title: Deep sequencing of hepatitis C virus hypervariable region 1 reveals no correlation between genetic heterogeneity and antiviral treatment outcome.
abstract: BACKGROUND
  Hypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable part of HCV genome and its translation product is a major target for the host immune response. Variability within HVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study was to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of PEG-IFN-α (pegylated interferon α) and ribavirin treatment.
  
  
  METHODS
  HVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype 1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche). Reads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using three different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mann-Whitney and Fisher's exact tests.
  
  
  RESULTS
  Complexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic substitutions were not significantly different between responders and non-responders, when analyzing viral populations at any of the three frequencies (≥1%, ≥2% and ≥5%). When clonal sample was used to determine pyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a frequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous variant present at frequency of 0.5%.
  
  
  CONCLUSIONS
  While deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients, there was no correlation between treatment outcome and any of the analyzed quasispecies parameters.
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, 3c Pawińskiego Street, 02-106 Warsaw, Poland. kcaraballo@wum.edu.pl.
authors.forename: Kamila Caraballo
authors.initials: KC
authors.lastname: Cortés
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Zagordi
authors.forename: Karol
authors.initials: K
authors.lastname: Perlejewski
authors.forename: Tomasz
authors.initials: T
authors.lastname: Laskus
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Maroszek
authors.forename: Iwona
authors.initials: I
authors.lastname: Bukowska-Ośko
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pawełczyk
authors.forename: Rafał
authors.initials: R
authors.lastname: Płoski
authors.forename: Hanna
authors.initials: H
authors.lastname: Berak
authors.forename: Andrzej
authors.initials: A
authors.lastname: Horban
authors.forename: Marek
authors.initials: M
authors.lastname: Radkowski
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D005260:Female; D018740:Genetic Heterogeneity; D014644:Genetic Variation; D016174:Hepacivirus / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use*; D008297:Male; D008875:Middle Aged; D011446:Prospective Studies; D011994:Recombinant Proteins / Q000627:therapeutic use; D012254:Ribavirin / Q000627:therapeutic use*; D016896:Treatment Outcome; D014759:Viral Envelope Proteins / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D012254:Ribavirin