Investigation of the Influenza Virus Replication on the Single Cell Level

Investigation of the Influenza Virus Replication on the Single Cell Level

Motivation

The annual demand of human influenza vaccines still relies on conventional production in embryonated eggs despite several disadvantages of this technology introduced more than 60 years ago. The establishment of scalable cell culture-based production processes, however, can be crucial to provide sufficient vaccine doses in light of an imminent pandemic thread. Besides process development, the optimization of the smallest production unit, a single infected cell, has an enormous potential to contribute to the progression of cell culture-based production technologies.

Monitoring cell growth and virus propagation in cell cultures yields only average cellular or viral characteristics. However, it has to be assumed that significant differences in the physiological status of cells, progress of infection, and cell-specific virus yields exist. Among genetically identical cells, cellular heterogeneity arises from variation in cell size and cell cycle, fluctuations of cellular constituents and stochastic effects in gene expression. The variability of influenza virions is caused by high mutation rates, diversity in virus particle composition as well as the occurrence of defective interfering particles.

Aim of the project

In this project, we utilize new and powerful technologies to investigate the variability of virus infections on the single cell level (Heldt and Kupke et al., 2015). Based on an improved understanding of the influenza virus replication, we finally want to identify bottlenecks limiting cell-specific virus yields, and develop strategies for optimization of host cells or virus strains for cell culture-based influenza vaccine production.

Figure: Single Cell Analysis. Due to the heterogeneity existent in cellular and viral populations, the infection of a single cell with a single virus particle (A) leads to significant differences in outcome of virus infections among single infected cells (B). Measuring cell populations yields solely averaged cellular or viral-derived characteristics, masking how these characteristics are distributed (C). Thus, the analysis of single cells allows the characterization of various cellular events or even the detection of rare subpopulations, which can have a profound impact in the long-term behavior of a population.

References

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)
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)
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)

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