title: A translational study of resistance emergence using sequential direct-acting antiviral agents for hepatitis C using ultra-deep sequencing.
abstract: OBJECTIVE
  Direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) have recently been developed and are ultimately hoped to replace interferon-based therapy. However, DAA monotherapy results in rapid emergence of resistant strains and DAAs must be used in combinations that present a high genetic barrier to resistance, although viral kinetics of multidrug-resistant strains remain poorly characterized. The aim of this study is to track the emergence and fitness of resistance using combinations of telaprevir and NS5A or NS5B inhibitors with genotype 1b clones.
  
  
  METHODS
  HCV-infected chimeric mice were treated with DAAs, and resistance was monitored using direct and ultra-deep sequencing.
  
  
  RESULTS
  Combination therapy with telaprevir and BMS-788329 (NS5A inhibitor) reduced serum HCV RNA to undetectable levels. The presence of an NS3-V36A telaprevir resistance mutation resulted in poor response to telaprevir monotherapy but showed significant HCV reduction when telaprevir was combined with BMS-788329. However, a BMS-788329-resistant strain emerged at low frequency. Infection with a BMS-788329-resistant NS5A-L31V mutation rapidly resulted in gain of an additional NS5A-Y93A mutation that conferred telaprevir resistance during combination therapy. Infection with dual NS5AL31V/NS5AY93H mutations resulted in poor response to combination therapy and development of telaprevir resistance. Although HCV RNA became undetectable soon after the beginning of combination therapy with BMS-788329 and BMS-821095 (NS5B inhibitor), rebound with emergence of resistance against all three drugs occurred. Triple resistance also occurred following infection with the NS3V36A/NS5AL31V/NS5AY93H triple mutation.
  
  
  CONCLUSIONS
  Resistant strains easily develop from cloned virus strains. Sequential use of DAAs should be avoided to prevent emergence of multidrug-resistant strains.
authors.affiliation: Center for Medical Specialist Graduate Education and Research, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Abe
authors.forename: C Nelson
authors.initials: CN
authors.lastname: Hayes
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Hiraga
authors.forename: Michio
authors.initials: M
authors.lastname: Imamura
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuge
authors.forename: Daiki
authors.initials: D
authors.lastname: Miki
authors.forename: Shoichi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ochi
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Chayama
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use*; D024882:Drug Resistance, Viral / Q000187:drug effects* / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy* / Q000235:genetics / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing; D051379:Mice; D009842:Oligopeptides / Q000627:therapeutic use*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D009842:Oligopeptides; D017361:Viral Nonstructural Proteins; C486464:telaprevir; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase