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.
Unit of Typing and Genetics of Mycobacteria, Institut Pasteur de Tunis, 13, Place Pasteur, B.P. 74, 1002 Tunis-Belvédère, Tunisie.
N
N
Ben Abdeljelil
B
B
Delmas
H
H
Mardassi
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics; D017930:Genes, Reporter; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D003698:Hepatitis Delta Virus / Q000235:genetics; D007243:Infectious bursal disease virus / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis; D014709:Vero Cells; D019065:Virus Assembly; D014779:Virus Replication
D016337:RNA, Catalytic; D012367:RNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins