The viral sequences are flanked by a T7 RNA polymerase promoter at the 5' end and a hepatitis delta ribozyme sequence at the 3' end, such that transcription by T7 RNA polymerase produces an RNA molecule with exact viral genomic termini. Transcription of the minigenome is dependent on the replication of the helper virus and is assayed by detection of the reporter gene product. These systems can be used as an assay for antiviral agents. A stock of minigenome-containing virus can be produced by collecting the supernatant fluid from transfected, virus infected cells, and passaging it onto fresh cell monolayers until the desired volume is obtained. Identical aliquots of this stock are then used to infect cells in a standard 96 well microtitre plate, in the presence or absence of various concentrations of potential antiviral compounds.
Antiviral activity is measured simply by assaying the cells for reporter gene expression.
Also, our assay has the potential to be automated, which cannot be done for traditional viral assays. This marks a significant advance for the potential detection of antiviral compounds against these viruses and, importantly, establishes that such an approach will be valid for all NNS viruses. We are now extending this system to cover other NNS viruses, and to use other reporter genes, in particular the luciferase gene which is the reporter of choice for large-scale automated screening. In, Reverse Genetics of RNA viruses. Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus.