title: Characterization of novel entecavir resistance mutations.
abstract: OBJECTIVE
  Entecavir (ETV) is approved for the treatment of chronic hepatitis B virus (HBV) infections, but the virus can acquire resistance to the drug. This requires lamivudine resistance mutations (LAMr) and at least one additional mutation. Here, we characterized two novel mutations, rtI163V and rtA186T, associated with viral breakthrough (VBT) in an ETV-refractory patient.
  
  
  METHODS
  HBV from an ETV-refractory patient was sequenced, and newly identified mutations were inserted into a replication-competent clone by mutagenesis. Clones were analyzed for replication efficacy and susceptibility to ETV in vitro. Chimeric mice with human hepatocytes were inoculated with the patient's serum at VBT, and monitored for viral mutation pattern using a next-generation sequencing approach.
  
  
  RESULTS
  RtI163V and rtA186T mutations were detected together with LAMr (rtL180M and rtM204V) at VBT. RtA186T plus LAMr reduced susceptibility to ETV more than 111.1-fold compared with the wild-type clone, while rtI163V plus LAMr resulted in a 20.4-fold reduction. RtA186T significantly reduced viral replication efficacy, while the rtI163V mutation rescued it. Interestingly, the viral mutation pattern in the chimeric mice indicated dominant (or selective) proliferation of a clone containing rtI163V and rtA186T mutations plus LAMr under ETV treatment. Three-dimensional docking simulation indicated that rtA186T reduced the binding affinity of the HBV polymerase to ETV.
  
  
  CONCLUSIONS
  VBT in this ETV-refractory patient is attributable to the novel ETV resistance mutations rtI163V and rtA186T. RtA186T was apparently responsible for ETV resistance but the selection of a clone with the double mutation plus LAMr suggests that rtI163V is required to sustain viral fitness.
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan.
authors: Sanae
authors: S
authors: Hayashi
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan.
authors: Shuko
authors: S
authors: Murakami
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan.
authors: Katsumi
authors: K
authors: Omagari
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan; Center for Gastroenterology, Teine Keijinkai Hospital, Sapporo, Japan.
authors: Takeshi
authors: T
authors: Matsui
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan.
authors: Etsuko
authors: E
authors: Iio
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan.
authors: Masanori
authors: M
authors: Isogawa
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan.
authors: Tsunamasa
authors: T
authors: Watanabe
authors: Department of Gastroenterology, Sapporo Kosei General Hospital, Hokkaido, Japan.
authors: Yoshiyasu
authors: Y
authors: Karino
authors: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Nagoya, Japan. Electronic address: ytanaka@med.nagoya-cu.ac.jp.
authors: Yasuhito
authors: Y
authors: Tanaka
mesh_terms: D000328:Adult; D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use*; D024882:Drug Resistance, Viral; D006147:Guanine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D019694:Hepatitis B, Chronic / Q000188:drug therapy*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; C413685:entecavir; D006147:Guanine; D012194:RNA-Directed DNA Polymerase
keywords: Antiviral resistance; Combination therapy; Entecavir; Entecavir resistance; HBV mutations; Hepatitis B treatment; Hepatitis B virus; Lamivudine; Viral breakthrough; rtA186T; rtI163V