Antigenic analysis of mutants
To determine whether high-frequency mutations near the RBS could drive antigenic drift of H9N2-AIV, antiserum preparation and reciprocal HI tests were performed as previously reported (Xia, Cui, et al., 2017). Here, three criteria (antigenic relatedness values (ARV, r) of HI and microneutralization titer, and antigenic map) were used to judge the difference of virul antigenicity.
The HI and microneutralization titers were used to calculate the “r” by the method of Archetti & Horsfall (1950):\(\text{AVR}\left(r\right)=\sqrt{r1\times r2}\times 100\%\) where r1 represents the ratio of the heterologous HI titer of virus 1 to the homologous titer of virus 1, and r2 represents the ratio of heterologous titer of virus 2 to the homologous titer of virus 2. Mutants where 0.67 ≤ r ˂ 1.5 were considered to be antigenically similar, whereas those with 0.5 ≤ r ˂ 0.67 were considered to possess small antigenic differences, and r ˂ 0.5 and r ≥ 1.5 were considered to contain significant antigenic differences. Antigen mapping were also performed by using the program AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap) as previously reported (Xia, Cui, et al., 2017).
Sites between non-adjacent unit were considered to have antigenic differences. The farther the distance, the greater the antigenicity difference. The mutation was been considered as significant if they were supported by both of the r value and antigen map.