Characterisation of rIBVs with replacement of ORFs 3a and 3b with hRluc.

<p>(A) Monolayers of CK cells were infected with wild type virus (Beau-R), control virus (BeauRΔ3ab) or P4 of rIBV BeauR-hRlucΔ3ab and cytopathic effect analysed by plaque assay. (B) Growth kinetics of Beau-R, BeauRΔ3ab and R1 of BeauR-hRlucΔ3ab in CK cells. Extracellular virus was harvested at various times post infection and the progeny virus titres determined by plaque assay. Error bars represent 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-hRlucΔ3ab. 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 for the sg mRNAs produced from the rIBVs. The new sg mRNA (labelled E), synthesised from the inserted TRS for expression of the E protein, is indicated with an open arrowhead. (E) 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-hRlucΔ3ab R1 and R2 were analysed by western blot with anti-IBV, anti-hRluc and anti-actin antibodies. Cell lysates from uninfected cells (M) were included as a negative control.