Synthesis and Anti-Influenza Virus Effects of Novel Substituted Polycyclic Pyridone Derivatives Modified from Baloxavir.

In this work, a series of novel substituted polycyclic pyridone derivatives were designed and synthesized as potent anti-influenza agents. The cytopathic effect (CPE) assay and cytotoxicity assay indicated that all of the compounds possessed potent anti-influenza virus activity and relatively low cytotoxicity; some of them inhibited the replication of influenza A virus (IAV) at picomolar concentrations. Further studies revealed that, at a concentration of 3 nM, three compounds (10a, 10d, and 10g) could significantly reduce the M2 RNA amounts and M2 protein expression of IAV and inhibit the activity of RNA-dependent RNA polymerase (RdRp). Among them, (R)-12-(5H-dibenzo[a,d][7]annulen-5-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (10a) was found to be a promising anti-influenza drug candidate with good human liver microsomal stability, as well as with better selectivity index and oral bioavailability than Baloxavir.

Organic Electrochemical Transistors as Versatile Tool for Real-Time and Automatized Viral Cytopathic Effect Evaluation.

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

Quantification of Infectious SARS-CoV-2 by the 50% Tissue Culture Infectious Dose Endpoint Dilution Assay.

A number of viral quantification methods are used to measure the concentration of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the traditional plaque-based assay allows for direct enumeration of replication competent lytic virions and remains the gold standard for the quantification of infectious virus, the 50% tissue culture infectious dose (TCID50) endpoint dilution assay allows for a more rapid, large-scale analysis of experimental samples. In this chapter, we describe a well-established TCID50 assay protocol to measure the SARS-CoV-2 infectious titer in viral stocks, in vitro cell or organoid models, and animal tissue. We also present alternative assays for scoring the cytopathic effect of SARS-CoV-2 in cell culture and comparable methods to calculate the 50% endpoint by serial dilution.

Upregulation of INF-γ, IL-6, and IL-8 expression during replication of turkey coronavirus in nonepithelial cells obtained from Meleagris gallopavo.

Mesenchymal stromal cells (MSCs) are considered multipotent progenitors with the capacity to differentiate into mesoderm-like cells in many species. The immunosuppressive properties of MSCs are important for downregulating inflammatory responses. Turkey coronavirus (TCoV) is the etiological agent of a poult mortality syndrome that affects intestinal epithelial cells. In this study, poult MSCs were isolated, characterized, and infected with TCoV after in vitro culture. The poult-derived MSCs showed fibroblast-like morphology and the ability to undergo differentiation into mesodermal-derived cells and to support virus replication. Infection with TCoV resulted in cytopathic effects and the loss of cell viability. TCoV antigens and new viral progeny were detected at high levels, as were transcripts of the pro-inflammatory factors INFγ, IL-6, and IL-8. These findings suggest that the cytokine storm phenomenon is not restricted to one genus of the family Coronaviridae and that MSCs cannot always balance the process.

Effect of Short Time of SARS-CoV-2 Infection in Caco-2 Cells.

Coronavirus disease 19 (COVID-19) clinical manifestations include the involvement of the gastrointestinal tract, affecting around 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children. In the present work, the consequence of a short time of viral absorption (5, 15, 30 and 60 min) was tested on the Caco-2 intestinal epithelial cell line. Our findings show that Caco-2 cells are highly permissive to SARS-CoV-2 infection, even after 5 min of viral inoculation at a multiplicity of infection of 0.1. No cytopathic effect was evident during the subsequent 7 days of monitoring; nevertheless, the immunofluorescence staining for the viral nucleocapsid confirmed the presence of intracellular SARS-CoV-2. Our findings highlight the very short time during which SARS-CoV-2 is able to infect these cells in vitro.

Rapid SARS-CoV-2 Adaptation to Available Cellular Proteases.

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.

Evolution of the SARS-CoV-2 spike protein in the human host.

Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants to better understand the evolution of the virus in humans. Spikes of both variants have the same mutation, N501Y, in the receptor-binding domains. This substitution confers tighter ACE2 binding, dependent on the common earlier substitution, D614G. Each variant spike has acquired other key changes in structure that likely impact virus pathogenesis. The spike from the Alpha variant is more stable against disruption upon binding ACE2 receptor than all other spikes studied. This feature is linked to the acquisition of a more basic substitution at the S1-S2 furin site (also observed for the variants of concern Delta, Kappa, and Omicron) which allows for near-complete cleavage. In the Beta variant spike, the presence of a new substitution, K417N (also observed in the Omicron variant), in combination with the D614G, stabilises a more open spike trimer, a conformation required for receptor binding. Our observations suggest ways these viruses have evolved to achieve greater transmissibility in humans.

Human Kidney Spheroids and Monolayers Provide Insights into SARS-CoV-2 Renal Interactions.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. METHODS: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. RESULTS: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. CONCLUSIONS: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and ß-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

Snake venom phospholipase A2s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2.

The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell-cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA2s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA2s on ß-coronaviruses. Thus, snake PLA2s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells.

Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication.

Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection.

Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.

Advances on Antiviral Activity of Morus spp. Plant Extracts: Human Coronavirus and Virus-Related Respiratory Tract Infections in the Spotlight.

(1) Background: Viral respiratory infections cause life-threatening diseases in millions of people worldwide every year. Human coronavirus and several picornaviruses are responsible for worldwide epidemic outbreaks, thus representing a heavy burden to their hosts. In the absence of specific treatments for human viral infections, natural products offer an alternative in terms of innovative drug therapies. (2) Methods: We analyzed the antiviral properties of the leaves and stem bark of the mulberry tree (Morus spp.). We compared the antiviral activity of Morus spp. on enveloped and nonenveloped viral pathogens, such as human coronavirus (HCoV 229E) and different members of the Picornaviridae family-human poliovirus 1, human parechovirus 1 and 3, and human echovirus 11. The antiviral activity of 12 water and water-alcohol plant extracts of the leaves and stem bark of three different species of mulberry-Morus alba var. alba, Morus alba var. rosa, and Morus rubra-were evaluated. We also evaluated the antiviral activities of kuwanon G against HCoV-229E. (3) Results: Our results showed that several extracts reduced the viral titer and cytopathogenic effects (CPE). Leaves' water-alcohol extracts exhibited maximum antiviral activity on human coronavirus, while stem bark and leaves' water and water-alcohol extracts were the most effective on picornaviruses. (4) Conclusions: The analysis of the antiviral activities of Morus spp. offer promising applications in antiviral strategies.

mSphere of Influence: Viruses-Pathogens or Expert Cell Biologists?

Chelsey C. Spriggs works in the field of DNA viral entry with a specific interest in virus-host interactions. In this mSphere of Influence article, she reflects on how two papers, "The HCMV assembly compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread" (D. J. Procter, A. Banerjee, M. Nukui, K. Kruse, et al., Dev Cell 45:83-100.e7, 2018, https://doi.org/10.1016/j.devcel.2018.03.010) and "Cytoplasmic control of intranuclear polarity by human cytomegalovirus" (D. J. Procter, C. Furey, A. G. Garza-Gongora, S. T. Kosak, D. Walsh, Nature 587:109-114, 2020, https://doi.org/10.1038/s41586-020-2714-x), impacted her research by reinforcing the scientific value in using viruses to understand cell biology.

SARS-CoV-2 Short-Time Infection Produces Relevant Cytopathic Effects in Vero E6 Cell Line.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly transmitted through respiratory droplets from positive subjects to susceptible hosts or by direct contact with an infected individual. Our study focuses on the in vitro minimal time of viral absorption as well as the minimal quantity of virus able to establish a persistent infection in Vero E6 cells. We observed that 1 min of in vitro virus exposure is sufficient to generate a cytopathic effect in cells after 7 days of infection, even at a multiplicity of infection (MOI) value of 0.01. Being aware that our findings have been obtained using an in vitro cellular model, we demonstrated that short-time exposures and low viral concentrations are able to cause infection, thus opening questions about the risk of SARS-CoV-2 transmissibility even following short contact times.

Antiviral Activity of Umifenovir In Vitro against a Broad Spectrum of Coronaviruses, Including the Novel SARS-CoV-2 Virus.

An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21-36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.

Endoplasmic reticulum stress and NF-kB activation in SARS-CoV-2 infected cells and their response to antiviral therapy.

Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1α-mediated transcriptional regulation of stress response genes with anti-inflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.

Complement Proteins as Soluble Pattern Recognition Receptors for Pathogenic Viruses.

The complement system represents a crucial part of innate immunity. It contains a diverse range of soluble activators, membrane-bound receptors, and regulators. Its principal function is to eliminate pathogens via activation of three distinct pathways: classical, alternative, and lectin. In the case of viruses, the complement activation results in effector functions such as virion opsonisation by complement components, phagocytosis induction, virolysis by the membrane attack complex, and promotion of immune responses through anaphylatoxins and chemotactic factors. Recent studies have shown that the addition of individual complement components can neutralise viruses without requiring the activation of the complement cascade. While the complement-mediated effector functions can neutralise a diverse range of viruses, numerous viruses have evolved mechanisms to subvert complement recognition/activation by encoding several proteins that inhibit the complement system, contributing to viral survival and pathogenesis. This review focuses on these complement-dependent and -independent interactions of complement components (especially C1q, C4b-binding protein, properdin, factor H, Mannose-binding lectin, and Ficolins) with several viruses and their consequences.

Evaluation of MERS-CoV Neutralizing Antibodies in Sera Using Live Virus Microneutralization Assay.

The microneutralization (MN) assay is a standard and important technique in virology, immunology, and epidemiology. It is a highly specific and sensitive assay for evaluating virus-specific neutralizing antibodies (nAbs) in human and animal sera. It provides the most precise answer to whether or not an individual or animal has antibodies that can neutralize or inhibit the infectivity of a specific virus strain. However, using live virus-based MN assay might require working under high containment facilities especially when dealing with high-risk pathogens such as the Middle East respiratory syndrome-coronavirus (MERS-CoV). In this chapter, we describe the isolation, amplification, and titration of MERS-CoV, as well as detailed MN assay to measure nAb levels in sera from different mammalian species.

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

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.