Potential conflict of interest: Nothing to report.
Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors
Article first published online: 13 DEC 2013
© 2013 by the American Association for the Study of Liver Diseases
Volume 59, Issue 2, pages 395–407, February 2014
How to Cite
Ramirez, S., Li, Y.-P., Jensen, S. B., Pedersen, J., Gottwein, J. M. and Bukh, J. (2014), Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors. Hepatology, 59: 395–407. doi: 10.1002/hep.26660
This study was supported by research grants from The Lundbeck Foundation (to S.R., Y.P.L., J.M.G., and J.B.), The Danish Cancer Society (to J.M.G. and J.B.), The Novo Nordisk Foundation (to Y.P.L., J.M.G., and J.B.), The Region H Research Fund (to S.R., J.M.G., and J.B.), The A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fondation (to J.B.), and the Danish Council for Independent Research, Medical Sciences (to S.R., Y.P.L., and J.B.). S.R. is the recipient of an Individual Postdoctoral Stipend from the Danish Council for Independent Research of Medical Sciences. The authors thank C.M. Rice (Rockefeller University, New York, NY) for providing valuable reagents.
- Issue published online: 29 JAN 2014
- Article first published online: 13 DEC 2013
- Accepted manuscript online: 2 AUG 2013 06:55AM EST
- Manuscript Accepted: 25 JUL 2013
- Manuscript Received: 12 JUN 2013
Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. Conclusion: Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment. (Hepatology 2014;59:395–407)