Immune protection analysis
Next, the ability of the two mutants (A168N and D201G) to break through the immune protection induced by the parent strain was analyzed by avaccinal protection test. There were no unexpected deaths observed during the study in all groups. There was also no infection in immunized control chickens (Vaccine_control group) and blank control group. No significant difference in the proportion of sick chickens with respiratory signs were observed (including cough and mouth breathing) among the 3 challenge control groups (CQY-2014, A168N and D201G group) at 3 ~ 6 day-post-challenge (d.p.c), which were between 50% ~ 70%. At 14 d.p.c, the pathological lesions of chickens in each of the 3 challenge and control groups were also similar, and about 20% ~ 30% of chickens had intestinal and tracheal congestion or hemorrhage. The results suggest that the 2 key mutations (A168N and D201G) influencing antigenicity did not exert a significant effect on H9N2-AIV pathogenicity.
Comparing the data between the 3 immune-challenge groups, it can be observed that the D201G mutation [Vaccine_(D201G) group] has the potential ability to break through the immune protection conferred by the parental virus. Recovery of challenge virus remained at 60% and 20% at 5 and 7 d.p.c, respectively (Figure 4 B and C). During the study 30% of chickens presented with signs of respiratory disease in Vaccine_(D201G) group. Groups challenged with other mutants had a significant decrease in re-isolation rate (20% ~30%) at 5 d.p.c. At 7 d.p.c., no virus was isolated and no clinical signs or pathological impairments were observed (Figure 4). The results suggested that the D201G mutation not only changes the antigenicity of H9N2-AIV, but also confers the ability to escape the host immune responses.