Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Genetic Recombination and Reconstitution of Infectious HCMV TB40-FLuc
2.2. Cell Culture and Virus Infection
2.3. Luminescence-Based and Fluorescence-Based HCMV Replication Assays
2.4. Neutral Red Assay
2.5. Quantitative Real-Time PCR (qPCR)
2.6. Indirect Immunofluorescence (IF) Analysis and Confocal Laser-Scanning Microscopy
2.7. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis
2.8. Antiviral Compounds
3. Results and Discussion
3.1. Generation of Recombinant HCMV BAC and Reconstitution of the TB40-FLuc Reporter Virus
3.2. Establishment of the Infection System with HCMV TB40-FLuc in Primary Human Fibroblasts
3.3. Replication Kinetics of HCMV TB40-FLuc in Single-Round or Multi-Round Settings
3.4. Characterization of Viral Protein Expression Patterns in HCMV TB40-FLuc-Infected Cells
3.5. Assessment of Antiviral Drug Activity in Two HCMV Reporter Systems (TB40-FLuc and AD169-GFP)
3.6. Determination of Reporter Sensitivity Comparing Different HCMV Infection Systems
3.7. Further Options for the Application of HCMV TB40-FLuc
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Tillmanns, J.; Kicuntod, J.; Ehring, A.; Elbasani, E.; Borst, E.M.; Obergfäll, D.; Müller, R.; Hahn, F.; Marschall, M. Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy. Pathogens 2024, 13, 645. https://doi.org/10.3390/pathogens13080645
Tillmanns J, Kicuntod J, Ehring A, Elbasani E, Borst EM, Obergfäll D, Müller R, Hahn F, Marschall M. Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy. Pathogens. 2024; 13(8):645. https://doi.org/10.3390/pathogens13080645
Chicago/Turabian StyleTillmanns, Julia, Jintawee Kicuntod, Antonia Ehring, Endrit Elbasani, Eva Maria Borst, Debora Obergfäll, Regina Müller, Friedrich Hahn, and Manfred Marschall. 2024. "Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy" Pathogens 13, no. 8: 645. https://doi.org/10.3390/pathogens13080645
APA StyleTillmanns, J., Kicuntod, J., Ehring, A., Elbasani, E., Borst, E. M., Obergfäll, D., Müller, R., Hahn, F., & Marschall, M. (2024). Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy. Pathogens, 13(8), 645. https://doi.org/10.3390/pathogens13080645