title: Similar prevalence of low-abundance drug-resistant variants in treatment-naive patients with genotype 1a and 1b hepatitis C virus infections as determined by ultradeep pyrosequencing.
abstract: OBJECTIVE
  Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.
  
  
  METHODS
  We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.
  
  
  RESULTS
  A total of 3.5 million high-quality reads of ≥ 200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.
  
  
  CONCLUSIONS
  The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.
authors.affiliation: Infectious Diseases and Geographic Medicine, Stanford University Medical School, Stanford, California, United States of America.
authors.forename: Severine
authors.initials: S
authors.lastname: Margeridon-Thermet
authors.affiliation: Infectious Diseases Discovery, Pharma Research Early Development, Roche, Nutley, New Jersey, United States of America; Pharma Development, Genentech, South San Francisco, California, United States of America.
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Pogam
authors.affiliation: Infectious Diseases Discovery, Pharma Research Early Development, Roche, Nutley, New Jersey, United States of America.
authors.forename: Lewyn
authors.initials: L
authors.lastname: Li
authors.affiliation: Infectious Diseases and Geographic Medicine, Stanford University Medical School, Stanford, California, United States of America.
authors.forename: Tommy F
authors.initials: TF
authors.lastname: Liu
authors.affiliation: Pharma Development, Genentech, South San Francisco, California, United States of America.
authors.forename: Nancy
authors.initials: N
authors.lastname: Shulman
authors.affiliation: Infectious Diseases and Geographic Medicine, Stanford University Medical School, Stanford, California, United States of America.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Shafer
authors.affiliation: Infectious Diseases Discovery, Pharma Research Early Development, Roche, Nutley, New Jersey, United States of America; Infectious Diseases Discovery, Pharma Research Early Development, Roche, Shanghai, China.
authors.forename: Isabel
authors.initials: I
authors.lastname: Najera
mesh_terms: D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000235:genetics*; D005787:Gene Frequency; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase