title: Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors.
abstract: Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5' untranslated region (5'UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of direct-acting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log10focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5'UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Long V
authors.initials: LV
authors.lastname: Pham
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Santseharay
authors.initials: S
authors.lastname: Ramirez
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Thomas H R
authors.initials: THR
authors.lastname: Carlsen
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Li
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark jbukh@sund.ku.dk.
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008826:Microbial Sensitivity Tests; D011480:Protease Inhibitors / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D020121:5' Untranslated Regions; D000998:Antiviral Agents; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; 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; C455041:NS4A cofactor peptide, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: HCV; antiviral agents; antiviral susceptibility testing; cell culture; direct-acting antivirals; genotype 1b; hepatitis C virus; liver disease