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.