Two single substitutions significantly change the antigenicity of H9N2-AIV
A total of seven substitutions (including six high-frequency mutation sites) in/near the RBS were tested using reciprocal HI and microneutralization test to support the hypothesis that the single high-frequency mutation site near the RBS drives antigenic drift of H9N2-AIV. The original data were suppored in Table S3 and Figure S1, and the Integration results were shown in Table 2. As the Table 2 showed, mutations that drive antigen drift were different, whether based on the r-values of HI and neutralizing titers, or the antigen maps. Only two mutations, A168N and D201G were strong supported by all criteria, and those two mutations were considered to significantly cause the antigenic differences. The other 5 mutations, which are only supported by partial criteria, are thought to exerte mild effects on antigenicity. Notably, at the 201 site, two single substitutions (either D to G and D to A) produced drastic phenotypic differences, with glycine (G) had a significant effect on antigenicity, while the substitution of alanine (A) had no significant effect. The visualizations in Figure S1 were even more obvious. The antigen distance between the two single substitutions at site 201 was more than 2 units in antigen map, with more than 4-fold HI and microneutralization titer change. By predicting the 3D structure of the HA protein, it was observed that the substitution of glycine at position 201 did not change the α helix structure, but significantly changed the surface structure (Figure 2 B). In conclusion, the A168N and D201G mutations near the RBS significantly affected the antigenicity of circulating H9N2-AIV.