title: Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.
abstract: Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual.
authors.affiliation: Clinical Virology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden.
authors.forename: Assar
authors.initials: A
authors.lastname: Bergfors
authors.affiliation: Clinical Virology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden; Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 305-8577, Tsukuba, Japan.
authors.forename: Daniël
authors.initials: D
authors.lastname: Leenheer
authors.affiliation: Clinical Virology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Bergqvist
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory Uppsala, Uppsala University, SE-751-23, Sweden.
authors.forename: Adam
authors.initials: A
authors.lastname: Ameur
authors.affiliation: Clinical Virology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden. Electronic address: johan.lennerstrand@medsci.uu.se.
authors.forename: Johan
authors.initials: J
authors.lastname: Lennerstrand
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy / Q000235:genetics* / Q000628:therapy; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Deep sequencing; Hepatitis C virus; NS5A; Pacific biosciences; RAV; Resistance