Evolution and Selection of IAV DIPs

Motivation

Influenza A virus (IAV) infection poses a global threat to human health. For antiviral therapy, the use of defective interfering (DI) particles (DIPs) might be a promising option. DIPs arise randomly during IAV replication and generally carry an internal deletion in one of their eight genomic viral RNA (vRNA) segments. In a co-infection with infectious standard virus (STV), the DI vRNA replication results in a suppression and interference with IAV replication. The antiviral activity was already demonstrated by rescuing mice and ferrets from a lethal IAV challenge. Previously, cell-culture based production of DIPs in a continuous cultivation system showed periodic oscillations in virus titers due to the dynamic interplay of DIP and STV propagation (Fig. 1,2). Furthermore, de novo generated DI vRNAs accumulated during the production run. We aim to use the continuous culture to study the evolution of IAV DIPs. 

Fig. 1: Experimental setup of a continuous two-stage bioreactor system. Cells are cultivated in a CB and continuously transferred to a VB, where DIP/STV propagation takes place. Moreover, fresh medium is fed to the CB and VB, and virus harvest is removed.

© MPI Magdeburg (BPE)

Fig. 2: IAV propagation in a continuous two-stage bioreactor system leads to periodic oscillations of virus titers. (1) After infection, STV replicates to high virus titers. (2) Co-infection of DIP and STV leads to an increased DIP replication. (3) DIPs impair STV replication. (4) At low virus titers, the chance of DIP replication is reduced heavily, subsequently, STV replicates again [3, 4

Aim of the project

For this concept, a two-stage bioreactor system is utilized. Cells are grown in a cell bioreactor and are continuously transferred to a virus bioreactor for DIP/STV propagation. To monitor STV/DIP dynamics, various virus quantification assays and real-time RT-qPCR are applied. Moreover, imaging flow cytometry is used to investigate intracellular virus replication and apoptosis induction at the single cell level. These data will be used to further refine existing mathematical models.

During the continuous passaging of virus, DI vRNAs are in a mutual competition for cellular and viral resources. Therefore, this set-up is also used to identify and select the “fittest” DIPs, which may serve as possible candidates for antiviral therapy.

 References

Pelz, L.; Rüdiger, D.; 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. bioRxiv (2021)
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), p. e72288 (2013)
Tapia, F.; Laske, T.; Wasik, M.; Rammhold, M.; Genzel, Y.; Reichl, U.: Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times—Insights From qPCR Measurements and Viral Dynamics Modeling. Frontiers in Bioengineering and Biotechnology 7, 275 (2019)
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)

 

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