Replication and packaging of an infectious bursal disease virus segment A-derived minigenome.

A synthetic cytomegalovirus (CMV) promoter-driven cDNA minigenome containing the enhanced green fluorescence protein (EGFP) gene as a reporter was derived from the genomic segment A of infectious bursal disease virus (IBDV). The 5'-end of the minigenome was fused to the transcription start site of the immediate early CMV promoter, and the hepatitis delta virus (HDV) ribozyme sequence was added at its 3'-end. We show that co-transfection of the minigenome with a plasmid encoding the IBDV RNA-dependent RNA polymerase VP1, results in a consistent increase of the EGFP expression, as measured by fluorescence microscopy and flow cytometry assays. Replication of the minigenome-derived transcript was evidenced by real-time RT-PCR analyses targeted to both the plus- and minus-sense strands. When cells were infected with IBDV and transfected with the plasmid carrying the minigenome, the minigenome was packaged and EGFP was found to be expressed in a second cycle of infection. These results show the potential use of this system as a new tool to characterize IBDV replication and genome packaging.