Fig 1. Schematic overview of selection and characterization of polymerase and NP mutations.

Based on a database search of clade 2.2.1 virus sequences, we selected a total of 46 single mutations that were detected either only in human viruses (Category 1) or were more prevalent in human viruses than in bird viruses (Category 2). Since some viruses contained multiple mutations, we examined the sequences in the two virus categories for intra-segment multiple mutations and selected 23 viruses with intra-segment multiple mutations. These single and intra-segment multiple mutations were also detected in inter-segment combinations of mutations in both Category 1 and Category 2 viruses. Therefore, we searched for inter-segment combinations of single and intra-segment multiple mutations from natural human isolates, and 16 inter-segment combinations of mutations in viruses from patients were selected and designated Inter-1 to Inter-16. A total of 85 single and multiple mutations, that were carried with PB2-627K in clade 2.2.1 isolates, were assayed for polymerase activity by minigenome assays. Of these 85 mutations, 29 were introduced into EG/D1/clade 2.2.1 (wt) viruses and their effect on progeny vRNA production in human cells was investigated. From the in vitro progeny vRNA production results, 4 virus mutants were selected and tested for virulence in mice.