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The global SARS-CoV-2 pandemic has resulted in widespread activities seeking to identify new anti-viral drugs that might be used to treat COVID-19 patients [1,2,3,4,5]. Remdesivir has emerged as a lead candidate with clear anti-viral activity in vitro [2).

Fig. 2
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Crystal violet staining for remdesivir and hydroxychloroquine. Cytotoxicity assay (Vero E6 seeded at 104 cells/well with no virus). Cytomorbidity assay (Vero E6 seeded at 400 cells/well with no virus). Viral CPE (Vero E6 seeded at 104 cells/well with virus MOI ≈ 0.01). After 4 days in culture 96 well plates were fixed and stained with paraformaldehyde and crystal violet, respectively. Plates were washed in water, dried and scanned, and for the data in Fig. 1, the dye was dissolved in methanol and read at OD595 nm. For the Cytotoxicity assay wells encircled in red show overt cytotoxicity. For the Cytomorbidity assay wells encircled in red show overt cell growth reduction. For viral CPE assay, wells encircled in red show inhibition of CPE indicating potential antiviral activity

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Table 1 The half maximal inhibitory dose (IC50), half maximal cytotoxic concentration (CC50), and half maximal cytomorbidity concentration (MC50) for each compound
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The close relationship between anti-viral activity and translation inhibition (inherent in the stress responses described above) can be seen with the use of the translation inhibitors, cycloheximide and didemnin B. These drugs provide selectivity indices of ≥ 10, when comparing viral CPE inhibition and cytotoxicity. However, concentrations that inhibited viral CPE again overlapped with those that caused cytomorbidity (Fig. 1, Cycloheximide, Didemnin B). The drug γ-mangostin would appear to have a small level of anti-viral activity with a low selectivity index, but again this activity overlapped with the cytomorbidity (Fig. 1, γ-mangostin). Linoleic acid is reported to contribute to anti-viral activity at 50 µM [39]; however, this drug shows clear cytomorbidity activity above ≈ 20 µM (Fig. 1, Linoleic acid). Thus, as for hydroxychloroquine, the assay results for these latter drugs provide no supportive data for anti-viral activity, instead they suggest these drugs inhibit viral replication non-specifically by impairing cellular activities. Nitazoxanide showed some anti-viral activity, but this coincided with cytotoxicity, providing an example of the conventional cytotoxicity control that would be used to argue that the drug has no specific anti-viral activity and has a selectivity index of 1 (Fig. 1, Nitazoxanide). Curiously, higher concentrations of nitazoxanide were needed to inhibit cell growth than were needed to induce cytotoxicity; likely an example of cell density associated toxicity.

The frequently used MTS assay, as expected, often gave results similar to those provided by the cytotoxicity assay. Importantly, the MTS assay did not provide a measure of cytomorbidity, presumably because mitochondria largely remain active even in stressed cells and/or cells in G0 (cytostasis). For oleuropein, cyclosporine A and γ-mangostin, cytomorbidity was associated with an increase in MTS activity (Fig. 1). The MTS bioassay may thus provide slightly misleading information in this context; i.e. increased mitochondrial activity, rather than indicating increased cell numbers, can sometimes be associated with stress or mild toxicity.

The CPE-based assay described herein has some inherent limitations. Drugs whose mechanism of action require induction of type I interferons, would be ineffective in this assay system as Vero E6 cells do not make type I interferons. The CPE-based assay also provides a low sensitivity read-out. Higher drug concentrations are likely needed to prevent virus-induced CPE (overwhelming infection resulting in cell death) than would be needed to inhibit viral replication as measured (for instance) by qRT-PCR of virus released into culture supernatants [40]. Although more sensitive anti-viral activities exist [40], the CPE-based assay represents a screening tool able rapidly and cheaply to identify promising anti-viral candidates. More sensitive assays could be also envisaged for assessing cytomorbidity, such as measuring activation of stress factors such as ATF3 [41], analyzing cell cycle perturbations by flow cytometry or cell growth kinetics using the IncuCyte live-cell analysis system. The cell line used herein, Vero E6, is a monkey kidney-derived cell line, whereas in humans ciliated airway cells and alveolar type II pneumocytes (AT-2 cells) are thought to be the primary targets for SARS-CoV-2 infection [42]. Drug metabolism and/or bioavailability in such cells may not be reflected in Vero E6 cells. However, although a number of human cell lines support SARS-CoV-2 infection, few if any exhibit the fulminant CPE seen in Vero E6 cells [43].

Conclusions

In conclusion, in vitro screening of anti-SARS-CoV-2 drugs should include not just a cytotoxicity control, but also a cytomorbidity control in order to identify potential false positives associated with anti-viral activity arising from non-specific stress responses or other disruptions of cellular activities/functions.