Seven high-frequency mutation sites were located near/in RBS
A total of 46 reported antigenic sites were curated form research papers in three databases (Table S1). A total of 2,927 full-length HA sequences were analyzed in this part of the study. Only 43.48% (20/46) of the sequences were hypervariable under the natural selection, most of which were conservative (Table S1). Interestingly, 14 of the 20 mutant antigenic sites (70%) are located in/near the HA RBS.
Figure 1 shows the specific mutations of each additional subclade, consisting of a total of 14 amino acid sites, which were also distributed in/near the RBS. Of those, 6 positions, including 164, 168, 171, 198, 200 and 201 (H9 numbering) near/in the RBS of H9N2-AIV were selected as high-frequency mutations sites. All of those seven sites were located on the surface of the HA protein head domain (Figure 2A). The seven mutations (R164Q, A168N, I171T, T198A, R200T, D201G and D201A) were selected as the pre-selection substitutions for subsequent antigenicity verification.