title: Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing.
abstract: OBJECTIVE
  The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail.
  
  
  METHODS
  Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy.
  
  
  RESULTS
  Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents.
  
  
  CONCLUSIONS
  Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nasu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Marusawa
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Ueda
authors.forename: Norihiro
authors.initials: N
authors.lastname: Nishijima
authors.forename: Ken
authors.initials: K
authors.lastname: Takahashi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Osaki
authors.forename: Yukitaka
authors.initials: Y
authors.lastname: Yamashita
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Inokuma
authors.forename: Takashi
authors.initials: T
authors.lastname: Tamada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Sato
authors.forename: Kazuharu
authors.initials: K
authors.lastname: Shimizu
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Chiba
mesh_terms: D000368:Aged; D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004359:Drug Therapy, Combination; D005260:Female; D018740:Genetic Heterogeneity; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006526:Hepatitis C / Q000188:drug therapy* / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000187:drug effects; D059645:Mutation Rate; D011092:Polyethylene Glycols / Q000627:therapeutic use; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000627:therapeutic use; D012254:Ribavirin / Q000627:therapeutic use; D016896:Treatment Outcome
chemical_list: D000998:Antiviral Agents; D018076:DNA, Complementary; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D012254:Ribavirin; C417083:peginterferon alfa-2b