Search Results (190)

Background/Objectives: Marine-derived bioactive compounds have attracted increasing interest due to their potential antiviral properties. This study investigated in vitro antiviral activity of oil extracted from the green-lipped mussel (Perna canaliculus, GLM) and low-molecular-weight (LMW) fucoidan from Undaria pinnatifida against three human viruses in mammalian cell systems. herpes simplex virus-1 (HSV-1), respiratory syncytial virus (RSV), and SARS-CoV-2. These marine compounds were selected with the longer-term aim of evaluating their combination as a potential synergistic antiviral strategy. Methods: Antiviral efficacy was assessed using complementary assay platforms, including plaque reduction assays in mammalian cell systems and a lentiviral pseudovirus system delivering a bioluminescent reporter gene in HEK293/ACE2 cells pseudotyped with the SARS-CoV-2 spike glycoprotein. Cytotoxicity was assessed in parallel, and the selectivity index (SI) was calculated as the ratio of CC₅₀ to IC₅₀ for each compound and virus tested. Results: GLM oil showed potential antiviral activity against SARS-CoV-2 pseudovirus (SI > 6.20), with limited activity against RSV (SI > 3.48) and HSV-1 (SI > 2.28). In contrast, LMW fucoidan did not demonstrate antiviral activity against any of the tested viruses. Conclusions: These findings support further investigation of GLM-derived bioactive compounds as potential antiviral agents, including studies to elucidate their mechanisms of action and in vivo studies to confirm their antiviral efficacy. Combination studies were not pursued in the present work as both compounds require further optimisation individually; however, future studies should evaluate their combined antiviral potential, as synergistic or additive effects remain plausible. Full article

Salivary gland infection by SARS-CoV-2 requires viral entry via routes and mechanisms that remain unresolved. This study examined the expression of the angiotensin-converting enzyme 2 (ACE2) receptor in salivary tissues and basal cell-derived human salivary progenitor cells (hS/PCs), an unstudied potential entry point for SARS-CoV-2. Multiple detection modalities, including immunocytochemistry, Western blotting, flow cytometry and RT-PCR, demonstrated a consistent lack of ACE2 protein and transcript in both tissue specimens and primary salivary epithelial cells. Antigen retrieval at pH 9 was determined to be optimal for immunodetection protocols, yet ACE2 remained undetectable. Small intestine tissue served as a positive control, confirming the validity of the methods and reagents we used. Considering there can be other receptors for SARS-CoV-2, flow cytometric analyses demonstrated that recombinant SARS-CoV-2 spike protein failed to bind to salivary epithelial cells, in contrast to HEK293 cells engineered to overexpress ACE2, which showed robust spike binding. Additional studies showed that patient-derived salivary cells, negative for ACE2, are not infected by the SARS-CoV-2 pseudovirus, while ACE2-positive cells are readily infected. These findings strongly support our conclusion that salivary cells do not serve as major targets for SARS-CoV-2 infection via ACE2, spike protein, or an alternate receptor. Thus, salivary cells are unlikely major targets for SARS-CoV-2 infection, either through direct exposure to viral particles in ductal fluids or via access to basal cells across the basement membrane. Full article

The pseudovirus neutralization (PNT) assay is an established high-throughput, robust, and efficient BSL-2 method for detecting neutralizing antibodies (NAbs) against SARS-CoV-2 with the correlates of protection previously established for the ancestral (Wuhan) strain. The PNT assay was validated using nonmatched ancestral sera with anti-JN.1 cross-NAbs, clinically matched JN.1 sera with anti-JN.1 NAbs, or nonmatched JN.1 sera with anti-LP.8.1, KP.2, or KP.3 cross-reacting NAbs. In line with predefined validation acceptance criteria, the PNT assay was precise, with %GCV ≤ 50 in ~90–100%/200 results (40 samples/strain). The acceptance criteria were met for linearity (slope ranged from 1.041 for ancestral sera with anti-JN.1 NAbs to 1.213 for JN.1 sera with anti-KP.2 NAbs), R² (0.9619–0.9944 for ancestral sera with anti–JN.1 NAbs), % relative bias, and total %GCV < 50 for almost all of the 15 serum samples tested for four virus strains. Human sera collected pre–COVID-19 had no detectable titer for tested Omicron JN.1 subvariants (<LLOQ) and all influenza and RSV clinical samples tested negative (<LLOQ) for SARS-CoV-2 and highly immunogenic for seasonal influenza or RSV post-vaccination, demonstrating the PNT assay specificity. Our data suggest this assay is suitable for assessing immune responses to ancestral and current SARS-CoV-2 strains and has potential for evaluating cross-reacting NAbs against emerging Omicron JN.1 subvariants. Full article

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

The COVID-19 pandemic highlighted the critical role of serological assays in understanding antiviral immune responses, monitoring vaccine efficacy, and informing public health strategies. This review provides a comprehensive overview of commonly used SARS-CoV-2 antibody detection methods, focusing on binding and neutralization assays. Antibody binding assays, including enzyme-linked immunosorbent assays (ELISAs), chemiluminescence immunoassays (CLIAs), lateral flow immunoassays (LFAs), and multiplex platforms, enable the rapid and high-throughput detection of immunoglobulin isotypes against various viral antigens. Neutralization assays, including live-virus, pseudovirus (PsV), and surrogate assays, offer functional insights into the ability of antibodies to prevent viral entry, though they often require higher biosafety levels and optimization. Serological assays, primarily antibody binding assays and several surrogate neutralization assays, received Emergency Use Authorization (EUA) during the pandemic, supporting seroprevalence efforts. Antibody binding assays and neutralization assays were also widely used in vaccine immunogenicity studies. Despite many standardization initiatives, assay standardization and data harmonization remain challenging and require further efforts. The choice of assay should be guided by study goals: antibody binding assays are preferred for high-throughput monitoring and epidemiological studies, while neutralization assays are essential for assessing functional immunity and variant-specific neutralization and protection. Full article

Background: The emergence of SARS-CoV-2 variants necessitates the development of effective adjuvants to enhance subunit vaccine immunogenicity. Safe adjuvants are essential to enhance the immunogenicity of SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines. Traditional Chinese medicine polysaccharides are attractive candidates due to their immunomodulatory properties. Methods: Female BALB/c mice (6–8 weeks) were immunized on days 0, 7, and 21 with an RBD protein (20 μg) alone or formulated with Poria cocos polysaccharide fraction PCP-I or PCP-II (200 μg), Isatis indigotica polysaccharide, or aluminum adjuvant; PBS served as a control. RBD-specific total IgG and subclasses were quantified by ELISA on day 7 after the third immunization. Neutralizing antibody titers were measured by a pseudovirus assay on days 14, 28, and 56 after the first immunization. Splenic CD19⁺ B cells were analyzed by flow cytometry, and antigen-stimulated IFN-γ and IL-4 spot-forming cells were quantified by ELISpot. Results: PCP-II significantly increased RBD-specific total IgG and IgG1 compared with RBD alone and other formulations, whereas IgG2a and IgG2b remained unchanged. Both PCP-I and PCP-II increased neutralizing titers versus RBD alone, and PCP-II showed an earlier and sustained increase in neutralizing responses through day 56. PCP-II showed a non-significant increase in splenic CD19⁺ B cell frequency. PCP-I and PCP-II markedly increased IFN-γ-secreting splenocytes without increasing IL-4, indicating enhanced antigen-specific cellular responses. Conclusion: In this comparative evaluation of traditional Chinese medicine polysaccharide candidates in a SARS-CoV-2 RBD subunit vaccine model, PCP-II showed the most prominent adjuvant activity. PCP-II enhanced antigen-specific humoral immunogenicity, improved neutralizing antibody responses, and was associated with increased IFN-γ-related cellular responses, supporting its potential as a candidate polysaccharide adjuvant for protein subunit vaccines. Full article

The development of novel antiviral nanomaterials is an important approach for addressing emerging viral threats. In this study, glycyrrhizic acid-modified gold nanoparticles (GA-Au NPs) were successfully synthesized and characterized, and their inhibitory effects against porcine reproductive and respiratory syndrome virus (PRRSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus were systematically evaluated. At non-cytotoxic concentrations, GA-Au NPs showed inhibitory activity against PRRSV in vitro. Stage-specific assays suggested that intracellular replication-related events were prominently affected, with additional inhibitory effects observed during adsorption, invasion, and release, whereas no direct virucidal activity was detected under the tested conditions. Furthermore, GA-Au NPs dose-dependently reduced SARS-CoV-2 pseudovirus infection-associated reporter signals in HEK-293T-ACE2 cells, supporting inhibitory activity in an additional viral model. In conclusion, GA-Au NPs represent a biocompatible antiviral nanomaterial with multi-stage inhibitory activity against PRRSV and inhibitory effects in a SARS-CoV-2 pseudovirus model, supporting their further evaluation as antiviral nanomaterials in enveloped virus-related models. Full article

Cytokine storm is a central pathogenic mechanism underlying sepsis-induced acute lung injury (SALI) and severe coronavirus disease 2019 (COVID-19), yet effective therapeutic strategies remain limited. Eupatorium lindleyanum DC. (EL), a traditional Chinese medicinal herb, has been reported to possess anti-inflammatory, antioxidant, and antiviral-related activities; however, its protective mechanisms in SALI and virus-associated inflammatory lung injury remain incompletely understood. In this study, an integrated strategy combining computational prediction and experimental validation was employed to investigate the therapeutic potential and underlying mechanisms of EL. The chemical constituents of EL were characterized by UPLC–Q–TOF/MS, followed by network pharmacology, molecular docking, and molecular dynamics analyses to predict key targets and signaling pathways. A cecal ligation and puncture (CLP)-induced SALI rat model was used to evaluate lung histopathology, pulmonary edema, cytokine production, and inflammatory signaling activation. In parallel, LPS-stimulated RAW264.7 macrophages were used to assess cytokine secretion and pathway regulation in vitro. In addition, a SARS-CoV-2 pseudovirus-induced mouse model was employed to further evaluate the in vivo relevance of the representative bioactive compound hyperoside in pseudovirus-associated lung injury. A total of 32 active compounds and 697 putative targets were identified, among which 116 were associated with sepsis and COVID-19. In vivo, EL markedly alleviated lung injury, reduced the lung coefficient and wet/dry ratio, and suppressed excessive production of proinflammatory cytokines and activation of key signaling proteins. In vitro, EL dose-dependently inhibited TNF-α and IL-6 secretion and regulated the PI3K–Akt and NF-κB signaling pathways. Notably, hyperoside showed favorable predicted interactions with PI3K–Akt pathway-related targets (EGFR, PI3K, and Akt), while molecular dynamics simulations supported stable interactions with several COVID-19-related targets, including ACE2, Mpro, and RdRp. Furthermore, hyperoside significantly alleviated SARS-CoV-2 pseudovirus-associated lung injury, reduced ACE2 protein expression, and downregulated EGFR, PI3K, and Akt mRNA levels in vivo. Collectively, these findings indicate that EL exerts protective effects through multi-component, multi-target, and multi-pathway mechanisms, and support its potential value for further investigation in SALI and virus-associated inflammatory lung injury. Full article

In the post-pandemic era, monitoring adaptive immunity of the population to emerging SARS-CoV-2 variants remains an important public health priority. To address this need, we developed a test that can simultaneously assess the neutralization ability of three SARS-CoV-2 variants. A panel of lentiviral pseudoviruses, each bearing the S-protein of different SARS-CoV-2 variants (Wuhan-Hu-1, BA.1, and XBB.1.5) and expressing a unique fluorescent protein (Clover, mRhubarb713, or mRuby3) was generated and used to transduce hACE2-overexpressing cells. The percentage of infected target cells for each variant was quantified via flow cytometry. Co-infection led to a minor reduction in the percentage of infected cells compared to mono-infection controls, confirming the robustness of the assay. We then applied the test to the analysis of human sera samples, which were collected in the Sirius Federal Territory (Russian Federation) and revealed the following: (1) sera collected in 2021 neutralized the Wuhan-Hu-1 variant and demonstrated cross-specificity to the BA.1 variant, but not to the XBB.1.5 variant; (2) sera collected after the Omicron emergence point neutralized Wuhan-Hu-1 and BA.1, and possessed a weak ability to neutralize the XBB.1.5. This assay provides a valuable tool for efficient profiling of humoral immunity and monitoring its development in response to ongoing viral diversity. Full article

Graphene-based nanomaterials exhibit exceptional physicochemical properties that facilitate a range of diverse biomedical applications, including liquid biopsy. In this study, graphene-based magnetic units, termed MAGU (MAGnetic Units), were specifically engineered for the selective isolation of exosomes. Total extracellular vesicles were first enriched using ultracentrifugation, followed by immunomagnetic capture of CD9⁺ exosomes. MAGU functionalized with anti-CD9 antibody (MAGU-anti-CD9) efficiently recovered a CD9-positive exosome subpopulation expressing canonical markers ALIX, CD147, TSG101, and Flotillin-1, thereby confirming selective isolation performance. To investigate viral associated signaling, 293T cells were transduced with SARS-CoV-2 spike pseudovirus. This pseudovirus was engineered to express the SARS-CoV-2 spike protein, enabling simulation of viral entry and assessment of potential alterations in the exosomal profile induced by viral binding. Exosomes released by pseudovirus-transduced 293T cells were analyzed and compared to those from non-transduced controls. The MAGU-anti-CD9 complex selectively isolated a defined subset of CD9-positive vesicles enriched in the multifunctional transmembrane glycoprotein CD147, which has been proposed as a cofactor in SARS-CoV-2 entry. Comprehensive molecular profiling of selectively captured exosome subpopulations is expected to further support the application of MAGU technology in virus–host interaction research and liquid-biopsy-based diagnostics. Full article

Requirements for novel effective antiviral agents against SARS-CoV-2 emphasizes the importance of robust in vitro screening platforms. We developed a test system based on spike-pseudotyped lentiviruses, carrying either luc+ or EGFP reporter genes as a payload, and a human non-small cell lung carcinoma (NSCLC) cell line, overexpressing ACE2 (H1299-hACE2). The cell origin makes our system resemble lung epithelium infection. Transmission electron microscopy confirmed that the spike glycoproteins on the pseudotyped lentiviral particles resemble native SARS-CoV-2 spike glycoproteins, thus validating their use in inhibitor screening. H1299-hACE2 cells showed significantly higher infection rate (p < 0.005) with spike-pseudotyped lentiviruses compared to parental H1299 cells, as determined by luciferase and fluorescence assays. The susceptibility of the stable H1299-hACE2 cell line to a broad panel of SARS-CoV-2 variants (Wuhan, Beta, Delta, Omicron) was assessed here for the first time in a unified experimental setting. Infection of H1299-hACE2 cells with SARS-CoV-2 induced cell fusion and syncytium formation with subsequent cell death. The developed pseudovirus-based assay was further used for assessment of the antiviral properties of derivatives of 1,7,7-trimethyl-[2.2.1]-bicycloheptane-potential spike protein inhibitors, which possess moderate activity against lentiviral particles. The H1299-hACE2/spike-pseudotyped lentivirus assay is, therefore, a reliable, high-efficiency platform for screening spike-mediated entry inhibitors. The cell line obtained during the development of the platform can be used to isolate and study new variants of SARS-CoV-2. Full article

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose major global health challenges despite extensive vaccination efforts. Variant escape, waning immunity, and reduced vaccine efficacy in immunocompromised populations underscore the urgent need for complementary antiviral therapeutics. Here, we report the design, synthesis, and biological evaluation of precision-engineered dermatan sulfate (DS)-mimetic glycopolymers as multi-targeted inhibitors of SARS-CoV-2. Guided by molecular docking and virtual screening, sulfation at the C2 and C4 positions of iduronic acid was identified as critical for binding to the viral spike protein and inhibiting host and viral enzymes, including heparanase (HPSE) and main protease (M^pro). Chemically synthesized DS disaccharides were covalently grafted onto polymer scaffolds via a post-modification strategy, yielding glycopolymers with well-defined assembly that form uniform nanoparticles under physiological conditions. Surface plasmon resonance and pseudovirus assays revealed strong binding to the viral spike protein (K_D ≈ 177 nM), potent viral neutralization, and minimal cytotoxicity. Cellular uptake studies further demonstrated efficient internalization of nanoparticles and intracellular inhibition of HPSE and M^pro. These results establish a modular, non-anticoagulant, and glycosaminoglycan-mimetic platform for the development of broad-spectrum antiviral agents to complement vaccination and enhance preparedness against emerging coronavirus variants. Full article

Background/Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can naturally infect a broad spectrum of animal species, with cats, minks, and ferrets being highly susceptible. There is a potential risk that infected animals could transmit viruses to humans. Moreover, SARS-CoV-2 continues to evolve via mutation and genetic recombination, resulting in the continuous emergence of new variants that have triggered a wave of reinfection. Therefore, safe and effective corona virus disease 2019 (COVID-19) vaccines for animals are still being sought. Methods: We generated three recombinant Modified vaccinia virus Ankara (MVAs) expressing the prefusion-stabilized S proteins, S6P, DS6P, and BA2S6P, targeting the full-length S protein genes of the ancestral, Delta, and Omicron BA.2 strains of SARS-CoV-2. Subsequently, the safety, immunogenicity, and protective efficacy of these MVA-based oral COVID-19 vaccine candidates were assessed in mice, minks, and cats. Results: These recombinant MVAs are safe in mice, minks, and cats. Oral or intramuscular vaccination with rMVA-S6P induced a robust SARS-CoV-2 neutralizing antibody (NA) response and conferred complete protection against the SARS-CoV-2 challenge in mice. Meanwhile, oral or intramuscular administration of these recombinant MVAs in combination induced a potent and durable NA response against homotypic SARS-CoV-2 pseudovirus in mice, minks, and cats, respectively. Conclusions: These findings suggest that the MVA-vectored vaccines are promising oral COVID-19 vaccine candidates for animals, and that the combined vaccination approach is an effective administration strategy for such vaccines. Full article

Objectives: The emergence of SARS-CoV-2 variants with enhanced immune evasion capabilities poses ongoing challenges for maintaining population-level immunity. This study aim to evaluate neutralizing antibody responses to the wild-type (WT) strain and the Omicron sublineage XBB.1.9 in the Israeli population using serum samples collected between August 2022 and January 2023, prior to widespread circulation of XBB.1.9. Methods: Pseudovirus-based microneutralization assays incorporating variant-specific spike proteins were employed to measure neutralizing geometric mean titers (GMTs) across subgroups categorized by age, gender, socioeconomic status, and geographic region. Results: Neutralizing titers against XBB.1.9 were significantly lower than those against WT across all demographic groups, with a 29-fold reduction in neutralization activity against XBB.1.9, underscoring the immune escape potential of XBB.1.9. For WT, older adults (≥65 years) exhibited higher titers than younger individuals (p < 0.01), whereas no significant age-related differences were observed for XBB.1.9 (p > 0.05). Regional disparities in WT immunity were identified, with higher titers in Northern Israel compared to Jerusalem and Southern regions. By contrast, XBB.1.9 neutralization showed no significant regional variation. Conclusions: These findings demonstrate substantially reduced neutralization of XBB.1.9 compared to WT and reveal disparities in WT immunity by age and region. The results emphasize the need for updated vaccines targeting immune-evasive variants and for tailored vaccination strategies to address regional and demographic gaps in protection. Full article

Neutralizing antibodies (nAbs) are key indicators of protection against SARS-CoV-2, and their measurement remains essential for monitoring vaccine responses and population immunity. While the plaque reduction neutralization test (PRNT) is the gold standard, it relies on replicative viruses and is not suited for high-throughput applications. Here, both an in-house and a commercial pseudovirus-based neutralization (PBN) assay were standardized and compared with PRNT to assess performance and concordance. The in-house PBN employed a VSV-ΔG pseudovirus encoding NanoLuc and displaying the SARS-CoV-2 Spike from the Wuhan or Omicron BA.1 variants in HEK293T-hACE2 cells, whereas the commercial assay (Integral Molecular, Philadelphia, PA, USA) used a lentiviral backbone with Renilla or GFP reporters and Wuhan or Omicron XBB.1.5/XBB.1.9 Spikes in Vero E6-ACE2-TMPRSS2 cells. Both assays showed strong correlations with PRNT, the commercial assay; moreover, they offered superior reproducibility and scalability, while the in-house version provided a cost-effective alternative suitable for BSL-2 settings. A total of 600 serum samples from vaccinated individuals were analyzed by commercial PBN at collection time points, from pre-vaccination to twelve months post–second dose, enabling large-scale screening, revealing marked differences in neutralization between Wuhan and Omicron XBB.1.5/1.9, and allowing unbiased classification of low, medium, and high responders using k-means clustering. The geometric mean titers (log₁₀ GMT) highlighted a ~1.5 log₁₀ (eightfold) reduction in neutralizing activity against Omicron, reflecting antibody waning and antigenic drift. Altogether, this study integrates assay standardization, PRNT comparison, and large-scale immune profiling, establishing a robust framework for harmonized pseudovirus-based neutralization testing. Full article