Search Results (106)

Background: A major challenge in antiviral development is the identification of novel virus–host interactions while ensuring therapeutic efficacy and safety. These challenges have renewed interest in phytochemicals derived from medicinal plants as alternative antiviral agents. Objectives: In this study, we investigated the antioxidant, antiviral, and immunomodulatory properties of a Mediterranean Urtica dioica extract (UdE) against SARS-CoV-2 using chemical, biochemical, and in vitro approaches. Methods: The physicochemical properties of UdE were characterized using microtiter assays and HPLC analysis. Cytocompatibility was evaluated in HEK293T, Vero E6, Caco-2, and Calu-3 cell lines while antioxidant activity was assessed using both chemical and cell-based assays. Antiviral activity was evaluated by assessing inhibition of SARS-CoV-2 receptor binding domain (RBD)–ACE2 interaction using ELISA, inhibition of SARS-CoV-2 main protease (Mpro) activity via FRET assay and inhibition of viral entry using SARS-CoV-2 S1 pseudovirus neutralization assay. Results: UdE (100 µg/mL) inhibited RBD–ACE2 binding by 94% and suppressed Mpro activity by 74%, while reducing moderate but significant inhibition of pseudovirus entry (33.6%) at 300 µg/mL dose level in ACE2 expressing HEK293T cells. Immunomodulatory analysis revealed significant suppression of IL-1β and IL-6 production, accompanied by increased TNF-α and IL-8 levels. Conclusions: Collectively, these findings highlight that UdE exhibits multi-target in vitro antioxidant, antiviral, and immunomodulatory activity against SARS-CoV-2; therefore, UdE represents a promising bioactive extract for the management of SARS-CoV-2 infection. Full article

Background/Objectives: The global rise in multidrug-resistant (MDR) bacteria, particularly methicillin-resistant and methicillin-susceptible Staphylococcus aureus (MRSA and MSSA), continues to pose a major public health challenge, including in Hawaii. This underscores the need to discover new antimicrobial agents from natural sources. Guided by teachings from a Buddhist master regarding the medicinal value of lichens, we investigated the endemic Hawaiian lichen Cladonia skottsbergii. Methods: Specimens of C. skottsbergii were collected from the Lotus Buddhist Monastery in Mountain View, Hawaii. A methanolic extract was prepared and purified using chromatographic techniques, and compound structures were elucidated through spectroscopic analyses and single-crystal X-ray diffraction. The antibacterial activity of the compounds was assessed against Gram-positive strains (MRSA, MSSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). Cytotoxicity was assessed using A549 (non-small cell lung cancer) and Vero E6 (non-tumorigenic) cell lines. Results: Three compounds were isolated: clarosione (1), a newly identified trichlorinated xanthone, and two known metabolites, (S)-usnic acid (2) and perlatolic acid (3). Compounds 2 and 3 demonstrated strong inhibitory effects against MRSA and MSSA. Their minimum inhibitory concentrations (MICs) ranged from 2 to 4 µg/mL, compared with vancomycin (0.5–1 µg/mL). Cytotoxicity testing showed higher sensitivity in A549 cells than in Vero E6 cells, resulting in favorable selectivity indices for the active compounds. Conclusions: In the current study, a new compound, clarosione (1) was discovered. This enhances our understanding of the constituents of C. skottsbergii and its potential antibacterial properties. Lichen-derived compounds may serve as lead candidates for further development, and further study is warranted. Full article

Recombinant baculovirus vectors are recognized as effective gene delivery systems for mammalian cells in vitro. However, their application in vivo has been limited due to inactivation by the host’s complement system. We developed a recombinant baculoviral vector derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), incorporating both CMV-IE and polyhedron promoter-driven green fluorescence protein (EGFP) (vAcMBac-CMV-IE-EGFP). We then evaluated the transduction efficiency and safety of vAcMBac-CMV-IE-EGFP at a high multiplicity of infection (MOI) across five distinct cell lines and in Sprague Dawley (SD) rats. In vitro, Sf9, HepG2, and Vero E6 cells showed high transduction rates (95.52 ± 4.86%, 80.53 ± 3.31%, and 80.87 ± 2.50%, respectively), significantly outperforming the other cell types tested, and cell viability remained largely unaffected even at an MOI of 1000. In vivo, EGFP expression was observed in the heart, liver, spleen, lungs, and kidneys of SD rats after tail vein injection. Direct injection of vAcMBac-CMV-IE-EGFP into the rat striatum also resulted in strong EGFP signals in neural tissues. These results demonstrate that a high-MOI baculovirus infection can serve as a remarkably efficient and versatile platform for gene delivery across diverse mammalian cell types as well as in various organs and neural tissues in animal models. This robust method might hold significant promise for future gene therapy applications. Full article

Objectives: Combining two pharmacophores into one molecule with multiple applications presents interest in the field of medicinal chemistry. Quinazolinones are among privileged scaffolds due to their wide biological activities, whereas hydrazones are versatile linkers with pharmacological potential. Thus, this article focused on a green method for the synthesis of new N-acyl-hydrazones of 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide and the exploration of their biological potential. Methods: The novel N-acyl-hydrazones (1a–1f) were synthesized under microwave irradiation, using various substituted salicylaldehydes and benzaldehydes. The products were characterized by FT-IR, ¹H-NMR, ¹³C-NMR, and HRMS. Their pharmacological profile was assessed by in silico methods and docking simulations. Biological evaluation included antioxidant, antimicrobial, and cytotoxic activities, as well as preliminary toxicity on Artemia franciscana. Results: Spectroscopic data indicated syn-E and anti-E isomers. Compound 1c showed the highest antioxidant activity. Antimicrobial assays indicated narrow-spectrum activity, with compounds 1a and 1b being most effective against C. albicans and S. aureus. Biofilm inhibition assays revealed that 1a and 1c interfered with microbial adhesion, highlighting their potential in combating biofilm-associated infections. Cytotoxicity tests on HT-29 and A431 cancer cell lines showed selective anticancer effects for compounds 1a–1d, with minimal toxicity on normal Vero cells, especially for 1b and 1d. Toxicity against Artemia franciscana correlated with in vitro cytotoxicity data, revealing low lethality for all N-acyl-hydrazones. Docking studies indicate that the antibacterial activity may involve inhibition of S. aureus DNA gyrase B, whereas the cytotoxic effects could be mediated by interaction with the EGFR kinase. Conclusions: These findings may increase the chances of identifying a lead compound in this class, supporting the further development of selected N-acyl-hydrazones and their pharmacological exploration. Full article

Serum-free suspension culture technology for animal cells involves the division and proliferation of cells in serum-free medium as single cells or cell clusters within shaking flasks or bioreactors. This approach enables large-scale cell culture, enhances the yield and quality of biopharmaceuticals, reduces costs, and broadens the applications of animal cells. Serum-free suspension culture of adherent cells (e.g., Madin–Darby canine kidney (MDCK), Chinese hamster ovary (CHO), Vero, baby hamster kidney (BHK-21), and human embryonic kidney (HEK293) cells) has been successfully achieved through direct or indirect adaptation, medium optimization, and genetic engineering. Additionally, novel suspension cell lines, such as duck embryonic stem (EB66) and human retinoblastoma (PER.C6) cells, have been developed as potential new substrates for biopharmaceutical production. This review examines animal cell suspension culture technology and its applications in viral vaccines, recombinant proteins, and monoclonal antibodies, providing insights into the development and utilization of this important technology. Full article

The COVID-19 pandemic has reignited interest in traditional medicinal plants as potential therapeutic agents. This study examined the chemical composition, cytotoxicity, and antimicrobial activity of essential oils from six Moroccan medicinal plants, namely, Eucalyptus globulus, Artemisia absinthium, Syzygium aromaticum, Thymus vulgaris, Artemisia alba, and Santolina chamaecyparissus, which are commonly used by the Moroccan population for COVID-19 prevention. The chemical composition of each essential oil was determined using gas chromatography–mass spectrometry (GC–MS) to identify key compounds. Cytotoxicity was evaluated in the Vero E6 cell line, which is frequently used in SARS-CoV-2 research, using the neutral red assay, with oil concentrations ranging from 25 to 100 µg/mL. Antimicrobial activity was tested against standard reference strains, including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 10231), and Bacillus subtilis (ATCC 6633), using the disc diffusion method. GC–MS analysis revealed significant components such as spathulenol (15%) and caryophyllene oxide (7.67%) in Eucalyptus globulus and eugenol (54.96%) in Syzygium aromaticum. Cytotoxicity assays indicated that higher concentrations of essential oils significantly reduced cell viability, with Thymus vulgaris showing the highest IC₅₀ (8.324 µM) and Artemisia absinthium the lowest (18.49 µM). In terms of antimicrobial activity, Eucalyptus globulus had the strongest effect, with a 20 ± 0.00 mm inhibition zone against Bacillus subtilis, whereas both Syzygium aromaticum and Artemisia herba-alba had a 12.25 ± 0.1 mm inhibition zone against the same strain. These findings suggest that these essential oils have significant therapeutic potential, particularly in combating antimicrobial resistance and exerting cytotoxic effects on viral cell lines. Further research is necessary to explore their mechanisms of action and ensure their safety for therapeutic use. Full article

Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging tick-borne infectious disease that poses a significant public health threat. SFTS virus (SFTSV) has a broad host range, including humans, cats, and natural reservoir species. Therefore, cultured cell lines derived from different mammalian species are useful for understanding the susceptibility of SFTSV in hosts. In this study, we evaluated pathogenicity and infectivity, focusing on cytopathic effect (CPE) induction and growth kinetics of SFTSV in several mammalian cell lines, including our original tiger-derived TLT, wild deer–derived DFKT and DFLT, and hedgehog-derived HHoVT. Following SFTSV infection, TLT, CRFK (cat), FCWF-4 (cat), and CPK (porcine) cells exhibited CPE, whereas Vero E6 (monkey), A549 (human), BHK-21 (hamster), DFKT, DFLT, and HHoVT cells did not. Infectious viral yields in the supernatants of TLT, CRFK, FCWF-4, Vero E6, and BHK-21 were higher than those of CPK, A549, DFLT, and DFKT. SFTSV infection in hedgehog-derived HHoVT cells was very limited. These observations suggest that features such as viral CPE and virus yield following SFTSV infection depend on cell type. It is noteworthy that TLT formed clear plaques that were easy to count, indicating that TLT cells are useful for the titration of infectious SFTSV by plaque-forming assay. Our results provide useful information and tools for further elucidating the mechanisms of SFTSV infectivity, proliferation, and pathogenicity using in vitro models. Full article

Fungi are a prolific source of diverse bioactive metabolites, yet many remain unexplored. Among these, depsidones are a rare class of compounds with significant biological potential, but they are seldom reported in mushrooms. This study investigated the medicinal fungus Ganoderma lucidum, known for its extensive therapeutic use in traditional medicine. Fruiting bodies were extracted using petroleum ether, ethyl acetate, n-butanol, and methanol. Extracts were screened phytochemically and assessed for total phenolic content and antioxidant activity using the DPPH assay. Ethyl acetate extract exhibited the highest phenolic yield and antioxidant potential and was subsequently evaluated for cytotoxicity against HepG2, HCT116, MCF7, and A549 cancer cell lines. It showed notable anticancer activity with minimal toxicity to normal Vero cells. UHPLC/Q-TOF-MS/MS analysis of G. lucidum ethyl acetate extract tentatively identified nine minor depsidones including mollicellin G, simplicildone I, mollicellin B, talaromyone B, simplicildone A, purpactin C, emeguisin B, mollicellin E, and simplicildone D on the basis of high-resolution negative-mode detection and characteristic MS/MS fragmentation patterns. Molecular docking revealed strong binding affinities between these compounds and cancer-related targets (AKT1, CDK2, ERK1, TNFα), with simplicildone D and mollicellin G demonstrating particularly high interactions. These findings provide mechanistic insights into the observed bioactivity and highlight G. lucidum as a promising source of therapeutic depsidones for future anticancer drug development. Full article

Bovine viral diarrhea virus (BVDV) is a globally significant pathogen affecting both domestic livestock and wildlife, including white-tailed deer (WTD; Odocoileus virginianus). While experimental infections have demonstrated WTD susceptibility to BVDV, natural infections and associated reproductive outcomes remain scarcely documented. Here, we report the first confirmed case of naturally occurring BVDV-1 infection associated with fetal mummification in farmed WTD in Florida. A two-year-old doe experienced a stillbirth involving two mummified fetuses, which were submitted for necropsy and laboratory diagnostics. Gross findings included diarrhea and underdeveloped eyes in the fetuses, along with small white nodules indicative of amnion nodosum. While not harmful, this condition suggests underlying fetal compromise or intrauterine stress. Virus isolation using Vero E6 and bovine turbinate cell lines, along with a reverse transcription PCR (RT-PCR) assay specifically developed in this study, confirmed the presence of BVDV-1 (Pestivirus bovis) RNA in both maternal and fetal samples, suggesting vertical transmission. Sanger sequencing of RT-PCR amplicons further verified the virus species as BVDV-1. Differential diagnostics for other pathogens, including bluetongue virus, epizootic hemorrhagic disease virus, Mycobacterium spp., and Toxoplasma gondii, were negative. These findings underscore the importance of using biosecurity measures and including BVDV in the differential diagnosis of abortions to reduce the risk of BVDV transmission and potential outbreaks on deer farms, particularly those close to cattle operations. The molecular tools developed in this study provide a robust framework for improved detection and monitoring of BVDV in both wildlife and livestock populations. Full article

Background: Modern viral vector production needs to consider process intensification for higher yields from smaller production volumes. However, innate antiviral immunity triggered in the producer cell may limit virus replication. While commonly used cell lines (e.g., Vero or E1A-immortalised cells) are already compromised in antiviral pathways, the redundancy of innate signaling complicates host cell optimization by genetic engineering. Small molecules that are hypothesized to target antiviral pathways (Viral Sensitizers, VSEs) added to the culture media offer a versatile alternative to genetic modifications to increase permissiveness and, thus, viral yields across multiple cell lines. Methods: To explore how the yield for a chimeric Zika vaccine candidate (YF-ZIK) could be further be increased in an intensified bioprocess, we used spin tubes or an Ambr15 high-throughput microbioreactor system as scale-down models to optimize the dosing for eight VSEs in three host cell lines (AGE1.CR.pIX, BHK-21, and HEK293-F) based on their tolerability. Results: Addition of VSEs to an already optimized infection process significantly increased infectious titers by up to sevenfold for all three cell lines tested. The development of multi-component VSE formulations using a design of experiments approach allowed further synergistic titer increases in AGE1.CR.pIX cells. Scale-up to 1 L stirred-tank bioreactors and 3D-printed mimics of 200 or 2000 L reactors resulted in up to threefold and eightfold increases, respectively. Conclusions: Addition of single VSEs or combinations thereof allowed a further increase in YF-ZIK titers beyond the yield of an already optimized, highly intensified process. The described approach validates the use of VSEs and can be instructive for optimizing other virus production processes. Full article

Artemisia serotina Bunge represents one of the endemic Artemisia L. species in flora of Central Asia. There is scant information on the phytochemistry and biological activity of this species. The aim of the present study was to analyze the chemical composition of essential oil from A. serotina (ASEO) growing in south Kazakhstan, together with the determination of its biological activity. ASEO isolation was carried out by hydrodistillation according to the State Pharmacopoeia of the Republic of Kazakhstan. Analysis of GC/MS data revealed that the most characteristic components of ASEO were irregular monoterpenes from three families: santolinane, artemisane, and lavandulane. The major compound was santolina alcohol (34.6%). Antimicrobial activity was studied against the reference bacterial and fungal strains using the recommended methods, allowing for an estimation of MIC (minimum inhibitory concentration). ASEO was most effective against Candida albicans (MIC = 2 mg/mL), exerting fungicidal activity. Thw MIC for bacterial species was higher, i.e., 4–16 mg/mL. Antiviral activity was tested against Coxsackievirus B3 (CVB3) and Human Herpesvirus type 1 (HHV-1) propagated in VERO cells. No antiviral effect against either virus was found at an ASEO concentration of 0.25 mg/mL, but a noticeable decrease in the intensity of HHV-1-related cytopathic effects was observed. Anticancer activity studies included several cancer cell lines. Cytotoxicity, cell cycle, thiol levels, and cell vitality were analyzed. Among the cancer cell lines tested, the breast cancer T47-D cell line exhibited the highest sensitivity to ASEO (IC₅₀ = 40.81 ± 4.21 µg/mL at 24 h; IC₅₀ = 33.17 ± 2.11 µg/mL at 48 h). The anticancer effect was suggested to be mainly due to the induction of cytostatic effects, accompanied by a disturbance of the intracellular redox balance. The obtained data provide novel information on the unique chemical composition of ASEO from south Kazakhstan, representing a new chemotype. Its bioactivity, including promising antifungal and anticancer properties, was demonstrated for the first time. Full article

SPR has been recently established as a powerful tool for studying various cellular processes in real time and without the use of labeling agents. So far, all studies in this area have been performed using the Kretschmann method for SPR excitation. In our studies, we used grating-based SPR. Here, we investigated the feasibility of this approach in a cell-based assay applied for antiviral drug screening. It was found that the continuous-flow SPR detection used in the conventional SPR can be replaced by sequential signal measurements of SPR slides removed from the medium at fixed hours after seeding. A protocol ensuring correct measurements was established. SPR detection was performed up to 48 h after seeding the VERO E6 cell line in three experiments, in which the cells were (i) compound-untreated, (ii) compound-treated, and (iii) infected with human coronavirus type 229E and compound-treated. Therefore, the temporal variation in the SPR signal was determined, induced by the cell coverage on the slide, the compound toxicity, and its antiviral action. MTT analysis and microscopic observations were used as reference methods. The remarkable agreement found in the results of SPR detection proved the effectiveness and reliability of grating-based SPR applied in cell-based assays. Full article

Background: Respiratory droplets are the main way in which the COVID-19 pandemic’s causal agent, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), spreads. Angiotensin-converting enzyme 2 (ACE2) receptors, especially in lung cells, allow the virus to enter host cells. However, ACE2 expression in intestinal cells has sparked worries about possible fecal transfer, particularly in poor-sanitation areas like India. Methods: Between July 2021 and July 2024, clinical (nasopharyngeal, saliva, and stool samples) and sewage samples were collected from outpatient departments and sewage treatment plants (STPs), respectively, from the high-population-density area under study in order to investigate SARS-CoV-2 transmission. Results: This proof-of-concept study analyzed clinical samples from n = 60 COVID-19-positive patients at a central Indian tertiary care hospital and n = 156 samples from hospital STPs. Variants of SARS-CoV-2 were found using qRT-PCR and Next-Generation Sequencing (NGS). Of the n = 37 qRT-PCR-positive patients who gave their assent, 30% had stool samples that tested positive for viral RNA. In 70% of positive NP and 65% of positive saliva samples, along with two stool samples from immunocompromised patients, the live virus was identified using Vero E6 cell lines. Although 18% of the tests reported qRT-PCR-positive results, no live virus was detected in sewage samples despite NGS validation. The detection of SARS-CoV-2 in the absence of confirmed clinical cases may indicate the silent circulation of the virus within the community, suggesting that sewage surveillance can serve as an early warning system before an outbreak occurs. Conclusions: These findings provide critical insights into the importance of continuous environmental surveillance, silent virus circulation, changes in viral epidemiology throughout the years, and strategies to mitigate coronavirus outbreaks. Full article

Despite extensive efforts, no highly effective antiviral molecule exists for treating moderate and severe COVID-19. Nanotechnology has emerged as a promising approach for developing novel drug delivery systems to enhance antiviral efficacy. Among these, polymeric nanomicelles improve the solubility, bioavailability, and cellular uptake of therapeutic agents. In this study, Pluronic F127-based nanomicelles were developed and evaluated for their antiviral activity against SARS-CoV-2. The nanomicelles, formulated using the direct dissolution method, exhibited an average size of 37.4 ± 8.01 nm and a polydispersity index (PDI) of 0.427 ± 0.01. Their antiviral efficacy was assessed in SARS-CoV-2-infected Vero E6 and Calu-3 cell models, where treatment with a 1:2 dilution inhibited viral replication by more than 90%. Cytotoxicity assays confirmed the nanomicelles were non-toxic to both cell lines after 72 h. In SARS-CoV-2-infected Calu-3 cells (human type II pneumocyte model), treatment with Pluronic F127-based nanomicelles containing atazanavir (ATV) significantly reduced viral replication, even under high MOI (2) and after 48 h, while also preventing IL-6 upregulation. To investigate their mechanism, viral pretreatment with nanomicelles showed no inhibitory effect. However, pre-exposure of Calu-3 cells led to significant viral replication reduction (>85% and >75% for 1:2 and 1:4 dilutions, respectively), as confirmed by transmission electron microscopy. These findings highlight Pluronic F127-based nanomicelles as a promising nanotechnology-driven strategy against SARS-CoV-2, reinforcing their potential for future antiviral therapies. Full article

Current antiviral treatments often target specific viral components, which can lead to the rapid emergence of drug-resistant mutants. Targeting host signaling pathways, including their associated cellular factors, that are important for virus replication is a novel approach toward the development of next-generation antivirals to overcome drug resistance. Various cellular receptor tyrosine kinases (RTKs) have previously been shown to play important roles in mediating viral replication including coronaviruses. In this study, we examined the roles of RTKs in SARS-CoV-2 replication in two cell lines, A549-ACE2 (human lung epithelial cells) and Vero-E6 (African Green Monkey kidney cell), via chemical inhibitors. We showed that the HER2 inhibitor Lapatinib significantly reduced viral replication in both cell lines, the TrkA inhibitor GW441756 was effective only in A549-ACE2 cells, while the EGFR inhibitor Gefitinib had little effect in either cell line. Lapatinib and GW441756 exhibited a high therapeutic index (CC₅₀/EC₅₀ > 10) in A549-ACE2 cells. Time-of-addition experiments indicated that Lapatinib may inhibit the early entry step, whereas GW441756 can affect post-entry steps of the viral life cycle. These findings suggest the important roles of HER2 and TrkA signaling in SARS-CoV-2 infection in human lung epithelial cells and support further investigation of RTK inhibitors as potential COVID-19 treatments. Full article