Search Results (291)

Background/Objectives: Viral respiratory infections have a significant impact on global health and the economy. While vaccines are effective in preventing infection, they might not be available or sufficient when used alone and must be complemented by specific therapeutic strategies. The development of new antiviral agents is increasingly important due to the continual emergence of novel respiratory pathogens. Previously we identified bovine lactoferrin (bLf)-derived tetrapeptides and peptidomimetics that showed potent in vitro activity against the influenza A virus in the picomolar range. Methods: Inspired by these results, in this study, we evaluated the antiviral potential of these compounds against HCoV-229E, a human coronavirus that can cause severe disease in immunocompromised individuals, using a compound repositioning approach. Results: Functional studies revealed that SK(N-Me)HS (3) interferes with viral entry and replication, while compound SNKHS (5) primarily blocks infection in the early stages. Biophysical analyses confirmed the occurrence of high-affinity binding to the viral spike protein, and computational studies suggested that the compounds target a region involved in conformational changes necessary for membrane fusion. Conclusions: These findings highlight these compounds as promising candidates for coronavirus entry inhibition and underscore the value of compound repurposing in antiviral development. Full article

The Akabane virus (AKAV) is a significant member of the Orthobunyavirus genus, with its envelope glycoprotein Gc, focusing on its molecular structural features, immunoregulatory mechanisms, and application value in pathogen diagnosis and vaccine design. As a key structural protein of AKAV, Gc mediates virus adsorption and neutralizing antibody recognition through the N-terminal highly variable region (HVR), while the C-terminal conserved region (CR) dominates the membrane fusion process, and its glycosylation modification has a significant regulatory effect on protein function. In clinical diagnostics, serological assays based on Gc proteins (e.g., ELISA, immunochromatographic test strips) have been standardized; in vaccine development, the neutralizing epitope of Gc proteins has become a core target for subunit vaccine design. Follow-up studies were deeply needed to analyze the structure-function interaction mechanism of Gc proteins to provide theoretical support for the construction of a new type of AKAV prevention and control system. Full article

Ovine herpesvirus 2 (OvHV-2) causes the fatal veterinary disease malignant catarrhal fever (MCF). Fusion is an essential step in the host cell entry of enveloped viruses and is an important target for vaccine development. OvHV-2 cannot be propagated in vitro, so a robust virus-free cell–cell membrane fusion assay is necessary to elucidate its entry mechanism. OvHV-2 cell–cell fusion requires three conserved herpesviral envelope glycoproteins: gB, gH, and gL. OvHV-2 fusion activity is detectable but low. We hypothesize that enhancing the cell surface expression of gB, which is the core herpesviral fusogen, will increase cell–cell fusion. We generated C-terminal truncation mutants of gB and determined their cell surface expression, subcellular distribution, and fusion activity. Two mutants, including one that lacked the entire cytoplasmic tail domain, failed to function in the cell–cell fusion assay, despite wild-type levels of surface expression. This suggests that the OvHV-2 gB cytoplasmic tail is critical for fusion. A gB mutant truncated at amino acid 847 showed increased surface expression and fusion relative to the wild type. This suggests that the robust fusion activity of gB847 is the result of increased surface expression. gB847 may be used in place of wild-type gB in an improved, more robust OvHV-2 fusion assay. Full article

The Chikungunya virus (CHIKV) continues to pose a significant global health challenge due to the absence of effective antiviral treatments and limited vaccine availability. This study employed a comprehensive in silico workflow, incorporating high-throughput virtual screening, binding free-energy calculations, ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis, and 200 ns molecular dynamics (MD) simulations, to identify new inhibitors targeting the E1–E2 glycoprotein complex, crucial for CHIKV entry and membrane fusion. Four promising candidates were identified from a library of 20,000 compounds, with CID 136801451 showing the most potent binding (docking score: −10.227; ΔG_bind: −51.53 kcal/mol). The top four compounds exhibited favorable ADMET profiles, meeting nearly all criteria. MD simulations confirmed stable binding and strong interactions between CID 136801451 and the E1–E2 complex, evidenced by consistently low RMSD values. These findings highlight CID 136801451 as a promising CHIKV entry inhibitor, warranting further in vitro and in vivo evaluation to advance the development of effective anti-CHIKV therapeutics. Full article

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development. Full article

Peptides designed to interfere with specific steps of viral infection mechanisms have shown promising antiviral potential. In this study, we investigated the ability of a synthetic peptide (peptide 303), derived from the fusion protein sequence of the Infectious Salmon Anemia Virus (ISAV), to inhibit membrane fusion mediated by the ISAV fusion peptide (ISAV-FP1). To assess this, we employed a model membrane system consisting of large unilamellar vesicles (LUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and cholesterol. Membrane fusion kinetics were monitored via R18 fluorescence dequenching. Additionally, the interaction of peptide 303 with lipid membranes was evaluated using fluorescence anisotropy measurements. The potential direct interaction between peptide 303 and ISAV-FP1 was further examined through Förster Resonance Energy Transfer (FRET) assays. Our results demonstrate that peptide 303 effectively inhibits ISAV-FP1-mediated membrane fusion. Furthermore, peptide 303 was shown to interact with lipid bilayers and with ISAV-FP1 itself. These findings suggest a dual inhibitory mechanism in which peptide 303 both prevents ISAV-FP1 binding to the membrane and directly interacts with the fusion peptide, thereby disrupting its fusogenic activity. Full article

Background: The transmembrane fusion (F) protein of RSV plays important roles in RSV pathogenesis as it mediates the fusion between the virus and the target cell membrane. During the fusion process, the F protein transits from a metastable state (prefusion, preF) to a stable state (postfusion, postF) after the merging of the virus and cell membranes. The majority of highly neutralizing antibodies induced by natural infection or immunization target the preF form, which makes it the preferred antigen for vaccine development. Methods: Here, we designed an effective RSV mRNA vaccine, STR-V003, consisting of mRNA encoding preF protein in lipid nanoparticles (LNPs). The immunogenicity, protection efficacy and toxicity were measured in multiple animal models. Results: STR-V003 demonstrated robust immunogenicity in both mice and cotton rats, inducing high levels of neutralizing antibodies and RSV preF-specific IgG antibodies and significantly reducing the RSV viral loads in the lung and nose tissue of challenged animals. In addition, STR-V003 did not show significant enhancement of lung pathology without causing vaccine-enhanced disease (VED). The repeated dose general toxicology studies and local tolerance studies of STR-V003 were evaluated in rats and non-human primate (NHP). Conclusions: STR-V003 demonstrates a favorable safety profile and induces robust protective immunity against RSV. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19) pandemic that devastated the world. While this is a respiratory virus, one feature of the SARS-CoV-2 infection was recognized to cause pathogenesis of other organs. Because the membrane fusion protein of SARS-CoV-2, the spike protein, binds to its major host cell receptor angiotensin-converting enzyme 2 (ACE2), which regulates a critical mediator of cardiovascular diseases, angiotensin II, COVID-19 is largely associated with vascular pathologies. The present study examined the pulmonary vasculature of COVID-19 patients using large sample sizes and provides mechanistic information through histological observations. We studied 56 postmortal lung samples from COVID-19 patients. The comparative group consisted of 17 postmortal lung samples from patients who died of influenza A virus subtype H1N1. The examination of 56 autopsy lung samples showed thickened vascular walls of small pulmonary arteries after 14 days of disease compared to H1N1 influenza patients who died before the COVID-19 pandemic started. Pulmonary vascular remodeling in COVID-19 patients was associated with hypertrophy of the smooth muscle layer, perivascular fibrosis, edema and lymphostasis, inflammatory infiltration, perivascular hemosiderosis, and neoangiogenesis. We found a correlation between the duration of hospital stay and the thickness of the muscular layer of the pulmonary arterial walls. These results demonstrate that COVID-19 significantly affected the pulmonary vasculature in fatal-course patients, also suggesting the need for careful follow-up in non-fatal cases, at risk of pulmonary hypertension. Full article

The influenza A viruses (IAV) are the principal pathogens for annual (seasonal) influenza, which cause world-wide outbreaks in poultry and pose a persistent threat to public health. The Hemagglutinin protein (HA) of IAV promotes virus infection by binding the host membrane receptor and mediating virus–host membrane fusion. Immunoprecipitation–mass spectrometry (IP-MS) provides global insights into IAV HA–host protein interactions. However, various experimental conditions might affect the identification of interactions. Here, we performed a serial IP-MS to compare interactors of IAV HA in accidental host human, chicken and reservoir host duck cells. We found that the positive ratio of interactors identified by the IP-MS was improved when the transfected HA plasmid had a similar expression level to HA proteins found in IAV virus infection. Comparing interactors in human, chicken and duck cells, we found that HA–interacting host factors might play a role in the susceptibility of accidental hosts (human and chicken) to IAV infection compared to reservoir hosts (duck). We then focused on the function of two heat shock proteins (HSPA5 and HSPA8), which interacted with IAV HA proteins in all three species (human, chicken and duck). We found that both HSPA5 and HSPA8 promoted the IAV replication by enhancing the viral attachment and internalization. These findings extend our knowledge about the mechanisms of IAV entry to host cells and provide target genes to create chickens resistant to avian influenza. Full article

The endosomal sorting complexes required for transport (ESCRT) comprise a fundamental cellular machinery with remarkable versatility in membrane remodeling. It is multifunctional in the multivesicular body (MVB) biogenesis, exosome formation and secretion, virus budding, cytokinesis, plasma membrane repair, neuron pruning, and autophagy. ESCRT’s involvement in cellular mechanisms extends beyond basic membrane trafficking. By directly interacting with autophagy-related (ATG) proteins and facilitating autophagosome-lysosome fusion, ESCRT ensures cellular homeostasis. Dysregulation in ESCRT function has been implicated in cancer, neurodegenerative disorders, and infectious diseases, underscoring its critical role in numerous pathologies. Hepatitis B virus (HBV) is an enveloped virus that exploits ESCRT and autophagy pathways for viral replication, assembly, and secretion. This review synthesizes recent mechanistic insights into ESCRT’s multifaceted roles, particularly focusing on its interactions with autophagy formation and the HBV lifecycle. Full article

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus, causing the hemorrhagic infectious disease of SFTS, with a case fatality rate up to 30% due to the absence of effective therapeutic interventions. Therefore, it is urgent to develop safe and effective therapeutic drugs to control this viral hemorrhagic fever. Methods: The activity of verteporfin (VP), screened from an FDA-approved drugs library, against SFTSV, was systematically evaluated in Huh7 cells in a wide range of concentrations. We performed time-of-addition experiments with VP, along with binding, endocytosis, and membrane fusion assays, to determine which part of the SFTSV life cycle VP has its effect on. The potential targets of VP were detected by a drug affinity responsive target stability (DARTS) assay. Results: VP exhibited a potent anti-SFTSV activity by blocking the initial viral binding to the target cells during viral entry via significantly inducing the degradation of the viral Gn protein. Conclusions: The VP-induced inhibition of SFTSV binding, the first step of viral invasion, suggested that VP might be an ideal and potent anti-SFTSV agent due to its prophylaxis and therapeutic effects on viral infection. Full article

The peptide TAT-I24, a fusion of the TAT peptide (amino acids 48–60) and the 9-mer peptide I24, has been previously shown to neutralize several double-stranded (ds) DNA viruses in vitro. We have now extended the testing to potentially sensitive RNA viruses and analyzed the antiviral effect of the peptide against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). In Vero E6 cells, TAT-I24 neutralized the human 2019-nCoV isolate (Wuhan variant) in a dose-dependent manner, while it was unable to neutralize two SARS-CoV-2 variants of concern, Delta and Omicron. Moreover, TAT-I24 could not significantly neutralize any of the SARS-CoV-2 variants in the human lung carcinoma cell line Calu-3, which provides an alternative entry route for SARS-CoV-2 by direct membrane fusion. Therefore, a possible dependence on virus uptake by endocytosis was investigated by exposing Vero E6 cells to chloroquine (CQ), an inhibitor of endosomal acidification. The Wuhan variant was highly sensitive to inhibition by CQ, an effect which was further enhanced by TAT-I24, while the Delta variant was less sensitive to inhibition by higher concentrations of CQ compared to the Wuhan variant. The microscopic analysis of COS-7 cells using a rhodamine-labeled TAT-I24 (Rho-TAT-I24) showed the endosomal localization of fluorescent TAT-I24 and co-localization with transfected GFP-Rab14 but not GFP-Rab5. As these proteins are found in distinct endosomal pathways, our results indicate that the virus entry pathway determines sensitivity to the peptide. Full article

Background: Endemic viruses are becoming increasingly the norm, and the development of a rapid and effective vaccine is necessary. Methods: Here, we used our StealthX^TM exosome platform to express either Influenza H3 (StealthX^TM-Hemagglutinin, STX-H3), SARS-CoV-2 Delta spike (StealthX^TM-Spike, STX-S) or respiratory syncytial virus proteins (StealthX^TM-RSV fusion protein, STX-RSV) protein on the membrane surface and facilitate their trafficking to the exosomes. Results: The administration of exosomes carrying one of the antigens by themselves resulted in a strong immune response with the production of a potent humoral and cellular immune response in mice. Interestingly, these effects were obtained with the administration of nanograms of protein and without adjuvant. We tested the possibility of manufacturing a multivalent vaccine by combining exosomes expressing either STX-H3, STX-RSV or STX-S exosomes in the same formulation, in a “mix and match” approach. Mice immunized with the cocktail vaccine showed an increased immune response against all three antigens received. Conclusions: The results further demonstrated that our STX trivalent cocktail vaccine elicited a strong immune response, and the magnitude of the responses was comparable to the single antigen administered individually. Our data show that our exosome platform has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation, to tackle the fast-evolving viral infections. Full article

Background: Lawsonia intracellularis (LI) is the agent of proliferative enteropathy in swine, a common disease that affects pigs for up to eight weeks after weaning. Aim: To evaluate the effectiveness of two novel subunit vaccines targeting outer membrane proteins on LI. Methods: The two vaccines included OMP2c.cVLP, where the OMP2c antigen was anchored on the surface of capsid virus-like particles (cVLP); and MBP.INVASc, where antigens were anchored to an MBP fusion protein. Groups of six mice, as proof of concept, and six piglets were immunized with either OMP2c.cVLP, MBP.INVASc., or PBS as a control using a prime-boost regime. Results: Both OMP2c.cVLP and MBP.INVASc subunit vaccines induced strong antigen-specific serum IgG and IgA responses. There were no significant differences in weight gain among the groups. Mild-to-moderate clinical signs of LI infection were observed, but vaccinated groups showed lower inflammatory scores and fewer animals tested positive for bacteria by immunohistochemistry. Although neither vaccine completely prevented clinical signs of LI infection, both effectively reduced inflammation and lowered the pathogen load, thereby mitigating the severity of the disease, particularly the MBP.INVASc vaccine. Conclusions: These findings suggest that both vaccines have the potential for further development and optimization to enhance their protective efficacy against LI infections. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV) cause serious economic losses to the swine industry worldwide. Both viruses show a tropism for macrophages, based on the use of specific entry mediators (e.g., Siglec-1 and CD163). Identifying additional mediators of viral entry is essential for advancing antiviral and vaccine development. In this context, monoclonal antibodies (mAbs) are valuable tools. This study employed a library of 166 mAbs targeting porcine alveolar macrophages (PAMs) to identify candidates capable of blocking early infection stages, including viral binding, internalization, and fusion. Immunofluorescence analysis revealed 74 mAbs with cytoplasmic staining and 70 mAbs with membrane staining. Fifteen reacted with blood monocytes as determined by flow cytometry. mAb blocking assays were performed at 4 °C and 37 °C to analyze the ability of mAbs to block PRRSV and/or ASFV infections in PAMs. The mAb 28C10 significantly blocked PRRSV (96% at 4 °C and 80% at 37 °C) and ASFV (64% at 4 °C and 81% at 37 °C) infections. The mAb 28G10B6 significantly blocked PRRSV (86% at 4 °C and 74% at 37 °C) and partially blocked ASFV (35% at 4 °C and 64% at 37 °C) infections. mAb 26B8F5-I only partially blocked PRRSV infection (65% at 4 °C and 46% at 37 °C). Western blotting and mass spectrometry identified the corresponding proteins as Siglec-1 (28C10; 250 kDa), MYH9 (28G10B6; 260 kDa), and ANXA1 (26B8F5-I; 37 kDa). Our findings are indicative that Siglec-1, MYH9, and ANXA1 play a role in PRRSV/ASFV entry into macrophages. Full article