A system for production of pure Influenza A virus defective interfering particles (DIPs) and assessment of their antiviral activity
by Najat Bdeir
Date of Examination:2021-07-19
Date of issue:2021-09-09
Advisor:Prof. Dr. Stefan Pöhlmann
Referee:Prof. Dr. Stefan Pöhlmann
Referee:Dr. Alexander Hahn
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EnglishInfluenza A virus (IAV) epidemics and pandemics constitute a major threat to human health.The ability of IAV to rapidly acquire mutations can render antivirals and vaccines ineffective andnovel antiviral strategies are urgently needed. IAV particles harbor eight genomic RNA segmentsand defective interfering RNAs (DI RNAs) are naturally occurring byproducts of viral genomereplication. These RNAs interfere with replication of the WT RNAs and, upon packaging intodefective interfering particles (DIPs), can inhibit spread of WT IAV. It has been postulated thatDIPs interfere with IAV infection by competing for cellular resources and inducing the interferon (IFN) response. However, the relative contribution of these processes to DIP antiviral activity hasbeen unclear and it was unknown whether DIPs harboring more than one DI RNA exert increasedantiviral activity. Further, development of DIPs for IAV treatment was hampered by the need touse WT IAV as helper virus for DIP amplification, which raises safety concerns. The goals of thisthesis were to remove these roadblocks as a step forward towards the development of DIPs as novel antivirals. Studies with a prototypic DI RNA, DI-244, revealed that providing the open reading framein trans that had been destroyed upon DI RNA generation allows to generate DIPs in the absenceof helper virus. The DIPs produced under those conditions suppressed IAV infection and antiviralactivity was not increased when particles were engineered to harbor more than one DI RNA.Further, it was revealed that any central deletion in the genomic IAV segments 1, 2 and 3 is sufficient to convert these RNAs into DI RNAs. Antiviral activity of these DI RNAs was inversely correlated to DI RNA length in the absence of a functional IFN response. However, in the presence of a functional IFN response, antiviral activity was independent from DI RNA length and was associated with the expression of interferon (IFN) stimulated genes (ISGs) but not IFN. In conclusion, this thesis reports a system to safely produce DIPs in the absence of WT virus and demonstrates that DIP antiviral activity might be mainly due to induction of ISG expression which may rely on a previously uncharacterized pathway since DIPs did not induce IFN expression.
Keywords: Key words: Influenza A virus (IAV), defective interfering particles (DIPs), DI244, IFN induction, ISG induction, replication interference.