Dokument

Summary:
Transmission of influenza viruses from aquatic birds to mammals is promoted by the adaptation of the viral proteins to the new host. This includes the PB2 subunit of the viral polymerase complex. This protein has been described as an important host range factor, able to modulate the virulence of influenza viruses. Several adaptive mutations in the PB2 subunit of various influenza-A subtypes have been described, such as D253N, Q591K, E627K, D701N, S714I and S714R. H9N2 influenza viruses are endemic in poultry in Asia and other parts of the world. Moreover these viruses have been occasionally transmitted to humans and are often involved in the generation of viruses causing zoonotic infections in humans by providing internal genes. H9N2 viruses have therefore the potential to cause a pandemic. This study was undertaken to analyse the role of the PB2 subunit in the adaptation of avian influenza virus of subtype H9N2 to mammals. In the first part of the thesis, the results demonstrated that PB2 mutations D253N, E627K, D701N, S714I and S714R increase the H9N2 polymerase activity in mammalian cells. Furthermore, mutations E627K, D701N and S714I/R also enhance viral growth in mammalian cells. Pathogenicity studies indicated that combination of mutations E627K-D701N-S714R increase the lethality of H9N2 virus in mice. The effects of the adaptive mutations have then been compared in H9N2, H1N1pdm09 and H7N7 viruses. The results have shown that the enhancement of the polymerase activity by the adaptive mutations is higher in the phylogenetically related H9N2 and H7N9 than in the non-related H7N7 and H1N1pdm09 viruses. In addition, analysis of heterologous polymerase complexes composed of H9N2, H1N1pdm09, H7N7, and H7N9 subunits provides further evidence for the concept that this enhancing effect is a specific trait of H9N2-PB2 without significant contribution of PA and PB1. From these observations, it can be concluded that the PB2 subunit of the H9N2 viruses is characterised by a particularly high adaptability to mammalian cells. In the second part of the thesis, the mechanisms by which E627K and D701N promote adaptation to a mammalian host were analysed. The results demonstrated that viruses bearing the avian signature 627E in PB2 are sensitive to RIG-I activation. This sensitivity is mediated by the destabilisation of the nucleocapsid by RIG-I, exposing thereby the double-stranded RNA required for RIG-I activation. In contrast viruses containing mutation E627K interfere with RIG-I activation, by stabilizing the association of the polymerase complex to the nucleocapsid. These observations indicate that PB2 mutation E627K modulates the inhibition of virus replication mediated by RIG-I. Furthermore, the data showed that mutation D701N promotes not only the nuclear import of newly synthesized PB2 protein, but also the nuclear import of PB2 bound to the incoming vRNPs.

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