The overall goal of this procedure is to identify and characterize small molecule antivirals against blue tongue virus or BTV using the CPE based cell viability assay. This is accomplished by first screening a library of antiviral compounds using the Cytopathic effect or CPE based assay with the cell titer glow reagent. Next, the antiviral efficacy is confirmed and the cytotoxicity of selected antivirals is evaluated using the dose response assay.
Finally, the time of addition assay is carried out to determine the possible viral lifecycle stage of selected antivirals leading to the possible mechanisms of action. Ultimately, results can be obtained that show novel antivirals with potent antiviral CIE and low cytotoxicity based on the dose response assay and time of addition assay. So the main advantage of this technique over the existing methods like TCI D 50 assay in a plaque assay is that this technique provides a sensitive, robust, and highly reproductive in the measurable method to screen and categorize antivirus against a certain viruses, including gluten virus, which induces measurable CP in infected cells cells In preparation for the CPE based assay from BSR cells maintained in supplemented DMEM use a microflow select dispenser to seed 20 microliters of cells at 5, 000 cells per well into a 384.
Well microplate incubate the cells for two to three hours to allow them to fully adhere to the plate, then add antiviral compounds at a final concentration of 10 micromolar to each well and mix completely. Use assay medium to dilute plaque purified and propagated BTV to the desired titer and add five microliters of BTV with denoted MOI to each well for the control. Well add five microliters of assay medium, put a wet box containing the assay plate into the incubator and incubate the infected cells for 72 hours at 37 degrees Celsius with 5%carbon dioxide and 80 to 95%humidity.
Then take the assay plate out of the incubator, next thaw and equilibrate CTG buffer and the Lyophilized CTG substrate to room temperature. Then reconstitute the homogenous CTG reagent solution by mixing the lyophilized enzyme substrate and the buffer reagent according to the manufacturer's instructions. After equilibrating the assay plates to room temperature for 15 minutes, use a dispenser to add 25 microliters of CTG reagents to each well incubate for 15 minutes in the dark before using a multi-mode reader with an integration time of 0.1 seconds to measure luminescent signals seed 5, 000 BSR cells per well in 384 well microplate for the dose response assay using 20 microliters for the antiviral efficacy assay and 25 microliters for the cytotoxicity assay.
After a two to three hour incubation, allocate eight replicates for each concentration of compound per assay as shown here. Assign the first column as the positive control without adding compound or virus and the last column as the negative control by adding virus only for the antiviral efficacy assay dilute compounds to 50 micromolar in assay medium and to the second column add 20 microliters per well. Using an eight channel semi-automatic pipette.
Mix the compound five times to a concentration of 25 micromolar. Next transfer 20 microliters of the mixture in the second column into column three and mix well resulting in a concentration of 12.5 micromolar. Repeat this twofold.
Serial dilution up to the 11th column, which will have a final concentration of 0.04 micromolar. Then add five microliters of medium to column one as the cell only positive control and five microliters per well of BTV to column 12 as A BTV infection only. Negative control add five microliters per well of BTV from column two through column 11 for the cytotoxicity assay, dilute the compound to an initial concentration of 200 micromolar.
Add 25 microliters per well of compound to column two and mix five times to bring the concentration to 100 micromolar. Carry out the twofold series dilution by transferring 25 microliters from column two to the neighboring column three and continue through column 12. After mixing column 12, aspirate and discard 25 microliters of the mixture.
Column one is the cell only control for both the antiviral and cytotoxicity assays. Put a wet box containing the assay plate in the incubator for 72 hours and take the plate out afterward. Then use the CT G kit to measure cell viability.
Use a biostatistic and graphic software to calculate the mean value standard deviation coefficient variations and percentage of cell viability for each compound concentration and carry out non-linear regression analysis to determine the EC 50 and CC 50 of each compound. Prepare for the time of addition or TOA assay by seeding 5, 000 cells per well in 15 microliters in column one through 24 of a 384. Well microplate for each compound.
Utilize all 24 columns with eight replicates for each time point. Assign column one as a cells only control by adding 10 microliters per well of assay medium mark column 24 as a BTV infection only. Negative control by adding five microliters per well of assay medium and five microliters per well of virus at an MOI of 0.01.
In a final volume of 25 microliters per well select the even numbered columns from column two to 22 as antiviral efficacy evaluation columns at different hours post infection or HPI in these columns, infect cells with five microliters per well of BTV at an MOI of 0.01 at different HPI also add five microliters per well of diluted compound at a final volume of 25 microliters per well for the denoted minus two and minus one HPI. Add the compound to BSR cells prior to BTV infection for zero HPI add the compound and BTV to the culture simultaneously. In parallel, designate the odd numbered columns from three to 23 as compound only controls corresponding to the different time points in these columns.
Add five microliters per well of assay medium and five microliters per well of diluted compound in a final volume of 25 microliters per well. After incubating the cells to 72 HPI in the incubator, take the assay plate out and use the cell titer glow kit to determine cell viability. Finally, use software that processes luminescent signals obtained via the multi-mode reader to determine the mean value.
S-T-D-E-V CV and percentage of protection cell viability for each treatment using the CPE assay to quantify cellular A TP in living cells, we previously identified several promising lead compounds with potent antiviral efficacy, low toxicity and high high selectivity. For example, compound 0 5 2 was determined to have an EC 50 of 0.27 plus or minus 0.12 micromolar and CC 50 of 82.69 micromolar both showing typical regressive curves. Under the nonlinear regression analyses analysis, the SI 50 of C 0 5 2 was determined that 306 based on its EC 50 and CC 50 values the nano molar scale antiviral efficacy, low toxicity, and consequently high SI 50 indicated that C 0 5 2 might be a potent and selective antiviral against BTV as seen here, cells were infected with BTV at an MOI of 0.01 in the presence of 10 different concentrations of C 0 5 2 and cell viability was determined at 72 HPI.
Using the CTG kit, each data point represents means and SD from five replicates. The TOA assay was used to determine the possible stages of the viral lifecycle targeted by compounds. When C 0 5 2 was added at one or two hours prior to BTV infection, the antiviral efficacy remained at the nano molar scale as seen in this figure.
In addition, cell viability decreased further when C 0 5 2 was added at 32 and 48 HPI, indicating that C 0 5 2 might act beyond the early stage of the viral lifecycle, such as virus entry. Since the first cycle of BTV viral replication is usually complete in infected cells within 24 HPI, our results suggested that C 0 5 2 might act at the late stages of the BTV viral lifecycle, such as virus replication, packaging, maturation, and egress. Meanwhile, it is also possible that C 0 5 2 acts on host cellular machinery that functions during later stages of the viral lifecycle.
After watching this video, you should have a good understanding of how to utilize the CPE best essay to identify and categorize the potential antivirus against the viruses that induce measurable CPE post infection.
