title: Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the Hepatitis C virus protease coding-region.
abstract: A new generation of drugs targeting the non-structural (NS) proteins of the Hepatitis C virus (HCV) will substantially increase treatment success rates, reducing global infections. Amongst the NS proteins, the NS3 protease represents an important drug target, responsible for liberation of mature NS proteins from the nascent HCV polyprotein and suppression of host innate immunity. Despite this, the evolutionary stability of the genomic locus encoding the NS3 protease is poorly characterized in chronic HCV infection. To address this shortfall, we developed a high-throughput amplicon pyrosequencing protocol and utilised it to monitor NS3 protease coding-sequence evolution for over a decade in two patients. Although patient-specific evolutionary trends were apparent, the protease amino acid population consensus remained stable with a massive excess of synonymous mutations observed, confirming this locus is under strong purifying selection during chronic infection within individual patients. No evidence for continuous immune escape was detected. Additionally, both patients failed protease inhibitor (PI) therapy and protease sequence diversity pre- and post-therapy were also assessed. No baseline resistance associated variants (RAVs) contributed to treatment failure. Significant reductions in viral diversity were observed post-PI therapy, indicating a population bottleneck occurred. The genetic vestiges of this bottleneck were still detectable 18months after therapy discontinuation. Although significant enrichment of the Q80L mutation was observed in one patient, genetic and phenotypic data reveal no detectable RAV persistence post-therapy failure. Together this investigation provides a sensitive and reproducible high-throughput framework to interrogate viral sequence diversity at high-resolution, with potential applications for routine monitoring of treatment regimens. This study also reveals novel insights into the evolutionary processes that shape NS3 sequence divergence in both chronic HCV infection and post PI-therapy failure.
authors: The School of Life Sciences and the NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: William L
authors: WL
authors: Irving
authors: Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg University, 69120 Heidelberg, Germany.
authors: Daniel
authors: D
authors: Rupp
authors: The School of Life Sciences and the NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: C Patrick
authors: CP
authors: McClure
authors: The School of Life Sciences and the NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: Lwin Mar
authors: LM
authors: Than
authors: The School of Life Sciences and the NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: Andrew
authors: A
authors: Titman
authors: The School of Life Sciences and the NIHR Nottingham Digestive Diseases Biomedical Research Unit, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors: Jonathan K
authors: JK
authors: Ball
authors: Divison of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Str. 7, 30625 Hannover, Germany.
authors: Eike
authors: E
authors: Steinmann
authors: Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg University, 69120 Heidelberg, Germany.
authors: Ralf
authors: R
authors: Bartenschlager
authors: Divison of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Str. 7, 30625 Hannover, Germany.
authors: Thomas
authors: T
authors: Pietschmann
authors: Divison of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Str. 7, 30625 Hannover, Germany. Electronic address: richard.brown@twincore.de.
authors: Richard J P
authors: RJ
authors: Brown
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000235:genetics*; D019143:Evolution, Molecular; D014644:Genetic Variation; D016174:Hepacivirus / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000188:drug therapy; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012189:Retrospective Studies; D017423:Sequence Analysis, RNA; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D009842:Oligopeptides; D011392:Proline; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide
keywords: Chronic infection; HCV NS3 protease; Pyrosequencing; RAV persistence