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Influenza-Virusproduktion in tierischen Zellkulturen : Untersuchungen zur Zellphysiologie


Schulze,  M.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Schulze, M. (2007). Influenza-Virusproduktion in tierischen Zellkulturen: Untersuchungen zur Zellphysiologie. Diploma Thesis, Technische Universität, Berlin.

When focusing on cell physiology in virus production processes two parameters should be clearly significant for high virus productivity: by high active cells, thus cells in S-phase, and time point of apoptosis. Both parameters were investigated in this work for an influenza A virus production process with MDCK cells growing on microcarriers First cell cultures with high and low S-phase content were compared for their cell cycle distribution at the time of infection (toi) by propidium iodide staining. The cells were infected with a moi of 0.025 with human influenza A virus and the course of infection was observed with HA-Assay. When the cells had a high S-phase content (>25 %) compared to low S-phase content (<10 %) the achieved virus titers were 30 % higher, indicating an impact of the S-phase on virus progression. Second, during influenza A infection of MDCK cells the time course of apoptosis was followed under different process conditions. Therefore, two different virus seeds of A/PR/8/34 (H1N1) from RKI and NIBSC were compared for their apoptotic behaviour. The induction of apoptosis in infected cells is a crucial event during virus infection. Additionally, the effect of moi on apoptosis development was analyzed. Apoptosis can be determined by a variety of methods. In this work the virus induced apoptosis was analyzed by labeling the strand breaks in the host cell DNA with immunofluorescent studies. This was done by labeling the DNA fragments (TUNEL-Assay) and viral nukleoproteins by FITC-conjugated monoclonal antibodies. This double staining procedure was established during this work and sample preparation was optimized and modified. The apoptosis levels and the course of infection over cultivation time were determined in parallel by flow cytometry. Determination of apoptosis was also accomplished by agarose gel electrophoresis. It could be clearly seen, that only infected cells showed apoptosis. Non- infected cells did not show increased apoptosis from possible signaling of infected cells. The differences seen for the two virus` seeds and from different moi indicated a possible antiviral mechanism of apoptosis. High apoptosis levels led to low virus titers. Especially at early infection times apoptosis levels seem to have a more significant impact.