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. 2022 Feb 5;12(3):e4314.
doi: 10.21769/BioProtoc.4314.

Cytopathic Effect Assay and Plaque Assay to Evaluate in vitro Activity of Antiviral Compounds Against Human Coronaviruses 229E, OC43, and NL63

Yanmei Hu  1 , Chunlong Ma  1 , Jun Wang  1
Affiliations

Cytopathic Effect Assay and Plaque Assay to Evaluate in vitro Activity of Antiviral Compounds Against Human Coronaviruses 229E, OC43, and NL63

Yanmei Hu et al. Bio Protoc. .

Abstract

Coronaviruses are important human pathogens, among which the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. To combat the SARS-CoV-2 pandemic, there is a pressing need for antivirals, especially broad-spectrum antivirals that are active against all seven human coronaviruses (HCoVs). For this reason, we are interested in developing antiviral assays to expedite the drug discovery process. Here, we provide the detailed protocol for the cytopathic effect (CPE) assay and the plaque assay for human coronaviruses 229E (HCoV-229E), HCoV-OC43, and HCoV-NL63, to identify novel antivirals against HCoVs. Neutral red was used in the CPE assay, as it is relatively inexpensive and more sensitive than other reagents. Multiple parameters including multiplicity of infection, incubation time and temperature, and staining conditions have been optimized for CPE and plaque assays for HCoV-229E in MRC-5, Huh-7, and RD cell lines; HCoV-OC43 in RD, MRC-5, and BSC-1 cell lines, and HCoV-NL63 in Vero E6, Huh-7, MRC-5, and RD cell lines. Both CPE and plaque assays have been calibrated with the positive control compounds remdesivir and GC-376. Both CPE and plaque assays have high sensitivity, excellent reproducibility, and are cost-effective. The protocols described herein can be used as surrogate assays in the biosafety level 2 facility to identify entry inhibitors and protease inhibitors for SARS-CoV-2, as HCoV-NL63 also uses ACE2 as the receptor for cell entry, and the main proteases of HCoV-OC43 and SARS-CoV-2 are highly conserved. In addition, these assays can also be used as secondary assays to profile the broad-spectrum antiviral activity of existing SARS-CoV-2 drug candidates.

Keywords: 229E; Antiviral; Human Coronavirus; NL63; OC43.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Flowchart for the CPE assay.
The whole process includes: cell seeding, to prepare cells in a 96-well plate for the experiments; stock virus titration, to optimize the assay conditions; and compound testing, for the evaluation of the antiviral activity of test compounds. Both stock virus titration and compound testing comprise the following steps: dilute the stock virus to obtain the desired MOI; infect the cells in the 96-well plate with a small volume (100 µL) of the diluted virus; incubate the 96-well plate in the incubator for 1-2 h, to facilitate viral attachment (virus adsorption); add the test compounds (only for the compound testing experiment); incubate the 96-well plate in the incubator to develop CPE; stain the cells with neutral red; extract neutral red from the cells; quantify neutral red by measuring absorbance; analyze data. A. Preparation of cells in the 96-well plate for the CPE assay; B. Major steps of stock virus titration in the CPE assay; C. Assessment of antiviral activity of test compounds in the CPE assay.
Figure 2.
Figure 2.. Flowchart for the plaque assay.
The whole process includes: cell seeding, to prepare cells in a 6-well plate for the experiments; stock virus titration, to optimize the assay conditions; and compound testing, for the evaluation of the antiviral activity of test compounds. Both stock virus titration and compound testing comprise the following steps: dilute the stock virus, to obtain the desired MOI; infect the cells in the 6-well plate with a small volume (500 µL) of the diluted virus; incubate the 6-well plate in the incubator for 1-2 h, to facilitate viral attachment (virus adsorption); add an avicel overlay containing the test compounds (only for the compound testing experiment); incubate the 6-well plate in the incubator, to allow plaque formation; stain the cells with crystal violet; analyze data. A. Preparation of cells in the 6-well plate for the plaque assay; B. Major steps of stock virus titration in the plaque assay; C. Assessment of antiviral activity of test compounds in the plaque assay.
Figure 3.
Figure 3.. CPE assay for HCoV-229E.
A. Titration of HCoV-229E in the MRC-5 cell line. B. Results of HCoV-229E titration in MRC-5 cell line in CPE assay. The signal to background (S/B) ratios are labeled for each virus dilution. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test). C. Determination of EC50 values for GC-376 and remdesivir against HCoV-229E in the CPE assay with the MRC-5 cell line. D. EC50 curve fitting for remdesivir obtained in GraphPad Prism 8. E. EC50 curve fitting for GC-376 obtained in GraphPad Prism 8. F. Titration of HCoV-229E in Huh-7 cell line. G. Titration of HCoV-229E in RD cell line. The images are representatives of three independent repeats.
Figure 4.
Figure 4.. CPE assay for HCoV-NL63.
A. Titration of HCoV-NL63 in the Vero E6 cell line. B. Results of HCoV-NL63 titration in Vero E6 cell line in CPE assay. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test). The S/B ratios are labeled for each virus dilution. C. Determination of EC50 values for GC-376 and remdesivir against HCoV-NL63 in the CPE assay. D. EC50 curve fitting for remdesivir obtained in GraphPad Prism 8. E. EC50 curve fitting for GC-376 obtained in GraphPad Prism 8. F. Titration of the HCoV-NL63 in Huh-7 cell line; G. Titration of HCoV-NL63 in MRC-5 cell line. H. Titration of HCoV-NL63 in RD cell line. The images are representatives of three independent repeats.
Figure 5.
Figure 5.. CPE assay for HCoV-OC43.
A. Titration of HCoV-OC43 in the RD cell line. B. Results of HCoV-OC43 titration in RD cell line in CPE assay. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test). The S/B ratios are labeled for each virus dilution. C. Determination of EC50 values for GC-376 and remdesivir against HCoV-OC43 in CPE assay; D. EC50 curve fitting for remdesivir obtained in Prism 8. E. EC50 curve fitting for GC-376 obtained in Prism 8. F. Titration of HCoV-OC43 in the MRC-5 cell line. G. Titration of HCoV-OC43 in the BSC-1 cell line. The images are representatives of three independent repeats.
Figure 6.
Figure 6.. Plaque reduction assay for HCoV-229E.
A. Titration of HCoV-229E in the RD cell line. B. Determination of EC50 value for remdesivir against HCoV-229E in plaque assay. C. Determination of EC50 value for GC-376 against HcoV-229E in plaque assay. D. EC50 curve fitting for remdesivir obtained in GraphPad Prism 8. E. EC50 curve fitting for GC-376 obtained in GraphPad Prism 8. F. Titration of HcoV-229E in the Huh-7 cell line. G. Titration of HCoV-229E in the MRC-5 cell line. The images are representatives of three independent repeats.
Figure 7.
Figure 7.. Plaque reduction assay for HCoV-NL63.
A. Titration of HCoV-NL63 in the RD cell line. B. Determination of EC50 value for remdesivir against HCoV-NL63 in plaque assay. C. Determination of EC50 value for GC-376 against HCoV-NL63 in plaque assay. D. EC50 curve fitting for remdesivir obtained in GraphPad Prism 8. E. EC50 curve fitting for GC-376 obtained in GraphPad Prism 8. F. Titration of HCoV-NL63 in the Huh-7 cell line. G. Titration of HCoV-NL63 in the MRC-5 cell line. H. Titration of HCoV-NL63 in the RD cell line. The images are representatives of three independent repeats.
Figure 8.
Figure 8.. Plaque reduction assay for HCoV-OC43.
A. Titration of HCoV-OC43 in the RD cell line. B. Determination of EC50 value for remdesivir against HCoV-OC43 in plaque assay. C. Determination of EC50 value for GC-376 against HCoV-OC43 in plaque assay. D. EC50 curve fitting for remdesivir obtained in GraphPad Prism 8. E. EC50 curve fitting for GC-376 obtained in GraphPad Prism 8. F. Titration of HCoV-OC43 in the MRC-5 cell line. G. Titration of HCoV-OC43 in the BSC-1 cell line. The images are representatives of three independent repeats.
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