In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide
Abstract
:1. Introduction
2. Materials and Methods
2.1. Production of Lentiviral Vectors Expressing NTCP
2.2. Establishment of Stable NTCP-Expressing Huh7.5 Cell Line (Huh7.5-NTCP)
2.3. Conventional Culture of Huh7.5 Cells and Huh7.5-NTCP Cells
2.4. Differentiation of Huh7.5 and Huh7.5-NTCP Cells in Human Serum
2.5. Infection of Cells with HBV
2.6. Culture of PXB Cells
2.7. Analysis of Pregenomic RNA (pgRNA)
2.8. Analysis of Covalently Closed Circular DNA (cccDNA)
2.9. Enzyme-Linked Immunosorbent Assays (ELISAs)
2.10. Aptamer-Binding Assay for the E Antigen (HBeAg)
2.11. Immunofluorescence Staining of Huh7.5 Cells Overexpressing NTCP
2.12. Flow Cytometry Analysis of NTCP Expression
2.13. Western Blotting
2.14. Nanoluciferase Reporter Luminescence Assay
2.15. Statistical Analysis
3. Results
3.1. Huh7.5 Cell Line Overexpressing NTCP (Huh7.5-NTCP Cells)
3.2. Human Serum Culture Enhanced Productive HBV Infection in Huh7.5-NTCP Cells
3.3. Huh7.5-NTCP Cells in a Human Serum Culture Serve as a Model for Long-Term HBV Infection
3.4. Human Serum Alters Hepatocyte Differentiation Markers in Huh7.5-NTCP
3.5. Involvement of NTCP and Possible Effect of Its N-Glycosylation on Viral Entry
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Le, C.; Sirajee, R.; Steenbergen, R.; Joyce, M.A.; Addison, W.R.; Tyrrell, D.L. In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide. Viruses 2021, 13, 97. https://doi.org/10.3390/v13010097
Le C, Sirajee R, Steenbergen R, Joyce MA, Addison WR, Tyrrell DL. In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide. Viruses. 2021; 13(1):97. https://doi.org/10.3390/v13010097
Chicago/Turabian StyleLe, Connie, Reshma Sirajee, Rineke Steenbergen, Michael A. Joyce, William R. Addison, and D. Lorne Tyrrell. 2021. "In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide" Viruses 13, no. 1: 97. https://doi.org/10.3390/v13010097