A198V HA mutation causes HI titer decrease significantly
We previously reported that the 15 mutations (as seen in Figure 1A) on the HA protein from the passaged virus occurred when the F/98 strain passaged continuously in SPF embryonated chicken eggs or in SPF chickens (Jin et al., 2018; Su et al., 2020). In order to evaluate the role of these mutations, 15 recombinant viruses, each containing a single HA mutation from the passaged viruses occurring antigenic drift in F/98 backbone, were generated. These viruses are rF/HAK131R, rF/HAQ133H, rF/HAS145N, rF/HAQ164L, rF/HAA168T, rF/HAG181E, rF/HAA198V, rF/HAM224K, rF/HAQ234L, rF/HAY264H, rF/HAG270R, rF/HAG274R, rF/HAK278E, rF/HAI386V, and rF/HAK399N. The serum against the paternal virus F/98 in chickens was used as the reference serum to analyze the antigenicity of the recombinant viruses by HI assay. Compared with the paternal virus F/98, the mutations S145N, Q164L, A168T, M224K, and Q234L slightly reduced HI titers. The mutation A198V exhibited 6.67-fold lower HI titers, which was antigenically distinct from F/98 (Figure 1B).
To further confirm the contribution of HA A198V mutation to the antigenic change of F/98 strain, 5 recombinant viruses possessing multiple mutations from HA protein of the 47thgeneration of F/98 strain (containing K131R+S145N+G181E+A198V) passaged in SPF embryonated chicken eggs (Jin et al., 2018) in F/98 backbone were generated, respectively, including rF/HA348 (K131R+S145N+G181E), rF/HA349 (K131R+S145N+A198V), rF/HA389 (K131R+G181E+A198V), rF/HA489 (S145N+G181E+A198V), and rF/HA47 (K131R+S145N+G181E+A198V). The results showed that the viruses possessing A198V mutation, including rF/HA349, rF/HA389, rF/HA489, and rF/HA47, displayed the same 6.67-fold lower HI titers as rF/HAA198V (Figure 1C). These results suggested that the HA mutation A198V is the key change related to the antigenicity of H9N2 virus.