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Joyce Itatani | Applications Scientist | Molecular Devices

Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices

Introduction

Viral infection of mammalian cells often reduces cell viability and causes visible effects upon the cells such as changes in shape or size, or fusion with adjacent cells. These changes are called cytopathic effects (CPE), and they can be assessed using light microscopy or imaging systems, or measured using more quantitative means.

The Viral ToxGlo Assay Kit from Promega measures cellular ATP, which is present in viable cells and provides an easy way to quantitate cell viability with a luminescence readout. Depletion of ATP due to viral-induced CPE leads to a reduction in luminescence signal, enabling quantitation of viral-induced CPE in host cells. Using a simple mix-and-read workflow, assay results are easily detected on the SpectraMax® iD5 Multi-Mode Microplate Reader and analyzed using SoftMax® Pro Software (Figure 1).

Here we demonstrate how the Viral ToxGlo assay is used to measure viral infectivity and tissue culture infective dose (TCID₅₀) in mammalian cells infected with viruses, as well as how compound antiviral potency is measured. The studies presented here were performed with two previously described models of virus infection: Madin-Darby canine kidney (MDCK) cells were infected with influenza A (H1N1) virus1, and MRC-5 human lung fibroblasts were infected with human coronavirus strain 229E (HCoV-229E)². Ribavirin³ and remdesivir⁴, two compounds with demonstrated anti-viral effects, were applied to virus-exposed cells, and antiviral potency for each was measured.

Figure 1. Viral ToxGlo assay workflow.

Materials

• Viral ToxGlo Assay Kit (Promega cat. #G8942)
• MDCK (NBL-2) cell line (ATCC cat. #CCL-34)
• MRC-5 cell line (ATCC cat. #CCL-171)
• Growth medium (for MDCK and MRC-5): MEM (Corning cat. #10-010-CV) + 10% fetal bovine serum (FBS, Avantor® Seradigm cat. #1500-500) + penicillin/ streptomycin (Thermo Fisher cat. #15070-063)
• Influenza A virus (H1N1), (ATCC cat. #VR-95PQ)
• Human coronavirus 229E (ATCC cat. #VR-740) Fluorescence Polarization (FP-FLUO) Detection Cartridge (Molecular Devices cat. #0200-7009
• Ribavirin (Sigma cat. #R-9644)
• Remdesivir (MedChemExpress cat. #HY-104077)
• 96-well white, tissue culture-treated, clear-bottom microplate (Corning cat. #3610)
• Nunc white sealing tape (ThermoFisher cat. #236272)
• SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices)

Method

TCID₅₀ determination

Viral infectivity and tissue culture infective dose (TCID) were determined by preparing serial dilutions of a virus stock and adding these to target cells for a specified exposure time. At the end of the exposure, the Viral ToxGlo assay was used to measure ATP, which serves as an indicator of cell viability. The TCID₅₀ or 50% cytopathic effect (CPE) is the virus dilution that reduces the measured cell viability by 50%. This value was used for subsequent studies on the potency of antiviral agents.

Cells were plated at 10,000 cells/well in 96-well white microplates with clear bottoms. Cells were allowed to attach and grow overnight at 37°C and 5% CO₂. An initial 1:1000 (H1N1) or 1:5 (HCoV-229E) dilution of the virus stock was made in culture media. Half-log (3.16-fold) serial dilutions were made in the cell plate by adding 46 μL of the initial virus dilution to wells (initial volume of 100 μL/well) of column 1. 46 μL was then transferred from column 1 to column 2, and so on through column 10. 46 μL was then removed from column 10 to maintain the volume of all experimental wells at 100 μL/well. No-virus and no-cell control wells were included. Cells were incubated with virus for three days (MDCK/H1N1) or six days (MRC-5/HCov-229E) at 37°C and 5% CO₂.

Cytopathic effect was determined using the Viral ToxGlo kit. At the end of the 3- or 6-day virus treatment, ATP detection reagent was added to assay wells, and the plates were incubated at room temperature for 10 minutes to allow cell lysis. Nunc white sealing tape was placed on the bottom of each clear-bottom microplate prior to reading in order to maximize luminescent signal. The plates were read on the SpectraMax iD5 reader using the settings shown in Table 1.

Table 1. SpectraMax iD5 reader settings for the Viral ToxGlo assay. Settings are specified in the plate section in SoftMax Pro Software. Read height is optimized by checking the box next to ‘Show Pre-Read Optimization Options’ under More Settings, and following the instructions that appear once the read is initiated.

Results were plotted as RLU vs. viral dilution factor, using a 4-parameter curve fit in SoftMax Pro Software, and the TCID₅₀ value for each virus on its respective cell line was obtained from these curves (Figure 2). A 999,000-fold dilution of the H1N1 virus stock reduced MDCK viability by 50%, while a similar reduction in viability was effected in MRC-5 cells by a 43-fold dilution of the HCoV-229E virus stock.

Figure 2. Concentration-response curves for H1N1 virus on MDCK cells (A) and HCoV-229E on MRC-5 cells (B). TCID₅₀ was observed at a dilution of 1:999,000 for H1N1 and 1:43 for HCoV-229E.

Determination of compound antiviral potency

To assess the effectiveness of the compounds ribavirin and remdesivir at reducing cytopathic effects on virus-treated cells, MDCK or MRC-5 cells were plated at 10,000 cells per well in 50 μL of medium in 96-well white microplates with clear bottoms. No-cell control wells containing culture medium only were included. Cells were allowed to attach and grow overnight in a 37°C, 5% CO₂ incubator.

To MDCK cells, 25 μL of a 1:3 serial dilution of ribavirin ranging from 1000 μM to 0.02 μM was added. MRC-5 cells received 25 μL of a 1:3 serial dilution of remdesivir ranging from 17 μM to 0.003 μM. Dilutions of the stock viruses that would give optimal cytopathic effects in excess of TCID₅₀ (1:1000 for H1N1 and 1:5 for HCov-229E) were added to MDCK or MRC-5 cells, respectively, in a volume of 25 μL per well. To determine off-target cytotoxicity of the compounds, ribavirin or remdesivir was added to cells without virus using the same compound dilution series described above. Treated cell plates were incubated at 37°C and 5% CO₂ for three days (MDCK) or six days (MRC-5).

After incubation with viruses and compounds, Viral ToxGlo ATP detection reagent was added to assay wells, and the plates were incubated at room temperature for 10 minutes to allow cell lysis. Nunc white sealing tape was placed on the bottom of the clear-bottom microplate before reading the luminescent signal on the SpectraMax iD5 reader (settings, Table 1).

Results were plotted as RLU vs. compound concentration, using a 4-parameter curve fit in SoftMax Pro Software, and the EC₅₀ value for each compound was obtained from the curves. Ribavirin partially rescued viability in MDCK cells exposed to H1N1 virus, with an EC₅₀ of 89 μM (Figure 3A). However, toxic effects were observed above 100 μM in cells treated with ribavirin alone, without virus. Viability of remdesivir-treated MRC-5 cells exposed to HCoV-229E was nearly fully rescued, with an EC₅₀ value of 215 nM (Figure 3B). Off-target effects of remdesivir were minimal, seen only above 16 μM.

Figure 3. Off-target (green) and on-target (red) effects of ribavirin on MDCK alone or treated with H1N1 virus (A) and of remdesivir on MRC-5 alone or treated with HCoV-229E virus (B). Ribavirin EC₅₀ = 89 μM; remdesivir EC₅₀ = 215 nM.

Conclusion

In contrast to time-consuming microscopic assessment of effects on cell viability induced by infection with virus, the Viral ToxGlo assay offers a simple mix-and-read format. Detection and analysis of results can be performed just 10 minutes after reagent is added to treated cells. The SpectraMax iD5 reader offers highly sensitive detection of this assay, and SoftMax Pro Software enables easy determination of the TCID₅₀ as well as on- and off-target effects of antiviral compounds.

References

1. Niles, A et al. Determine Viral-Induced Cytopathic Effect Using a Luminescent Assay: https://www.promega.com/ resources/pubhub/determine-viral-induced-cytopathic-effectusing- a-luminescent-assay/
2. Funk, CJ et al. Infection of human alveolar macrophages by human coronavirus strain 229E. J. Gen. Virol. (2012), 93, 494–503
3. Khalili, JS et al. Novel coronavirus treatment with ribavirin: Groundwork for an evaluation concerning COVID-19. J. Med. Virol. 2020; 1–7.
4. Parang, K et al. Comparative antiviral activity of remdesivir and anti-HIV nucleoside analogs against human coronavirus 229E (HCoV-229E). Molecules 2020, 25, 2343.

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