Comparison of growth characteristics of the mutants
The virus, CQY-2014, was generated from a wild-type H9N2-AIV using reverse genetics. Each of the high-frequency mutation sites (R164Q, A168N, I171T, T198A, R200T, D201G and D201A) were introduced into the CQY-2014 backbone individually, and there were no unexpected mutations in the sequencing of each virus (Figure 3 A). The titer of the parental CQY-2014 virus in chicken embryos reached 8.5log10 EID50/mL. Observed titers in MDCK cells were slightly lower at 6.5log10 TCID50/mL. Compared with the parental virus, titers of the seven mutants were slightly lower in both CEK cells and chicken embryos (Table 1). However, no significant differences among growth curves were observed (Figure 3 B).