0×10(-4) vs 55.0×10(-4) nt substitution/site, respectively, p<0.0001), but phylogenetic trees did not evidence compartmentalization between the two compartments. At baseline RAMs (i.e. V36A, T54A) were detected very low levels (range: 0.31-0.52%) in both specimen types. However, phylogenetic analyses revealed that the viral variants carrying these mutations at baseline were different from those that became fixed at breakthrough, when combined V36M+R155K, conferring high-level resistance to TVR, were observed. The frequency of resistance-associated variants declined after withdrawal of drug selective pressure.
  
  
  CONCLUSIONS
  UDPS allowed extensive evaluation of quasispecies compartmentalization and of their dynamics after withdrawal of TVR. Plasma and liver NS3 quasispecies, including low level RAMs, do not show significant difference.
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bartolini
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Selleri
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Anna Rosa
authors.initials: AR
authors.lastname: Garbuglia
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Giombini
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Taibi
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Lionetti
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy.
authors.forename: Gianpiero
authors.initials: G
authors.lastname: D'Offizi
authors.affiliation: "L. Spallanzani" National Institute of Infectious Diseases, Via Portuense 292, 00149 Rome, Italy. Electronic address: maria.capobianchi@inmi.it.
authors.forename: Maria R
authors.initials: MR
authors.lastname: Capobianchi
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use*; D024882:Drug Resistance, Viral; D014644:Genetic Variation; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008099:Liver / Q000821:virology; D008137:Longitudinal Studies; D009842:Oligopeptides / Q000494:pharmacology / Q000627:therapeutic use*; D010949:Plasma / Q000821:virology; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D009842:Oligopeptides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C486464:telaprevir
keywords: Diversity; HCV; NS3; Resistance associated mutations; Ultra-deep pyrosequencing; Viral quasispecies