Technologies

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luciferase-Neomycin gene-replicon F. BHK cell line that constitutively expresses DENV-1 structural proteins (C-preM-E) (Unpublished - Confidential)

Our cell-based panels of secondary screening assays were developed to screen for inhibitors of flaviviruses. Specifically, our assay could be used to examine if inhibitors of West Nile virus (WNV) could also inhibit other flaviviruses such as Dengue (DENV), Yellow fever (YFV), Saint Louis encephalitis (SLEV), Japanese encephalitis (JEV), Murray Valley encephalitis (MVEV) and Tick-borne encephalitis viruses (TBEV). Currently, there is only one type of cell-based assay available for screening flaviviral inhibitors. These assays are limited in their overall efficiency since only a single enzyme or protein can be tested and monitored for any potential assays. These assays are highly labor-intensive and impossible to use when screening compound libraries in large quantities.