PMID- 28348150 OWN - NLM STAT- MEDLINE DCOM- 20180226 LR - 20220110 IS - 1098-6596 (Electronic) IS - 0066-4804 (Print) IS - 0066-4804 (Linking) VI - 61 IP - 6 DP - 2017 Jun TI - Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors. LID - 10.1128/AAC.00037-17 [doi] LID - e00037-17 AB - 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 log(10) focus-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. CI - Copyright (c) 2017 American Society for Microbiology. FAU - Pham, Long V AU - Pham LV AD - 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. FAU - Ramirez, Santseharay AU - Ramirez S AD - 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. FAU - Carlsen, Thomas H R AU - Carlsen THR AD - 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. FAU - Li, Yi-Ping AU - Li YP AD - 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. FAU - Gottwein, Judith M AU - Gottwein JM AD - 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. FAU - Bukh, Jens AU - Bukh J AD - 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. LA - eng PT - Journal Article DEP - 20170524 PL - United States TA - Antimicrob Agents Chemother JT - Antimicrobial agents and chemotherapy JID - 0315061 RN - 0 (5' Untranslated Regions) RN - 0 (Antiviral Agents) RN - 0 (Carrier Proteins) RN - 0 (Intracellular Signaling Peptides and Proteins) RN - 0 (NS3 protein, hepatitis C virus) RN - 0 (NS4A cofactor peptide, Hepatitis C virus) RN - 0 (Protease Inhibitors) RN - 0 (Viral Nonstructural Proteins) RN - EC 2.7.7.48 (NS-5 protein, hepatitis C virus) SB - IM MH - 5' Untranslated Regions/genetics MH - Antiviral Agents/*pharmacology MH - Carrier Proteins/antagonists & inhibitors MH - Cell Line, Tumor MH - Hepacivirus/*drug effects/genetics/growth & development MH - Humans MH - Intracellular Signaling Peptides and Proteins MH - Microbial Sensitivity Tests MH - Protease Inhibitors/*pharmacology MH - Viral Nonstructural Proteins/*antagonists & inhibitors/*genetics PMC - PMC5444172 OTO - NOTNLM OT - HCV OT - antiviral agents OT - antiviral susceptibility testing OT - cell culture OT - direct-acting antivirals OT - genotype 1b OT - hepatitis C virus OT - liver disease EDAT- 2017/03/30 06:00 MHDA- 2018/02/27 06:00 PMCR- 2017/11/24 CRDT- 2017/03/29 06:00 PHST- 2017/01/21 00:00 [received] PHST- 2017/03/15 00:00 [accepted] PHST- 2017/03/30 06:00 [pubmed] PHST- 2018/02/27 06:00 [medline] PHST- 2017/03/29 06:00 [entrez] PHST- 2017/11/24 00:00 [pmc-release] AID - AAC.00037-17 [pii] AID - 00037-17 [pii] AID - 10.1128/AAC.00037-17 [doi] PST - epublish SO - Antimicrob Agents Chemother. 2017 May 24;61(6):e00037-17. doi: 10.1128/AAC.00037-17. Print 2017 Jun.