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.