A198V HA mutation may contribute to escape from neutralizing-antibodies
As the Figure 2A shown, amino acid position 198 is located in the sialic acid binding domain as one of the receptor binding sites, suggesting that A198V substitution might play a key role in the evolution of H9N2 viruses. In order to study the role of the mutation A198V in escaping from neutralizing antibodies, microneutralization (MN) assay was performed, which is more sensitive than HI assay (Segovia,Franca,Bahnson,Latorre-Margalef, & Stallknecht, 2019). In comparison to the parental F/98 strain, the rF/HAA198Vvirus exhibited 8-fold reduction in MN titers to anti-F/98 serum (Figure 2B), and 8-fold reduction to homologous anti-rF/HAA198Vserum (Figure 2C), which suggested that the A198V mutation promoted the rF/HAA198V virus escape from anti-F/98 or anti-rF/HAA198V serum. Further, we comprehensively compared the serological cross-reactivity induced by the H9N2 viruses A/Chicken/Guangdong/SS/1994 (GD/SS/94, original HA A198), A/Chicken/Jiangsu/YZ618/2012 (JS/YZ618/12, original HA T198) (R. Zhu et al., 2018), and rF/HAA198V in chickens. Results showed that the rF/HAA198V virus displayed 4-fold reduction of MN titers to anti-GD/SS/94 serum compared to the GD/SS/94 virus (Figure 2D); the rF/HAA198V virus displayed 8-fold reduction of MN titers to anti-JS/YZ618/12 serum (Figure 2E) compared to the JS/YZ618/12 virus. However, compared to the viruses GD/SS/94 and JS/YZ618/12, the rF/HAA198V virus displayed 4- and 8-fold reductions of HI titers to homologous anti-rF/HAA198V serum, respectively (Figure 2F). These results showed that HA A198V mutation could decrease the readout of MN titers to anti-H9N2 sera significantly, even to anti- rF/HAA198V serum, indicating that this substitution could promote viral escape from neutralizing-antibodies.