title: Deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype 1b hepatitis C virus.
abstract: Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy.
  
  
  OBJECTIVE
  In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Mitsuaki
authors: M
authors: Sato
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan maekawa@yamanashi.ac.jp.
authors: Shinya
authors: S
authors: Maekawa
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Nobutoshi
authors: N
authors: Komatsu
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Akihisa
authors: A
authors: Tatsumi
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Mika
authors: M
authors: Miura
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Masaru
authors: M
authors: Muraoka
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Yuichiro
authors: Y
authors: Suzuki
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Fumitake
authors: F
authors: Amemiya
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Shinichi
authors: S
authors: Takano
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Mitsuharu
authors: M
authors: Fukasawa
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Yasuhiro
authors: Y
authors: Nakayama
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Tatsuya
authors: T
authors: Yamaguchi
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Tomoyoshi
authors: T
authors: Uetake
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Taisuke
authors: T
authors: Inoue
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Tadashi
authors: T
authors: Sato
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Minoru
authors: M
authors: Sakamoto
authors: Department of Microbiology, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Atsuya
authors: A
authors: Yamashita
authors: Department of Microbiology, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Kohji
authors: K
authors: Moriishi
authors: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan.
authors: Nobuyuki
authors: N
authors: Enomoto
mesh_terms: D000328:Adult; D000368:Aged; D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D024882:Drug Resistance, Viral; D005260:Female; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000627:therapeutic use; D010802:Phylogeny; D011480:Protease Inhibitors / Q000627:therapeutic use*; D012254:Ribavirin / Q000627:therapeutic use; D016415:Sequence Alignment; 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; D016898:Interferon-alpha; D009842:Oligopeptides; D011480:Protease Inhibitors; D012254:Ribavirin; 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