Figure 2. Schematic overview of polymerase screens and characterization of selected mutations.

(a) Schematic overview of polymerase screens. Plasmid libraries possessing random mutations in PB2(1–587), PB2(588–759), PB1 or PA were used to generate the respective HA-deficient virus libraries. 293 Cells expressing HA were infected with the virus libraries and screened by FACS analysis for cells with increased levels of GFP expression. Individual GFP-positive cells were incubated and retested for GFP expression. (b) Schematic overview of the number of viruses and mutants analysed. FACS screens yielded 3,744 and 3,168 GFP-positive samples for the pilot and full screens, respectively; virus amplification and GFP expression were confirmed for 1,485 and 713 of these. The latter samples were further analysed by qRT–PCR analysis: we detected 208 viruses with M vRNA levels higher than those of the control virus. A total of 90 candidates with the highest increases in viral replication were characterized by deep sequencing of the polymerase and NP genes. For the pilot study, 96 randomly selected samples were analysed by Sanger sequencing. From the mutants identified in the pilot and full screens, 85 were characterized in viral replication assays in cultured cells. Of those, 11 mutations were introduced into authentic TY93/H5N1 virus and tested for their effects on replication in human and avian cells, and for virulence in mice.