Search Results (509)

This review describes a novel method for increasing the effectiveness of inactivated enveloped whole-virus vaccines by targeting them for extensive uptake by antigen-presenting cells (APCs). Several inactivated whole-virus vaccines with dense glycan shields display suboptimal effectiveness because the multiple carbohydrate chains (glycans) on the virus mask immunogenic peptides and surround the virus with a negative electrostatic charge that decreases uptake by APCs. It is postulated that engineering such vaccinating viruses to present the carbohydrate antigen “α-gal epitope” on the glycan shields will immunocomplex them with the anti-Gal antibody; thus, it will target them for robust uptake by APCs. Anti-Gal is an abundant natural antibody in humans, constituting ~1% of human circulating immunoglobulins. The ligand of anti-Gal is the α-gal epitope, which is naturally synthesized in non-primate mammals and New World monkeys by the glycosylation enzyme α1,3galactosyltransferase. This enzyme is encoded by the GGTA1-gene. Viral vaccines presenting multiple α-gal epitopes on their glycan shield bind anti-Gal and activate the complement system to produce complement chemotactic cleavage peptides C5a and C3a that induce extensive recruitment of APCs to vaccine injection sites. The virion-bound anti-Gal further targets the viral vaccine for robust uptake by APCs, following binding of its Fc “tail” to Fcγ-receptors on APCs. The efficacy of this method was studied in anti-Gal-producing mice with α-gal presenting inactivated influenza virus vaccine and with gp120 of HIV presenting this epitope. These studies indicated that virus vaccines engineered to present α-gal epitopes increase anti-virus antibody production and virus-specific T-cell activation by 15- to 100-fold in comparison to the same vaccines lacking α-gal epitopes. It is suggested that α-gal presenting inactivated SARS-CoV-2 virus vaccines can induce a similar protective long-term immune memory against S- M-, E-, and N-viral proteins. Furthermore, immune-escaping variants of the mutated S-protein may be destroyed by antibodies to M and E proteins, and cells infected with such variants may be killed by cytotoxic T cells specific to peptides of the N-protein. Such an anti-M-, E-, and N-protein immune protection may prevent expansion of these variants and thus may avoid the need for immunization with COVID-19 vaccines every 6 months or following the appearance of new variants. A similar potent immunization may be achieved with an inactivated Ebolavirus vaccine engineered to present α-gal epitopes on the glycan shield. The resulting immune response to the various Ebolavirus proteins also may contribute to cross-reactive protection against other Ebolavirus species containing proteins with evolutionarily conserved structures. An effective method for the preparation of a whole-virus vaccine presenting α-gal epitopes is by arming it with the GGTA1-gene inserted into the viral genome. Such virions will present multiple α-gal epitopes on their glycan shield, which will amplify their immunogenicity instead of reducing it in the wild-type virus. Full article

Influenza viruses, due to their simple genomic structure and potent immunostimulatory capacity, have been extensively explored for applications in cancer immunotherapy and viral vector vaccine development. However, wild-type influenza viruses possess inherent risks of lethal pathogenicity and transmissibility, which limit their direct application. Special cold-adapted influenza strains have been widely used in live attenuated vaccines, which rely on specific amino acid mutations. With the advancement in synthetic biology and reverse genetics technologies, a variety of next-generation attenuated influenza virus have been developed, including genome-recoded viruses, miRNA-targeted viruses, viruses containing premature termination codons, and proteolysis-targeting recombinantviruses. This study systematically summarized the synthetic biology-based strategies for generating a next-generation method for the attenuated influenza virus, critically discussed the advantages and limitations of each strategy, and further analyzed their applications and challenges in cancer therapy and viral vector vaccine development. By synthesizing current research progress, this review aimed to provide a theoretical basis for constructing safer, more stable, and more controllable influenza virus engineering platforms, and to offer new insights for the design of attenuated influenza virus suitable for tumor therapy and novel vaccine delivery. Full article

H3N2 subtype avian influenza virus (AIV) is prevalent in poultry and wild birds and typically causes asymptomatic or mild respiratory infections. However, genetic reassortment between H3N2 and other AIV subtypes generates novel strains capable of crossing the species barrier, posing a threat to both poultry and public health. In this study, nine H3N2 AIVs were isolated from ducks in live poultry markets (LPMs) in Guangxi, southern China, during 2022–2024. Phylogenetic analysis revealed that all eight gene segments of the nine isolates were clustered within the Eurasian lineage, with internal genes derived from multiple subtypes, including H1, H2, H3, H4, H5, H6, H7, and H9. These findings indicate complex gene reassortment of H3N2 AIVs in Guangxi. Importantly, the PB2 genes of certain isolates were closely related to those of highly pathogenic H5 subtype viruses, suggesting that H3N2 AIVs may contribute internal genes to H5 viruses. Three representative isolates (LZD44, NND98, and NND100) were assessed for pathogenicity in SPF chickens and mice. All three strains successfully replicated in the respiratory tissues of both species. Notably, the LZD44 virus, which harbored the mammalian-adaptive mutations PB2-MVV and NP-I353V, presented significantly higher virulence in chickens and mice than the other two strains. These results demonstrate that H3N2 subtype AIVs are capable of replicating in certain tissues of chickens and mice without prior adaptation, underscoring a potential risk for cross-species transmission. Consequently, sustained surveillance of H3N2 subtype AIVs is essential to prevent the spillover of novel recombinants into the human population. Full article

Viruses cause a great number of infectious diseases with medical, veterinary, agricultural, social and economic impact. Their unique mechanisms to spread, overcome and resist the existing countermeasures require innovative and smart antiviral strategies such as the effective disinfection of enclosed environments with ensured broad-spectrum efficacy and minimized risks associated with handling liquid biocides. Formic acid (FA) is a well-established natural acaricide used in beehives with an antiviral potential; however, its application in a liquid form is hindered by severe corrosiveness and rapid, uncontrolled evaporation. This study describes a novel formulation of FA, using a cryogel carrier for achieving a vapor-phase inactivation of viruses, thus eliminating the need for direct contact between the disinfectant and the pathogen. Firstly, a poly(N-isopropylacrylamide) (PNIPAm) cryogel was synthesized by a procedure involving cryogenic treatment, photochemical crosslinking, and freeze-drying, and then the cryogel was swollen with 65% FA or _ddH₂O as a control. After an exposure of a panel of animal and human viruses to FA, evaporated by the polymer carrier for time intervals between 15 min and 12 h, they were neutralized completely as follows: Poliovirus (PV) as a surrogate for major bee viral pathogens for 60 min by 5.1 ∆lg; Feline calicivirus (FCV) for 60 min by 5.3 ∆lg; Adenovirus 5 (AdV5) for 12 h by 4.0 ∆lg; and Influenza virus A (IAV) for 15 min by 5.1 ∆lg. Results were recorded after titration, 48–72 h incubation, cytopathic effect estimation and NR uptake assay. Our results suggest that 65% FA, when delivered via the PNIPAm cryogel matrix, acts as a powerful agent for fumigation-like disinfection. This “dry” delivery strategy offers significant practical advantages: it eliminates the need for open liquid containers, prevents spill-related hazards, and provides an alternative for controlled, long-term release of active vapors. Full article

To address the lack of a commercially available vaccine for goose circovirus (GoCV), we developed and evaluated a prokaryotically expressed subunit vaccine targeting the viral capsid (Cap) protein. A truncated Cap protein (GoCV-ΔCap) was expressed in Escherichia coli (E. coli) and formulated with aluminum hydroxide as a subunit vaccine (GoCVsubvac). Goslings were primed intramuscularly (i.m.) with high (75 µg) or low (15 µg) doses GoCVsubvac, followed by a boost 14 days later. At 14 days post-boost, goslings were challenged with GoCV and were administered a bivalent inactivated vaccine against Newcastle disease virus (NDV) and H9-subtype Avian influenza virus (AIV). Using our established gosling pathogenicity model, vaccine efficacy was evaluated via body weight, lesions, viral load, antibody titers, cytokine responses, and interference with NDV/AIV immunity. Results demonstrated that the GoCV-ΔCap vaccine, especially the high-dose formulation, provided effective immunoprotection. It elicited robust humoral and cellular immune responses, reduced lymphoid pathology, and decreased the viral detection rate in lymphoid tissues from 100% (5/5) in infected controls to 40% (2/5). Importantly, it alleviated GoCV-induced immunosuppression and preserved the immunogenicity of co-administered vaccines. This novel subunit vaccine is a promising candidate for controlling GoCV disease (GoCVD). Full article

This work focuses on rapid, catalyst-free synthesis of a new series of acetylenic phosphonates as promising building blocks for creating antiviral and anticancer agents. A comprehensive assessment of the biological activity of the synthesized compounds was conducted. Dialkyl phosphonates 4d, 4e, and 4g were found to exhibit pronounced antiproliferative activity against human cancer cell lines, with the greatest IC₅₀ = 6 μg/mL against the K562 cell line. Further studies revealed that these compounds cause significant disorganization of the actin cytoskeleton, leading to the loss of stress fibers and reduced cell motility. In contrast, diamide derivatives demonstrated a more favorable safety profile, with low cytotoxicity and moderate antiviral activity against influenza A (H1N1) virus, among which compound 6b achieved a selectivity index of 5 with IC₅₀ = 56.9 μg/mL. Screening studies of both dialkyl and diamide acetylenic phosphonates revealed some features of the interaction with kinase and nonkinase targets used for drug development and provide a basis for the subsequent rational design of novel selective anticancer agents based on the acetylenic phosphonate scaffold. Full article

The innate immune system, particularly the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway, is a major early defense barrier against influenza A virus infection. However, excessive immune responses can trigger lethal cytokine storms and severe immune-mediated pathology. In this study, we performed a genome-wide CRISPR/dCas9 gene activation screen in human lung epithelial (A549) cells by using an A/Puerto Rico/8/1934 (H1N1) reporter virus, and identified the ubiquitin-specific protease USP17L13 as a novel negative regulator of innate immunity that promotes influenza virus replication. Overexpression of USP17L13 significantly enhanced the replication of multiple subtypes of influenza viruses in A549 cells, including a human pandemic H1N1 virus, seasonal H3N2 viruses, as well as a globally circulating clade, 2.3.4.4b, of the highly pathogenic avian H5N1 virus. Transcriptomic analysis demonstrated that USP17L13 suppresses host antiviral defenses by downregulating nuclear factor kappa B (NF-κB) signaling and arachidonic acid metabolism, while upregulating pathways associated with ribosomal translation and oxidative phosphorylation to facilitate viral production. Mechanistically, USP17L13 attenuates the host interferon (IFN) response by promoting the degradation of the key viral RNA sensors, RIG-I, and melanoma differentiation-associated protein 5 (MDA5). Further analysis revealed that USP17L13 is inducible by type I and type II interferons as well as inflammatory cytokines, suggesting that it may act as a negative-feedback regulator to limit excessive inflammation. Collectively, our findings identify USP17L13 as a previously unrecognized proviral host factor and provide new insight into how host deubiquitinases shape influenza virus-host interactions, with potential implications for host-directed approaches to controlling excessive inflammation during viral infection and improving influenza vaccine production. Full article

The hemagglutinin (HA) of influenza A/H3N2 virus evolves rapidly, with glycosylation driving immune evasion. However, how host microenvironmental cues influence this process remains poorly understood. We identified a novel N-linked glycosylation site at position 110 (N110) in contemporary H3N2 viruses (NSS genotype) that enhances viral fitness by increasing receptor-binding signal, HA cleavage, and replication. Remarkably, hypoxia, which mimics the respiratory tract microenvironment, significantly augments N110 glycosylation. Mechanistically, we identified the B4GAT1-B4GALT1 complex as the key mediator of this modification. Hypoxia upregulates their expression and strengthens their interaction with HA. In ferret models, N110-glycosylated viruses exhibit heightened pathogenicity and evade ancestral antibodies. Furthermore, immunization with N110-containing HA confers broad-spectrum protection, whereas reciprocal immunization is ineffective. Our findings reveal hypoxia-driven glycosylation as a previously unrecognized mechanism of H3N2 adaptation, providing critical insights for vaccine efficacy and highlighting the importance of integrating microenvironmental factors into future antiviral strategies. Full article

The H5N1 subtype of highly pathogenic avian influenza (HPAI) poses a major zoonotic threat due to its high fatality rate and capacity for cross species transmission. In early 2025, Argentina detected a novel triple reassortant A(H5N1) virus in Chaco Province, combining Eurasian, North American, and South American lineage segments. Genomic analyses of subsequent outbreaks in Buenos Aires and Entre Ríos confirmed persistence of this reassortant and additional HA substitutions (T204K, P251S) potentially linked to increased mammalian receptor affinity. Although PB2 sequences lacked canonical mammalian-adaptive markers (E627K, Q591K, D701N), all contained I292M, a mutation associated with human adaptation. Phylogenetic analyses revealed distinct genotypes and increasing divergence. These findings indicate ongoing viral evolution and adaptation within Argentina, emphasizing the urgent need for sustained genomic surveillance, timely data sharing, and integrated One Health strategies to mitigate zoonotic and socioeconomic risks associated with H5N1 spread in South America. Full article

Background: Hemagglutinin (HA) is the primary surface protein of the influenza A virus, determining its subtype and antigenic properties. Traditional subtype classification methods rely on DNA or amino acid sequence analysis, which does not account for protein spatial folding. Methods: In this work, we propose EpitopeGNN—a graph neural network (GNN) that constructs a residue interaction network (RIN) from the 3D structure of HA and classifies the virus subtype. The model was trained on 249 structures from the Protein Data Bank (PDB), containing H1N1, H3N2, H5N1, and other subtypes. Results: After rigorous sequence redundancy reduction (92% identity), the model maintained 95–100% accuracy on non-redundant data, significantly outperforming sequence-only baselines (the best baseline achieved 85% for multi-class and 92.3% for binary classification). A significant correlation was found between the obtained structural embeddings and phylogenetic distances (r = 0.38, p < 0.001), confirming their biological relevance and opening opportunities for structural monitoring of virus evolution, as well as rapid analog searching for novel strains. Conclusions: We developed a new graph neural network that classifies influenza A virus subtypes directly from the 3D structure of hemagglutinin using residue interaction networks and physicochemical features, which can serve as a foundation for predicting influenza virus receptor specificity and epitope immunogenicity. Full article

H5 subtype avian influenza virus (AIV) can infect both chickens and ducks, leading to substantial economic losses. Nevertheless, certain strains cause silent infections in ducks. In this study, a goose-origin clade 2.3.4.4h H5N6 AIV was isolated, which caused high mortality in mixed-gender white leghorn chickens but no deaths in mixed-gender mallard ducks. After independent serial in vitro passage in duck embryo fibroblasts (DEFs) and in vivo passage in specific-pathogen-free (SPF) ducks, the DEF-passage 10 (P10) virus induced markedly higher mortality rates and viral loads in SPF ducks compared to the DEF-P1 virus and the original parental virus prior to passage. Similarly, the in vivo-passaged P3 and P4 viruses exhibited significantly higher mortality rates than the P1 virus in SPF ducks, with 100% mortality and markedly increased viral titers in the organs. A whole-genome SNP analysis identified seven high-frequency mutations in the M1, NA and NS1 proteins. The NS1-F161L substitution virus exhibited significantly increased mortality rates, viral loads in multiple tissues, and a robustly induced innate immune response in ducks. Furthermore, dynamic evolutionary variations in the NS1 protein among global H5 avian influenza viruses revealed that the NS1-F161L substitution became dominant in clade 2.3.4.4b viruses in 2021 and subsequent years. Collectively, our findings demonstrate that host-driven adaptation can rapidly increase the pathogenicity of H5N6 AIVs in ducks and identify NS1-F161L as a critical virulence marker. These results offer novel insights relevant to the molecular surveillance, virulence prediction, and risk assessment of circulating H5 AIVs in waterfowl. Full article

Background/Objectives: Influenza A virus (IAV) infection triggers robust inflammation and acute lung injury. Andrographolide, a primary active compound from Andrographis paniculata, can mitigate IAV-induced inflammation; however, its precise mechanisms remain poorly elucidated. This study aimed to define its host-directed protective effects and molecular mechanisms. Methods: We used a lethal IAV (H1N1, PR8) model in BALB/c mice and infected A549 cells. Survival, lung pathology, cytokines, and viral titers were measured. Lung RNA sequencing identified dysregulated signaling pathways. PI3K/AKT and pyroptosis pro-teins were analyzed by Western blot. The PI3K/AKT axis was functionally validated with the AKT inhibitor in vivo and AKT1 siRNA in vitro. Results: Andrographolide improved survival, attenuated body weight loss, and reduced lung pathology and inflammatory cytokine levels in IAV-infected mice, without exhibiting direct antiviral activity. Consistent with the in vivo findings, andrographolide enhanced cell viability and suppressed cytokine secretion in infected cells. RNA sequencing revealed marked upregulation of the PI3K/AKT signaling pathway in the lungs of treated mice, as confirmed by increased PI3K and AKT phosphorylation. Furthermore, andrographolide downregulated the expression of key pyroptosis-executing proteins, including cleaved caspase-3 and the gasdermin E (GSDME) N-terminal fragment. These protective effects were substantially abrogated by an AKT inhibitor and AKT1 siRNA. Conclusions: These findings reveal a novel host-directed mechanism by which andrographolide alleviates IAV-induced immunopathology by activating the PI3K/AKT pathway, thereby suppressing caspase-3/GSDME-dependent pyroptosis. Thus, this axis represents a promising target for controlling excessive inflammation in severe influenza. Full article

Avian influenza infections cause substantial economic losses in the poultry industry and raise public health concerns due to viral adaptation and cross-species transmission. The frequent antigenic drift of influenza viruses further complicates the prevention and treatment of avian respiratory infections. In this study, we generated high-affinity heavy-chain variable domain (VHH) nanobodies from naïve alpaca/camelid VHH libraries using phage display combined with H9N2 influenza A virus (IAV)-infected Madin-Darby Canine Kidney (MDCK) cells. Based on binding affinity and neutralization potential, we identified seven hemagglutinin (HA)-specific and two neuraminidase (NA)-specific VHHs. Molecular docking predicted the interaction sites of HA-specific VHHs (L1-2, L1-4, A5) and NA-specific VHHs (L1-3, L2-2), providing mechanistic insights. Notably, the three HA-specific VHHs (L1-2, L1-4, A5) showed cross-reactivity to representative HA subtypes (H1, H3, and influenza B), indicating recognition of conserved epitopes across divergent influenza strains. For the first time, these camelid nanobodies were fused to the chicken IgY Fc domain, and the expression cassette was integrated into the Saccharomyces cerevisiae genome, achieving a secretion yield of 15–20 mg/L of VHH-Fc antibodies. Experimental validation confirmed that the three HA-specific VHHs-Fc constructs effectively blocked viral infection, while the two NA-specific VHH-Fc constructs (L1-3, L2-2) inhibited NA activity, demonstrating the functional efficacy of the yeast-secreted VHH–IgY Fc platform. This novel IgY Fc fusion approach offers a scalable platform with enhanced stability, extended circulation potential, and applicability in poultry. Full article

Background: In complex biological processes, there exists a tipping point (pre-disease state) when the system undergoes a sudden and dramatic shift to a contrasting state. Accurate detection of the pre-disease state is critical for preventive medicine. However, precise detection of the pre-disease state proves challenging due to the clinical single-sample problem. Methods: To address this challenge, in this study, we introduce a novel single-sample pre-disease state detection method based on the change in local network enrichment level. Results: We validated the proposed method on five independent real datasets, including one influenza virus infection time-course dataset and four tumor datasets. Experimental results confirmed that the proposed method can accurately identify the pre-disease state prior to overt disease onset. Further analysis verified key genes identified by the proposed method in pre-disease state are associated with viral infection and immune dysregulation for the influenza dataset, and tumor metastasis for the tumor datasets. Conclusions: These results demonstrate that this method is a robust and biologically interpretable tool for single-sample pre-disease state detection, with great potential for clinical translation in individualized preventive medicine. Full article

The ongoing panzootic of the highly pathogenic avian influenza (HPAI) H5N1 virus, dominated by clade 2.3.4.4b, constitutes a significant global threat to wildlife, animal health, and public health. Once characterized by sporadic outbreaks, H5N1 has evolved into a sustained, year-round infection with an expanded host range that now includes numerous mammalian species. Its high pathogenicity is primarily driven by the acquisition of a polybasic haemagglutinin cleavage site, enabling systemic viral spread, alongside emerging endothelial and neurotropic properties that contribute to severe disease and high mortality in mammals. Although zoonotic transmission remains limited, H5N1 continues to accumulate mutations associated with mammalian adaptation, particularly within the haemagglutinin and polymerase complex. Notably, recent outbreaks in U.S. dairy cattle highlight the emergence of novel mammalian reservoirs with increased human exposure risk. Concurrently, vaccination strategies are advancing beyond traditional adjuvanted inactivated vaccines toward next-generation platforms, including mRNA and virus-like particle vaccines, designed for rapid deployment and broader immune protection. However, ongoing viral evolution, constrained vaccine availability, and gaps in coordinated surveillance underscore the urgent need for an integrated One Health approach to reduce panzootic risk. Full article