Characterisation of rIBVs with replacement of Gene 5 with eGFP or hRluc.

<p>(A) Monolayers of CK cells were infected with wild type virus (Beau-R), control virus (BeauRΔ5ab) or P4 of rIBVs BeauR-eGFPΔ5ab, BeauR-hRlucΔ5ab or BeauR-IBVhRlucΔ5ab and cytopathic effect analysed by plaque assay. (B) Growth kinetics of Beau-R, BeauRΔ5ab and rIBVs in CK cells. Extracellular virus was harvested at various times post infection and the progeny virus titres determined by plaque assay. Error bars represent standard deviation (SD) of results from three experiments assayed in triplicate. (C) Northern blot analysis of purified mRNA extracted from CK cells infected with 1×10⁵ pfu wild type, parental virus, Beau-R (WT) and replicates R1 and R2 of P4 BeauR-eGFPΔ5ab (left panel) or BeauR-hRlucΔ5ab and BeauR-IBVhRlucΔ5ab (right panel). Purified mRNA was not quantified prior to loading on gel and is shown only to demonstrate that sg mRNAs were generated as expected. RNAs were hybridised with a DNA probe specific to the IBV 3′ UTR. Closed arrowheads indicate the sizes of sg mRNAs from Beau-R and open arrowheads indicating the altered sizes of the sg mRNAs from the rIBVs. (D) Western blot analysis of reporter gene expression. Proteins in cell lysates of CK cells infected with wild type, parental virus, Beau-R (WT) or P4 BeauR-eGFPΔ5ab (top panel) or BeauR-hRlucΔ5ab and BeauR-IBVhRlucΔ5ab (lower panel) were analysed by western blot analysis using anti-IBV, anti-eGFP or anti-hRluc and anti-actin antibodies. Cell lysates from uninfected cells (M) were included as a negative control.