title: A near-full length genotypic assay for HCV1b.
abstract: A near-full genome genotypic assay for HCV1b was developed, which may prove useful to investigate antiviral drug resistance, given new combination therapies for HCV1 infection. The assay consists of three partially overlapping PCRs followed by Sanger population or Illumina next-generation sequencing. Seventy-seven therapy-naïve samples, spanning the entire diversity range of currently known HCV1b, were used for optimization of PCRs, of which ten were sequenced using Sanger and of these ten, four using Illumina. The median detection limits for the three regions, 5'UTR-NS2, E2-NS5A and NS4B-NS5B, were 570, 5670 and 56,670 IU/ml respectively. The number of Illumina reads mapped varied according to the software used, Segminator II being the best performing (81%). Consensus Illumina and Sanger sequencing results accord largely (0.013% major discordances). Differences were due almost exclusively to a larger number of ambiguities (presumably minority variants) scored by Illumina (1.50% minor discordances). The assay is easy to perform in an equipped laboratory; nevertheless, it was difficult to reach high sensitivity and reproducibility, due to the high genetic viral variability. This assay proved to be suitable for detecting drug resistance mutations and can also be used for epidemiological research, even though only a limited set of samples was used for validation.
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. Electronic address: lize.cuypers@uzleuven.be.
authors.forename: Lize
authors.initials: L
authors.lastname: Cuypers
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Joke
authors.initials: J
authors.lastname: Snoeck
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: Vrancken
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Lien
authors.initials: L
authors.lastname: Kerremans
authors.affiliation: Debiopharm SA, Che. Messidor 5-7, P.O. Box 5911, 1002 Lausanne, Switzerland.
authors.forename: Grégoire
authors.initials: G
authors.lastname: Vuagniaux
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Jannick
authors.initials: J
authors.lastname: Verbeeck
authors.affiliation: Department of Hepatology, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium.
authors.forename: Frederik
authors.initials: F
authors.lastname: Nevens
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium; Centro de Malária e Outras Doencas Tropicais and Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisbon, Portugal.
authors.forename: Ricardo J
authors.initials: RJ
authors.lastname: Camacho
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium; Centro de Malária e Outras Doencas Tropicais and Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisbon, Portugal.
authors.forename: Anne-Mieke
authors.initials: AM
authors.lastname: Vandamme
authors.affiliation: Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Sonia
authors.initials: S
authors.lastname: Van Dooren
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification* / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D000998:Antiviral Agents
keywords: Drug resistance; Full-genome sequencing; Genotyping; HCV1b; Illumina; NGS