West Nile Virus and Dengue Virus Screening Assays
As a primary screening assay, to analyze multiple viral protein targets simultaneously through large compound libraries.As a secondary screening assay, to study the mode of action of inhibitors or the assay can be adapted to screen for inhibitors targeting individual viral proteins i.e. target-based secondary profilingcDNA clones from human pathogenic RNA of West Nile viruses, therefore compounds identified from such an assay may have direct relevance to human disease.
Rapid primary screening of large compound libraries for potential inhibitors of WNV and other flaviviruses.Secondary screening to characterize functional activity as well as activity against the target species being tested.Engineering targeted mutations into WNV, resulting in attenuated viral strains that could potentially be used for vaccine development.
Our secondary screening assay is high throughput and sensitive which makes for rapid screening and identification of potential inhibitors in libraries with a large amount of compounds possible. We use "reverse genetic systems", which are cDNA clones of RNA viruses, and developed them for use in our genetic cell-based screening assays. Comparisons were performed on three cell based high throughput screening assays for WNV drug discovery: 1) an assay with a replicon-containing cell line that allows screening for inhibitors of viral replication; 2) a replicon bearing VLP infection assay that allows screening for inhibitors of viral entry as well as replication; and 3) a full-length reporting virus infection assay that allows screening for inhibitors of any step(s) of the viral life cycle, including entry, replication, and virion assembly. All three assays were converted into a 384-well format, validated with known WNV inhibitors, and now offer speed and sensitivity which make them ideal for screening libraries with large numbers of compounds. The following assays are available: A. WNV replicon with both Renilla luciferase (Rluc) and Neomycin Phosphotransferase (Neo) B. WNV VLPs containing a Renilla luciferase expressing replicon (Rluc-VLP) C. BHK-21 cell line containing a WN virus replicon (Rluc-Neo-Rep) D. Plasmid for DENV-1 replicon containing Renilla luciferase reporter E. Vero cell line containing DENV-1 Renilla luciferase-Neomycin gene-replicon F. BHK cell line that constitutively expresses DENV-1 structural proteins (C-preM-E) (Unpublished - Confidential)
United States
7,355,033
USA
