Efficient hepatitis c virus genotype 1b core-NS5A recombinants permit efficacy testing of protease and NS5A inhibitors
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Efficient hepatitis c virus genotype 1b core-NS5A recombinants permit efficacy testing of protease and NS5A inhibitors. / Pham, Long V.; Ramirez Almeida, Santseharay; Carlsen, Thomas H R; Li, Yi-Ping; Gottwein, Judith M.; Bukh, Jens.
In: Antimicrobial Agents and Chemotherapy, Vol. 61, No. 6, e00037-17, 2017.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Efficient hepatitis c virus genotype 1b core-NS5A recombinants permit efficacy testing of protease and NS5A inhibitors
AU - Pham, Long V.
AU - Ramirez Almeida, Santseharay
AU - Carlsen, Thomas H R
AU - Li, Yi-Ping
AU - Gottwein, Judith M.
AU - Bukh, Jens
PY - 2017
Y1 - 2017
N2 - 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 log10 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.
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 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 log10 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.
KW - Antiviral agents
KW - Antiviral susceptibility testing
KW - Cell culture
KW - Direct-Acting antivirals
KW - Genotype 1b
KW - HCV
KW - Hepatitis c virus
KW - Liver disease
U2 - 10.1128/AAC.00037-17
DO - 10.1128/AAC.00037-17
M3 - Journal article
C2 - 28348150
AN - SCOPUS:85019768101
VL - 61
JO - Antimicrobial Agents and Chemotherapy
JF - Antimicrobial Agents and Chemotherapy
SN - 0066-4804
IS - 6
M1 - e00037-17
ER -
ID: 180609711