Analysis of immune protection conferred by the parental strain against single mutants
To identify whether the key antigenic single mutations can break through the immune protection induced by the parental strain, the parental virus CQY-2014 was selected as the vaccine strain and prepared as an oil-adjuvant inactivated vaccine, as previously reported (Xia, Yao, et al., 2017). Commercial un-immunized 28-day-old Partridge Shank broilers (n = 80) were randomly divided into 8 groups, 4 groups were subcutaneously immunized with 105EID50 CQY-2014 inactivated vaccine. At 21 day-post-immunization (d.p.i), birds of 3 immune-challenge groups and 3 challenge control groups were challenged with 106EID50 of CQY-2014, CQY-A168N, or CQY-D201G virus by eye and nose drip, respectively. The birds of immune control group were received only the CQY-2014 vaccine. Birds of blank control group were mock infected with phosphate-buffered saline (PBS) buffer at the corresponding time points.
Chicken feeding and sample collection were carried out as previously described (Xia, Cui, et al., 2017). Briefly, birds in each group were held in separate biosafety level 2+ (BSL2+) isolators and monitored daily for appetite, activity, fecal output, conjunctivitis, cyanosis of the comb, ruffled feathers and dyspnea. Tracheal swabs from each group were collected at 3, 5 and 7 day-post-challenge (d.p.c) for virus isolation. At 14 d.p.c, all remaining birds were euthanized and necropsied for pathological observation.