title: Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus.
abstract: This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p<0.001). On X/preCore genotyping, all samples were genotype A, whereas polymerase/surface yielded genotypes A (80%), D (16.7%), and F (3.3%) (p = 0.036). Genotype changes in polymerase/surface were observed in four patients during natural quasispecies dynamics and in two patients during treatment. There were no genotype changes in X/preCore. Quasispecies complexity was higher in X/preCore than in polymerase/surface (p = 0.004). The results provide evidence of genotype mixtures and differential genotype proportions in the polymerase/surface and X/preCore regions. The genotype dynamics in HBV infection and the different patterns of quasispecies complexity in the HBV genome suggest a new paradigm for HBV genotype classification.
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Andrea
authors.initials: A
authors.lastname: Caballero
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.|Liver Diseases Unit, Vall d'Hebron Research Institute, Barcelona, Spain.|Roche Diagnostics S.L., Sant Cugat del Vallès, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Gregori
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Homs
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Tabernero
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Gonzalez
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.|Liver Diseases Unit, Vall d'Hebron Research Institute, Barcelona, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Quer
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Blasi
authors.affiliation: Liver Diseases Unit, Vall d'Hebron Research Institute, Barcelona, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Casillas
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Nieto
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.|Liver Unit, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Mar
authors.initials: M
authors.lastname: Riveiro-Barciela
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.|Liver Unit, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Esteban
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.|Liver Unit, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Buti
authors.affiliation: Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Rodriguez-Frias
mesh_terms: D000328:Adult; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D016679:Genome, Viral; D005838:Genotype; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D010802:Phylogeny; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA; D014758:Viral Core Proteins / Q000235:genetics; D055815:Young Adult
chemical_list: D004279:DNA, Viral; D014758:Viral Core Proteins