Adaption aviärer Influenzaviren vom Subtyp H5N1 an die Maus durch Mutationen in der Viruspolymerase

The sporadic transmission of HPAIV of subtype H5N1 from poultry to humans and other mammals is associated with a high mortality rate of about 59%. Although H5N1 viruses have been studied extensively, the underlying mechanisms that confer the high morbidity and mortality of these viruses, in comparison to other HPAIV like A/FPV/Rostock/34 (H7N1), are still not fully understood. The aim of this work was elucidate the role of two mutations (D701N and S714I or S714R) in the PB2 subunit of the viral polymerase that were found in H5N1 isolates of humans and poultry and are associated with an increase in host-range and pathogenicity for mammals. The human H5N1 isolate A/Thailand/1 (Kan-1)/04 was used as a model to study the effect of the mentioned mutations. 1. It can be shown, that all introduced mutations, D701N, S714I or S714R in the PB2 protein are capable of increasing the activity of the viral polymerase in mammalian cells significantly. In combination with D701N, either mutation S714I or S714R increases the polymerase activity even further. 2. Recombinant Influenza A viruses bearing the respective mutations alone or in combination were generated and used in growth kinetics and animal experiments. 3. In recombinant viruses, the mutation D701N alone is responsible for a significantly faster replication of viruses in cells of mammalian origin. The mutations S714I or S714R alone do not enhance viral replication. Only a combination of either substitution at position 714 with 701N results in an enhanced replication. 4. In contrast to mammalian cells, mutations S714I and S714R enhance polymerase activity in avian cells, while D701N has no positive effect. However, this enhanced polymerase activity does not lead to an increased replication rate in avian cells. 5. To assess the influence of the aforementioned mutations on pathogenicity, infection studies in mice were done. Here it could be observed, that mutation D701N alone is the major contributor to an increase in pathogenicity. Again, mutations S714I and S714R, in combination with 701N, increase pathogenicity even further. If position 701 is avian-like however, mutant viruses with S714I or S714R are attenuated. 6. While performing the growth kinetics in mammalian cells, viruses with avian-like 701D often mutated to the mammalian-like 701N or even at other, adaption-related positions in the PB2 subunit, indicating a strong selective pressure.