Chicken experiments
A total of 24 three-week-old SPF chickens were divided into three
groups: the F/98 vaccine group including 12 chickens, and the
rF/HAA198V vaccine group including 12 chickens, which
were immunized with the emulsion vaccine of the F/98 virus, and the
rF/HAA198V virus, respectively. At day 21
post-vaccination, chickens were bled from the wing vein for sera, and HI
reactions against the F/98 virus, or the rF/HAA198Vvirus was performed, respectively. Then, six chickens from each group
were challenged intranasally and intratracheally with
106 EID50 of the F/98 virus, or the
rF/HAA198V virus. Chickens were monitored daily for
morbidity and mortality after challenge. At days 3 and 5 post-challenge,
tracheal and cloacal swabs from challenged chickens were collected in 1
mL of PBS containing antibiotics. After one freeze-thaw cycle, the swabs
were centrifuged at 3000 rpm for 10 min. A 0.2 mL supernatant was taken
to inoculate 10-day-old SPF chicken eggs. Viral shedding in the trachea
and cloacal was evaluated via HA titers of the allantoic cavity of SPF
chicken eggs at day 5 post-inoculated according to the standard of
HA≥23.