title: Next generation sequencing of the hepatitis C virus NS5B gene reveals potential novel S282 drug resistance mutations.
abstract: Identifying HCV drug resistance mutations (DRMs) is increasingly important as new direct acting antiviral therapies (DAA) become available. Tagged pooled pyrosequencing (TPP) was originally developed as cost-effective approach for detecting low abundance HIV DRMs. Using 127 HCV-positive samples from a Canadian injection drug user cohort, we demonstrated the suitability and efficiency of TPP for evaluating DRMs in HCV NS5B gene. At a mutation identification threshold of 1%, no nucleoside inhibitor DRMs were detected among these DAA naïve subjects. Clinical NS5B resistance to non-nucleoside inhibitors and interferon/ribavirin was predicted to be low within this cohort. S282T mutation, the primary mutation selected by sofosbuvir in vitro, was not identified while S282G/C/R variants were detected in 9 subjects. Further characterization on these new S282 variants using in silico molecular modeling implied their potential association with resistance. Combining TPP with in silico analysis detects NS5B polymorphisms that may explain differences in treatment outcomes.
authors.affiliation: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Hezhao
authors.initials: H
authors.lastname: Ji
authors.affiliation: Department of Pathobiology, University of Guelph, Guelph, Canada.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kozak
authors.affiliation: Arthur Labatt Family School of Nursing, Western University, London, Canada.
authors.forename: Mia J
authors.initials: MJ
authors.lastname: Biondi
authors.affiliation: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Pilon
authors.affiliation: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Vallee
authors.affiliation: Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Ben Binhua
authors.initials: BB
authors.lastname: Liang
authors.affiliation: Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: David
authors.initials: D
authors.lastname: La
authors.affiliation: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Kim
authors.affiliation: Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Gary
authors.initials: G
authors.lastname: Van Domselaar
authors.affiliation: Department of Epidemiology and Community Medicine, University of Ottawa, Ottawa, Canada.
authors.forename: Lynne
authors.initials: L
authors.lastname: Leonard
authors.affiliation: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: Sandstrom
authors.affiliation: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada. Electronic address: james.brooks@phac-aspc.gc.ca.
authors.forename: James
authors.initials: J
authors.lastname: Brooks
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D015331:Cohort Studies; D024882:Drug Resistance, Viral; D005260:Female; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D050505:Mutant Proteins / Q000235:genetics*; D020125:Mutation, Missense; D015819:Substance Abuse, Intravenous / Q000150:complications; D016896:Treatment Outcome; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D050505:Mutant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Drug resistance mutation; HCV; In silico molecular modeling; NS5B; Pyrosequencing