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Novel production system for influenza A virus-derived defective interfering particles and analysis of antiviral activity

dc.contributor.advisorPöhlmann, Stefan Prof. Dr.
dc.contributor.authorArora, Prerna
dc.titleNovel production system for influenza A virus-derived defective interfering particles and analysis of antiviral activityde
dc.contributor.refereePöhlmann, Stefan Prof. Dr.
dc.description.abstractengInfluenza A virus (IAV) increases morbidity and mortality rates and novel antivirals are needed to combat the virus. Errors of the viral polymerase lead to the generation of defective RNAs. These DI-RNAs may interfere with wild-type (wt) IAV infection and may be packaged into defective interfering particles (DIPs), which exhibit antiviral activity. DIPs inhibit IAV infection by competing with wt IAV for cellular and viral resources required for genome replication (replication interference) and by inducing interferon (IFN). DI-244 is a prototypic DI-RNA derived from IAV genomic segment 1 that harbours a large central deletion, it exerts potent antiviral activity and is considered for the development as antiviral. However, it is unclear whether DI-244 inhibits IAV via replication interference and/or IFN induction. Moreover, there is no system available to produce DI-244 in the absence of wt IAV, which raises safety concerns. The goal of this thesis was to close these research gaps by engineering MDCK cells to express codon optimized PB2 (PB2opt). The PB2 open reading frame is destroyed in DI-244 and this defect should be complemented by the PB2 provided in trans. Indeed, MDCK-PB2opt cells in absence of wt IAV were able to produce DI-244 merely from plasmids. The generated DI-244 exerted strong antiviral activity against H1N1 and H3N2 IAV, but not against a dissimilar virus (vesicular stomatitis virus (VSV)). Furthermore, MDCK-PB2opt cells were successfully used to quantify DI-244 infectivity and thus constituted a useful tool to study how DI-244 inhibits IAV infection. This research revealed that any deletion in IAV genomic segment 1 could convert it into a DI-RNA and the antiviral activity was inversely correlated with DI-RNA length in the absence of a functional IFN system. In the presence of a functional IFN system, DI-244 induced a robust, partially STAT1-independent anti-IAV activity that was not determined by DI-RNA length and was more potent than DIP-mediated replication interference. Interestingly, RNAseq analysis and quantitative RT-PCR revealed that DI-244 induced expression of IFN-stimulating genes (ISGs) but not IFN, suggesting that DIPs might stimulate ISG expression via a novel pathway. In summary, the present study reports a system that allows production of DIPs in the absence of wt IAV and provides evidence that induction of the IFN system is a major contributor to DIP antiviral activity. Though, the induction of the IFN system does not involve DIP stimulated expression of IFN but direct induction of ISG
dc.contributor.coRefereeWalter, Lutz Prof. Dr.
dc.contributor.thirdRefereeWeber, Friedemann Prof. Dr.
dc.subject.engInfluenza A virusde
dc.subject.engdefective interfering particlesde
dc.subject.engreplication interferencede
dc.subject.enginterferon inductionde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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