Background

Humans have been battling viruses throughout history. Viral epidemics and pandemics have caused devastating economic, social, and political unrest, in addition to significant morbidity and mortality (Shang et al., 2021). There are seven human coronaviruses: SARS-CoV (Gagneur et al., 2002), MERS-CoV (Zumla et al., 2015), and SARS-CoV-2 (Hui et al., 2020), which cause severe acute respiratory syndrome; and four common human coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, which cause a significant portion of upper and lower respiratory tract infections in humans worldwide (Mesel-Lemoine et al., 2012). Although vaccines are the mainstay for viral prophylaxis, antiviral drugs are essential complements and play a vital role in viral disease containment, especially during the lag period between the initial viral outbreak and the delivery of effective vaccines. Furthermore, vaccines may lose potency and become ineffective when mutations emerge among circulating viruses (Collier et al., 2021; Lopez Bernal et al., 2021; Williams and Burgers, 2021). As the third coronavirus outbreak in human history, the COVID-19 pandemic is a timely reminder of the urgent need for broad-spectrum antiviral drugs that can be rapidly deployed for the prevention and treatment of emerging and re-emerging viral diseases.

We have routinely used the CPE and plaque assays to test antivirals against many different types of viruses, including influenza, enterovirus, and coronavirus (Hu et al., 2017a, 2017b, 2017c, 2017d, 2018, 2021a, 2021b; Li, F. et al., 2016, 2017; Ma et al., 2019, 2020a, 2020b; Musharrafieh et al., 2019a, 2019b, 2020; Smail et al., 2021; Wang et al., 2018; Xia et al., 2021; Zhang et al., 2018, 2019, 2020). The CPE and plaque assays have several key advantages over alternative methods. First, these assays measure viral infectivity instead of a given viral protein or gene, as both assays directly report the potency of testing compounds in inhibiting the replication of infectious viruses. Second, CPE and plaque assays are more reproducible than the TCID₅₀ assay (Nadgir et al., 2013). Third, both CPE and plaque assays are relatively inexpensive compared to RT-qPCR (Corman et al., 2020), immunoassays (Liu et al., 2005; Payne et al., 2006), viral flow cytometry (Brussaard et al., 2000), or Transmission Electron Microscope (TEM) (Roingeard, 2008), and do not require expensive reagents or specialized instruments. In this study, we have established two cell-based antiviral assays, the CPE assay and the plaque assay for HCoV-229E, HCoV-NL63, and HCoV-OC43. These assays can be used as alternatives for the primary screening of SARS-CoV-2 antivirals in BSL-2 facilities, and also as secondary assays to characterize the broad-spectrum antiviral activity of potential drug candidates. The CPE assay is appropriate for high-throughput screenings in a 96-well plate format, while the plaque assay is comparatively labor-intense and time-consuming, and is more suitable as a secondary assay to confirm the antiviral activity of initial hits identified from the primary CPE assay (Ratnam et al., 1995). Overall, these assays are expected to expedite the discovery of new antivirals against coronaviruses in BSL-2 facilities. In this report, we describe the detailed protocols for utilizing the CPE assay (flowchart illustrated in Figure 1) and the plaque assay (flowchart illustrated in Figure 2) to test antiviral activity of drug candidates against HCoV-229E, HCoV-NL63, and HCoV-OC43. Multiple parameters for the assays have been optimized, including the cell lines, multiplicity of infection, incubation time and temperature, and staining conditions. The optimized CPE and plaque assays conditions for HCoV-229E, HCoV-OC43 and HCoV-NL63 are listed in Table 1. Both assays have been calibrated with the positive control compounds remdesivir (Wang et al., 2020) and GC-376 (Ma et al., 2020c). Alternatively, CPE and plaque assays protocols for HCoV-229E, HCoV-OC43, and HCoV-NL63 can be found elsewhere (Schmidt et al., 1979; Gerna et al., 1980; Herzog et al., 2008; Bracci et al., 2020; Hirose et al., 2021; Schirtzinger et al., 2021).

Table 1. Cell lines and incubation temperature and time for HCoV-229E, HCoV-OC43, and HCoV-NL63 in the CPE and plaque assays in this protocol.

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. 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.