2.8  Efficacy of rH120-S1/YZ vaccination

2.8.1  Viral challenge
In total, 30 one-day-old SPF chickens were divided into three groups of 10 chickens. The three groups were intranasally vaccinated with the rH120 and rH120-S1/YZ strains at a dose of 104 EID50/200 μL, and the control group received PBS. Two weeks post-vaccination, the chickens in each group were challenged with the QX-like strain, rIBYZ, at 106 EID50/200 μL via eye drop and the intranasal route. Any clinical signs, as well as the percentage of morbidity and mortality were recorded for 14 days. The dead chickens were also examined for gross tracheal, lung, and kidney lesions.

2.8.2  Antibody response measured by an enzyme-linked immunosorbent assay (ELISA)
45 1-day-old SPF chickens were divided into three groups (n = 15 per group). After vaccinating with the rH120 and rH120-S1/YZ strains at a dose of 104 EID50/200 μL, the sera of 10 birds from each group were randomly collected and antibodies were tested using an ELISA created in our lab at 7, 14, 21, and 28 days post-vaccination (dpv). ELISA plates were coated with antigen of culture supernatants of inactivated IBV rMJ , a vero cells adaption strain domesticated from its parental strain of IBYZ in our lab. Individual chicken sera collected from different groups diluted to 1:200 were loaded and the plates were incubated at 37 °C for 60 min. IBV-specific IgG was detected with anti-chicken IgY (IgG) (whole molecule)-peroxidase antibodies produced in rabbits (Sigma-Aldrich, Germany). After incubated with 3,3′,5,5′-Tetramethylbenzidine(TMB) substrate for 15 min and terminated with 2 mol/L H2SO4 solution, the absorbance at 490 nm was measured using a Model 680 microplate reader (Bio-Rad, USA).