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Influenza viruses continually evolve new antigenic variants, through mutations in epitopes of their major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Antigenic drift potentiates the reinfection of previously infected individuals, but the contribution of this process to variability in annual epidemics is not well understood. Here, we link influenza A(H3N2) virus evolution to regional epidemic dynamics in the United States during 1997-2019. We integrate phenotypic measures of HA antigenic drift and sequence-based measures of HA and NA fitness to infer antigenic and genetic distances between viruses circulating in successive seasons. We estimate the magnitude, severity, timing, transmission rate, age-specific patterns, and subtype dominance of each regional outbreak and find that genetic distance based on broad sets of epitope sites is the strongest evolutionary predictor of A(H3N2) virus epidemiology. Increased HA and NA epitope distance between seasons correlates with larger, more intense epidemics, higher transmission, greater A(H3N2) subtype dominance, and a greater proportion of cases in adults relative to children, consistent with increased population susceptibility. Based on random forest models, A(H1N1) incidence impacts A(H3N2) epidemics to a greater extent than viral evolution, suggesting that subtype interference is a major driver of influenza A virus infection ynamics, presumably via heterosubtypic cross-immunity.

Phylogenetic and discrete-trait evolutionary inference depend heavily on an appropriate characterization of the underlying character substitution process. In this paper, we present random-effects substitution models that extend common continuous-time Markov chain models into a richer class of processes capable of capturing a wider variety of substitution dynamics. As these random-effects substitution models often require many more parameters than their usual counterparts, inference can be both statistically and computationally challenging. Thus, we also propose an efficient approach to compute an approximation to the gradient of the data likelihood with respect to all unknown substitution model parameters. We demonstrate that this approximate gradient enables scaling of sampling-based inference, namely Bayesian inference via Hamiltonian Monte Carlo, under random-effects substitution models across large trees and state-spaces. Applied to a dataset of 583 SARS-CoV-2 sequences, an HKY model with random-effects shows strong signals of nonreversibility in the substitution process, and posterior predictive model checks clearly show that it is a more adequate model than a reversible model. When analyzing the pattern of phylogeographic spread of 1441 influenza A virus (H3N2) sequences between 14 regions, a random-effects phylogeographic substitution model infers that air travel volume adequately predicts almost all dispersal rates. A random-effects state-dependent substitution model reveals no evidence for an effect of arboreality on the swimming mode in the tree frog subfamily Hylinae. Simulations reveal that random-effects substitution models can accommodate both negligible and radical departures from the underlying base substitution model. We show that our gradient-based inference approach is over an order of magnitude more time efficient than conventional approaches.

The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.

Birth-death models play a key role in phylodynamic analysis for their interpretation in terms of key epidemiological parameters. In particular, models with piecewise-constant rates varying at different epochs in time, to which we refer as episodic birth-death-sampling (EBDS) models, are valuable for their reflection of changing transmission dynamics over time. A challenge, however, that persists with current time-varying model inference procedures is their lack of computational efficiency. This limitation hinders the full utilization of these models in large-scale phylodynamic analyses, especially when dealing with high-dimensional parameter vectors that exhibit strong correlations. We present here a linear-time algorithm to compute the gradient of the birth-death model sampling density with respect to all time-varying parameters, and we implement this algorithm within a gradient-based Hamiltonian Monte Carlo (HMC) sampler to alleviate the computational burden of conducting inference under a wide variety of structures of, as well as priors for, EBDS processes. We assess this approach using three different real world data examples, including the HIV epidemic in Odesa, Ukraine, seasonal influenza A/H3N2 virus dynamics in New York state, America, and Ebola outbreak in West Africa. HMC sampling exhibits a substantial efficiency boost, delivering a 10- to 200-fold increase in minimum effective sample size per unit-time, in comparison to a Metropolis-Hastings-based approach. Additionally, we show the robustness of our implementation in both allowing for flexible prior choices and in modeling the transmission dynamics of various pathogens by accurately capturing the changing trend of viral effective reproductive number.

In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.

BACKGROUND: Our previous study established a 2-dose regimen of high-dose trivalent influenza vaccine (HD-TIV) to be immunogenically superior compared to a 2-dose regimen of standard-dose quadrivalent influenza vaccine (SD-QIV) in pediatric allogeneic hematopoietic cell transplant (HCT) recipients. However, the durability of immunogenicity and the role of time post-HCT at immunization as an effect modifier are unknown. METHODS: This phase II, multi-center, double-blinded, randomized controlled trial compared HD-TIV to SD-QIV in children 3-17 years old who were 3-35 months post-allogeneic HCT, with each formulation administered twice, 28-42 days apart. Hemagglutination inhibition (HAI) titers were measured at baseline, 28-42 days following each dose, and 138-222 days after the second dose. Using linear mixed effects models, we estimated adjusted geometric mean HAI titer ratios (aGMR: HD-TIV/SD-QIV) to influenza antigens. Early and late periods were defined as 3-5 and 6-35 months post-HCT, respectively. RESULTS: During 3 influenza seasons (2016-2019), 170 participants were randomized to receive HD-TIV (n = 85) or SD-QIV (n = 85). HAI titers maintained significant elevations above baseline for both vaccine formulations, although the relative immunogenic benefit of HD-TIV to SD-QIV waned during the study. A 2-dose series of HD-TIV administered late post-HCT was associated with higher GMTs compared to the early post-HCT period (late group: A/H1N1 aGMR = 2.16, 95% confidence interval [CI] = [1.14-4.08]; A/H3N2 aGMR = 3.20, 95% CI = [1.60-6.39]; B/Victoria aGMR = 1.91, 95% CI = [1.01-3.60]; early group: A/H1N1 aGMR = 1.03, 95% CI = [0.59-1.80]; A/H3N2 aGMR = 1.23, 95% CI = [0.68-2.25]; B/Victoria aGMR = 1.06, 95% CI = [0.56-2.03]). CONCLUSIONS: Two doses of HD-TIV were more immunogenic than SD-QIV, especially when administered ≥6 months post-HCT. Both groups maintained higher titers compared to baseline throughout the season. CLINICAL TRIALS REGISTRATION: NCT02860039.

Aim: Mineralo-organic particles, naturally present in human body fluids, participate in ectopic calcification and inflammatory diseases. These particles coexist with influenza A virus (IAV) in the same microenvironment during viral infection. Our objective was to investigate the functional consequences of the potential interactions between these particles and the virions. Materials & methods: We used in vitro models, including electron microscopy, fluorescence microscopy, hemagglutination assay and viral infection assays to examine the interactions. Results: Mineralo-organic particles bind to IAV virions through interactions involving particle-bound fetuin-A and mineral content, effectively engaging viral hemagglutinin. These interactions result in hindered viral infection. Conclusion: These findings uncover the novel interactions between mineralo-organic particles and IAV, highlighting the impact of virus microenvironment complexity. Article highlights In vitro mineralo-organic particles, produced in controlled settings, enabled study of particle−virion interactions with physiological implications. These particles are capable of binding to influenza A virus and reduce infection in mammalian cells. Mineralo-organic particles exhibit the ability to inhibit influenza A virus-induced hemagglutination and disrupt viral attachment to cells by engaging in interactions between particle-associated fetuin-A and viral HA. Mineralo-organic particles are capable of engaging with virions, thereby presenting a particle-mediated host factor that influences infectious disease outcomes. [ABSTRACT FROM AUTHOR]

Ageing is a major risk factor that contributes to increased mortality and morbidity rates during influenza A virus (IAV) infections. Macrophages are crucial players in the defense against viral infections and display impaired function during ageing. However, the impact of ageing on macrophage function in response to an IAV infection remains unclear and offers potential insight for underlying mechanisms. In this study, we investigated the immune response of young and aged human monocyte-derived macrophages to two different H1N1 IAV strains. Interestingly, macrophages of aged individuals showed a lower interferon response to IAV infection, resulting in increased viral load. Transcriptomic data revealed a reduced expression of stimulator of interferon genes (STING) in aged macrophages albeit the cGAS-STING pathway was upregulated. Our data clearly indicate the importance of STING signaling for interferon production by applying a THP-1 STING knockout model. Evaluation of mitochondrial function during IAV infection revealed the release of mitochondrial DNA to be the activator of cGAS-STING pathway. The subsequent induction of apoptosis was attenuated in aged macrophages due to decreased STING signaling. Our study provides new insights into molecular mechanisms underlying age-related immune impairment. To our best knowledge, we are the first to discover an age-dependent difference in gene expression of STING on a transcriptional level in human monocyte-derived macrophages possibly leading to a diminished interferon production. [ABSTRACT FROM AUTHOR]

Viroporins possess important potential as antiviral targets due to their critical roles during virus life cycles, spanning from virus entry to egress. Although the antiviral amantadine targets the M2 viroporin of influenza A virus, successful progression of other viroporin inhibitors into clinical use remains challenging. These challenges relate in varying proportions to a lack of reliable full‐length 3D‐structures, difficulties in functionally characterising individual viroporins, and absence of verifiable direct binding between inhibitor and viroporin. This review offers perspectives to help overcome these challenges. We provide a comprehensive overview of the viroporin family, including their structural and functional features, highlighting the moldability of their energy landscapes and actions. To advance the field, we suggest a list of best practices to aspire towards unambiguous viroporin identification and characterisation, along with considerations of potential pitfalls. Finally, we present current and future scenarios of, and prospects for, viroporin targeting drugs. [ABSTRACT FROM AUTHOR]

Pathogen encounter can result in epigenetic remodeling that shapes disease caused by heterologous pathogens. Here, we examined innate immune memory in the context of commonly circulating respiratory viruses. Single-cell analyses of airway-resident immune cells in a disease-relevant murine model of SARS-CoV-2 recovery revealed epigenetic reprogramming in alveolar macrophages following infection. Post-COVID-19 human monocytes exhibited similar epigenetic signatures. In airway-resident macrophages, past SARS-CoV-2 infection increased activity of type I interferon (IFN-I)-related transcription factors and epigenetic poising of antiviral genes. Viral pattern recognition and canonical IFN-I signaling were required for the establishment of this innate immune memory and augmented secondary antiviral responses. Antiviral innate immune memory mounted by airway-resident macrophages post-SARS-CoV-2 was necessary and sufficient to ameliorate secondary disease caused by influenza A virus and curtailed hyperinflammatory dysregulation and mortality. Our findings provide insights into antiviral innate immune memory in the airway that may facilitate the development of broadly effective therapeutic strategies. [Display omitted] • Airway-resident macrophages (AMF) establish durable epigenetic memory post SARS2 • Chromatin loci of ISGs retain increased accessibility and are decorated with H3K4me1 • AMFs hyper-induce ISGs upon secondary antiviral recall responses • Innate immune memory in AMF ameliorates secondary influenza A virus disease The therapeutic potential of antigen-independent innate immune memory (IIM) is of particular relevance in the context of respiratory viruses with pandemic potential. Lercher et al. find that antiviral IIM in alveolar macrophages following SARS-CoV-2 infection ameliorates disease caused by a secondary unrelated pathogen, influenza A virus. [ABSTRACT FROM AUTHOR]

Background: Aggregated samples such as oral fluids (OFs) display an animal friendly and time and cost-efficient sample type for swine Influenza A virus (swIAV) monitoring. However, further molecular and biological characterization of swIAV is of particular significance. The reportedly inferior suitability of aggregated samples for subtyping of swIAV presents a major drawback compared to nasal swabs, still considered the most appropriate sample type for this purpose (Garrido-Mantilla et al. BMC Vet Res 15(1):61, 2019). In addition, the viral load in the original sample, storage conditions and characteristics of different swIAV strains might further compromise the eligibility of aggregated samples for molecular detection and subtyping. Therefore, the present study aimed to evaluate the suitability of stabilizing media to minimize the degradation of viral RNA and thus increase the detection and subtyping rate of swIAV by RT-qPCR in spiked OFs under different conditions (virus strain, storage temperature and viral load in the original sample) over a time span of 14 days. Results: The use of stabilizing media in spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to OFs without stabilizers (OR = 46.1, p < 0.001). In addition, swIAV degradation over time was significantly reduced in samples suspended with stabilizer (OR = 5.80, p < 0.001), in samples stored at 4 °C (OR = 2.53, p < 0.001) and in samples spiked with the avian derived H1N2 subtype (OR = 2.26, p < 0.01). No significant differences in swIAV RNA detection and degradation of swIAV RNA in spiked OFs over time were observed between the three different stabilizing media. Conclusion: Addition of stabilizers and storage of samples under cooled conditions significantly improved detection and subtyping of swIAV in spiked OFs. [ABSTRACT FROM AUTHOR]

The present study aimed to determine the dynamics of influenza A virus (IAV) infection in two endemically infected farms (F1 and F2), where a longitudinal follow-up of piglets was performed from birth to 8–12 weeks of age. During the study, a highly virulent isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was introduced on both farms. This allowed us to examine the impact of such introduction on the patterns of infection, disease, and the antibody response of pigs to IAV infection. The introduction of the new PRRSV strain coincided with a change in the dynamics of IAV infection on both farms. In F1, the cumulative incidence of IAV increased from 20% before the outbreak to 67.5%, together with the existence of animals that tested positive for IAV (RT‒qPCR) in nasal swabs for two or more consecutive samples. In F2, the cumulative incidence of IAV increased from 50% before the PRRSV outbreak to 70%, and the proportion of prolonged IAV shedders increased sharply. Additionally, some animals were infected with the same IAV twice during the observation period. In contrast to previous reports, our study revealed that prolonged shedding was not related to the titres of maternally derived antibodies at the time of infection but was significantly (p < 0.05) related to PRRSV infection status. Notably, both before and after the PRRSV outbreak, a high proportion of IAV-infected piglets did not seroconvert, which was significantly (p < 0.05) related to the hemagglutination inhibition titres against IAV when infected. [ABSTRACT FROM AUTHOR]

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals. Synopsis: The cryo-EM structure of the influenza A virus Pol-NS2 complex shows a highly ordered barrel-like hexamer. This study explains how the NS2-FluAPol interaction inhibits viral transcription and promotes genome replication. The structure of the full-length NS2 as part of the FluPol-NS2 complex is resolved at ~3.0 Å. NS2 forms a pseudo two-fold domain-swapped dimer. The binding of NS2 to FluAPol impairs FluAPol transcriptase activity by hampering FluAPol-Pol II CTD interactions. NS2 stalls FluAPol in the replicase conformation. The cryo-EM structure of the influenza A virus Pol-NS2 complex shows a highly ordered barrel-like hexamer. This study explains how the NS2-FluAPol interaction inhibits viral transcription and promotes genome replication. [ABSTRACT FROM AUTHOR]

There is increasing evidence that thirdhand exposure to e-cigarette vapor (e-vapor) can have detrimental effects on the lungs. However, whether maternal exposure during pregnancy results in harmful changes to the offspring is unknown. Using two different e-cigarette settings (low vs. high power), BALB/c mice were subjected to thirdhand e-vapor (e-vapor deposited onto towels, towels changed daily) in the absence or presence of nicotine, before, during, and after pregnancy. Male adult offspring were then infected with mouse-adapted influenza A virus (A/PR/8/34 H1N1; Flu) and lung and bone marrow immune cell responses were assessed 7 days postinfection. Maternal thirdhand exposure to low-power (MLP) or high-power (MHP) e-vapor with nicotine (MLP + NIC and MHP + NIC, respectively) increased the percentage of lung immune cells and neutrophils in the bone marrow. Interestingly, Flu-infected offspring from MLP + NIC and MHP + NIC groups had lower percentages of lung alveolar macrophages and more pronounced increases in neutrophils in the bone marrow, when compared with offspring from MSham Flu controls. Flu infection also decreased the percentage of lung CD4+ T cells and increased the percentage of lung CD8+ T cells, irrespective of maternal exposure (MLP −/+ NIC and MHP −/+ NIC). Significantly, both MLP + NIC and MHP + NIC resulted in blunted activation of lung CD4+ T cells, but only MLP + NIC caused blunted activation of lung CD8+ T cells. Together, we show for the first time that maternal thirdhand exposure to e-vapor results in significant, long-lived effects on lung and bone marrow immune cell responses in offspring at baseline and response to Flu infection. NEW & NOTEWORTHY: Maternal exposure to environmental residues of e-cigarette use has significant effects on immune cell responses in the lungs and bone marrow of offspring at both baseline and in response to influenza A virus (Flu) infection. [ABSTRACT FROM AUTHOR]

Prim-O-glucosylcimifugin (POG) is a chromone derived primarily from Saposhnikovia divaricata (Turcz) Schischk and Cimicifuga simplex. Previous research has shown that POG possesses antibacterial, anticancer, anti-inflammatory, antioxidant, anticonvulsant, antipyretic, and analgesic properties. However, the specific impact of POG on influenza-virus-induced pneumonia is not well understood. In this study, we investigated the protective effects and underlying mechanisms of POG in pneumonia caused by influenza A virus (IAV). In vitro, POG was found to have a protective effect against infections caused by the respiratory viruses respiratory syncytial virus (RSV), human coronavirus OC43, and influenza A virus. POG inhibited A/FM/1/1947(H1N1) infection with an EC50 ranging from 3.01 to 10.43 in vitro. Intraperitoneal infection of mice with POG at a dose of 5 or 10 mg/kg resulted in a reduction in IAV-induced pneumonia, as evidenced by decreased pulmonary edema, improved lung histopathology, and reduced inflammatory cell accumulation. At the higher dose (10 mg/kg), POG treatment significantly increased survival rates, decreased viral titres in the lungs, improved lung histology, and reduced lung inflammation in IAV-infected mice. POG also effectively alleviated pulmonary fibrosis by reducing the levels of fibrotic markers (hydroxyproline [Hyp] and transforming growth factor β1 [TGF-β1]) and suppressing the expression of alpha smooth muscle actin (α-SMA), p focal adhesion kinase (p-FAK), and TGF-β1 in lung tissues. In addition, POG inhibited the expression of the RELA proto-oncogene (RELA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), and mitogen- and stress-activated protein kinase 2 (MSK2) in lung tissues. These results indicate that POG may have a protective effect against IAV-induced pneumonia by downregulating the TGF-β1/PI3KCD/MSK2/RELA signalling pathway in the lungs. [ABSTRACT FROM AUTHOR]

Viral infectious diseases are spread worldwide. Search for natural antiviral therapy from natural resources to overcome the serious drawbacks of synthetic chemicals is imperative. The antiviral activity against influenza virus subtype H9N2, Herpes simplex II (HSV II), Adenovirus (AdVs), and Newcastle virus (NDV) was assessed using the cytopathic effect (CPE) inhibition assay. Phytochemical investigation of Eriocephalus africanus var. africanus aerial parts led to the isolation and identification of bioactive compounds including a mixture of aromatic hydrocarbons 1 , 3 β -friedelinol 2 , eicosanol 3 , 1-hexadecanol 4 , β -sitosterol 5 , undecanol 6 , pectolinarigenin 7 , cirsilineol 8 , chrysoeriol 9 , kaempferol 10 , tricin 11 , luteolin 12 , 2,4- dihydroxyacetophenone 13 , ferulic acid 14 , β -sitosterol glucoside 15 , afzelin 16 , hesperidin 17 , kaempferitrin 18 , d-(+)–pinitol 19 and 1,5–dimethyl citrate 20. The antiviral assessment revealed promising activity for the total extract against H9N2, HSV II, NDV, and AdVs with selectivity index (SI) = 12.4, 20.8, 27.4, and 12.9, respectively. The Petroleum ether fraction (PE) had promising anti-H9N2, NDV and the adenovirus with SI = 12.1, 17.0, and 19.1, respectively. Molecular modeling study proved promising activity for β -sitosterol with H9N2 (ΔG = -7.17 Kcal/mol), kaempferitrin with HSV-II (ΔG = -7.64 Kcal/mol), 3 β -friedelinol with AdVs (ΔG = -7.63 kcal/mol), and pectolinarigenin with NDV (ΔG = -7.55 Kcal/mol.). The top 4 candidates showed promising druglikeness and drug-score with no potential toxicity except for pectolinarigenin and kaempferitrin. They showed low to high probability for intestinal absorption, no BBB permeation with zero PAINS alerts and ease for synthetic accessibility. This may establish the promising potential of such compounds for further development into clinical candidates. [Display omitted] • Chromatographic isolation and spectroscopic identification of the bioactive compounds from the aerial parts of Eriocephalus africanus var. africanus cultivated in Egypt. • In silico and in vitro antiviral activity for Eriocephalus africanus total methanol extract as well as other solvent fractions together with the previously isolated essential oil against influenza a virus subtype H9N2, Herpes simplex II, Adeno virus and Newcastle virus. • Estimation of ADME/Tox for isolated compounds. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza A(H5N1) detected in dairy cows raises concerns about milk safety. The effects of pasteurization-like temperatures on influenza viruses in retail and unpasteurized milk revealed virus resilience under certain conditions. Although pasteurization contributes to viral inactivation, influenza A virus, regardless of strain, displayed remarkable stability in pasteurized milk. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) vaccines have revolutionized the fight against infectious diseases and are poised to transform other therapeutic areas. Lipid nanoparticles (LNP) represent the most successful delivery system for mRNA. While the mRNA-LNP products currently in clinics are stored as frozen suspensions, there is evidence that freeze-drying mRNA-LNP into dry powders can potentially enable their storage and handling at non-freezing temperatures. Previously, we successfully applied thin-film freeze-drying (TFFD) to transform a polyadenylic acid [poly(A)]-LNP formulation from a liquid suspension to dry powders. The poly(A)-LNP were structurally multilamellar spheres without blebs, but the mRNA vaccines in clinics are comprised of mRNA-LNP that are structurally spheres surrounded by a unilamellar lipid bilayer, with some containing blebs, and it was reported that the presence of blebs increases the sensitivity of mRNA-LNP to freeze-drying-induced stress. In the present study, using an influenza A virus hemagglutinin (HA) mRNA in LNP that were structurally similar to that in the COVID-19 mRNA vaccines currently in clinic, we studied the effect of TFFD on the physical properties, internal structure, as well as immunogenicity of the HA mRNA-LNP vaccine. We concluded that TFFD can be utilized to prepare dry powders of the HA mRNA-LNP, but a sufficient amount of excipients were needed to minimize changes in the physical properties, structure, and immunogenicity of the HA mRNA-LNP vaccine. [Display omitted] • The HA mRNA-LNP vaccine used in this study contained nanoparticles with unilamellar lipid bilayer, some with blebs. • TFFD was used to transform the HA mRNA-LNP vaccine from liquid to dry powders while maintaining its immunogenicity. • Sufficient excipients were needed to minimize the effect of the freeze-drying stress on the HA mRNA-LNP. [ABSTRACT FROM AUTHOR]

Seasonal influenza A virus (IAV), particularly the H1N1 subtype, poses a significant public health threat because of its substantial morbidity and mortality rates worldwide. Understanding the immune response to H1N1 is crucial for developing effective treatments and vaccines. In this study, we deciphered the single-cell transcriptomic landscape of peripheral blood mononuclear cells (PBMCs) from H1N1-infected humans and lung tissue samples from H1N1-infected mice by mining HIN1-related single-cell RNA sequencing data from the GEO database. We observed similar changes in immune cell composition following H1N1 infection, with an increase in macrophages but a decrease in T cells in both species. Moreover, significant transcriptional changes in bystander immune cells upon H1N1 infection were identified, with the upregulation of the chemokine CCL2 in human PBMCs and increased expression of interferon-stimulated genes such as Ifit3, Ifit1 and Isg15 in mouse pulmonary immune cells. Intercellular cross-talk analysis highlighted enhanced interactions among bystander immune cells during H1N1 infection, with neutrophils in humans and macrophages in mice showing the most remarkable increases in interaction intensity. Transcription factor analysis revealed the conserved upregulation of key antiviral regulons, including STAT1 and IRF7, in T cells across both species, highlighting their pivotal roles in antiviral defense. These results suggest that humans and mice exhibit common immune responses to H1N1 infection, underscoring the similarity of vital immune mechanisms across species. The conserved immune mechanisms identified in this study provide potential therapeutic targets for enhancing antiviral immunity. Our research underscores the importance of understanding species-specific and conserved immune responses to H1N1 and offers insights that could inform the development of novel antiviral therapies and improve clinical outcomes for individuals affected by influenza. [ABSTRACT FROM AUTHOR]

We describe a novel biosafety aerosol chamber equipped with state-of-theart instrumentation for bubble-bursting aerosol generation, size distribution measurement, and condensation-growth collection to minimize sampling artifacts when measuring virus infectivity in aerosol particles. Using this facility, we investigated the effect of relative humidity (RH) in very clean air without trace gases (except ~400 ppm CO2) on the preservation of influenza A virus (IAV) infectivity in saline aerosol particles. We characterized infectivity in terms of 99%-inactivation time, t99, a metric we consider most relevant to airborne virus transmission. The viruses remained infectious for a long time, namely t99 > 5 h, if RH < 30% and the particles effloresced. Under intermediate conditions of humidity (40% < RH < 70%), the loss of infectivity was the most rapid (t99 ∾ 15-20 min, and up to t99 ∾ 35 min at 95% RH). This is more than an order of magnitude faster than suggested by many previous studies of aerosol-borne IAV, possibly due to the use of matrices containing organic molecules, such as proteins, with protective effects for the virus. We tested this hypothesis by adding sucrose to our aerosolization medium and, indeed, observed protection of IAV at intermediate RH (55%). Interestingly, the t99 of our measurements are also systematically lower than those in 1-mL droplet measurements of organic-free saline solutions, which cannot be explained by particle size effects alone. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies are an effective method of treating acute infectious diseases. However, knowing which of the produced antibodies in the vast number of human antibodies can cure the disease requires a long time and advanced technology. The previously introduced iBRAB method relies on studied antibodies to design a broad-spectrum antibody capable of neutralizing antigens of many different Influenza A viral strains. To evaluate the antigen-binding fragment as an applicable drug, the therapeutic antibody profiles providing guidelines collected from clinically staged therapeutic antibodies were used to access different measurements. Although the evaluated values were within an accepted range, the modification in the amino acid sequence is required for better properties. Thus, using the steered molecular dynamics (SMD) simulation to determine the binding capacity of amino acids in the functional region, the profile of interacted amino acids of Fab with the antigen was established for modified reference. As a result, the model was modified with amino acids elimination at positions 96–97 in the heavy chain and 26–27, 91, 96–97, and 102–103 in the light chain, which has better Therapeutic Antibody Profiler evaluations than the original designation. Thus again, SMD simulation is a promising computational approach for post-modification in rational drug design. [ABSTRACT FROM AUTHOR]

Ferroptosis is a novel form of cell death caused by the accumulation of lipid peroxides in an iron‐dependent manner. However, the precise mechanism underlying the exploitation of ferroptosis by influenza A viruses (IAV) remains unclear. The results demonstrate that IAV promotes its own replication through ferritinophagy by sensitizing cells to ferroptosis, with hemagglutinin identified as a key trigger in this process. Hemagglutinin interacts with autophagic receptors nuclear receptor coactivator 4 (NCOA4) and tax1‐binding protein 1 (TAX1BP1), facilitating the formation of ferritin‐NCOA4 condensates and inducing ferritinophagy. Further investigation shows that hemagglutinin‐induced ferritinophagy causes cellular lipid peroxidation, inhibits aggregation of mitochondrial antiviral signaling protein (MAVS), and suppresses the type I interferon response, thereby contributing to viral replication. Collectively, a novel mechanism by which IAV hemagglutinin induces ferritinophagy resulting in cellular lipid peroxidation, consequently impairing MAVS‐mediated antiviral immunity, is revealed. [ABSTRACT FROM AUTHOR]

Background: Fei-Yan-Qing-Hua decoction (FYQHD) is derived from the well-known Ma Xing Shi Gan decoction, which was documented in Zhang Zhong Jing’s “Treatise on Exogenous Febrile Disease” during the Han Dynasty. Although FYQHD has been used in the treatment of pneumonia and has demonstrated clinical efficacy for decades, the underlying mechanism by which FYQHD protects against influenza virus infection through modulation of gut flora remains unclear. Here, we examined the regulatory impacts of FYQHD on an influenza virus-infected mouse model and explored the mechanisms involved. Methods: An infectious mouse model was created by intranasal instillation of influenza A virus (IAV). The effectiveness of FYQHD was assessed through various measures, including weight loss, lung wet/dry ratio, oxidative stress levels, viral load in lung tissues, and intestinal injuries. Changes in gut microbiota and SCFA production were also examined. Results: The results showed that FYQHD significantly reduced viral load, increased the production of type I interferon (IFN-I), and restored the integrity of the intestinal barrier following IAV challenge. Additionally, FYQHD significantly corrected the dysbiosis of gut microbiota induced by influenza virus infection, enhancing the abundance of SCFA-producing bacteria and acetate production. However, the depletion of gut microbiota significantly attenuated the protective effects of FYQHD against influenza virus infection. In vitro, the antiviral effect of acetate was demonstrated through the upregulation of concentrations of IFN-β. Conclusion: FYQHD attenuates influenza virus-induced lung and intestinal injuries by boosting the host antiviral response through increasing the abundance of Lachnospiraceae_NK4A136 and Roseburia, along with elevated acetate levels. The study advances our understanding of the therapeutic mechanisms of FYQHD and provides a theoretical basis for the application of FYQHD in the treatment of influenza. [ABSTRACT FROM AUTHOR]

Introduction: The influenza virus is recognized as the primary cause of human respiratory diseases, with the current influenza vaccine primarily offering strainspecific immunity and limited protection against drifting strains. Considering this, the development of a broad-spectrum influenza vaccine capable of inducing effective immunity is considered the future direction in combating influenza. Methods: The present study proposes a novel mRNA-based multi-epitope influenza vaccine, which combines three conserved antigens derived from the influenza A virus. The antigens consist of M2 ion channel's extracellular domain (M2e), the conserved epitope of located in HA2 of hemagglutinin (H1, H3, B), and HA1 of hemagglutinin. At the same time, trimeric sequences and ferritin were conjugated separately to investigate the immune effects of antigen multivalent presentation. Results: Immunization studies conducted on C57BL/6 mice with these vaccines revealed that they can elicit both humoral immunity and CD4+ and CD8+ T cell responses, which collectively contribute to enhancing cross-protective effects. The virus challenge results showed that vaccinated groups had significantly reduced lung damage, lower viral loads in the lungs, nasal turbinates, and trachea, as well as decreased levels of pro-inflammatory cytokines. Conclusion: These findings clearly demonstrate the wide range of protective effects provided by these vaccines against H1N1 and B influenza viruses. The present finding highlights the potential of mRNA-based influenza vaccines encoding conserved proteins as a promising strategy for eliciting broadspectrum protective humoral and cellular immunity against H1N1 and B influenza viruses. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) is known for causing seasonal epidemics ranging from flu to more severe outcomes like pneumonia, cytokine storms, and acute respiratory distress syndrome. The innate immune response and inflammasome activation play pivotal roles in sensing, preventing, and clearing the infection, as well as in the potential exacerbation of disease progression. This study examines the complex relationships between donor-specific characteristics and cytokine responses during H3N2 IAV infection using an ex vivo model. At 24 h post infection in 31 human lung explant tissue samples, key cytokines such as interleukin (IL)-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) were upregulated. Interestingly, a history of lung cancer did not impact the acute immune response. However, cigarette smoking and programmed death-ligand 1 (PD-L1) expression on macrophages significantly increased IL-2 levels. Conversely, age inversely affected IL-4 levels, and diabetes mellitus negatively influenced IL-6 levels. Additionally, both diabetes mellitus and programmed cell death protein 1 (PD-1) expression on CD3+/CD4+ T cells negatively impacted TNF-α levels, while body mass index was inversely associated with IFN-γ production. Toll-like receptor 2 (TLR2) expression emerged as crucial in mediating acute innate and adaptive immune responses. These findings highlight the intricate interplay between individual physiological traits and immune responses during influenza infection, underscoring the importance of tailored and personalized approaches in IAV treatment and prevention. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) can cause seasonal epidemics and global pandemics, posing serious threats to public health, making a deeper understanding of its biological characteristics and effective countermeasure strategies essential. Autophagy not only maintains cellular homeostasis but also plays an important role in host defense against IAV infection. However, the relationship between IAV and autophagy is complex, and effective antiviral drugs are not yet available. Natural products have shown excellent potential in disease control due to their diversity and multi-targeting. This review focuses on the relationship between IAV and autophagy and discusses the potential of targeting autophagic pathways for the development of new antiviral therapies. Particularly, the use of plant extracts as autophagy modulators has garnered attention due to their non-toxic nature and cost-effectiveness, which provides strong support for the development of future antiviral drugs that can help to inhibit viral infections and slow down disease progression. [ABSTRACT FROM AUTHOR]

Melanin concentrating hormone (MCH) controls many brain functions, such as sleep/wake cycle and memory, and modulates the inflammation response. Previous studies have shown that influenza A virus (IAV) infection-induced neuroinflammation leads to central nervous damage. This study investigated the potential effects of MCH against neuroinflammation induced by IAV infection and its mechanism. MCH (1 and 2 mg/ml) was administrated for 5 consecutive days before IAV infection. Pentobarbital-induced sleep tests, an open-field test, and a Morris water maze were performed to measure sleep quality, spatial learning and memory ability. Neuronal loss and microglial activation were observed with Nissl staining and immunofluorescence assay. The levels of inflammatory cytokines and the expression of the JNK/ERK signaling pathway were examined by ELISA and western blot. IAV infection led to poor sleep quality, impaired the ability of spatial learning and memory, caused neuronal loss and microglial activation in mice's hippocampus and cortex. Meanwhile the level of inflammatory cytokines increased, and the JNK/ERK signaling pathway was activated after IAV infection. MCH administration significantly alleviated IAV-induced neuroinflammation, cognitive impairment, and sleep disorder, decreased the levels of inflammatory cytokines, and inhibited neuronal loss and microglial activation in the hippocampus and cortex by regulating the JNK/ERK signaling pathway. Therefore, MCH alleviated the neuroinflammation, spatial learning and memory impairment, and sleep disorder in IAV-infected mice by regulating the JNK/ERK signaling pathway. Highlights: IAV infection could cause the sleep disorder, impairment of the spatial learning and memory. IAV infection could cause the neuronal loss and activation of microglia, increase the inflammatory cytokines to result in neuroinflammation. MCH alleviated the cognitive impairment and sleep disorder in IAV-infected mice. MCH inhibited the neuron damage, microglia activation, and inflammatory cytokines expression, to alleviate the neuroinflammation in IAV-infected mice. MCH alleviated the neruoinflammation in IAV-infected mice by regulating the JNK/ERK signaling pathway. [ABSTRACT FROM AUTHOR]

We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018–2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020–2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country. [ABSTRACT FROM AUTHOR]

Background: Influenza A viruses pose a significant public health threat globally and are characterized by rapid evolution of the hemagglutinin (HA) gene causing seasonal epidemics. The aim of this study was to investigate the evolutionary dynamics of A(H3N2) circulating in Bhutan during 2022 and 2023. Methods: We analysed 166 whole‐genome sequences of influenza A(H3N2) from Bhutan, obtained from the GISAID database. We employed a Bayesian Markov Chain Monte Carlo (MCMC) framework, with a curated global dataset of HA sequences from regions with significant migration links to Bhutan. Phylogenetic, temporal, and phylogeographic analyses were conducted to elucidate the evolutionary dynamics and spatial dissemination of the viruses. Results: Our phylogenetic analysis identified the circulation of influenza A(H3N2) Clade 3C.2a1b.2a.2 in Bhutan during 2022 and 2023, with viruses further classified into three subclades: 2a.3 (39/166), 2a.3a.1 (58/166) and 2a.3b (69/166). The TMRCA estimates suggest that these viral lineages originated approximately 1.93 years prior to their detection. Phylogeographic analysis indicates introductions from the United States in 2022 and Australia in 2023. The mean evolutionary rate across all gene segments was calculated to be 4.42 × 10−3 substitutions per site per year (95% HPD: 3.19 × 10−3 to 5.84 × 10−3), with evidence of purifying selection and limited genetic diversity. Furthermore, reassortment events were rare, with an estimated rate of 0.045 events per lineage per year. Conclusion: Our findings show that primary forces shaping the local evolution of the influenza A(H3N2) in Bhutan are largely stochastic, with only sporadic instances of adaptive change, and thus underscore the importance of continuous surveillance to mitigate the impact of evolving strains. [ABSTRACT FROM AUTHOR]

Simple Summary: The influenza virus is a global problem for humanity due to its high variability. Research is ongoing worldwide to develop a so-called universal vaccine to control this infection. Chimeric proteins are one of the platforms for the development of such a vaccine. Within this platform, the least variable proteins of the influenza virus are used so that the vaccine can protect against many strains. In this work, we investigated the possibility of enhancing the efficacy of a candidate vaccine protein by introducing an additional fragment of influenza virus into the construct. Antigen-specific antibody and T-cell immune responses in mice to vaccination were evaluated. Animals were challenged with a lethal dose of influenza viruses to assess the efficacy of the candidate vaccine proteins. It was found that the introduction of new conserved antigens into the construct affects the formation of antibodies to other vaccine antigens but does not affect the protective efficacy of the candidate protein. Conserved influenza virus proteins, such as the hemagglutinin stem domain (HA2), nucleoprotein (NP), and matrix protein (M), are the main targets in the development of universal influenza vaccines. Previously, we constructed a recombinant vaccine protein Flg-HA2-2-4M2ehs containing the extracellular domain of the M2 protein (M2e) and the aa76–130 sequence of the second HA subunit as target antigens. It demonstrated immunogenicity and broad protection against influenza A viruses after intranasal and parenteral administration. This study shows that CD8+ epitopes of NP, inserted into a flagellin-fused protein carrying M2e and HA2, affect the post-vaccination immune humoral response to virus antigens without reducing protection. No differences were found between the two proteins in their ability to stimulate the formation of follicular Th in the spleen, which may contribute to a long-lasting antigen-specific humoral response. The data obtained on Balb/c mice suggest that the insertion of CTL NP epitopes into the flagellin-fused protein carrying M2e and HA2 reduces the antibody response to M2e and A/H3N2. In C57Bl6 mice, this stimulates the formation of NP-specific CD8+ Tem and virus-specific mono- and multifunctional CD4+ and CD8+ Tem in the spleen and completely protects mice from influenza virus subtypes A/H1N1pdm09 and A/H3N2. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) infection contributes to high annual morbidity and mortality, thus necessitating measures aimed at protecting against the disease. Alcohol-based disinfectants are commonly used to inactivate IAV, but they have several undesirable properties. In search of other means which would inactivate IAV, we focused on the effect of alkaline solutions on IAV. We found the viral infectivity remarkably decreased with treatment of an alkaline solution at pH 12.0 for 1 min, where destruction of the viral spikes was observed using an electron microscope. A more detailed examination revealed that the infectivity of IAV was remarkedly reduced by brief treatment with the alkaline solution at pH 11.75 or above, most likely due to the degradation of viral hemagglutinin protein. These results show that at a high pH, the haemagglutinin protein is degraded, resulting in very rapid inactivation of IAV. [ABSTRACT FROM AUTHOR]

Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (β), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAV) are dynamic and highly mutable respiratory pathogens that present persistent public health challenges. Inflammasomes, as components of the innate immune system, play a crucial role in the early detection and response to infections. They react to viral pathogens by triggering inflammation to promote immune defences and initiate repair mechanisms. While a strong response is necessary for early viral control, overactivation of inflammasomes can precipitate harmful hyperinflammatory responses, a defining characteristic observed during severe influenza infections. The Absent in Melanoma 2 (AIM2) inflammasome, traditionally recognised for its role as a DNA sensor, has recently been implicated in the response to RNA viruses, like IAV. Paradoxically, AIM2 deficiency has been linked to both enhanced and reduced vulnerability to IAV infection. This review synthesises the current understanding of AIM2 inflammasome activation during IAV and explores its clinical implications. Understanding the nuances of AIM2's involvement could unveil novel therapeutic avenues for mitigating severe influenza outcomes. [ABSTRACT FROM AUTHOR]

Influenza is an important zoonotic disease that persistently threatens global public health. While it is widely acknowledged that probiotics can modulate the host response to protect the host against infectious disease, the prophylactic efficacy on respiratory viral infection and the detailed mechanism remains elusive. Lactobacillus, the most commonly used probiotic widely applied in food production, has garnered significant attention. In our study utilizing both C57BL/6 and BALB/c mouse models, we explored the protective effect against two strains of influenza virus, A/Mink/China/01/2014(H9N2) and A/California/04/2009(H1N1), through the administration of Lactiplantibacillus plantarum strain 16 (L. plantarum 16) and Lacticaseibacillus rhamnosus strain P118 (L. rhamnosus P118), aiming to identify robust probiotic strains with antiviral properties. Our findings indicate that administering L. plantarum 16 or L. rhamnosus P118 alone does not provide sufficient protection against influenza. However, the co-administration of L. plantarum 16 and L. rhamnosus P118 dramatically reduces viral titers in the respiratory tract and lung, thereby markedly alleviating the clinical symptoms, improving prognosis, and reducing mortality. The mechanisms underlying this effect involve the modulation of host gut microbiota and metabolism through the co-administration of L. plantarum 16 and L. rhamnosus P118, resulting in enrichment of Firmicutes and enhancement of phenylalanine-related metabolism, ultimately leading to an augmentation of the antiviral immune response. Notably, we identified that the circulating metabolic molecule 2-Hydroxycinnamic acid plays a significant role in combating influenza. Our data suggest the potential utility of L. plantarum 16 and L. rhamnosus P118 two-bacterium or 2-Hydroxycinnamic acid in preventing influenza. [ABSTRACT FROM AUTHOR]

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) headfocused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability. [ABSTRACT FROM AUTHOR]

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirm ed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species. [ABSTRACT FROM AUTHOR]

Objective: To compare the epidemic trends of different types of influenza viruses and the clinical characteristics of patients, so as to provide reference for influenza prevention and control. Methods: This was descriptive research. The human monitoring data collected from the Influenza Research Database (IRD) from 2006 to 2016 were used to descriptively analyze the distribution of influenza viruses in terms of time, geography, gender and age. The positive samples were divided into three groups based on the type of pathogen (H1N1 influenza A viruses, H3N2 influenza A viruses, and influenza B viruses). Compared and analyzed the distribution and clinical characteristics among groups. Results: There were statistically significant differences in the positive rates among different countries (p< 0.001). The proportion of positive samples gradually decreased with age. The proportion of oseltamivir resistance was significantly higher in H1N1-positive patients compared with that in H3N2-positive patients (p< 0.001). Significant differences were observed in the vaccination status among H1N1, H3N2 and influenza B viruses (p< 0.001). Cough was common in all cases with H1N1, H3N2 and influenza B infections, while cough, fever and running nose occurred more frequently in influenza B-positive cases than those of H1N1-positive and H3N2-positive cases (p< 0.001). Conclusion: People aged 0-18 years are the major susceptible population to influenza, and H1N1 influenza viruses are the main pathogens of infection in this population, with major clinical manifestations of fever, cough and headache. The findings in this study highlight the necessity to strengthen the protection for this age group in clinical practice. [ABSTRACT FROM AUTHOR]

Studying influenza A viruses (IAVs) requires secondary experimental procedures to detect the presence of the virus in infected cells or animals. The ability to generate recombinant (r)IAV using reverse genetics techniques has allowed investigators to generate viruses expressing foreign genes, including fluorescent and luciferase proteins. These rIAVs expressing reporter genes have allowed for easily tracking viral infections in cultured cells and animal models of infection without the need for secondary approaches, representing an excellent option to study different aspects in the biology of IAV where expression of reporter genes can be used as a readout of viral replication and spread. Likewise, these reporter-expressing rIAVs provide an excellent opportunity for the rapid identification and characterization of prophylactic and/or therapeutic approaches. To date, rIAV expressing reporter genes from different viral segments have been described in the literature. Among those, rIAV expressing reporter genes from the viral NS segment have been shown to represent an excellent option to track IAV infection in vitro and in vivo, eliminating the need for secondary approaches to identify the presence of the virus. Here, we summarize the status on rIAV expressing traceable reporter genes from the viral NS segment and their applications for in vitro and in vivo influenza research. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) infection while primarily affecting the lungs, is often associated with cardiovascular complications. However, the mechanisms underlying this association are not fully understood. Here, we investigated the potential role of FBXL19, a member of the Skp1–Cullin-1–F-box family of E3 ubiquitin ligase, in IAV-induced cardiac inflammation. We demonstrated that FBXL19 overexpression in endothelial cells (ECs) reduced viral titers and IAV matrix protein 1 (M1) levels while increasing antiviral gene expression, including interferon (IFN)-α, -β, and -γ and RANTES (regulated on activation normal T cell expressed and secreted) in the cardiac tissue of IAV-infected mice. Moreover, EC-specific overexpression of FBXL19 attenuated the IAV infection-reduced interferon regulatory factor 3 (IRF3) level without altering its mRNA level and suppressed cardiac inflammation. Furthermore, IAV infection triggered cellular senescence programs in the heart as indicated by the upregulation of p16 and p21 mRNA levels and the downregulation of lamin-B1 levels, which were partially reversed by FBXL19 overexpression in ECs. Our findings indicate that EC-specific overexpression of FBXL19 protects against IAV-induced cardiac damage by enhancing interferon-mediated antiviral signaling, reducing cardiac inflammation, and suppressing cellular senescence programs. NEW & NOTEWORTHY: Our study reveals a novel facet of IAV infection, demonstrating that it can trigger cellular senescence within the heart. Intriguingly, upregulation of endothelial FBXL19 promotes host innate immunity, reduces cardiac senescence, and diminishes inflammation. These findings highlight the therapeutic potential of targeting FBXL19 to mitigate IAV-induced cardiovascular complications. [ABSTRACT FROM AUTHOR]

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket. Modulation of calcium binding in the neuraminidase low-affinity calcium-binding pocket suggests a novel role of calcium during cross-species transmission of Influenza A viruses. [ABSTRACT FROM AUTHOR]

Influenza A virus outbreaks pose a significant burden to global health. However, detecting and subtyping the virus is challenging due to its high mutation rate, and it is difficult to distinguish viable viruses that cause infection from clinical samples. Here is a novel PMAxx-LAMP assay for the classification of viable and non-viable Influenza A virus together with differentiation between H1N1 and H3N2 subtypes. Our loop-mediated isothermal amplification (LAMP) primers displayed limits of detection of 102 copies/μL for both subtypes, high specificity, reproducibility, and good linearity with correlation coefficients of 0.9762 and 0.9901 for H1N1 and H3N2, respectively. PMAxx effectively distinguished between viable and non-viable viruses at an optimal concentration of 10 μM. In the LAMP method, the clinical specificity and sensitivity for the H1N1 subtype were both 100%, while for the H3N2 subtype, they were 94.44 and 92.31%, respectively. With the PMAxx-LAMP assay, the clinical specificity and sensitivity were 68% and 65% for the H1N1 subtype, and 81% and 69% for the H3N2 subtype, respectively. The reduced clinical specificity and sensitivity observed in the PMAxx-LAMP assay can be attributed to the presence of non-viable viruses, which are labeled with PMAxx and subsequently eliminated from positive samples. The PMAxx-LAMP offers potential benefits in determining the stage of infection, enabling early screening of patients, and shortening the window period for virus detection. The simplicity and efficiency of this assay make it suitable for use in resource limited settings and point-of-care (POCT) applications. [ABSTRACT FROM AUTHOR]

This study described the results of a surveillance program monitoring circulation of influenza A viruses among domestic pigs (Sus domesticus) in Kazakhstan during 2018--2021. PCR data derived from 2,513 samples (nasopharyngeal swabs) collected from swine on large pig complexes and peasant farms located in different regions of Kazakhstan revealed that about 5% of samples were positive for influenza A virus RNA. This result suggested low levels of influenza A virus circulation in Kazakhstan. Subtyping of a set of samples revealed that the main strains circulating in 2018--2019 were A/H1N1 and A/H3N2. Surveillance conducted in 2020--2021 identified only A/H1N1 viruses in swine. The PCR data were confirmed by isolation of six strains: five influenza A/H1N1 viruses and one A/H3N2 virus. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic had a significant impact on the global influenza vaccination and the epidemics of seasonal influenza. To further explore the molecular epidemiology of influenza viruses and assess vaccine effectiveness, we collected influenza cases in Wuhan during the 2022–2023 influenza season. Among 1312 clinical samples, 312 samples tested positive for influenza viruses using reverse transcription polymerase chain reaction. These positive samples included 146A/H1N1 subtypes (46.8%), 164A/H3N2 subtypes (52.6%) and 2 influenza B virus types (0.6%). Based on the whole genome sequence information of hemagglutinin (HA) and neuraminidase (NA) from 27A/H1N1 influenza virus strains and 26A/H3N2 influenza virus strains obtained in this study, a phylogenetic analysis was conducted. The analysis revealed that all A/H1N1 strains belonged to the evolutionary branch 6B.1A.5a.2a, and they exhibited specific substitutions at positions K71Q, Q206E, E241A, and R276K. Similarly, all A/H3N2 strains were classified into the 3C.2a1b.2a.1a subclade and displayed amino acid substitutions at positions S172H, N175Y, I176T, K187N, and S214P. Notably, the A/H3N2 strains also acquired a new potential glycosylation site at position N174. Using an epitope model, the predicted vaccine effectiveness was assessed for the A/H1N1 and A/H3N2 strains. The predicted vaccine effectiveness against the Wuhan influenza epidemic strain was over 85% for the A/H1N1 vaccine strain. However, the effectiveness against the A/H3N2 vaccine strain was only 48.7%. To further verify the protection of influenza vaccine against circulating influenza viruses in the region, we conducted in vivo and in vitro animal studies. The results of in vitro neutralization experiment showed that rabbit serum antibodies inoculated with quadrivalent isolated influenza vaccine had neutralization ability against all 24 isolated influenza viruses. In vivo experiments showed that vaccinated mice had fewer lung lesions when infected with the influenza strain circulating in Wuhan, suggesting that vaccination can effectively reduce the occurrence of severe lung damage. These findings emphasize the importance of accurately predicting seasonal influenza strains for effective influenza prevention and control, especially during the co‐circulation of SARS‐CoV‐2 and influenza viruses. This study provides valuable information on the seasonal influenza virus in Wuhan during the COVID‐19 pandemic and serves as a basis for vaccine prediction and updates. [ABSTRACT FROM AUTHOR]

Background: The rapid production of influenza vaccines is crucial to meet increasing pandemic response demands. Here, we developed plant-made vaccines comprising centralized consensus influenza hemagglutinin (HA-con) proteins (H1 and H3 subtypes) conjugated to a modified plant virus, tobacco mosaic virus (TMV) nanoparticle (TMV-HA-con). Methods: We compared immune responses and protective efficacy against historical H1 or H3 influenza A virus infections among TMV-HA-con, HA-con protein combined with AddaVax™ adjuvant, and whole-inactivated virus vaccine (Fluzone®). Results: Immunogenicity studies demonstrated robust IgG, IgM, and IgA responses in the TMV-HA-con and HA-con protein vaccinated groups, with relatively low induction of interferon (IFN)-γ+ T-cell responses across all vaccinated groups. The TMV-HA-con and HA-con protein groups displayed partial protection (100% and 80% survival) with minimal weight loss following challenge with two H1N1 strains. The HA-con protein group exhibited 80% and 100% survival against two H3 strains, whereas the TMV-HA-con groups showed reduced protection (20% survival). The Fluzone® group conferred 20–100% survival against two H1N1 strains and one H3N1 strain, but did not protect against H3N2 infection. Conclusions: Our findings indicate that TMV-HA and HA-con protein vaccines with adjuvant induce protective immune responses against influenza A virus infections. Furthermore, our results underscore the potential of plant-based production using TMV-like nanoparticles for developing influenza A virus candidate vaccines. [ABSTRACT FROM AUTHOR]

Background: Within the last decades industrial swine herds in Europe grown significantly, creating an optimized reservoir for swine influenza A viruses (swIAV) to become enzootic, particularly in piglet producing herds among newborn, partly immunologically naïve piglets. To date, the only specific control measure to protect piglets from swIAV is the vaccination of sows, which provides passive immunity through maternally derived antibodies in colostrum of vaccinated sows. Interruption of infection chains through management practices have had limited success. This study focused on weaned piglets in five enzootically swIAV infected swine herds in North-West and North-East Germany and aimed to better understand swIAV infection patterns to improve piglet protection and reduce zoonotic risks. Participating farms fulfilled the following inclusion criteria: sow herd with ≥ 400 sows (actual size 600–1850 sows), piglets not vaccinated against influenza A virus and a history of recurrent respiratory problems associated with continuing influenza A virus infection. Influenza vaccination was performed in all sow herds, except for one, which discontinued vaccination during the study. Results: First swIAV detections in weaned piglets occurred at 4 weeks of age in the nursery and continued to be detected in piglets up to 10 weeks of age showing enzootic swIAV infections in all herds over the entire nursery period. This included simultaneous circulation of two subtypes in a herd and co-infection with two subtypes in individual animals. Evidence for prolonged (at least 13 days) shedding was obtained in one piglet based on two consecutive swIAV positive samplings. Possible re-infection was suspected in twelve piglets based on three samplings, the second of which was swIAV negative in contrast to the first and third sampling which were swIAV positive. However, swIAV was not detected in nasal swabs from either suckling piglets or sows in the first week after farrowing. Conclusions: Predominantly, weaned piglets were infected. There was no evidence of transmission from sow to piglet based on swIAV negative nasal swabs from sows and suckling piglets. Prolonged virus shedding by individual piglets as well as the co-circulation of different swIAV subtypes in a group or even individuals emphasize the potential of swIAV to increase genetic (and potentially phenotypic) variation and the need to continue close monitoring. Understanding the dynamics of swIAV infections in enzootically infected herds has the overall goal of improving protection to reduce economic losses due to swIAV-related disease and consequently to advance animal health and well-being. [ABSTRACT FROM AUTHOR]

Background: Influenza is an acute respiratory infection caused by influenza viruses, with influenza A virus (IAV) being the most common and most likely to progress to critically ill cases leading to death. Pneumocystis jiroveci is an opportunistic lung-causing fungus that occurs most often in immunocompromised individuals and can cause Pneumocystis jiroveci pneumonia (PJP). It is rare for both diseases to occur in the same patient. Methods: Appropriate laboratory tests, chest computed tomography (CT), bronchoalveolar lavage fluid, secondgeneration macro gene sequencing, and pathogenetic tests to clarify the diagnosis. Results: G test and LDH were high, and chest CT showed rapidly progressive interstitial pneumonia, which was confirmed by bronchoalveolar lavage fluid and macrogenomic second-generation sequencing (mNGS) to be a mixed infection of H. influenzae type A virus and Pneumocystis jiroveci. Conclusions: In rapidly progressive interstitial pneumonia, bronchoalveolar lavage and mNGS should be done early to clarify the presence of infection with specific pathogenic organisms. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) causes highly contagious respiratory disease worldwide, so prevention and control of IAV is extremely important. However, overuse of neuraminidase inhibitor (NAI) drugs leads to drug resistance. To explore the up-to-date geographical distribution and evolution of drug-resistant mutations (DRMs) in the NA protein of IAV, 81,492 near full-length NA sequences downloaded from NCBI and GISAID databases, including 34,481 H1N1 and 46,622 H3N2, were processed and analyzed. Our results showed the annual number of NA sequences from 2011 to 2019 continuously increased. Meanwhile, almost 85% of sequences were from developed countries in North America, Europe and Asia. Clustering analysis demonstrated H3N2 varied more than H1N1. Notably, H3N2 exhibited a higher frequency of DRMs than H1N1, with prevailing DRMs mainly located at non-active sites within the NA protein. Phylogenetic analyses showed NA harboring DRMs collected in the same year and from the same location clustered together, which may be related to the local economic level, clinical monitoring of DRMs and research level. Consequently, it is imperative to enhance global surveillance targeting drug resistance in IAV infections which can mitigate the transmission of drug-resistant strains. In summary, our research provides valuable insights for clinical medication while establishing a robust scientific basis for IAV prevention and treatment strategies to improve overall efficacy. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) significantly impact animal and human health due to their zoonotic potential. A growing body of evidence indicates that the host's long noncoding RNAs (lncRNAs) play crucial roles in regulating host–virus interactions during IAV infection. However, numerous lncRNAs associated with IAV infection have not been well characterised. Here, in this study, we identify the LINC01197 as an antiviral host factor. LINC01197 was significantly upregulated after IAV infection, which is controlled by the NF-κB pathway. Functional analysis revealed that overexpression of LINC01197 inhibited IAV replication and virus production, while knockdown of LINC01197 facilitated IAV replication. Mechanistically, LINC01197 directly interacts with poly(A) binding protein cytoplasmic 1 (PABPC1), which in turn sequesters and restricts its functions. This work shows that LINC01197 acts as a protein decoy, suppressing IAV replication and playing a key role in controlling IAV replication. [ABSTRACT FROM AUTHOR]

Influenza viruses continually evolve new antigenic variants, through mutations in epitopes of their major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Antigenic drift potentiates the reinfection of previously infected individuals, but the contribution of this process to variability in annual epidemics is not well understood. Here, we link influenza A(H3N2) virus evolution to regional epidemic dynamics in the United States during 1997--2019. We integrate phenotypic measures of HA antigenic drift and sequence-based measures of HA and NA fitness to infer antigenic and genetic distances between viruses circulating in successive seasons. We estimate the magnitude, severity, timing, transmission rate, age-specific patterns, and subtype dominance of each regional outbreak and find that genetic distance based on broad sets of epitope sites is the strongest evolutionary predictor of A(H3N2) virus epidemiology. Increased HA and NA epitope distance between seasons correlates with larger, more intense epidemics, higher transmission, greater A(H3N2) subtype dominance, and a greater proportion of cases in adults relative to children, consistent with increased population susceptibility. Based on random forest models, A(H1N1) incidence impacts A(H3N2) epidemics to a greater extent than viral evolution, suggesting that subtype interference is a major driver of influenza A virus infection ynamics, presumably via heterosubtypic cross-immunity. [ABSTRACT FROM AUTHOR]

During in silico research, based on molecular docking, dynamics simulation, network pharmacology and ADMET (absorption, distribution, metabolism, excretion, and toxicity) property, 31 potential compounds are obtained from seven Chinese medicinal herbs containing coumarins. Moreover, mechanism researches discover these compounds treat influenza A virus through key targets TLR4, MMP9, and IL6 (high fever, acute respiratory distress syndrome), MAPK1 and MAPK3 (MAPK signaling pathway, viral RNP export and viral protein expression), CASP3 (apoptosis), EP300 and CREBBP (viral myocarditis, chemoattraction of monocytes and macrophages, T cell activation antibody response). Further experimental verification finds that 22 of 31 compounds have various degrees of anti‐influenza virus activities. Moreover, 12 of 22 compounds have better biological activities (IC50=0.1609–54.95 μM) than oseltamivir (IC50=114.3 μM). 3 of 12 compounds have better antioxidant activities (IC50=2.087–3.796 μM) than vitamin C (IC50=5.004 μM). Therefore, ellagic acid, hyperoside, and fraxetin are promising multi‐target lead compounds for influenza virus therapy. Meanwhile, the present study offers compelling evidence for the development of novel antiviral medications. [ABSTRACT FROM AUTHOR]