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.