title: High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease.
abstract: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution.
  
  
  
  To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant.
  
  
  
  HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization.
  
  
  
  We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization.
  
  
  
  HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.
authors.affiliation: Centre for Human Virology, University of Birmingham, Birmingham, UK.
authors.forename: Ditte L
authors.initials: DL
authors.lastname: Hedegaard
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.
authors.forename: Damien C
authors.initials: DC
authors.lastname: Tully
authors.affiliation: Centre for Human Virology, University of Birmingham, Birmingham, UK.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Rowe
authors.affiliation: NIHR Birmingham Liver Biomedical Research Unit, University of Birmingham, UK.
authors.forename: Gary M
authors.initials: GM
authors.lastname: Reynolds
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Bean
authors.affiliation: Centre for Human Virology, University of Birmingham, Birmingham, UK.
authors.forename: Ke
authors.initials: K
authors.lastname: Hu
authors.affiliation: Centre for Human Virology, University of Birmingham, Birmingham, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Davis
authors.affiliation: NIHR Birmingham Liver Biomedical Research Unit, University of Birmingham, UK.
authors.forename: Annika
authors.initials: A
authors.lastname: Wilhelm
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.
authors.forename: Colin B
authors.initials: CB
authors.lastname: Ogilvie
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Power
authors.affiliation: School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tarr
authors.affiliation: Liver Unit, Birmingham Childrens' Hospital, Birmingham, UK.
authors.forename: Deirdre
authors.initials: D
authors.lastname: Kelly
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.
authors.forename: Todd M
authors.initials: TM
authors.lastname: Allen
authors.affiliation: Centre for Human Virology, University of Birmingham, Birmingham, UK. Electronic address: p.balfe@bham.ac.uk.
authors.forename: Peter
authors.initials: P
authors.lastname: Balfe
authors.affiliation: Centre for Human Virology, University of Birmingham, Birmingham, UK; NIHR Birmingham Liver Biomedical Research Unit, University of Birmingham, UK; Institute for Advanced Study, Technische Universität München, Lichtenbergstrasse 2a, D-85748 Garching, Germany.
authors.forename: Jane A
authors.initials: JA
authors.lastname: McKeating
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology; D002451:Cell Compartmentation / Q000187:drug effects; D058625:End Stage Liver Disease / Q000209:etiology / Q000378:metabolism / Q000821:virology; D005260:Female; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D019698:Hepatitis C, Chronic / Q000150:complications / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D008099:Liver / Q000473:pathology / Q000821:virology; D008297:Male; D008875:Middle Aged; D012367:RNA, Viral / Q000032:analysis; D017423:Sequence Analysis, RNA / Q000379:methods; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D007372:Interferons
keywords: Compartmentalization; ESLD; Evolution; Hepatitis C; Innate immunity