A novel technique for in-vivo assay of viral regulatory regions in genomes of animal RNA viruses

A novel in-vivo assay system for mapping and analyzing regulatory signals which promote transcription and expression of viral RNA genomes is described. The system was developed using infectious bursal disease virus (IBDV), a double-stranded RNA virus and Vero cells which are permissive for IBDV, as a model. The model system consisted of engineered modifications of an enhancer-less pGL3-Promoter vector such that deleted lengths of the 5' noncoding region of genome segment A of IBDV were positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the luciferase (LUC) reporter gene. Transient transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in endogenous generation of recombinant viral RNA-LUC containing the 5' terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the Vero cell lysates allowed the localization of promoter activity in the 5'-terminal 32 base pairs of genomic segment A of IBDV. Because the viral RNA transcripts produced in-vivo can be either plus-sense or minus-sense, the system can be used to assay for regulatory regions for transcription or replication for any animal RNA virus.