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キーワード:
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要旨:
For cell culture based Influenza vaccine production processes virus yield optimization is of crucial importance. A well characterized factor that limits Influenza replication in vivo is the type I interferon (IFN) response. Therefore, we want to analyse whether the induction of type I IFN and subsequent expression of antiviral genes is also of importance in a Madin-Darby canine kidney (MDCK) cell culture based Influenza production process.
qRT-PCR and luciferase reporter assays were used to determine the expression levels of IFN-beta and Mx1 in MDCK cells infected with different Influenza viruses. All strains tested showed significant induction of IFN-beta, but the levels, as well as the kinetics of induction and the amount of resulting Mx1 expression varied strain dependently. As a first approach to examine the influence of IFN-beta expression on virus yield and virus replication dynamics, we transiently transfected MDCK cells with a plasmid containing the sequence of A/PR/8/34 NS1, which acts as a type I IFN antagonists. Infection of these cells with different Influenza strains showed at least 5 fold reduced levels of IFN-beta induction. For A/PR/8/34 delNS1 this inhibition of IFN induction resulted in faster virus production and slightly higher maximal virus yield compared to control cells. However, for virus strains with functional NS1 no significant effect of NS1-overexpression on virus yield or replication kinetics was found. A possible explanation could be that in cell culture with MDCK cells and MDCK-adapted Influenza viruses, expression of IFN and IFN-stimulated genes is not a yield limiting factor. This is also supported by experiments showing that pre-treatment of MDCK cells with IFN-containing medium does not affect virus yield in our system.
