Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Viruses
2.3. Drugs
2.4. Quantification of HEV RNA
2.5. Luciferase Assay
2.6. Time-of-Addition Assay
2.7. Immunofluorescence Assay
2.8. Validation of the Anti-HEV Activity of the Ritonavir and Ribavirin Combination Therapy
2.9. Cell Viability Assay
2.10. Evaluation of the Efficacy of Ritonavir and Ribavirin Combination Therapy in a Cell Culture System
2.11. Evaluation of the Efficacy of Ritonavir and Ribavirin Combination Therapy Using PLC/PRF/5 Cells That Robustly Produce HEV
2.12. LDH Cytotoxicity Assay
2.13. Calculation of the Degree of Synergism with Ritonavir and Ribavirin Combination Therapy
2.14. Statistical Analyses
3. Results
3.1. Ritonavir Blocks HEV Internalization
3.2. Validation of the Anti-HEV Activity of Ritonavir and Ribavirin Combination Therapy against HEV Using the eHEV-nanoKAZ System
3.3. Evaluation of the Efficacy of Ritonavir and Ribavirin Combination Therapy for Inhibiting Virus Growth in PLC/PRF/5 Cells Inoculated with eHEV-3 or eHEV-4
3.4. Evaluation of the Efficacy of Ritonavir and Ribavirin Combination Therapy for Inhibiting Virus Growth in PLC/PRF/5 Cells Robustly Producing eHEV-3 or eHEV-4
3.5. The Degree of Synergism of Ritonavir and Ribavirin Combination Therapy
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Treatment | Intracellular HEV RNA * | |
---|---|---|
eHEV-3 | eHEV-4 | |
No drug treatment | 5.9 × 108 copies/well (mean) | 1.1 × 108 copies/well (mean) |
Ribavirin (40 μM) | 1.2 × 108 copies/well (mean) | 4.4 × 107 copies/well (mean) |
Ribavirin (40 μM) + Ritonavir (5 μM) | Undetectable in all wells | Undetectable in all wells |
Ribavirin (40 μM) + Ritonavir (10 μM) | Undetectable in all wells | Undetectable in all wells |
Ribavirin (40 μM) + Ritonavir (20 μM) | Undetectable in all wells | Undetectable in all wells |
Ribavirin (40 μM) + Ritonavir (35 μM) | Undetectable in all wells | Undetectable in all wells |
Treatment | LDH Release (Mean ± SD) * | |
---|---|---|
eHEV-3 | eHEV-4 | |
No drug treatment | 23.1% ± 7.1% | 2.7% ± 1.0% |
Ribavirin (40 μM) | 19.5% ± 5.4% | 2.7% ± 1.0% |
Ribavirin (40 μM) + Ritonavir (5 μM) | 4.6% ± 1.9% | 3.4% ± 1.8% |
Ribavirin (40 μM) + Ritonavir (10 μM) | 1.9% ± 0.3% | 1.9% ± 0.3% |
Ribavirin (40 μM) + Ritonavir (20 μM) | 3.9% ± 0.2% | 2.7% ± 1.2% |
Ribavirin (40 μM) + Ritonavir (35 μM) | 13.0% ± 2.2% | 12.8% ± 0.8% |
Treatment | Intracellular HEV RNA * | |
---|---|---|
eHEV-3 | eHEV-4 | |
No drug treatment | 1.4 × 108 copies/well (mean) | 2.5 × 108 copies/well (mean) |
Ribavirin (40 μM) | 1.6 × 107 copies/well (mean) | 2.3 × 107 copies/well (mean) |
Ribavirin (40 μM) + Ritonavir (5 μM) | 2.0 × 107 copies/well (mean) | 1.5 × 107 copies/well (mean) |
Ribavirin (40 μM) + Ritonavir (10 μM) | Undetectable in all wells | Undetectable in all wells |
Ribavirin (40 μM) + Ritonavir (20 μM) | Undetectable in all wells | Undetectable in all wells |
Ribavirin (40 μM) + Ritonavir (35 μM) | Undetectable in all wells | Undetectable in all wells |
Treatment | LDH Release (Mean ± SD) * | |
---|---|---|
eHEV-3 | eHEV-4 | |
No drug treatment | 15.9% ± 1.5% | 8.1% ± 2.4% |
Ribavirin (40 μM) | 10.9% ± 0.9% | 3.2% ± 1.0% |
Ribavirin (40 μM) + Ritonavir (5 μM) | 4.0% ± 1.4% | 3.0% ± 1.8% |
Ribavirin (40 μM) + Ritonavir (10 μM) | 3.4% ± 4.0% | 2.2% ± 0.4% |
Ribavirin (40 μM) + Ritonavir (20 μM) | 1.9% ± 0.4% | 3.6% ± 0.1% |
Ribavirin (40 μM) + Ritonavir (35 μM) | 4.6% ± 2.0% | 9.8% ± 2.1% |
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Primadharsini, P.P.; Nagashima, S.; Takahashi, M.; Murata, K.; Okamoto, H. Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin. Viruses 2022, 14, 2440. https://doi.org/10.3390/v14112440
Primadharsini PP, Nagashima S, Takahashi M, Murata K, Okamoto H. Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin. Viruses. 2022; 14(11):2440. https://doi.org/10.3390/v14112440
Chicago/Turabian StylePrimadharsini, Putu Prathiwi, Shigeo Nagashima, Masaharu Takahashi, Kazumoto Murata, and Hiroaki Okamoto. 2022. "Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin" Viruses 14, no. 11: 2440. https://doi.org/10.3390/v14112440
APA StylePrimadharsini, P. P., Nagashima, S., Takahashi, M., Murata, K., & Okamoto, H. (2022). Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin. Viruses, 14(11), 2440. https://doi.org/10.3390/v14112440