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 directacting 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.

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

“…In vitro testing is required for characterization of RASs. In constrast to enzyme and replicon assays, infectious HCV culture systems allow investigation of the complete genome and viral life cycle, and results obtained in such systems reflect in vivo data …”

“…Recombinants had NS3P of the genotypes(isolates) 1a(TN), 1b(DH1), 2a(JFH1), 2b(DH8), 3a(S52), 3a(DBN), 4a(ED43), 5a(SA13), and 6a(HK6a) (Supporting Fig. ).…”

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals

“…Full-length sequence includes 1b (Con1, J4, and DH1), 3a (S52), 4a (ED43) and 5a (SA13), and 7a (QC69) (Gottwein et al, 2009; Li Y.P. et al, 2014; Pham et al, 2017; Li et al, 2018). The evolutionary history was inferred by using the neighbor-joining method in the freeware Molecular Evolutionary Genetics Analysis (MEGA), version 7 (Kumar et al, 2016).…”

Desenho vacinal por imunoinformática contra HCV: Caracterização química e predição de epítopos de células T

“…Dessa forma, a heterogeneidade no genoma, causada pela alta taxa de replicação sem o fator de reparo, gera a possibilidade de classificar seis genótipos, nomeados de 1 à 6 pela ordem de descoberta. Possuindo diversos subtipos, mais de 50 categorizados e descritos atualmente, representados por letras do alfabeto (1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a e 6a) e apresenta muitas quasiespecies, sendo outro fator importante para a progressão da infectividade (Bulut et al, 2021;Pham, 2017). O genótipo 1 é o mais comum em todo o mundo e o subtipo mais é o subtipo 1b, já no Brasil os genótipos 1, 2 e 3 são encontrados com maior frequência, sendo em números menores ou mesmo rara a infecção por outros tipos (Sawada et al, 2011).…”