Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives

Abstract

Background The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or “cytomorbidity”, as distinct from cytotoxicity or loss of viability. Methods A simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays. Results The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity. Conclusions We describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.

Identification of potential COVID-19 treatment compounds which inhibit SARS Cov2 prototypic, Delta and Omicron variant infection

“…SARS coronaviruses are known to replicate and induce cytopathy in several susceptible cell lines ( Kaye, 2006 ) and Vero E6 is one of the first cell lines used to isolate SARS CoV and SARS CoV2( Ksiazek et al, 2003 ; Ogando et al, 2020 ). Many drug screening studies have used Vero E6 as the target cell line to determine the antiviral effects of the candidate drugs ( Riva et al, 2020 ; Yan et al, 2021 ). As discussed above, the ability of chloroquine to inhibit SARS-CoV2 infection varied depending upon the cell line used for screening (( Liu et al, 2020 ), ( Ho et al, 2021 ), and failed in clinical trials ( Hoffmann et al, 2020b ).…”

Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

“…Many assays measure SARS-CoV-2-induced host cell destruction (cytopathic effect, CPE) or host cell viability for the identification of antiviral agents [Bojkova et al 2020; Riva et al, 2020; Touret et al, 2020; Zhang et al, 2020; Ellinger et al, 2021; Van Damme et al, 2021; Yan et al, 2021a]. However, such assays are not suitable for SARS-CoV-2 culture systems that do not display virus-induced cytotoxicity [Caccuri et al, 2020; Liao et al, 2020; Bielarz et al, 2021; Wurtz et al, 2021].…”

Synthesis and Antiviral Properties against SARS-CoV-2 of Epoxybenzooxocino[4,3-b]Pyridine Derivatives

“…The SARS-CoV-2 virus strain used in this work was obtained from a clinical sample by the authors themselves and registered under the name hCoV-19/Kazakhstan/20679/2020. The nucleotide sequence of the whole genome of this strain was determined, and the results are published in [41]. The nucleotide sequence of this virus genome was deposited in the GISAID database (https://www.gisaid.org/, accessed on 17 January 2022) with the number EPI_ISL_454501.…”