Assay for stage-specific inhibitors of Vaccinia orthopox Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2142-01_Inhibitor_SinglePoint_HTS_Activity
Assay Overview: Orthopoxviruses are a genus of viruses that include monkeypox, variola (the causative agent of smallpox) and vaccinia, the prototypical orthopoxvirus which was used in the world-wide vaccination program that eradicated smallpox . Smallpox was once the most deadly human pathogen, and is estimated to have killed more than 300 million people. Following the eradication of smallpox, more ..
BioActive Compounds: 12
Keywords: Vaccina, orthopoxvirus
Assay Overview: Orthopoxviruses are a genus of viruses that include monkeypox, variola (the causative agent of smallpox) and vaccinia, the prototypical orthopoxvirus which was used in the world-wide vaccination program that eradicated smallpox . Smallpox was once the most deadly human pathogen, and is estimated to have killed more than 300 million people. Following the eradication of smallpox, routine vaccination was discontinued in the 1970s, and there has consequently been a precipitous decline in population immunity to smallpox and other orthopoxviruses. There are currently no FDA-licensed drugs to treat infected individuals, which is a significant concern given the threat of orthopoxvirus weaponization and the rise in reports of humans infected with monkeypox, which is endemic to Central and Western Africa . Potent and validated inhibitors of this assay will be of significant interest to public and will provide the development of initial compound leads into therapeutic agents, either as a single agent or as a combination therapy with existing anti-viral agents.
In collaboration with the Connor lab, a high throughput assay using vaccinia virus model system was developed. The assay used recombinant viruses (coupled reporter system) that robustly monitor viral gene expression and viral spread to identify novel anti-orthopoxviral compounds. The first infection evaluates the stage gene expression in Vaccinia using the fluorescent Venus protein while the late infection uses the fluorescent mCherry protein reporter.
Expected Outcome: Compounds that show a statistically significant inhibition of the mCherry late expression fluorescence signal, and that also show a significant window of lower inhibition of the Venus early expression fluorescence signal, will be considered active.
 Moss B (2007) Poxviridae: The Viruses and Their Replication. In: Knipe DMH, P.M., editor. Fields Virology. 5 ed: Lippincott Williams & Wilkins.
 Rimoin AW, Kisalu N, Kebela-Ilunga B, Mukaba T, Wright LL et al. (2007) Endemic human monkeypox, Democratic Republic of Congo, 2001-2004. Emerg Infect Dis 13(6): 934-937
A549 cells were plated at a concentration of approximately 6,000 cells per well in clear-bottom black-walled plates. Compounds were added to the assay plate followed by addition of virus (LREV) that expresses the Venus fluorescent protein early in viral ininfection. During the late viral infection, mCherry was also added to the plate at an MOI of 10 (~ 60,000 plaque forming units (PFU) of virus). Following virus addition, plates were incubated in a tissue-culture incubator for 12-18 hours. At increasing times postinfection, fluorescence from both Venus and mCherrry was determined using a plate-reader and standard conditions for observing Venus fluorescence (EX515/EM530) and mCherry fluorescence (EX587/EM610). In each plate, positive controls (virus infection in the presence of 1% DMSO only) and an inhibitory drug control (AraC at 500 nM) was included and used to normalize the data.
1a) A549 cell 4 uL
1b) DMEM medium Control 4 uL
1000 cell/well of A549 ad let cells attach for 3 hrs at 37C in CO2 incubator, DMEM medium in negative control columns
2a) Control Inhibitor
2b) Offline Library 23 nL
23 nL Control Inhibitor, Intraplate 16-pt titration: AraC for mCherry detection & Actinomycin D for Venus detection
3) Viral Infection 2 uL 5 MOI of VAC-LREV virus in DMEM medium is dispensed by Bioraptor
4) Incubation for viral replication Overnight 37C in CO2 incubator
5) Envision Detection Over 18 hrs more Venus read: Exc 510nm/Em590nm & mCherry read: Exc 545nm/Em615nm
<= 33% in the mCherry channel and Venus - mCherry channel must be >= 40%.
Samples passing both criteria are considered Active (2)
Sample passing the 33% threshold for mCherry but not passing the criteria for Venus - mCherry will be considered Inconclusive (3)
Samples passing neither criteria will be considered Inactive (1)
Additionally, samples which pass both criteria but which have unacceptable reproduciblity between replicates will be considered Inconclusive (3)
The score was derived by fitting the mean mCherry values to a 1-100 scale
Categorized Comment - additional comments and annotations
Data Table (Concise)