title: Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation.
abstract: Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome.
  
  
  
  Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.
authors.affiliation: Systems Medicine, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia.
authors.forename: B D
authors.initials: BD
authors.lastname: Betz-Stablein
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.|SIB Swiss Institute of Bioinformatics, Basel, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Töpfer
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Littlejohn
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Yuen
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Colledge
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.
authors.forename: V
authors.initials: V
authors.lastname: Sozzi
authors.affiliation: Austin Health, The University of Melbourne, Melbourne, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Angus
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.|Department of Gastroenterology, St. Vincent Hospital, Melbourne, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Thompson
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Revill
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.|SIB Swiss Institute of Bioinformatics, Basel, Switzerland.
authors.forename: N
authors.initials: N
authors.lastname: Beerenwinkel
authors.affiliation: Victorian Infectious Disease Reference Laboratory (VIDRL), Melbourne, Australia.
authors.forename: N
authors.initials: N
authors.lastname: Warner
authors.affiliation: Systems Medicine, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia luciani@unsw.edu.au.
authors.forename: F
authors.initials: F
authors.lastname: Luciani
mesh_terms: D000368:Aged; D000998:Antiviral Agents / Q000627:therapeutic use; D019295:Computational Biology; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000150:complications / Q000235:genetics* / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008103:Liver Cirrhosis / Q000601:surgery* / Q000821:virology; D016031:Liver Transplantation; D008297:Male; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral