Evaluation of Neutralizing Activity against Omicron Subvariants in BA.5 Breakthrough Infection and Three-Dose Vaccination Using a Novel Chemiluminescence-Based, Virus-Mediated Cytopathic Assay.

Neutralizing potency of humoral immune responses induced by prior infection or vaccination is vital for protecting of individuals and population against severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, the emergence of viral variants that can evade neutralization by vaccine- or infection-induced immunity is a significant public health threat and requires continuous monitoring. Here, we have developed a novel scalable chemiluminescence-based assay for assessing SARS-CoV-2-induced cytopathic effect to quantify the neutralizing activity of antisera. The assay leverages the correlation between host cell viability and ATP levels in culture to measure the cytopathic effect on target cells induced by clinically isolated, replication-competent, authentic SARS-CoV-2. With this assay, we demonstrate that the recently arisen Omicron subvariants BQ.1.1 and XBB.1 display a significant decrease in sensitivity to neutralization by antibodies elicited from breakthrough infections with Omicron BA.5 and from receipt of three doses of mRNA vaccines. Thus, this scalable neutralizing assay provides a useful platform to assess the potency of acquired humoral immunity against newly emerging SARS-CoV-2 variants. IMPORTANCE The ongoing global pandemic of SARS-CoV-2 has emphasized the importance of neutralizing immunity in protecting individuals and populations against severe respiratory illness. In light of the emergence of viral variants with the potential to evade immunity, continuous monitoring is imperative. A virus plaque reduction neutralization test (PRNT) is a "gold standard" assay for analyzing neutralizing activity for authentic viruses that form plaques, like influenza virus, dengue virus, and SARS-CoV-2. However, this method is labor intensive and is not efficient for performing large-scale neutralization assays on patient specimens. The assay system established in this study allows for the detection of a patient's neutralizing activity by simply adding an ATP detection reagent, providing a simple evaluation system for neutralizing activity of antisera as an alternative to the plaque reduction method. Our extended analysis of the Omicron subvariants highlights their increasing capability to evade neutralization by both vaccine- and infection-induced humoral immunity.

In vitro evaluation of the activity of teriflunomide against SARS-CoV-2 and the human coronaviruses 229E and OC43

Previous data have suggested an antiviral effect of teriflunomide, including against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the agent underlying the ongoing COVID-19 pandemic. We undertook an in vitro investigation to evaluate the inhibitory activity of teriflunomide against SARS-CoV-2 in a cell-based assay. Teriflunomide was added to Vero (kidney epithelial) cells that had been infected with SARS-CoV-2. A nucleocapsid immunofluorescence assay was performed to examine viral inhibition with teriflunomide and any potential cytotoxic effect. The 50% effective concentration (EC(50)) for teriflunomide against SARS-CoV-2 was 15.22 μM. No cytotoxicity was evident for teriflunomide in the Vero cells (i.e., the 50% cytotoxic concentration [CC(50)] was greater than the highest test concentration of 100 μM). The data were supported by additional experiments using other coronaviruses and human cell lines. In the SARS-CoV-2-infected Vero cells, the prodrug leflunomide had an EC(50) of 16.49 μM and a CC(50) of 54.80 μM. Our finding of teriflunomide-mediated inhibition of SARS-CoV-2 infection at double-digit micromolar potency adds to a growing body of evidence for a broad-ranging antiviral effect of teriflunomide.

Evaluation of Viral Inactivation on Dry Surface by High Peak Power Microwave (HPPM) Exposure.

Previous research has shown that virus infectivity can be dramatically reduced by radio frequency exposure in the gigahertz (GHz) frequency range. Given the worldwide SARS-CoV-2 pandemic, which has caused over 1 million deaths and has had a profound global economic impact, there is a need for a noninvasive technology that can reduce the transmission of virus among humans. RF is a potential wide area-of-effect viral decontamination technology that could be used in hospital rooms where patients are expelling virus, in grocery and convenience stores where local populations mix, and in first responder settings where rapid medical response spans many potentially infected locations within hours. In this study, we used bovine coronavirus (BCoV) as a surrogate of SARS-CoV-2 and exposed it to high peak power microwave (HPPM) pulses at four narrowband frequencies: 2.8, 5.6, 8.5, and 9.3 GHz. Exposures consisted of 2 µs pulses delivered at 500 Hz, with pulse counts varied by decades between 1 and 10,000. The peak field intensities (i.e. the instantaneous power density of each pulse) ranged between 0.6 and 6.5 MW/m2 , depending on the microwave frequency. The HPPM exposures were delivered to plastic coverslips containing BCoV dried on the surface. Hemagglutination (HA) and cytopathic effect analyses were performed 6 days after inoculation of host cells to assess viral infectivity. No change in viral infectivity was seen with increasing dose (pulse number) across the tested frequencies. Under all conditions tested, exposure did not reduce infectivity more than 1.0 log10. For the conditions studied, high peak power pulsed RF exposures in the 2-10 GHz range appear ineffective as a virucidal approach for hard surface decontamination. © 2023 Bioelectromagnetics Society.

Intestinal Shedding of SARS-CoV-2 in Children: No Evidence for Infectious Potential.

The clinical courses of COVID-19 in children are often mild and may remain undiagnosed, but prolonged intestinal virus shedding has been documented, thus potentially enabling fecal-oral transmission. However, the infectious potential of SARS-CoV-2 viruses excreted with feces has remained unclear. Here, we investigated 247 stool specimens from 213 pediatric patients to assess the prevalence of intestinal SARS-CoV-2 shedding in hospitalized children without or with COVID-19 and determined the infectious capacity of stool-borne viruses. Upon RT-qPCR screening, the infectivity of virus-positive samples was tested in cell culture using the Vero-E6 permissive cell line. SARS-CoV-2 RNA was detected by RT-qPCR in 32 (13%) stool specimens, but the analysis of virus-positive samples in cell culture revealed no cytopathic effects attributable to SARS-CoV-2-related cell damage. Our findings do not support the notion of potential fecal-oral SARS-CoV-2 spreading, thus questioning the role of hygienic measures designed to prevent this mode of viral transmission.

Differential Cell Line Susceptibility to the SARS-CoV-2 Omicron BA.1.1 Variant of Concern.

The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased viral load. Omicron exhibited a shift in tropism infecting the upper respiratory tract compared to other variants of concern which have tropism for the lower respiratory tract. The tropism of omicron variants in cell lines of different hosts and tissue origins still remains unclear. Considering this, we assessed the susceptibility of different cell lines to the SARS-CoV-2 omicron BA.1.1 variant and permissiveness among different cell lines for omicron replication. Susceptibility and permissiveness of a total of eleven cell lines, including six animal cell lines and five human cell lines for omicron BA.1.1 infection, were evaluated by infecting individual cell lines with omicron BA.1.1 isolate at a 0.1 multiplicity of infection. Virus replication was assessed by observation of cytopathic effects followed by viral load determination by real-time PCR assay and virus infectivity determination by TCID50 assay. The characteristic cytopathic effect, increased viral load, and productive omicron replication was detected in Vero CCL-81, Vero E6, Vero/hSLAM, MA-104, and Calu-3 cells. Although LLC MK-2 cells showed an increased TCID50 titer at the second infection, the viral load did not show much difference in both infections. Caco-2 cells did not show evident CPE, but they supported omicron replication at a low level. A549, RD, MRC-5, and BHK-21 cells supported omicron BA.1.1 replication without the CPE. This is the first study on the comparison of susceptibility of different cell lines to Omicron variant BA.1.1, which might be useful for future studies on emerging SARS-CoV-2 variants.

Viral cytopathic effect in maxillary sinus epithelium and SARS-CoV-2: Pitfalls in diagnostic characterization.

Viral cytopathic effects (VCE) are a well-known phenomenon associated with SARS-CoV-2 infection, especially in the cells associated with the lungs. Because maxillary sinus epithelium expresses angiotensin-converting enzyme 2, cells associated with it are more likely to become infected with SARS-CoV-2 and develop VCE. If VCE is seen in with background of a confirmatory COVID-19 diagnosis, then connecting both become quite convincing. However, the diagnostic problem is expected when a similar VCE is seen without any confirmatory diagnosis of CODIV-19. We reported a biopsy sample of maxillary sinusitis in a COVID-19 negative patient. Histopathological examination revealed a pathognomonic VCE in the localized proliferating pseudostratified ciliated epithelium. The only confirmatory aspect linking this VCE with the SARS-CoV-2 was the detection of virus particles at the tissue level. In the present paper, pitfalls and recommendations for future research on this topic are discussed.

Cytopathic Effect (CPE )-Based Drug Screening Assay for SARS-CoV-2.

Identification of an effective antiviral for the treatment of COVID-19 is considered one of the holy grails in the bid to end the pandemic. However, the novelty of SARS-CoV-2, along with the little knowledge available about its infection characteristics at the beginning of this pandemic, challenges the scientific world on how one may be able to promptly identify promising drug candidates from a myriad of compound libraries. Here, we describe a cytopathic effect (CPE)-based drug screening assay for SARS-CoV-2 which allows for rapid assessment of drug compound libraries through pre- or posttreatment drug screening procedures and evaluation using a light microscope. By comparing the virus-induced CPE of the drug-treated cells against the vehicle and drug controls, potent drug candidates can be quickly identified for further downstream studies.

Gastrointestinal complications (gangrene or perforation) after corona virus disease 2019 - A series of ten patients.

During the recent second wave of corona virus disease 2019 (COVID-19) pandemic in India, we managed a series of gastrointestinal complications in patients with COVID-19. We aim to highlight the key presentation and clinical course and emphasize the lessons we learnt from our series of such patients. A case review of ten consecutive patients with either bowel gangrene or perforation who were managed at our centre from March 20, 2021 to June 10, 2021. Clinical-demographic details, possible etiology, radiological findings, management and outcomes have been described. Of the 10 patients, 2 presented with bowel gangrene and 8 with perforation. In our series, all these patients were diagnosed with the help of computed tomography (CT) abdomen during the 3rd week after diagnosis of COVID-19. All had received steroid medication. Both patients with bowel gangrene and 4 of 8 patients with perforation underwent surgery, while 4 were managed non-operatively. Barring one patient, all the operated patients succumbed within 5 days of surgery after rapid clinical deterioration. Non-operative management in selected patients with perforation including placement of percutaneous drains, bowel rest and antibiotics was successful. Emergency surgery for COVID-19 related intestinal gangrene or perforation was associated with high mortality in our series. Non-operative management which avoids the added stress of a major emergency surgery particularly in patients just recovering from COVID-19 may be considered in stable patients in whom perforation appears to be contained.

Antiviral potential of garlic (Allium sativum) and its organosulfur compounds: A systematic update of pre-clinical and clinical data.

BACKGROUND: Garlic (Allium sativum L.) is a common herb consumed worldwide as functional food and traditional remedy for the prevention of infectious diseases since ancient time. Garlic and its active organosulfur compounds (OSCs) have been reported to alleviate a number of viral infections in pre-clinical and clinical investigations. However, so far no systematic review on its antiviral effects and the underlying molecular mechanisms exists. SCOPE AND APPROACH: The aim of this review is to systematically summarize pre-clinical and clinical investigations on antiviral effects of garlic and its OSCs as well as to further analyse recent findings on the mechanisms that underpin these antiviral actions. PubMed, Cochrane library, Google Scholar and Science Direct databases were searched and articles up to June 2020 were included in this review. KEY FINDINGS AND CONCLUSIONS: Pre-clinical data demonstrated that garlic and its OSCs have potential antiviral activity against different human, animal and plant pathogenic viruses through blocking viral entry into host cells, inhibiting viral RNA polymerase, reverse transcriptase, DNA synthesis and immediate-early gene 1(IEG1) transcription, as well as through downregulating the extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway. The alleviation of viral infection was also shown to link with immunomodulatory effects of garlic and its OSCs. Clinical studies further demonstrated a prophylactic effect of garlic in the prevention of widespread viral infections in humans through enhancing the immune response. This review highlights that garlic possesses significant antiviral activity and can be used prophylactically in the prevention of viral infections.

Virological and genomic analysis of SARS-CoV-2 from a favipiravir clinical trial cohort.

INTRODUCTION: Several clinical studies have reported the efficacy of favipiravir in reducing viral load and shortening the duration of symptoms. However, the viability of SARS-CoV-2 in the context of favipiravir therapy and the potential for resistance development is unclear. METHODS: We sequenced SARS-CoV-2 in nasopharyngeal specimens collected from patients who participated in a randomized clinical trial of favipiravir at hospitals across Japan between March and May 2020. Paired genomes were sequenced from those who remained RT-PCR-positive 5-8 days into favipiravir therapy. Daily nasopharyngeal specimens from 69 patients who were RT-PCR-positive at randomization were examined for a cytopathic effect (CPE). RESULTS: Some strains early in the trial belonged to clade 19 B, whereas the majority belonged to clade 20 B. The median time from the disease onset to negative CPE was 9 days. CPE was strongly correlated with the time from disease onset, viral load, age, and male sex. Among 23 patients for whom paired genomes were available, all except one had identical genomes. Two mutations were observed in one patient who received favipiravir, neither in the RdRp gene. CONCLUSIONS: The SARS-CoV-2 genome distribution in this clinical trial conducted in Japan reflected the early influx of strains from China followed by replacement by strains from Europe. CPE was significantly associated with age, male sex, and viral loads but not with favipiravir therapy. There was no evidence of resistance development during favipiravir therapy.

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.

COVID-19 impact on the liver.

The coronavirus disease 2019 (COVID-19) pandemic imposed arestructuring of global health systems by rethinking spaces used for the care of these patients and the additions of intensive care, infectious diseases and pneumology departments. This paper provides evidence on the presence of severe acute respiratory syndrome coronavirus 2 in hepatocytes and its direct cytopathic activity, as well as the degree of liver damage due to drug toxicity, inflammation and hypoxia in COVID-19. A review of clinical trials has quantified liver damage through both pathology and biochemistry studies. Additionally, we briefly present the results of a study conducted in our clinic on 849 patients admitted for COVID-19 treatment, of which 31 patients had pre-existing chronic liver disease and 388 patients had values above the normal limit for alanine aminotransferase, aspartate aminotransferase, and total bilirubin. It was observed that patients with abnormal liver tests were significantly statistically older, had more comorbidities and had a higher percentage of unfavourable evolution (death or transfer to intensive care). The conclusion of this paper is that the main causes of liver damage are direct viral aggression, coagulation dysfunction and endothelial damage, and patients with impaired liver function develop more severe forms of COVID-19 which requires special care by a multidisciplinary team that includes a hepatologist.

High-throughput screening assays for SARS-CoV-2 drug development: Current status and future directions.

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic, a panel of assays has been developed and applied to screen collections of approved and investigational drugs for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity in a quantitative high-throughput screening (qHTS) format. In this review, we applied data-driven approaches to evaluate the ability of each assay to identify potential anti-SARS-CoV-2 leads. Multitarget assays were found to show advantages in terms of accuracy and efficiency over single-target assays, whereas target-specific assays were more suitable for investigating compound mechanisms of action. Moreover, strict filtering with counter screens might be more detrimental than beneficial in identifying true positives. Thus, developing novel HTS assays acting simultaneously against multiple targets in the SARS-CoV-2 life cycle will benefit anti-COVID-19 drug discovery.

A Cytopathic Effect-Based Tissue Culture Method for HCoV-OC43 Titration Using TMPRSS2-Expressing VeroE6 Cells.

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2.

Drug repurposing is a rapid approach to identify therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drugs and 49 investigational drugs. The anti-SARS-CoV-2 activities of 230 of these confirmed compounds, of which 38 are approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA-approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set is a useful resource for drug repurposing efforts, including design of new drug combinations for clinical trials for SARS-CoV-2.

Susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2 infection.

Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes - needed for invading epithelial cells - were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl. Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

Insights into forsythia honeysuckle (Lianhuaqingwen) capsules: A Chinese herbal medicine repurposed for COVID-19 pandemic.

Background: In December 2019, a novel coronavirus, SARS-CoV-2 caused a series of acute atypical respiratory diseases worldwide. However, there is still a lack of drugs with clear curative effects, and the clinical trial research of vaccines has not been completely finished. Purpose: LH capsules are approved TCM patent medicine that are widely used for the treatment of respiratory tract infectious diseases caused by colds and flu. On April 12, 2020, LH capsules and granules were officially repurposed by the China Food and Drug Administration (CFDA) for patients with mild COVID-19 based on their safety and efficacy demonstrated through multicentre, randomized, controlled clinical trials. We hope to conduct a comprehensive review of it through modern pharmacy methods, and try to explain its possible mechanism. Methods: Using the full names of LH capsules Lianhuaqingwen, Lianhua Qingwen andSARS-COV-2, COVID-19 as the keywords of the search terms, systemically search for existing related papers in various databases such as Web of Science and PubMed. And completed the collection of clinical data in ClinicalTrials.gov and Chinese Clinical Trial Registry. Last but not least, we have sorted out the anti-inflammatory and antiviral mechanisms of LH capsules through literature and Selleck. Results: This review systematically sorted out the active ingredients in LH capsules. Furthermore, the related pharmacological and clinical trials of LH capsule on SARS-CoV-2, IAV and IBV were discussed in detail. Moreover, the present review provides the first summary of the potential molecular mechanism of specific substances in LH capsules involved in resistance to SARS-COV-2 infection and the inhibition of cytokine storm syndrome (CSS) caused by IL-6. Conclusion: This review summarizes the available reports and evidence that support the use of LH capsules as potential drug candidates for the prevention and treatment of COVID-19. However, TCM exerts its effects through multiple targets and multiple pathways, and LH capsules are not an exception. Therefore, the relevant mechanisms need to be further improved and experimentally verified.

Natural and Nature-Derived Products Targeting Human Coronaviruses.

The ongoing pandemic of severe acute respiratory syndrome (SARS), caused by the SARS-CoV-2 human coronavirus (HCoV), has brought the international scientific community before a state of emergency that needs to be addressed with intensive research for the discovery of pharmacological agents with antiviral activity. Potential antiviral natural products (NPs) have been discovered from plants of the global biodiversity, including extracts, compounds and categories of compounds with activity against several viruses of the respiratory tract such as HCoVs. However, the scarcity of natural products (NPs) and small-molecules (SMs) used as antiviral agents, especially for HCoVs, is notable. This is a review of 203 publications, which were selected using PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar, evaluates the available literature since the discovery of the first human coronavirus in the 1960s; it summarizes important aspects of structure, function, and therapeutic targeting of HCoVs as well as NPs (19 total plant extracts and 204 isolated or semi-synthesized pure compounds) with anti-HCoV activity targeting viral and non-viral proteins, while focusing on the advances on the discovery of NPs with anti-SARS-CoV-2 activity, and providing a critical perspective.

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules.

Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects.

Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus production and cytopathic effects. Further analysis reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.