title: Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy.
abstract: Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC).
  
  
  
  Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes).
  
  
  
  Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion.
  
  
  
  The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects.
  
  
  
  Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.
authors.affiliation: Department of Medicine III, RWTH-University Hospital Aachen, Aachen, Germany; Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
authors.forename: Elham
authors.initials: E
authors.lastname: Shirvani-Dastgerdi
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
authors.forename: Benjamin Y
authors.initials: BY
authors.lastname: Winer
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
authors.forename: Toni
authors.initials: T
authors.lastname: Celià-Terrassa
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
authors.forename: Yibin
authors.initials: Y
authors.lastname: Kang
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; Liver Unit, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Tabernero
authors.affiliation: Medical Care Centre, Dr Stein and Colleagues, Mönchengladbach, Germany.
authors.forename: Eray
authors.initials: E
authors.lastname: Yagmur
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; Liver Unit, Vall d'Hebron Institute of Research (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Rodríguez-Frías
authors.affiliation: Roche Diagnostics SL, Sant Cugat del Vallès, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Gregori
authors.affiliation: Department of Medicine III, RWTH-University Hospital Aachen, Aachen, Germany.
authors.forename: Tom
authors.initials: T
authors.lastname: Luedde
authors.affiliation: Department of Medicine III, RWTH-University Hospital Aachen, Aachen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Trautwein
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ploss
authors.affiliation: Department of Medicine III, RWTH-University Hospital Aachen, Aachen, Germany. Electronic address: frank.tacke@gmx.net.
authors.forename: Frank
authors.initials: F
authors.lastname: Tacke
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000627:therapeutic use; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics; D004359:Drug Therapy, Combination; D005260:Female; D015684:Gene Products, pol / Q000235:genetics*; D005814:Genes, Viral; D006147:Guanine / Q000031:analogs & derivatives / Q000627:therapeutic use; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D019694:Hepatitis B, Chronic / Q000188:drug therapy* / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D000068698:Tenofovir / Q000627:therapeutic use; D014779:Virus Replication / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; C413685:entecavir; D006147:Guanine; D000068698:Tenofovir; D012194:RNA-Directed DNA Polymerase
keywords: Carcinoma; Drug resistance; Exosomes; Hepatitis B surface antigens; Hepatitis B virus; Hepatocellular; Mutations