Serial 10-fold dilutions from 105 copies to 109 copies per 0.1 mL of virus of the recombinant strain, rH120-S1/YZ, and its parental strains (rH120 and rIBYZ) were inoculated into the allantoic cavity of 10-day-old embryonated SPF chicken eggs. For each dilution, 0.1 mL of the virus solution was injected into each egg and 10 eggs were used for each dilution. PBS was used as a negative control. After a 24 h incubation, the dead embryos were considered nonspecific deaths and discarded. The embryos were examined for the presence of specific lesions caused by the virus after a 144-h incubation. Dead and live embryos that displayed IBV infectious signs (e.g., dwarfing, curling, and stunting) were considered positive samples. The 50% embryo infectious dose (EID50) of these rIBVs was calculated using the Reed-Muench method [37].