Assays for the Identification of Novel Antivirals against Bluetongue Virus

Summary

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Abstract

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC₅₀) or effective concentration (EC₅₀), as well as its range of cytotoxicity (CC₅₀). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Introduction

BTV is a prototype double-stranded RNA virus in the genus Orbivirus, family Reoviridae. BTV is one of the most important diseases of domestic livestock, including sheep, goat, cattle and other domestic animals, with $3 billion/year loss worldwide^1,2. The exotic BTV serotype is an important animal pathogen listed in the "USDA High Consequence Livestock Pathogens." Recently, the re-emerging of BTV has caused a major outbreak of disease in cattle and sheep in several countries across Northern Europe^3,4. As a result of its economic significance and as a model system, BTV has been the subject of extensive molecular, genetic and structural studies, and several vaccines have been developed. However, due to the lack of proper assays for antiviral drug discovery, there are no antivirals available against BTV.

In a recent high throughput screening (HTS) campaign using BTV as the model system, we developed, optimized and validated a CPE-based assay to identify potential broad-spectrum antivirals against arboviruses⁵. CPE-based assay is a well-recognized assay that has been used in antiviral drug discovery against a number of viruses that induced rapid and observable CPE/apoptosis^5-7. In our system, post BTV infection, CPE is evident in vertebrate cells, including HeLa, BSR, and HEK 293T⁸. BTV-induced CPE could be monitored and quantified using various cell viability detection methods, including the CellTiter Glo cell viability reagent kit (CTG kit)⁹. This kit determines the number of viable cells in culture based on quantitation of cellular ATP presented, which signals the presence of metabolically active living cells. Under optimized conditions, the CPE-based assay presented here showed its feasibility with the "mix and measure" one step protocol, and flexibility with stable luminescent signals. Meanwhile, toxic compounds reducing cell viability will be excluded in this CPE-based assay. The CPE-based assay showed its robustness and reliability for antiviral drug discovery against BTV, and has been used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR), which leads to the identification of six novel cluster of potential antiviral lead compound(s)⁵.

When a potential antiviral compound has been identified using the CPE-based assay, it will need to be subjected to the ten-concentration dose-response assay to determine the range of antiviral efficacy and cytotoxicity². The antiviral efficacy, represented as the 50% inhibitory concentration (IC₅₀) or the 50% effective concentration (EC₅₀), is the concentration of a drug which inhibits virus-induced CPE halfway between the baseline and maximum. The cytotoxicity of the antivirals, i.e. the 50% cytotoxicity concentration (CC₅₀), is the concentration of a drug inducing 50% of cytotoxicity between the baseline and maximum. The selective index (SI), denoted as 50% SI (SI₅₀) is calculated from CC₅₀/IC₅₀ which determines the specificity of the antiviral against virus-induced CPE. The IC₅₀ (or EC₅₀), CC₅₀ and SI₅₀ values are critical measures to determine whether an antiviral compound is potent and selective for further drug development.

When an antiviral showed no overt toxicity in vitro, yet prevented virus induced CPE and the productive viral life-cycle, it is important to characterize its MoA². We initiated such characterization by carrying out ToA assay to determine the possible step(s) of viral life-cycle that is affected by the antiviral. Generally, antiviral compound were added to cells at different times pre- or post-virus infection. If the antivirals were added to the infected cells post to its target step during the course of infection, it would result in lower activity when compared to the one which was added prior to the step. Thus, ToA study is critical for determining the antiviral efficacy of a compound, and its potential target, either on the viral life-cycle or the host machinery involved in the viral life-cycle.

For all three assays, cell viability was determined using the CTG kit following manufacturer's instruction⁵. This detection system outputs adequate luminescence signals that could be analyzed using various in-house software. Each assay was validated and performed at least in triplicate with eight replicas. For all the obtained data, three parameters, including mean value (AVE), standard deviation (STDEV), and co-efficient variation (CV) were analyzed to determine the robustness of the assay. Once the robustness of the assay has been determined, the data will be further analyzed and plotted using various biostatics and graphic tools².

Protocol

1. Cells, Virus and the Antiviral Compounds

1. Maintain BSR cells, a derivative of baby hamster kidney (BHK) cells¹⁰, in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS), 100U/ml penicillin and 100 μg/ml streptomycin.
2. For all three assays, plate cells in DMEM with 1% FCS, 100U/ml penicillin and 100 μg/ml streptomycin, as optimized previously⁵. This medium is referred as assay medium for all three assays.
3. Incubate all cells in the incubator at 37 °C, with 5% CO₂ and 80-95% humidity.
4. Plaque-purify and propagate the type 10 BTV (BTV-10) as described previously⁸. Dilute BTV-10 in assay medium for each designated assays.
5. Dissolve all testing compounds in DMSO to form a stock with concentration at 10 mM. Store the stocks at -20 °C.
6. Dilute compounds to desired concentrations using assay medium for the designated assays².

2. CPE-based Assay Using CTG Kit

1. Seed BSR cells into a 384-well microplate (black; format by 16 x 24) via MicroFlo select dispenser. The seeding density is 5,000 cells/well, and the seeding volume is 20 μl for antiviral efficacy analysis.
2. Incubate cells for 2-3 hr till cells get adherent to the plate thoroughly.
3. Add antiviral compound with a final concentration of 10 μM to each well. Mix it completely.
4. Dilute BTV to desired titer and add 5 μl BTV with denoted MOI to each well. For the control well, add 5 μl of assay media. Incubate the infected cells for 72 hr at 37 °C, with 5% CO₂ and 80-95% humidity.
5. At 72 h.p.i., thaw and equilibrate CTG buffer and the lyophilized CTG substrate to room temperature prior to use. Reconstitute the homogeneous CTG reagent solution by mixing the lyophilized enzyme/substrate and the buffer reagent according to the manufacturer's instructions.
6. Equilibrate the assay plates to room temperature for 15 min.
7. Add an equal volume (25 μl) of CTG reagents to each well by a dispenser. After incubating for 15 min at room temperature in darkness, measure luminescence signals using a multi-mode reader with an integration time of 0.1 sec.

3. Dose-response Assay

1. Seed BSR cells into a 384-well microplate (black; format by 16 x 24) via a dispenser, with a seeding density of 5,000 cells/well in a seeding volume of 20 μl for antiviral efficacy assay and 25 μl for cytotoxicity assay, respectively.
2. Incubate cells for 2-3 hr at 37 °C, with 5% CO₂ and 80-95% humidity till cells are well attached to the plate.
3. Process assay in a quarter area of the 384-well plate for each compound (equivalent to a 96-well plate), as shown in Table 1. Allocate eight replicates for each concentration in a single assay. Assign the first column as the positive control without adding compound and virus, and the last column (12^th) as negative control by adding virus only without compounds.
4. Dilute compounds to 50 μM in assay medium and add to the 2^nd column at 20 μl/well for antiviral efficacy assay. Mix the compound five times using 8-channel semi-automatic pipette to a concentration of 25 μM.
5. Using 8-channel semi-automatic pipette, transfer 20 μl mixture in 2^nd column to the next column (3^rd), mix well to form a concentration of 12.5 μM. Repeat this process by transferring 20 μl mixture from 3^rd column to the next column (4^th) to form another two-fold dilution with a concentration of 6.25 μM. Repeat this two-fold serial dilution till the 11^th column. Aspirate and discard 20 μl of the mixture in 11^th column after adding and mixing the compound.
6. Add BTV-10, based on MOI of 0.01, to each well from column 2 to column 11 with a volume of 5 μl/well. After adding the virus, the final compound concentration in each column should be: 20 μM in column 2, 10 μM in column 3, and continued with a two-fold dilution down to column 11 with a final concentration of 0.04 μM.
7. Add 5 μl of medium to column 1 as cell only control (positive) and 5 μl/well of BTV to column 12 as BTV infection only control (negative).
8. Dilute compounds to an initial concentration of 200 μM for cytotoxicity assay. Similarly, add 25 μl/well of compound to column 2 and mixed 5x with 8-channel semi-automatic pipette to a concentration of 100 μM. Do not add BTV.
9. Carry out the two-fold series dilution by aspirating 25 μl from column 2 to the neighboring column 3, and continued till the last column (12^th). At column 12, after mixing, aspirate and discard 25 μl of mixture. The final concentration at column 2 should be 100 μM and the 12^th column should be at 0.2 μM. The column 1 is the cell only control.
10. For both antiviral efficacy and cytotoxicity assays, incubate the plates at 37 °C, with 5% CO₂ and 80-95% humidity for 72 hr post treatment. Measure cell viability using the CTG kit as described above (protocol steps 2.5-2.7).

4. Time-of-Addition (ToA) Assay

1. Seed BSR cells from column 1-24 in a 384-well microplate (black; format by 16 x 24) via a dispenser at 5,000 cells/well and the seeding volume is 15 μl/well.
2. For each compound, utilize all twenty-four columns with eight replicas for each time-point in a half of the 384-well plate (Table 2). Assign column 1 as cells only control by adding 10 μl/well assay medium. Mark column 24 as BTV infection only control (negative control) by adding 5 μl/well assay medium and 5 μl/well virus at MOI of 0.01 to a final volume of 25 μl/well.
3. Select the even-numbered columns from column 2-22 as antiviral efficacy evaluation column at different hours post infection (h.p.i.). In these columns, infect cells with 5 μl/well BTV at MOI of 0.01. At different h.p.i., also add 5 μl/well diluted compound to each well to form a final volume of 25 μl/well. For the denoted -2 and -1 h.p.i. add compound to BSR cells prior to BTV infection. For 0 h.p.i., add the compound and BTV to the culture simultaneously.
4. In parallel, designate the odd-numbered columns from column 3-23 as compound only controls, of which compound were added at different time-points as designated (-2, -1, 0, 1, 2, 4, 8, 16, 24, 48 h.p.i.). In these columns, add 5 μl/well assay medium and 5 μl/well diluted compound to form a final volume of 25 μl/well.
5. After treatment, incubate cells at 37 °C, 5% CO₂ with 80-95% humidity.
6. Determine cell viability at 72 h.p.i. using the CellTiter-Glo kit as described previously (protocol steps 2.5-2.7).

5. Data Analysis

1. Process all data first using proper in-house software based on the luminescent signals obtained via the multi-mode reader. Determine the mean value (Average), standard deviation (STDEV) for each treatment as well as the coefficient variations (CV), which requires a value no greater than 10%.
2. Transfer the processed data from the above software to a biostatic and graphic software tool. Carry out the non-linear regression analysis to determine the values of EC₅₀ and CC₅₀.

Results

1. Antiviral efficacy of compound

The cell-based CPE assay was developed, optimized and validated in vitro using the luminescent-based CTG kit to identify novel antivirals against BTV as described previously^2,5. The ten-dose response assay was employed to reflect the antiviral efficacy and cytotoxicity of an identified lead compound by measuring the number of metabolically viable cells in culture based on quantitation of cellular ATP presented in the living cells^5,11

Discussion

For the initial identification of antiviral hits, one of the key steps for antiviral drug discovery and development is to develop robust assays, which includes selecting a quantifiable marker, developing a simple protocol, obtaining sufficient signals and less than 10% CV. Most biochemical or cell-based screens are designed to provide a chemical starting point based upon the most robust, simple and inexpensive assay, due to the required reproducibility in the screening process and the potentially large number of molecule...

Materials

Name	Company	Catalog Number	Comments
DMEM medium	Gibco	1134218	For cell culture
FBS	Gibco	16000044	For cell culture
0.05% Trypsin-EDTA	Gibco	1000185	For cell culture
DPBS	Gbico	1049769	For cell culture
CellTiter-Glo (CTG) kit	Promega	TB288	For cell viability measurement
70% ethanol	Fisher	S25309B	Diluted from 95%
Antiviral huashilcompounds	NIH MLSMR and de novo synthesis
BTV-10	ATCC	VR-187
BSR cell	Developed in house
Synergy-II multi-mode microplate reader	BioTek		For luminescent signal reading
MicroFlo select dispenser	BioTek		Adding cells, virus, and reagents
384-well flat-bottom microplate	CORNING	28908031	For cell culture
Gen. 5 software	BioTek		For analysis of reading outputs from Synergy-II multi-mode microplate reader
GraphPad Prism 5	GraphPad	Version 5	For biostatic analysis and plot