After being inoculated with the recombinant strains, typical IB lesions in the chick embryos from each group were observed, including amniotic membrane thickening, dwarfing, stunted growth, curling, or death of the embryo (Fig. 2 A). The EID50 of the virus was calculated according to the Reed-Muench method. The titers of the rH120, rIBYZ, and rH120-S1/YZ strains were determined to be 107.3 EID50/mL, 106.8 EID50/mL, and 108.4 EID50/mL, respectively (Fig. 2B).
Fig. 2 The biological characteristics of the rIBVs in SPF chicken embryos. (A) Pathogenicity of recombinant IBVs in infected chick embryos. Five 10-old-day embryonated SPF chicken eggs per group were inoculated with 0.2 mL virus diluent of rH120, rIBYZ, or rH120-S1/YZ. Chick embryo lesions were observed at 144 h post-inoculation at 37 °C. (B) EID50 of the recombinant IBVs, rH120, rIBYZ, and rH120-S1/YZ in SPF chicken embryonated eggs. (C) The multicycle growth kinetics of the recombinant IBVs, rH120, rIBYZ, and rH120-S1/YZ in SPF chicken embryonated eggs. All data are presented as the mean ± standard deviation (SD). Some of the error bars are too small to be seen. The qRT-PCR was carried out with three replicates. Markers of statistical difference were acquired by comparing between rH120 group and rH120-S1/YZ group. ***Indicates an extremely significant difference at P ≤ 0.001; **Indicates a highly significant difference at 0.001 < P ≤ 0.01; *Indicates a significant difference at 0.01 < P ≤ 0.05.