Vero E6 Research Articles

In-vitro Cytotoxicity of Extracts of Selected Malaria Medicinal Plants Used by Traditional Healers of Kericho East Sub-county, Kenya

Background: Malaria is a fatal disease which affects people of all ages; especially pregnant women, young children <5 years, and the elderly because of their weakened immune systems. The currently used anti-malarial drugs have been linked to a variety of negative side effects including the parasite resistance. Additionally, the costs associated with the conventional malaria management approach are arguably high, particularly for people living in low-income countries, highlighting the need for alternative and complementary approaches. Medicinal plants therefore are a viable alternative since they are arguably less expensive and easily accessible. However, there is limited information on safety and efficacy of the plants. This study was designed to investigate the cytotoxic activities of polar and non-polar crude extracts solvents of selected plants used by traditional healers in Kericho East Sub-County to treat malaria. Materials and Methods: Plants studied included Pittosporum viridiflorum (stem barks), Phytolacca dodecandra (Leaves), and Gardenia ternifolia (roots barks). Plant parts selected were collected from Kericho East Sub-county; Kapsoit, Kaitui, and Fort-Ternan. Their crude extracts were obtained from hexane, dichloromethane (DCM), Methanol (MeOH), and 5% H2O/MeOH. In vitro cytotoxic effects and safety of the studied plants' extracts were identified using mammalian Vero E6 cells. Results: Most of the plants tested yielded impressive cytotoxicity results, indicating that therapeutic doses could be achieved at safe concentrations. However, P. viridiflorum hexane, DCM, MeOH, and 5% H2O/MeOH crude extracts were toxic to the cultured cells expressing the mean CC50 ± SE of 65.11±0.40, 25.63±0.23, 87.94 ±0.59 and 98.54±0.66 μg/ml, respectively. Conclusion: G. ternifolia and P. dodecandra have offered hope in the treatment of malaria since their crude extracts have demonstrated no toxicity. The study found P. viridiflorum crude extracts to be toxic but there is the possibility of isolating safe nontoxic compound/s because they were less toxic at lower doses. This study therefore identified potential plants that could be used to develop novel anti-plasmodial agents.

Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece.

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (μ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of μ-tp and KDEL to the ACE2 protein (ACE2 μK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 μK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 μK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.

Favipiravir and ivermectin show in vitro synergistic antiviral activity against SARS-CoV-2

Despite the urgent need for effective antivirals against SARS-CoV-2 to mitigate the catastrophic impact of the COVID-19 pandemic, favipiravir and ivermectin are among the common repurposed drugs that have been provisionally used in some countries. There have been clinical trials with mixed results, and therefore, it is still inconclusive whether they are effective or should be dismissed. It is plausible that the lack of clear-cut clinical benefits was due to the finding of only marginal levels of in vivo antiviral activity. An obvious way to improve the activity of antivirals is to use them in synergistic combinations. The in vitro antiviral activity of the combinations of favipiravir, ivermectin, niclosamide, and chloroquine against SARS-CoV-2 was assessed in Vero E6 cells and the lung epithelial cell, Calu-3. Here we show that favipiravir and ivermectin had synergistic effects against SARS-CoV-2 in Vero E6 cells. In addition, we found that favipiravir had an additive effect with niclosamide, another repurposed anti-parasitic drug with anti-SARS-CoV-2 activity. However, the anti-SARS-CoV-2 activity of favipiravir was drastically reduced when evaluated in Calu-3 cells. This suggested that this cell type might not be able to metabolize favipiravir into its active form and that this deficiency in some cell types may affect the in vivo efficacy of this drug. Favipiravir and ivermectin show the best synergistic effect. This combination is being tested in a randomized controlled clinical trial (NCT05155527).

The study of the course of the infectious process in lungs of cats during experimental infection with SARS-CoV-2

Aim. To study and characterise the features of the course of coronavirus infection in a domestic cat model, as well as to assess the danger of SARSCoV-2 isolated in Russia for these animals based on the data obtained, with the possibility of extrapolating to wild felines.Material and Methods. For the study, model animals – Felis silvestris catus – were divided into 3 groups. The viral strain of SARS-Cov-2/human/RUS/Nsk-FRCFTM-1/2020 was cultivated on Vero E6 cell culture and then administered intranasally in a volume of 200 μl, 105TCID50/ml, to animals from the first and second groups. Model animals from the first group were euthanized and lung fragments were taken on 6–day post infection (d.p.i.), animals from the second group – on 14 d.p.i. The third group – the control group, was injected with saline in an equivalent volume. Histological sections colored with hematoxylin and eosin were investigated for pathomorphological changes in lung tissue using light microscopy.Results. According to the obtained results the pathomorphological picture in the lungs of experimental animals indicates the course of interstitial pneumonia for both 6 and 14 d.p.i. In addition, it has been determined that by 14 d.p.i. the growth of fibrous tissue in the lungs begins to be visualised, indicating the beginning of light regenerative processes.Conclusion. The study showed that cats are susceptible to the SARS-CoV-2 virus, and these model animals are characterized by the emergence of clinical manifestations and morphological patterns in the lungs, which correspond to interstitial pneumonia.

SARS-CoV-2 in freshwaters used for irrigation in the agro-urban environment of Milan (Northen Italy)

Abstract Background During the COVID-19 pandemic, water has played a key role in epidemiological monitor of SARS-CoV-2. Our study aimed to verify if flood spillways activity during heavy rain events could be a source of SARS-CoV-2 contamination of a rural canal and consequently of human infection. Methods A total of 12 water samples (2L) were collected in four sampling campaigns conducted in 2022 (April, September, November, and December) during heavy rainfall. An automated sampler located in an irrigation stream collected 250mL of water at intervals of 10’ for 80’ since the spillways activation (sample A), then every 20’ for 160’ (sample B), and finally every 30’ for 240’ (sample C). Each sample was pre-filtered and concentrated by tangential flow ultrafiltration according to an in-house protocol. The presence of SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV, internal control) RNA was investigated by real time RT-PCR. SARS-CoV-2 viability was tested on VERO E6 cell culture, verifying the cytopathic effects and the viral load by real time RT-PCR every 24 hours for 7 days. Results PMMoV RNA was detected in all specimens assessing RNA quality. Although SARS-CoV-2 RNA was detected in all samples, except in those of November and in April sample C, there was no evidence of cytopathic effect or active replication in cell culture. RNA concentrations were generally low (0.27-6.95 gc/µL) but slightly higher in samples A than in B and C. Conclusions Despite the limited number of samples collected during the experimental campaign and due to the severe drought that affected Northen Italy, our results suggest that spillways activation could be a source of SARS-CoV-2 contamination in freshwaters, but there was no evidence of risk to human health. The detection of small concentrations may be associated with the circulation, during the monitoring, of the Omicron variants, which rarely cause gastrointestinal symptoms. Key messages • Spillways activation could be a source of SARS-CoV-2 contamination. • Both environmental diffusion and infectivity of SARS-CoV-2 were investigated to better understand the risk of disease for humans.

Viricide activity of antimicrobial blue light (405 nm) against the SARS-CoV-2 and influenza virus

Abstract Background Over the years, antimicrobial blue light (aBL) at a 405 nm wavelength has emerged as a potential alternative treatment for reducing the environmental contamination. It is well accepted that aBL is much less detrimental to host cells than UV-C irradiation and can be used in the presence of people. This study aims to determine the viricide activity, against the SARS-CoV-2 and influenza virus types A and B, of the nUV-A radiation, using a customized nUV-A ceiling lamp System. Methods The experiment took place in March 2023. An nUV-A ceiling lamp prototype, composed of 9 LEDs at 405 nm, was fixed on a metal scaffold, on which the outline of a multiwell plate (24 wells) was traced to carry out the tests correctly. Each plate well was inoculated with 100 µL of SARS-CoV2 or influenza virus types A or B suspension. The multiwell plate was irradiated at 32 cm for 45 and 90 minutes, with an emitted energy dose of 12 J/cm2 and 24 J/cm2. Three samples were inoculated with viruses and subjected to the action of nUV-A as per protocol, and 3 samples were inoculated but not treated with nUV-A to determine viral titer after recovery and examined immediately after inoculation. The re-collected suspensions were used to inoculate a wells plate into which the VERO E6 cell cultures were fixed and then incubated for 3 days at 37° C. Results The results showed that after a 45 min exposition, the measurable log10 mean reduction was 1.50 for SARS-CoV2, 0.13 for influenza virus type A and 0.12 for type B; after 90 min the log10 mean reduction was 2.33 for SARS-CoV2, 0.16 for influenza virus type A and 0.17 for type B. Conclusions Tests have shown that aBL can be effective in reducing the viral load of the SARS-CoV2 virus and, to a lesser extent, the influenza A/B viruses. Disinfection with aBL appears effective and avoid the adverse effects of using UV-C. Further studies will evaluate how to improve the results on influenza viruses and the application on other viruses such as RSV. Key messages • NearUV-A lamp has proven to reduce the virucide activity of all viruses, more for sars-cov2. • Using aBL avoids the adverse UV-C’s effects.

Antiproliferative activity of ethyl acetate fraction of Euphorbia ingens against prostate cancer cell line: An in silico and in vitro analysis

Current prostate cancer (PCa) treatments often lead to severe side effects, prompting the exploration of safer alternatives. Euphorbia ingens is a medicinal plant used for cancer treatment in African communities; however, there is no scientific validation of its anticancer activity. This study therefore evaluated the antiproliferative activity of E. ingens on the DU-145 human PCa cell line. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) bio-assay was used to determine the antiproliferative activity of E. ingens ethyl acetate-, and water-fractions. Only the ethyl acetate fraction was considered to be active. It inhibited DU-145 cell growth selectively without toxicity to non-cancerous Vero E6 cells. The IC50 was 9.71 ± 0.4 µg/ml and had a selectivity index of 8.26, indicating promising efficacy and cellular safety. Qualitative phytochemical screening identified the presence of phenols, terpenoids, flavonoids, tannins, sterols, and saponins in the ethyl acetate fraction. An additional 18 chemical compounds with potential synergistic roles were identified through gas chromatography-mass spectrometry analysis. Network pharmacology was then employed to predict molecular targets and mechanisms of action for drug-like chemical compounds, implicating key targets such as ESR1, IL6, MMP9, CDK2, MAP2K1, AR, PRKCD, CDK1, CDC25B, and JAK2 with regulation of PI3K/Akt, MAPK, and p53 signaling pathways suggested to be the potential mechanism of action. Further, gene expression analysis of selected targets through reverse transcription-quantitative polymerase chain reaction revealed downregulation of AR and BCL2 levels, along with upregulation of p53 and caspase-3 in fraction-treated DU-145 cells compared to the 0.2 % DMSO-negative control. In conclusion, the findings of this study validate the traditional use of E. ingens in cancer management. The identified drug-like compounds, their targets, and associated signaling pathways could serve as a foundation for developing novel strategies for prostate cancer management. However, we recommend additional in vitro and in vivo studies to further substantiate these findings.

Hantavirus: an overview and advancements in therapeutic approaches for infection.

Hantaviruses are a significant and emerging global public health threat, impacting more than 200,000 individuals worldwide each year. The single-stranded RNA viruses belong to the Hantaviridae family and are responsible for causing two acute febrile diseases in humans: Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Currently, there are no licensed treatments or vaccines available globally for HTNV infection. Various candidate drugs have shown efficacy in increasing survival rates during the early stages of HTNV infection. Some of these drugs include lactoferrin, ribavirin, ETAR, favipiravir and vandetanib. Immunotherapy utilizing neutralizing antibodies (NAbs) generated from Hantavirus convalescent patients show efficacy against HTNV. Monoclonal antibodies such as MIB22 and JL16 have demonstrated effectiveness in protecting against HTNV infection. The development of vaccines and antivirals, used independently and/or in combination, is critical for elucidating hantaviral infections and the impact on public health. RNA interference (RNAi) arised as an emerging antiviral therapy, is a highly specific degrades RNA, with post-transcriptional mechanism using eukaryotic cells platform. That has demonstrated efficacy against a wide range of viruses, both in vitro and in vivo. Recent antiviral methods involve using small interfering RNA (siRNA) and other, immune-based therapies to target specific gene segments (S, M, or L) of the Hantavirus. This therapeutic approach enhances viral RNA clearance through the RNA interference process in Vero E6 cells or human lung microvascular endothelial cells. However, the use of siRNAs faces challenges due to their low biological stability and limited in vivo targeting ability. Despite their successful inhibition of Hantavirus replication in host cells, their antiviral efficacy may be hindered. In the current review, we focus on advances in therapeutic strategies, as antiviral medications, immune-based therapies and vaccine candidates aimed at enhancing the body's ability to control the progression of Hantavirus infections, with the potential to reduce the risk of severe disease.

Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism.

SARS-CoV-2 subverts host cell processes to facilitate rapid replication and dissemination, and this leads to pathological inflammation. We used niclosamide (NIC), a poorly soluble anti-helminth drug identified initially for repurposed treatment of COVID-19, which activates the cells' autophagic and lipophagic processes as a chemical probe to determine if it can modulate the host cell's total lipid profile that would otherwise be either amplified or reduced during SARS-CoV-2 infection. Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of SARS-CoV-2 infected Vero E6 cells, especially with triglycerides, which were elevated early during virus replication, but decreased thereafter, as well as plasmalogens, which were elevated at later timepoints during virus replication, but were also elevated under normal cell growth. These findings suggested a complex interplay of lipid profile reorganization involving plasmalogen metabolism. We also observed that NIC treatment of both low and high viral loads does not affect virus entry. Instead, NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which we found elevated during virus infection in the absence of NIC, resulting in a significant reduction in the production of infectious virions. Unexpectedly, at higher viral loads, NIC treatment also resulted in elevated triglyceride levels, and induced significant changes in phospholipid metabolism. We posit that future screens of approved or new partner drugs should prioritize compounds that effectively counter SARS-CoV-2 subversion of lipid metabolism, thereby reducing virus replication, egress, and the subsequent regulation of key lipid mediators of pathological inflammation.

Porphyrin-derived carbon dots for an enhanced antiviral activity targeting the CTD of SARS-CoV-2 nucleocapsid

BackgroundSince effective antiviral drugs for COVID-19 are still limited in number, the exploration of compounds that have antiviral activity against SARS-CoV-2 is in high demand. Porphyrin is potentially developed as a COVID-19 antiviral drug. However, its low solubility in water restricts its clinical application. Reconstruction of porphyrin into carbon dots is expected to possess better solubility and bioavailability as well as lower biotoxicity. Methods and resultsIn this study, we investigated the antiviral activity of porphyrin and porphyrin-derived carbon dots against SARS-CoV-2. Through the in silico analysis and assessment using a novel drug screening platform, namely dimer-based screening system, we demonstrated the capability of the antivirus candidates in inhibiting the dimerization of the C-terminal domain of SARS-CoV-2 Nucleocapsid. It was shown that porphyrin-derived carbon dots possessed lower cytotoxicity on Vero E6 cells than porphyrin. Furthermore, we also assessed their antiviral activity on the SARS-CoV-2-infected Vero E6 cells. The transformation of porphyrin into carbon dots substantially augmented its performance in disrupting SARS-CoV-2 propagation in vitro. ConclusionsTherefore, this study comprehensively demonstrated the potential of porphyrin-derived carbon dots to be developed further as a promisingly safe and effective COVID-19 antiviral drug.

Improved Culture Methods for Human Coronaviruses HCoV-OC43, HCoV-229E, and HCoV-NL63.

HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are four of the seven known human coronaviruses (HCoVs) and, unlike the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, these four so-called seasonal HCoVs generally cause mild upper-respiratory-tract illness. As Biosafety Level 2 (BSL-2) pathogens, the seasonal HCoVs are more accessible and can be used as surrogates for studying the highly pathogenic HCoVs. However, scientists have for many years found these difficult to study because of the lack of a universal culture system and the inability of typical culture methods to yield high-titer infectious stocks. We have developed assays to grow and quantify infectious virus and viral RNA for HCoV-OC43, -229E, and -NL63. We identified which immortalized cell lines should be used to optimize the replication of HCoV-OC43, -229E, and -NL63 in order to generate high titers (Vero E6, Huh-7, and LLC-MK2 cells, respectively). Here we present protocols for improved propagation and quantification of each seasonal HCoV. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Growth of HCoVs Basic Protocol 2: Quantification of HCoV by plaque assay Basic Protocol 3: Quantification of HCoV RNA products of replication Basic Protocol 4: Concentrating HCoVs via ultracentrifugation.

In-cell Western assay to quantify infection with pathogenic orthohantavirus Puumala virus in replication kinetics and antiviral drug testing

Hemorrhagic fever with renal syndrome (HFRS) represents a serious zoonotic disease caused by orthohantaviruses in Eurasia. A specific antiviral therapy is not available. HFRS is characterized by acute kidney injury (AKI) with often massive proteinuria. Infection of kidney cells may contribute to the clinical picture. However, orthohantaviral replication in kidney cells is not well characterized. Therefore, we aimed to perform a reliable high-throughput assay that allows the quantification of infection rates and testing of antiviral compounds in different cell types. We quantified relative infection rates of Eurasian pathogenic Puumala virus (PUUV) by staining of nucleocapsid protein (N protein) in an in-cell Western (ICW) assay. Vero E6 cells, derived from the African green monkey and commonly used in viral cell culture studies, and the human podocyte cell line CIHP (conditionally immortalized human podocytes) were used to test the ICW assay for replication kinetics and antiviral drug testing. Quantification of infection by ICW revealed reliable results for both cell types, as shown by their correlation with immunofluorescence quantification results by counting infected cells. Evaluation of antiviral efficacy of ribavirin by ICW assay revealed differences in the toxicity (TC) and inhibitory concentrations (IC) between Vero E6 cells and podocytes. IC5O of ribavirin in podocytes is about 12-fold lower than in Vero E6 cells. In summary, ICW assay together with relevant human target cells represents an important tool for the study of hantaviral replication and drug testing.

In vitro and in vivo anti-inflammatory and antiophidic effects of the extract and fraction of Eugenia uniflora

Ethnopharmacological relevanceEugenia uniflora Linn, popularly known as ‘pitanga’, is a native plant endemic to Brazil that belongs to the Myrtaceae family. Its traditional use (leaves infusion) has been reported for the treatment of different diseases, including hypertension, inflammation, and as a diuretic agent. Considering the snakebite problem and the rich molecule repertoire of this herbal species, studies that evaluate its antiophidic potential are relevant for a broad social impact. Aim of the studyThis approach aims to evaluate the anti-inflammatory and antiophidic potential in vitro and in vivo of the extract (aqueous) and a fraction (ethyl acetate) of E. uniflora leaves against Bothrops leucurus and Bothrops brazili venoms. Materials and methodsExtract and fraction from E. uniflora leaves were obtained by turbo-extraction and partitioning. The cytotoxicity was assayed on normal cell lines (Vero E6 and 3T3) using the 3-methyl-[4-5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The anti-inflammatory activity of the aqueous extract was analyzed in vivo in the zymosan-induced air pouch model, and the leukocytes migration and other molecular inflammatory mediators quantified (myeloperoxidase, total protein, pro-inflammatory cytokine, malondialdehyde, and glutathione). In vitro, the antiophidic effect was evaluated by the ability of the E. uniflora extract and fraction to inhibit the enzymatic action (proteolytic, phospholipase A2, and hyaluronidase) of B. leucurus and B. brazili venoms. In addition, the antiophidic action in vivo was investigated after treatment with E. uniflora extract and fraction (50, 100, and 200 mg/kg) in the B. leucurus venom-induced paw edema with an evaluation of the antiedematogenic effect and quantification of myeloperoxidase (MPO) and pro-inflammatory cytokine levels. ResultsThe E. uniflora leaves extract (7.8–125 mg/mL) revealed no toxicity in cell culture, but reduced MTT by 47% at the highest concentration (250 mg/mL) in Vero E6 cells. In contrast, the E. uniflora fraction (7.8–250 mg/mL) showed no cytotoxicity for both cell lines. In the air pouch model, E. uniflora leaves extract demonstrated anti-inflammatory activity, reducing cell migration, MPO activity, protein, malondialdehyde, and proinflammatory cytokines, and increased glutathione levels. Evaluating the antiophidic action in vitro, E. uniflora extract and fraction inhibited the proteolytic, phospholipase, and hyaluronidase effects of B. leucurus and B. brazili venoms at low concentrations. In addition, the extract and fraction also demonstrated in vivo antiophidic activity by reducing edema in the first 0.5 h after treatment, besides reducing MPO and pro-inflammatory cytokines levels. ConclusionE. uniflora leaves extract showed cytotoxicity only at the highest concentration while the fraction revealed no toxic effect in vitro. This approach showed for the first time that the aqueous extract and ethyl acetate fraction of E. uniflora leaves has similar antiophidic action in vitro and in vivo, with antiedematogenic and anti-inflammatory effects and the ability to inhibit the enzymatic action of B. leucurus and B. brazili venoms. Therefore, this study points to the presence of bioactive components in the leaves of E. uniflora useful for the treatment of inflammatory disorders and ophidian accidents, expanding the therapeutic potential of this herbal species.

A novel N95 respirator with chitosan nanoparticles: mechanical, antiviral, microbiological and cytotoxicity evaluations

BackgroundIt is known that some sectors of hospitals have high bacteria and virus loads that can remain as aerosols in the air and represent a significant health threat for patients and mainly professionals that work in the place daily. Therefore, the need for a respirator able to improve the filtration barrier of N95 masks and even inactivating airborne virus and bacteria becomes apparent. Such a fact motivated the creation of a new N95 respirator which employs chitosan nanoparticles on its intermediate layer (SN95 + CNP).ResultsThe average chitosan nanoparticle size obtained was 165.20 ± 35.00 nm, with a polydispersity index of 0.36 ± 0.03 and a zeta potential of 47.50 ± 1.70 mV. Mechanical tests demonstrate that the SN95 + CNP respirator is more resistant and meets the safety requisites of aerosol penetration, resistance to breath and flammability, presenting higher potential to filtrate microbial and viral particles when compared to conventional SN95 respirators. Furthermore, biological in vitro tests on bacteria, fungi and mammalian cell lines (HaCat, Vero E6 and CCL-81) corroborate the hypothesis that our SN95 + CNP respirator presents strong antimicrobial activity and is safe for human use. There was a reduction of 96.83% of the alphacoronavirus virus and 99% of H1N1 virus and MHV-3 betacoronavirus after 120 min of contact compared to the conventional respirator (SN95), demonstrating that SN95 + CNP have a relevant potential as personal protection equipment.ConclusionsDue to chitosan nanotechnology, our novel N95 respirator presents improved mechanical, antimicrobial and antiviral characteristics.

A new alkaloid from Pancratium maritimum - Structure elucidation using computer-assisted structure elucidation (CASE) and evaluation of cytotoxicity and anti-SARS-CoV-2 activity

Pancratium maritimum L. (or ‘sea daffodil’) is one of the most studied plant species in the Amaryllidaceae family. Recently, due to the advancement of new probabilistic methods and spectroscopic techniques, the potential for phytochemical investigations on isomeric compounds has significantly improved. In this study, application of these techniques in combination with conventional phytochemical analysis led to the isolation and identification of a new alkaloid named 2β,10bα-dihydroxy-9-O-demethylhomolycorine from P. maritimum, together with 17 known ones. Assessment of anti-SARS-CoV-2 activity and cytotoxicity on a Vero E6 cell line for all compounds revealed four compounds with weak anti-SARS-CoV-2 potency at non-cytotoxic concentrations, namely of 9-O-demethylhomolycorine (EC50 = 44 µM), galanthamine (EC50 = 70 µM), 1-O-acetyl-norpluviine (EC50 = 80 µM) and 1-O-acetyl-10-O-methylpseudolycorine (EC50 = 47 µM). On the other hand, cytotoxicity was observed for 2β,10bα-dihydroxy-9-O-demethylhomolycorine (CC50 = 13.28 µM), haemanthamine (CC50 = 0.76 µM), 6α- and 6β-haemanthidine (CC50 = 5.43 µM), 11-hydroxyvittatine (CC50 = 10.57 µM) and pseudolycorine (CC50 = 3.41 µM).

Antiviral Effect of 5'-Arylchalcogeno-3-aminothymidine Derivatives in SARS-CoV-2 Infection.

The understanding that zidovudine (ZDV or azidothymidine, AZT) inhibits the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and that chalcogen atoms can increase the bioactivity and reduce the toxicity of AZT has directed our search for the discovery of novel potential anti-coronavirus compounds. Here, the antiviral activity of selenium and tellurium containing AZT derivatives in human type II pneumocytes cell model (Calu-3) and monkey kidney cells (Vero E6) infected with SARS-CoV-2, and their toxic effects on these cells, was evaluated. Cell viability analysis revealed that organoselenium (R3a-R3e) showed lower cytotoxicity than organotellurium (R3f, R3n-R3q), with CC50 ≥ 100 µM. The R3b and R3e were particularly noteworthy for inhibiting viral replication in both cell models and showed better selectivity index. In Vero E6, the EC50 values for R3b and R3e were 2.97 ± 0.62 µM and 1.99 ± 0.42 µM, respectively, while in Calu-3, concentrations of 3.82 ± 1.42 µM and 1.92 ± 0.43 µM (24 h treatment) and 1.33 ± 0.35 µM and 2.31 ± 0.54 µM (48 h) were observed, respectively. The molecular docking calculations were carried out to main protease (Mpro), papain-like protease (PLpro), and RdRp following non-competitive, competitive, and allosteric inhibitory approaches. The in silico results suggested that the organoselenium is a potential non-competitive inhibitor of RdRp, interacting in the allosteric cavity located in the palm region. Overall, the cell-based results indicated that the chalcogen-zidovudine derivatives were more potent than AZT in inhibiting SARS-CoV-2 replication and that the compounds R3b and R3e play an important inhibitory role, expanding the knowledge about the promising therapeutic capacity of organoselenium against COVID-19.

Suramin inhibits SARS-CoV-2 nucleocapsid phosphoprotein genome packaging function

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines – a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals.

The BioCascade-VIVAS system for collection and delivery of virus-laden size-fractionated airborne particles

The size of virus-laden particles determines whether aerosol or droplet transmission is dominant in the airborne transmission of pathogens. Determining dominant transmission pathways is critical to implementing effective exposure risk mitigation strategies. The aerobiology discipline greatly needs an air sampling system that can collect virus-laden airborne particles, separate them by particle diameter, and deliver them directly onto host cells without inactivating virus or killing cells. We report the use of a testing system that combines a BioAerosol Nebulizing Generator (BANG) to aerosolize Human coronavirus (HCoV)-OC43 (OC43) and an integrated air sampling system comprised of a BioCascade impactor (BC) and Viable Virus Aerosol Sampler (VIVAS), together referred to as BC-VIVAS, to deliver the aerosolized virus directly onto Vero E6 cells. Particles were collected into four stages according to their aerodynamic diameter (Stage 1: >9.43 μm, Stage 2: 3.81–9.43 μm, Stage 3: 1.41–3.81 μm and Stage 4: <1.41 μm). OC43 was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses of samples from all BC-VIVAS stages. The calculated OC43 genome equivalent counts per cm3 of air ranged from 0.34 ± 0.09 to 70.28 ± 12.56, with the highest concentrations in stage 3 (1.41–3.81 μm) and stage 4 (<1.41 μm). Virus-induced cytopathic effects appeared only in cells exposed to particles collected in stages 3 and 4, demonstrating the presence of viable OC43 in particles <3.81 μm. This study demonstrates the dual utility of the BC-VIVAS as particle size-fractionating air sampler and a direct exposure system for aerosolized viruses. Such utility may help minimize conventional post-collection sample processing time required to assess the viability of airborne viruses and increase the understanding about transmission pathways for airborne pathogens.

Use of recombinant S1 protein with hFc for analysis of SARS-CoV-2 adsorption and evaluation of drugs that inhibit entry into VERO E6 cells

The significant number of deaths and infection caused by the new coronavirus SARS-CoV-2 has created an urgent demand for effective and readily available drugs for the treatment of COVID-19. However, the requirements for biosafety level 3 (NB-3) laboratories for experiments with the virus has made it very challenging for such research to meet this demand. It is known that angiotensin-converting enzyme 2 (ACE2), located on the surface of host cells, serves as the viral receptor for the spike (S) protein of SARS-CoV-2. This protein is a tetramer subdivided into S1 and S2 regions, with the former containing the receptor-binding domain (RBD). Therefore, drugs that interfere with the interaction between the spike and the receptor (as well as accessory proteins) or suppress their expression could inhibit the entry and spread of SARS-CoV-2 between cells. In this context, we standardized the use of recombinant SARS-CoV-2 S1 Protein with hFc (human Fc) for the analysis of binding in VERO E6 cells by flow cytometry, aiming to provide a new tool for identifying drugs and neutralizing antibodies, thus eliminating the need for NB-3 laboratories. Because minocycline (MCL), nimesulide (NMS), and berberine (BBR) have effects related to the ACE2 receptor, inhibit inflammation, and do not suppress the adaptive immune response (crucial for patient recovery), we investigated whether these drugs prevent the absorption of the spike protein into the host cell. For this purpose, we used VERO E6 cells under control conditions, pre-treated with these drugs and exposed to recombinant SARS-CoV-2 S1 Protein with hFC. We found that an exposure time of 30 min and a concentration of 10 μg/mL of spike S1 caused a strong signal detected by flow cytometry, using the secondary anti-hFc antibody conjugated with Alexa Fluor 647. Pre-treatment of cells with BBR for 30 min suppressed the signal from spike-positive cells, suggesting that this alkaloid interferes with spike adsorption on ACE2. The pre-incubation of spike protein with BBR did not alter its adsorption and internalization, indicating that BBR does not directly interact with spike protein. The ACE2 inactivation with a specific antibody inhibited spike protein adsorption and internalization. Furthermore, the pharmacological treatments did not alter the expression of ACE2. Exposure to spike protein increased IFNγ levels and the treatments with MCL and NMS were effective in inhibiting this increase. Taken together, we standardized a technique for analyzing the adsorption of SARS-CoV-2 and studying molecules that inhibit this process. Additionally, we demonstrated that BBR blocks spike entry bypre-binding to the host cell,and that the ACE2 receptor inactivation prevents Spike protein adsorption and penetration into cells.

Discovery of new non-covalent and covalent inhibitors targeting SARS-CoV-2 papain-like protease and main protease

Global coronavirus disease 2019 (COVID-19) pandemic still threatens human health and public safety, and the development of effective antiviral agent is urgently needed. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are vital proteins in viral replication and promising therapeutic targets. Additionally, PLpro also modulates host immune response by cleaving ubiquitin and interferon-stimulated gene product 15 (ISG15) from ISGylated host proteins. In this report, we identified [1,2]selenazolo[5,4-c]pyridin-3(2H)-one and benzo[d]isothiazol-3(2H)-one as attractive scaffolds of PLpro and Mpro inhibitors. The representative compounds 6c and 7e exhibited excellent PLpro inhibition with percent inhibition of 42.9% and 44.9% at 50 nM, respectively. The preliminary enzyme kinetics experiment and fluorescent labelling experiment results determined that 6c was identified as a covalent PLpro inhibitor, while 7e was a non-covalent inhibitor. Molecular docking and dynamics simulations revealed that 6c and 7e bound to Zn-finger domain of PLpro. Compounds 6c and 7e were also identified to potent Mpro inhibitors, and they exhibited potent antiviral activities in SARS-CoV-2 infected Vero E6 cells, with EC50 value of 3.9 μM and 7.4 μM, respectively. In addition, the rat liver homogenate half-life of 6c and 7e exceeded 24 h. These findings suggest that 6c and 7e are promising led compounds for further development of PLpro/Mpro dual-target antiviral drugs.