Fig. 4.

Adsorption and internalization assays.

Vero cells were cultured in 96-well plates and infected with the recombinant IBVs and two parental strains (10⁵ × EID₅₀) for 1 h at 4 °C or 1 h at 37 °C. The experiments were conducted based on three replicates. (a) Infected cells were fixed and cELISA was performed as described in the Materials and Methods. Results are shown as viral load adsorption on cell equivalents relative to EID₅₀ compared with that in the Beaudette strain (100%). (b) Vero cells were cultured in 24-well plates and infected with the recombinant IBVs and parental strains (10⁶ × EID₅₀) for 1 h at 4 °C. Total RNA was extracted from the infected cells before quantifying the viral loads using real-time RT-PCR. Data were normalized against the GAPDH expression levels and viral RNA was calculated relative to that in the Beaudette strain (100%). The experiments were conducted based on three replicates. (c) cELISA was performed to evaluate the viral load internalized into cells. (d) Real-time RT-PCR was also used to evaluate the viral load internalized into cells. The experiments were conducted based on three replicates. Differences were considered significant at P < 0.05 using ANOVA followed Tukey’s multiple comparison tests.