Oral immunization with virus-like particles elicits Ebola virus-specific humoral and cellular immune responses in mice.

Mechanistic insights into flavonoid-mediated regulation of human influenza virus replication.

The antibody response to influenza virus infection targets numerous viral proteins, with those specific for the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins being the most extensively studied. Antibodies to HA and NA function as independent immune correlates of protection and form the basis of most vaccine approaches. In contrast, the contribution of antibodies to other viral proteins such as the nucleoprotein (NP) remains unclear, and existing studies have reached conflicting conclusions. In this work, we examined three human monoclonal NP antibodies isolated from plasmablasts of two individuals naturally infected with influenza A virus (IAV) during the 2018-2019 influenza season. These antibodies showed strong binding to NP across a broad range of IAV strains, yet none were able to limit weight loss or mortality when delivered prophylactically to mice. Further characterization demonstrated that the antibodies did not neutralize infection, and although they bound to the surface of infected MDCK cells, they did not induce cellular antiviral responses. While the broad cross-reactivity of these monoclonal antibodies is attractive for next-generation vaccine design, strategies that aim to elicit similar responses must either enhance their antiviral potency or clarify their role in modulating viral replication and disease.

Influenza A virus (IAV) remains a significant public health threat due to its high variability and pathogenicity. Systematic identification of viral-host interfaces is critical for developing targeted therapies. In the present study, TurboID proximity labeling was applied to map interactomes of 10 H1N1 viral proteins in human alveolar epithelial cells A549. Key interactions were validated via co-immunoprecipitation (Co-IP), confocal microscopy, dual-luciferase reporter assays, and functional studies. Results showed that Hemagglutinin (HA) and polymerase acidic (PA) directly bound mitochondrial antiviral-signaling protein (MAVS), significantly suppressing interferon-beta (IFN-β) promoter activity and impairing phosphorylation of TANK-binding kinase 1 (TBK1)/signal transducer and activator of transcription (STAT) signaling proteins. HA exploited integrin alpha-2 (ITGA2) as a novel entry cofactor, with their interaction confirmed by bidirectional Co-IP and co-localization. ITGA2 silencing markedly reduced viral titers. Tripartite motif-containing protein 56 (TRIM56) restricted H1N1 replication by binding viral nucleoprotein (NP) and inducing proteasomal degradation via K48-linked ubiquitination. As a conclusion, this study delineates H1N1's coordinated tactics to hijack host pathways, identifying MAVS, ITGA2, and TRIM56 as pivotal nodes for combination therapies. TurboID-driven interactomics provides a promising framework for system-level antiviral discovery.

CD4+ T helper (Th) cells play a central role in orchestrating protective immune responses during infection and vaccination. Activation of naïve CD4+ T cells is initiated by the recognition of antigenic peptides presented on MHC class II (MHC-II) molecules, leading to their differentiation into distinct effector subsets. In the context of influenza virus infection, the quality of CD4+ T-cell responses is a critical determinant of both cellular and humoral immunity. Given the limited breadth of protection conferred by current influenza vaccines, which primarily induce strain-specific neutralizing antibodies, there is growing interest in harnessing CD4+ T-cell responses targeting conserved viral epitopes to achieve broader and more durable protection. Identifying potent T-cell epitopes within viral antigens is therefore critical for designing effective vaccines that elicit robust, durable, and cross-protective Th-cell and antibody responses. In this study, using a mouse model of influenza virus infection, we identified CD4+ T-cell epitopes within the hemagglutinin (HA) and nucleoprotein (NP) of the H1N1 strain. We generated two NP peptide/MHC-II tetramers, NP264-274/I-Ab and NP418-428/I-Ab, which enabled in vivo tracking and characterization of epitope-specific CD4+ T cells during infection. These epitope-specific T cells exhibited distinct T-cell receptor (TCR) repertoires and differentiation patterns toward either Th1 or T follicular helper (Tfh) lineages, suggesting that epitope specificity shapes the quality of the T-cell response. Our findings provide new insights into the epitope-dependent functional diversity of Th-cell responses and offer valuable tools for the rational design of broadly protective influenza vaccines.

Ma Xing Shi Gan Decoction reduces influenza A virus nucleoprotein levels in association with modulation of lactate-HMGB1 lactylation and autophagosome accumulation.

Several orthoebolaviruses have been established, including Orthoebolavirus zairense [for Ebola virus (EBOV)], Orthoebolavirus sudanense [for Sudan virus (SUDV)], and Orthoebolavirus bundibugyoense [for Bundibugyo virus (BDBV)]. Orthoebolaviruses are highly virulent pathogens that cause severe disease in humans. However, most existing vaccines primarily target EBOV, and fail to provide robust cross-protection against other lethal orthoebolaviruses. The development of a broad-spectrum vaccine has the potential to efficiently mitigate outbreaks caused by multiple orthoebolaviruses. In this study, we developed and evaluated a broad-spectrum mRNA vaccine, designated [GPs+NP]@LNP, formulated with a single lipid nanoparticle (LNP) platform to encapsulate a mixed mRNA payload encoding the glycoproteins (GPs) of EBOV, BDBV, and SUDV, along with the nucleoprotein (NP) of EBOV. This rational antigenic combination was designed to engage both humoral and cellular arms of the immune system. Consistent with this design, the compatibility of humoral immunity-driving GPs and cellular immune-dominating NPs supports complementary immune responses, which together are associated with broad and durable protection against EBOV, BDBV, and SUDV in animal models. Antigenic coordination within a single LNP formulation may therefore represent an effective strategy to optimize vaccine immunogenicity, protective effect, durability, and safety. Collectively, these findings highlight [GPs+NP]@LNP as a promising approach for the development of next-generation vaccines targeting multiple pathogenic orthoebolaviruses.

Background. The highly mutable influenza virus causes severe annual infections worldwide and results in substantial socioeconomic losses. The spread of infection could be effectively controlled by cross-protective vaccines and universal diagnostic test systems based on the nucleoprotein (NP) as one of the most conserved viral antigens. However, NP also undergoes slow evolutionary changes, and little is known about the influence of these mutations on its antigenicity and immunogenicity. Methods. We expressed the full-length recombinant 6xHis-tagged NPs of ten evolutionary distant influenza A strains of different subtypes in E. coli BL21(DE3) cells and purified these proteins by immobilized metal affinity chromatography. The obtained antigens were identified by mass spectrometry and serological methods. NPs served as antigens for three immunizations of BALB/c mice (15 µg/animal at 14-day interval) and as capturing proteins in ELISA at 2 µg/mL, in order to study the effect of adaptive mutations on the antigenic and immunogenic properties of NPs. Results. A pronounced cross-reactivity of anti-NP antibodies induced in mice by immunization with different NPs was revealed. At the same time, we observed the differences in the humoral immunogenicity of NP, which are in line with the accumulation of evolutionarily driven NP mutations. In general, antibody affinity to heterologous NPs was reduced, indicating the differences in the specificity of anti-NP immunoglobulins, which may be caused by evolutionarily determined variability of immunogenic epitopes leading to the emergence of escape mutations. Conclusions. Overall, our results reflect the slightly evolving nature of the NP antigen, which influences the specificity spectrum of anti-NP antibodies and should be considered as a limitation for the development of NP-based cross-protective vaccines and test systems.

Evaluation of immune response in calves vaccinated with baculovirus vectored inactivated vaccine expressing 2.3.4.4b H5 protein of highly pathogenic Avian Influenza virus.

Lassa virus (LASV), the causative agent of Lassa fever (LF), poses a significant public health concern in endemic regions due to its high morbidity and mortality. Rapid and accurate diagnosis is critical for effective clinical management and containment during LF outbreaks. However, the genetic diversity of LASV, encompassing at least seven distinct lineages, poses a major challenge for the development of broadly reactive diagnostic tools. Therefore, there is an urgent need to develop detection methods that are effective across diverse lineages. To address this challenge, we generated a panel of cross-reactive monoclonal antibodies (mAbs) targeting the LASV nucleoprotein (NP). Several mAbs exhibited broad reactivity across LASV lineages I-VII in different immunoassays such as enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence assay. Utilizing these broadly reactive mAbs, we developed an antigen-capture sandwich ELISA capable of detecting LASV NP from all seven lineages with high sensitivity. Our findings highlight a set of novel mAbs with broad cross-reactivity across lineage. In addition, we demonstrated their utility in a sandwich ELISA format for pan-LASV detection. This pan-LASV diagnostic approach offers a promising tool for improved LF diagnosis in both clinical and epidemiological settings.

The host RNA helicase DDX6 restricts avian influenza virus replication by targeting viral NP and modulating ISG15.

NPmut- and M2e-decorated T4 phage nanoparticles induce mucosal immunity and cross-group protection against influenza A virus.

The continuous antigenic shift and drift of influenza A virus (IAV) result in the emergence of novel strains and drug resistance. Accordingly, it is urgent to develop novel antiviral drugs that target host factors. Here, we revealed that tyrosine kinase LYN (Lck/Yes-related novel protein tyrosine kinase) interacts directly with nucleoprotein (NP) and reduces the replication and virulence of IAV in vitro and in vivo, in a kinase-dependent manner. By directly catalyzing the tyrosine phosphorylation of NP at Y10/40/97 sites, LYN impairs the interaction of NP with viral RNA and polymerase proteins, as well as the oligomerization of NP, and thus negatively modulates the assembly of the viral ribonucleoprotein complex. The NPY10/40/97F mutation significantly increases the pathogenicity of IAV in mice. Furthermore, the LYN-specific agonist MLR-1023 showed promising therapeutic effects against IAV. Collectively, our findings suggest that LYN is a novel host kinase restricting IAV replication, and a promising target for anti-influenza drug development.IMPORTANCEThe nucleoprotein (NP) of influenza A virus (IAV) is a highly conserved, multifunctional, and the most abundant viral protein in infected cells, which makes NP a promising target for the development of anti-influenza drugs. Phosphorylation plays a crucial role in the stability and functionality of NP. However, few kinases have been identified that directly phosphorylate NP. In this study, LYN (Lck/Yes-related novel protein tyrosine kinase) was identified as a novel kinase that directly catalyzes the tyrosine phosphorylation of IAV NP and thus restricts the replication and virulence of IAV. The LYN-specific agonist MLR-1023 exhibited promising protective efficacy against lethal IAV infection. Our findings discovered LYN and LYN-agonist as potential drug candidates targeting IAV NP, which will facilitate the development of host-directed antiviral therapies.

Influenza A virus (IAV) infection induces a type I interferon (IFN) response in host cells, which exerts antiviral effects by upregulating interferon-stimulated genes (ISGs). However, the ISG response to IAV remains incompletely understood. Here, we systematically identify antiviral ISGs in A549 cells following type I and type II IFN treatment. We demonstrate that ring finger protein 213 (RNF213) strongly inhibits IAV replication in vitro. Notably, RNF213 knockout (KO) mice exhibit heightened susceptibility to IAV infection, with exacerbated disease severity. Mechanistically, RNF213, which is induced by both IFN signaling and IAV infection, amplifies IFN production by enhancing melanoma-differentiation-associated gene 5 (MDA5) signaling. We further show that RNF213 interacts with MDA5 via its AAA+ ATPase and E3 ligase domains, promoting K63-linked polyubiquitination of MDA5 at Lys137 and Lys743. Additionally, RNF213 mediates K48-linked polyubiquitination of viral nucleoprotein (NP), targeting it for proteasomal degradation. Our findings identify RNF213 as a critical antiviral ISG that bridges innate immune activation and direct viral restriction. Our study reveals a dual mechanism of RNF213 in antiviral immunity, highlighting its potential as a therapeutic target against influenza.

Infants are significantly more susceptible to respiratory infection, often resulting in increased morbidity and hospitalization, and occasionally death. This susceptibility is partially explained by the developing nature of the thymus in human infants at-and for several months after-birth. However, the contribution of T cells produced in this thymic microenvironment to infant immune responses has received minimal investigation. Here, we utilized a previously described mouse model (Foxn1Δ/Δ), which exhibits a persistently immature thymus. Through further characterization, we have determined that adult Foxn1Δ/Δ mice retain some unique T cells observed in neonatal mice including CD8αß+ γδ T cells and CD8 T cells displaying a memory-like phenotype. For this reason, we assessed the potential of these neonatal-like T responses to 2 pathogens, which disproportionately affect neonates, Bordetella pertussis (Bp) and influenza. Utilizing these infections, we demonstrate that T cells generated in an incompletely developed thymus fail to control or mount an effective response against Bp. We also observe that Foxn1Δ/Δ mice control acute influenza infection, a response that does not require IL-17. However, the Foxn1Δ/Δ mice fail to generate an influenza nucleoprotein (NP) specific CD8+ T cell response, which is likely associated with their inability to fully clear the infection. Together, these data suggest that Foxn1Δ/Δ mice can be utilized to study the generation, function, and persistence of some unique T cells made in a neonatal-like thymus.

Multiple outbreaks of Ebola virus in West Africa have posed significant threats to global public health owing to its high pathogenicity and fatality rates. Current treatments for Ebola Virus Disease are limited, underscoring the imperative for novel antiviral therapies. VP30, a critical RNA synthesis factor, interacts with nucleoprotein (NP) to facilitate Ebola viral genome transcription and replication. Notably, the host ubiquitin-ligase retinoblastoma-binding protein 6 (RBBP6) binds to VP30 at the same interface as NP, thereby inhibiting VP30-NP interactions and indicating that targeting this interface could advance antiviral drug development. In this study, we engineered six peptide mutants through amino acid substitutions at key VP30 binding sites. These mutants were fused to DNA-binding protein from starved cells 4 (DPS4) to assemble nanoparticles, enabling surface display of the peptides. Antiviral effects were evaluated using minigenome and transcription and replication-competent virus-like particles (trVLPs) systems. Among the variants, RPL1 and NPL3 peptides exhibited relatively strong apparent affinities with the VP30 and potent antiviral activity by disrupting Ebola viral genome transcription and replication. To elucidate the binding details between the peptides and VP30, we determined crystal structures of complexes between RPL1 or NPL3 peptides and VP30 via X-ray crystallography. Concurrently, molecular dynamics (MD) simulations revealed the dynamic binding processes of these peptides to VP30. Structural analyses confirmed that the peptides bind to the VP30/NP interface and compete with NP. Our findings demonstrate that DPS4-fusion peptides effectively deliver peptides into cells as nanoparticles and inhibit VP30-NP interactions, presenting a novel antiviral strategy for Ebola virus.

Influenza A virus (IAV) infection constitutes a major public health threat. Severe influenza virus infection can induce intense inflammatory responses and lung injury, leading to serious clinical symptoms or even death. The utility of current anti-influenza drugs is often limited by side effects and the emergence of drug-resistant strains. Based on the critical role of L-type voltage-gated calcium channels (L-VGCCs) in influenza virus replication, this study investigates the antiviral activity and mechanism of verapamil, a classic L-type calcium channel antagonist, against H1N1-UI182 virus. Verapamil, an L-type calcium channel blocker, is widely used in the treatment of cardiovascular diseases and has a well-established safety profile. Through molecular dynamics (MD) simulation and network pharmacology analysis, we predicted the stable binding mode of verapamil to the target protein (PDB id: 6JPA) and its potential multi-target network. In vitro, verapamil exhibited antiviral activity against H1N1-UI182 in MDCK cells, enhancing the survival rate of infected cells and reducing viral nucleoprotein (NP) expression. In a lethal H1N1-UI182 infection mouse model, verapamil treatment markedly improved survival rates, alleviated weight loss and lung pathological damage, exhibiting a dose-dependent protective effect. Lung tissue analysis showed that verapamil effectively reduced the lung index and viral load, suppressed the activation of the Nuclear factor kappa B (NF-κB) signaling pathway, and decreased the expression of key inflammatory factors, thereby mitigating the cytokine storm. A comparison of administration regimens indicated that pre-treatment yielded optimal efficacy, suggesting verapamil acts primarily during the early stage of the viral life cycle. This study systematically elucidates that verapamil exerts antiviral and immunomodulatory effects by regulating the NF-κB pathway. Network pharmacology analysis suggested the potential involvement of multiple targets and pathways, including EGFR, SRC, and phospholipase D signaling, providing hypotheses for future mechanistic investigation. This paper supports a drug repurposing strategy against drug-resistant influenza viruses and highlights its significant potential for clinical translation.

Inhibition of influenza a virus infection by natural isoquinoline alkaloid neferine targeting virus nucleoprotein.

PCBP2 inhibits antiviral innate immune responses via the MAVS-mediated signaling pathway in severe fever with thrombocytopenia syndrome.

Objective To establish a stable cell line overexpressing integrin β3(ITGB3), aiming to overcome the limited replication efficiency and low viral yields of Hantaan virus (HTNV) in conventional cell culture systems. Methods A lentiviral vector encoding ITGB3 was constructed and packaged into viral particles. Vero E6 and A549 cells were transduced and selected with puromycin to generate stable lines. ITGB3 expression was verified by quantitative RT-qPCR, Western blot, and immunofluorescence staining, and cell viability was evaluated using the CCK-8 assay. Following HTNV infection, viral replication was assessed by measuring viral RNA copies, nucleoprotein (NP) expression, and virus titers. Results ITGB3 was stably and highly expressed in Vero E6 and A549 cells, with no significant impact on cell proliferation. After HTNV infection, the levels of viral RNA replication and the expression of NP protein were both significantly upregulated in the ITGB3-overexpressing group, and the virus titers were markedly higher than those in the control group. Conclusion The established ITGB3 overexpressing cell line promotes HTNV replication and propagation, providing a reliable and efficient model for investigating HTNV pathogenesis and facilitating the development of antiviral drugs.