Molecular Biology
Understanding viral replication is not only crucial for the development of new antiviral drugs but can also lead to novel biotechnological strategies for the optimization of vaccine and viral vector production. Thus, the research team “Molecular Biology” (MolBio) of the Bioprocess Engineering department is analyzing the intracellular viral life cycle steps and virus-host cell interactions on the molecular level. It is our aim to identify bottlenecks of virus replication, or determinants of efficient virus spread and pathogenesis. Furthermore, we are aiming for the development of strategies to increase cell-specific virus yields in animal cell culture. For instance, we investigate the impact of so-called defective interfering particles (DIPs) on the quality of seed virus and virus production yields, and explore options to use DIPs and their antiviral responses for new therapeutic and preventive approaches.
For the analysis of the complex interplay between influenza viruses and their host cells, sophisticated analytical methods such as real-time RT-qPCR and imaging flow cytometry in combination with mathematical modeling are essential. Moreover, single-cell analysis is an important novel tool to study details regarding the design of highly efficient vaccine production processes. In particular, we observed that the majority of individually infected cells is non-productive or releases relatively few progeny virions. However, some cells produce very high virus titers. We were able to demonstrate that this high cell-to-cell heterogeneity in influenza A virus infection is caused by stochastic fluctuations that are intrinsic to viral replication and by extrinsic noise, which can originate from cellular factors. In conclusion, the detailed investigation of virus replication in production cell lines using state-of-the-art analytical tools and mathematical modeling paves the way to optimize virus production by the targeted design of cell lines and virus strains and supports development of new antiviral strategies.
References
Pelz, L.; Dogra, T.; Marichal-Gallardo, P.; Hein, M. D.; Hemissi, G.; Kupke, S. Y.; Genzel, Y.; Reichl, U.: Production of antiviral “OP7 chimera” defective interfering particles free of infectious virus. Applied Microbiology and Biotechnology 108, 97 (2024)
Rüdiger, D.; Piasecka, J.; Küchler, J.; Pontes, C.; Laske, T.; Kupke, S. Y.; Reichl, U.: Mathematical model calibrated to in vitro data predicts mechanisms of antiviral action of the influenza defective interfering particle “OP7”. iScience 27 (4), 109421 (2024)
Pelz, L.; Piagnani, E.; Marsall, P.; Wynserski, N.; Hein, M. D.; Marichal-Gallardo, P.; Kupke, S. Y.; Reichl, U.: Broad-Spectrum Antiviral Activity of Influenza A Defective Interfering Particles against Respiratory Syncytial, Yellow Fever, and Zika Virus Replication In Vitro. Viruses 15 (9), 1872 (2023)
Dogra, T.; Pelz, L.; Boehme, J. D.; Küchler, J.; Kershaw, O.; Marichal-Gallardo, P.; Baelkner, M.; Hein, M. D.; Gruber, A. D.; Benndorf, D. et al.; Genzel, Y.; Bruder, D.; Kupke, S. Y.; Reichl, U.: Generation of “OP7 chimera” defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice. Scientific Reports
13, 20936 (2023)
Rüdiger, D.; Pelz, L.; Hein, M. D.; Kupke, S. Y.; Reichl, U.: Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture. PLoS Computational Biology 17, e1009357 (2021)
Hein, M. D.; Chawla, A.; Cattaneo, M.; Kupke, S. Y.; Genzel, Y.; Reichl, U.: Cell culture–based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system. Applied Microbiology and Biotechnology 105, pp. 7251 - 7264 (2021)
Pelz, L.; Rüdiger, D.; Dogra, T.; Alnaji, F. G.; Genzel, Y.; Brooke, C. B.; Kupke, S. Y.; Reichl, U.: Semi-continuous propagation of influenza A virus and its defective interfering particles: analyzing the dynamic competition to select candidates for antiviral therapy. Journal of Virology 95 (24), e01174-21 (2021)
Hein, M. D.; Arora, P.; Marichal-Gallardo, P.; Winkler, M.; Genzel, Y.; Pöhlmann, S.; Schughart, K.; Kupke, S. Y.; Reichl, U.: Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles. BMC Biology 19 (1), 91 (2021)
Rand, U.; Kupke, S. Y.; Shkarlet, H.; Hein, M. D.; Hirsch, T.; Marichal-Gallardo, P.; Cicin-Sain, L.; Reichl, U.; Bruder, D.: Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity. Cells 10 (7), 1756 (2021)
Hein, M. D.; Kollmus, H.; Marichal-Gallardo, P.; Püttker, S.; Benndorf, D.; Genzel, Y.; Schughart, K.; Kupke, S. Y.; Reichl, U.: OP7, a novel influenza A virus defective interfering particle: production, purification, and animal experiments demonstrating antiviral potential. Applied Microbiology and Biotechnology 105 (1), pp. 129 - 146 (2021)
Kupke, S. Y.; Ly, L.-H.; Börno, S. T.; Ruff, A.; Timmermann, B.; Vingron, M.; Haas , S.; Reichl, U.: Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication. Viruses 12 (1), 71 (2020)
Rüdiger, D.; Kupke, S. Y.; Laske, T.; Zmora, P.; Reichl, U.: Multiscale modeling of influenza A virus replication in cell cultures predicts infection dynamics for highly different infection conditions. PLoS Computational Biology 15 (2), e1006819 (2019)
Laske, T.; Bachmann, M.; Dostert, M.; Karlas, A.; Wirth, D.; Frensing, T.; Meyer, T. F.; Hauser, H.; Reichl, U.: Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth. PLoS Computational Biology 15 (4), e1006944 (2019)
Kupke, S. Y.; Riedel, D.; Frensing, T.; Zmora, P.; Reichl, U.: A Novel Type of Influenza A Virus-Derived Defective Interfering Particle with Nucleotide Substitutions in Its Genome. Journal of Virology 93 (4), 01786-18 (2019)
Wasik, M.; Eichwald, L.; Genzel, Y.; Reichl, U.: Cell culture-based production of defective interfering particles for influenza antiviral therapy. Applied Microbiology and Biotechnology 102 (3), pp. 1167 - 1177 (2018)
Frensing, T.; Kupke, S. Y.; Bachmann, M.; Fritzsche, S.; Gallo Ramirez, L. E.; Reichl, U.: Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells. Applied Microbiology and Biotechnology 100 (16), pp. 7181 - 7192 (2016)
Laske, T.; Heldt, F. S.; Hoffmann, H.; Frensing, T.; Reichl, U.: Modeling the intracellular replication of influenza A virus in the presence of defective interfering RNAs. Virus Research 213, pp. 90 - 99 (2016)
Bachmann, M.; Breitwieser, T.; Lipps, C.; Wirth, D.; Jordan, I.; Reichl, U.; Frensing, T.: Impaired antiviral response of adenovirus-transformed cell lines supports virus replication. Journal of General Virology 97 (2), pp. 293 - 298 (2016)
Heldt, F. S.; Kupke, S. Y.; Dorl, S.; Frensing, T.; Reichl, U.: Single-cell analysis and stochastic modelling unveil large cell-to-cell variability in influenza A virus infection. Nature Communications 6, 8938 (2015)
Frensing, T.: Defective interfering viruses and their impact on vaccines and viral vectors. Biotechnology Journal 10 (5), pp. 681 - 689 (2015)
Frensing, T.; Pflugmacher, A.; Bachmann, M.; Peschel, B.; Reichl, U.: Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production. Applied Microbiology and Biotechnology 98 (21), pp. 8999 - 9008 (2014)
Frensing, T.; Heldt, S.; Pflugmacher, A.; Behrendt, I.; Jordan, I.; Flockerzi, D.; Genzel, Y.; Reichl, U.: Continuous Influenza Virus Production in Cell Culture Shows a Periodic Accumulation of Defective Interfering Particles. PLoS One 8 (9), e72288 (2013)
Heldt, S.; Frensing, T.; Pflugmacher, A.; Gröpler, R.; Peschel, B.; Reichl, U.: Multiscale Modeling of Influenza A Virus Infection Supports the Development of Direct-Acting Antivirals. PLoS Computational Biology 9 (11), e1003372 (2013)
Heldt, F. S.; Frensing, T.; Reichl, U.: Modeling the intracellular dynamics of influenza virus replication to understand the control of viral RNA synthesis. Journal of Virology 86 (15), pp. 7806 - 7817 (2012)
Seitz, C.; Isken, B.; Heynisch, B.; Rettkowski, M.; Frensing, T.; Reichl, U.: Trypsin promotes efficient influenza vaccine production in MDCK cells by interfering with the antiviral host response. Applied Microbiology and Biotechnology 93 (2), pp. 601 - 611 (2012)
Frensing, T.; Seitz, C.; Heynisch, B.; Patzina, C.; Kochs, G.; Reichl, U.: Efficient influenza B virus propagation due to deficient interferon-induced antiviral activity in MDCK cells. Vaccine 29 (41), pp. 7125 - 7129 (2011)
Seitz, C.; Frensing, T.; Höper, D.; Kochs, G.; Reichl, U.: High yields of Influenza A virus in MDCK cells are promoted by an insufficient IFN-induced antiviral state. Journal of General Virology 91 (7), pp. 1754 - 1763 (2010)
Heynisch, B.; Frensing, T.; Heinze, K.; Seitz, C.; Genzel, Y.; Reichl, U.: Differential activation of host cell signaling pathways through infection with two variants of influenza A /Puerto Rico /8/34 (H1N1) in MDCK cells. Vaccine 28 (51), pp. 8210 - 8218 (2010)
