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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]

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]

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]

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]

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]

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]

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]

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]

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]

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: 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]

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]

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]

Prior research has indicated that the gut-lung-axis can be influenced by the intestinal microbiota, thereby impacting lung immunity. Rifaximin is a broad-spectrum antibacterial drug that can maintain the homeostasis of intestinal microflora. In this study, we established an influenza A virus (IAV)-infected mice model with or without rifaximin supplementation to investigate whether rifaximin could ameliorate lung injury induced by IAV and explore the molecular mechanism involved. Our results showed that IAV caused significant weight loss and disrupted the structure of the lung and intestine. The analysis results of 16S rRNA and metabolomics indicated a notable reduction in the levels of probiotics Lachnoclostridium, Ruminococcaceae_UCG-013, and tryptophan metabolites in the fecal samples of mice infected with IAV. In contrast, supplementation with 50 mg/kg rifaximin reversed these changes, including promoting the repair of the lung barrier and increasing the abundance of Muribaculum, Papillibacter and tryptophan-related metabolites content in the feces. Additionally, rifaximin treatment increased ILC3 cell numbers, IL-22 level, and the expression of RORγ and STAT-3 protein in the lung. Furthermore, our findings demonstrated that the administration of rifaximin can mitigate damage to the intestinal barrier while enhancing the expression of AHR, IDO-1, and tight junction proteins in the small intestine. Overall, our results provided that rifaximin alleviated the imbalance in gut microbiota homeostasis induced by IAV infection and promoted the production of tryptophan-related metabolites. Tryptophan functions as a signal to facilitate the activation and movement of ILC3 cells from the intestine to the lung through the AHR/STAT3/IL-22 pathway, thereby aiding in the restoration of the barrier. Key points: • Rifaximin ameliorated IAV infection-caused lung barrier injury and induced ILC3 cell activation. • Rifaximin alleviated IAV-induced gut dysbiosis and recovered tryptophan metabolism. • Tryptophan mediates rifaximin-induced ILC3 cell activation via the AHR/STAT3/IL-22 pathway. [ABSTRACT FROM AUTHOR]

ABSTRACT Aim Methods and Results Conclusion Backyard production systems (BPS) represent an interface of contact between people, domestic and wild animals. Studies conducted in Chile during the last decade have provided extensive evidence of influenza A virus (IAV) circulation in backyard poultry and swine. The aim of this study was to investigate exposure practices of humans to animal‐origin IAV within backyards.Backyard farmers and household members of a total of 101 BPS in the proximity of wetlands located throughout Chile were interviewed between 2021 and 2022. Data were collected on the nature of human–animal contacts through participation in productive activities conducted within backyards, which was used to estimate participants' exposure risk to animal‐origin IAV. Additionally, RT‐qPCR and serologic IAV active surveillance was carried out in backyard animals. Multilinear regression was used to identify factors associated with exposure risk. Overall, IAV prevalence was 10.1% (95% CI: 4.7%–15.5%) and seroprevalence was 43.5% (95% CI: 29.7%–54.2%), both at the BPS level. Of 180 interviewees, 86% reported participating regularly in poultry or swine exposure activities within the backyard. A greater participation of male participants was observed when evaluating swine exposure activities, while female participation was greater for some activities related to poultry handling. Handwashing was a very extended hygiene practice; however, the use of personal protective equipment was uncommon. Different factors related to participants, households and backyards were associated with an increased exposure risk of participants to animal‐origin IAV: (i) older age, (ii) less years of education, (iii) no off‐farm work, (iv) greater backyard production value and (v) greater household consumption of backyard products.These results indicate the circulation of IAV in BPS and the frequent human–animal contact at this interface, highlighting the need for awareness campaigns and educational programmes aimed at backyard farmers on prevention and biosecurity measures in the management of backyard animals. [ABSTRACT FROM AUTHOR]

Influenza B virus (IBV) stands as a paradox, often overshadowed by its more notorious counterpart, influenza A virus (IAV). Yet, it remains a captivating and elusive subject of scientific inquiry. Influenza B is important because it causes seasonal flu outbreaks that can lead to severe respiratory illnesses, including bronchitis, pneumonia, and exacerbations of chronic conditions like asthma. Limitations in the influenza B virus's epidemiological, immunological, and etiological evolution must be addressed promptly. This comprehensive review covers evolutionary epidemiology and pathogenesis, host-virus interactions, viral isolation and propagation, advanced molecular detection assays, vaccine composition and no animal reservoir for influenza B virus. Complex viral etiology begins with intranasal transmission of influenza B virus with the release of a segmented RNA genome that attacks host cell machinery for transcription and translation within the nucleus and the release of viral progeny. Influenza B virus prevalence in domesticated and wild canines, sea mammals, and birds is frequent, yet there is no zoonosis. The periodic circulation of influenza B virus indicates a 1-3-year cycle for monophyletic strain replacement within the Victoria strain due to frequent antigenic drift in the HA near the receptorbinding site (RBS), while the antigenic stability of Yamagata viruses portrays a more conservative evolutionary pattern. Additionally, this article outlines contemporary antiviral strategies, including pharmacological interventions and vaccination efforts. This article serves as a resource for researchers, healthcare professionals, and anyone interested in the mysterious nature of the influenza B virus. It provides valuable insights and knowledge essential for comprehending and effectively countering this viral foe, which continues to pose a significant public health threat. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between serum programmed cell death molecule 5 (PDCD5), CXC chemokine ligand 8 (CXCL8), human cartilage glycoprotein 39 (YKL-40) and disease condition and clinical outcome in influenza A virus pneumonia. Methods: 165 patients with influenza A virus pneumonia who were admitted to Jilin Fifth People's Hospital from March 2021 to May 2023 were selected as observation group, and 50 healthy subjects who underwent physical examination during the same period were selected as control group. The PDCD5, CXCL8 and YKL-40 of patients and subjects were detected, and the differences of each index level between groups were compared, and the levels of each index of patients with influenza A virus pneumonia in different conditions were compared. The clinical outcomes of patients were statistically analyzed, and patients were divided into death group and survival group, the levels of PDCD5, CXCL8 and YKL-40 were compared between two groups, the risk factors and protective factors of death in patients with influenza A virus pneumonia were analyzed by univariate and multivariate Logistic analysis. Results: The levels of PDCD5, CXCL8 and YKL-40 in observation group were higher than those in control group (P<0.05). The levels of PDCD5, CXCL8 and YKL-40 in severe group were higher than those in mild group (P<0.05). After follow-up observation, 39 patients died, and the levels of PDCD5, CXCL8 and YKL-40 in death group were higher than those in survival group (P<0.05). Univariate analysis showed that, there was no significant difference in gender, body mass index (BMI), smoking history, drinking history, hypertension history, diabetes history, hyperlipidemia history and course of disease between two groups (P>0.05). The age, PDCD5, CXCL8 and YKL-40 in death group were higher than those in survival group, and the forced vital capacity(FVC), peak expiratory flow (PEF) and maximum middle expiratory flow rate (MMEF) were lower than those in survival group (P<0.05). Logistic multivariate analysis showed that, age, PDCD5, CXCL8 and YKL-40 were independent risk factors for the prognosis of patients with influenza A virus pneumonia, and FVC, PEF and MMEF were protective factors for the prognosis of patients. Conclusion: Serum PDCD5, CXCL8 and YKL-40 are involve in the occurrence and progression of influenza A virus pneumonia, and are closely relate to the clinical outcome of patients, together with age, they constitute a risk factor for the prognosis of patients, while FVC, PEF and MMEF are protective factors for the prognosis of patients [ABSTRACT FROM AUTHOR]

This study compared the safety and pharmacokinetics of a single oral dose of onradivir, an inhibitor of polymerase basic protein 2 in influenza A virus, in patients with hepatic impairment and healthy participants with normal hepatic function. Eight participants with mild hepatic impairment (Child‐Pugh A), eight participants with moderate hepatic impairment (Child‐Pugh B), and eight healthy matched controls were enrolled in this open‐label, parallel‐group clinical trial. After the administration of 600 mg of onradivir, pharmacokinetic parameters were calculated for each cohort and compared. Onradivir was generally well tolerated by all participants. No serious adverse events (AEs) and no deaths were reported during the study. Six patients with moderate hepatic impairment and three patients with mild hepatic impairment reported AEs, all of which were mild and quickly resolved. Compared with the normal liver function group, the maximum concentration, area under the curve from time zero to the last measurable concentration, and area under the curve from time zero to infinity were 103%, 68.5%, and 69.2% higher, respectively, in the mild hepatic impairment group. In the moderate hepatic impairment group, these increases were 101%, 197%, and 204%, respectively. Overall, there were clinically relevant differences in onradivir exposure between patients with mild or moderate hepatic impairment and normal controls. These data imply that onradivir dose adjustment is warranted in patients with mild or moderate hepatic impairment. The trial is registered at ClinicalTrials.gov (CT.gov identifier: NCT05856513). [ABSTRACT FROM AUTHOR]

In March 2023, our pediatric intensive care unit (PICU) retrospectively examined six cases of pediatric necrotizing tracheobronchitis (NTB), focusing on co-infections with influenza A virus (IAV) and Staphylococcus aureus (S. aureus). This study aimed to elucidate NTB's clinical characteristics, diagnostics, and therapeutic approaches. Diagnostics included symptom assessment, microbiological testing that confirmed all patients were positive for IAV H1N1 with a predominant S. aureus co-infection, and bronchoscopy. The patients predominantly exhibited fever, cough, and dyspnea. Laboratory analysis revealed decreased lymphocyte counts and elevated infection markers like C-reactive protein and procalcitonin. Chest computed tomography (CT) scans detected tracheobronchial obstructions in half of the cases, while bronchoscopy showed severe mucosal congestion, edema, necrosis, and purulent-hemorrhagic exudates. Treatments encompassed comprehensive strategies like oxygen therapy, intubation, bronchoscopic interventions, thoracentesis, oseltamivir, and a regimen of antibiotics. Our findings suggested potential correlations between clinical markers, notably lymphocyte count and procalcitonin, and clinical interventions such as the number of rescues and intensive care unit (ICU) duration. This research highlights the importance of early detection and the role of bronchoscopy and specific markers in assessing NTB, advocating for continued research in larger cohorts to better understand its clinical trajectory and refine treatment approaches for this challenging pediatric disease. [ABSTRACT FROM AUTHOR]

Introduction: Influenza A virus (IAV) infection is a global respiratory disease, which annually leads to 3-5 million cases of severe illness, resulting in 290,000-650,000 deaths. Additionally, during the past century, four global IAV pandemics have claimed millions of human lives. The epithelial lining of the trachea plays a vital role during IAV infection, both as point of viral entry and replication as well as in the antiviral immune response. Tracheal tissue is generally inaccessible from human patients, which makes animal models crucial for the study of the tracheal host immune response. Method: In this study, pigs were inoculated with swine- or human-adapted H1N1 IAV to gain insight into how host adaptation of IAV shapes the innate immune response during infection. In-depth multi-omics analysis (global proteomics and RNA sequencing) of the host response in upper and lower tracheal tissue was conducted, and results were validated by microfluidic qPCR. Additionally, a subset of samples was selected for histopathological examination. Results: A classical innate antiviral immune response was induced in both upper and lower trachea after infection with either swine- or human-adapted IAV with upregulation of genes and higher abundance of proteins associated with viral infection and recognition, accompanied by a significant induction of interferon stimulated genes with corresponding higher proteins concentrations. Infection with the swine-adapted virus induced a much stronger immune response compared to infection with a human-adapted IAV strain in the lower trachea, which could be a consequence of a higher viral load and a higher degree of inflammation. Discussion: Central components of the JAK-STAT pathway, apoptosis, pyrimidine metabolism, and the cytoskeleton were significantly altered depending on infection with swine- or human-adapted virus and might be relevant mechanisms in relation to antiviral immunity against putative zoonotic IAV. Based on our findings, we hypothesize that during host adaptation, IAV evolve to modulate important host cell elements to favor viral infectivity and replication. [ABSTRACT FROM AUTHOR]

Background and Aim: Free-grazing duck (FGD) raising is a unique domestic duck production system that is widely practiced in several Asian countries, including Thailand. FGD is a significant reservoir for influenza A viruses (IAVs). In this study, we genetically characterized IAV-H10N6 and IAV-H10N7 isolated from avian influenza surveillance in FGDs in Thailand. Materials and Methods: We collected 640 swab samples from 29 FGD flocks located in 6 provinces of Thailand. IAVs were isolated from swab samples using egg inoculation. Hemagglutination test-positive samples were then subjected to IAV detection. Viral RNA was subjected to IAV detection using real-time reverse-transcription polymerase chain reaction (rRT-PCR) specific to matrix (M) gene. IAV subtypes were identified using the RT-PCR assay specific to all hemagglutinin and neuraminidase subtypes. Whole-genome sequencing of IAVs was performed to genetically characterize IAV-H10N6 and IAV-H10N7. Results: Our results showed that 41 (6.41%) samples tested positive for IAV using rRT-PCR specific to the M gene. Among these, only two IAVs were subtypes as IAV-H10N6 and IAV-H10N7 and were subjected to whole-genome sequencing. IAV-H10N6 and IAV-H10N7 belonged to the Eurasian lineage and did not show any evidence of reassortment from the North American lineage. The viruses exhibited low-pathogenic characteristics and preferred binding to avian-type receptors. Genetic analysis revealed no mutations in PB2 and M genes, unlike human IAV-H10N3 and IAV-H10N8, which exhibited increased virulence in mammals. Conclusion: IAV-H10N6 and IAV-H10N7 viruses have less potential as zoonotic viruses. However, IAV in FGDs should be monitored for novel reassortant or zoonotic viruses. This study provides information on the genetic characteristics and diversity of IAV-H10N6 and IAV-H10N7 that are circulated in FGDs in Thailand. [ABSTRACT FROM AUTHOR]

Objective: The aim of the study was to evaluate respiratory syncytial virus (RSV) infections in cases followed in the pediatric intensive care unit (PICU). Materials and Methods: The study was designed as a prospective cohort in 6 PICUs. There were 3 groups: only RSV (+), RSV (v+) who were positive for another viral agent(s) in addition to RSV, and RSV (b+) who were positive for a bacterial agent(s) in addition to RSV. Results: A total of 119 cases were included in the study, 67 (56.3%) of whom were male. The RSV (+) group had a lower pH compared to the other groups and a higher rate of acute bronc hiolitis/bronchitis diagnoses compared to the RSV (v+) group. The RSV (v+) group had higher bicarbonate levels, higher creatinine levels, longer hospital stays, and higher Pediatric Risk of Mortality-3 scores (PRISM-3) compared to the RSV (+) group. Cases with RSV (b+) were younger and also had lower body weight compared to the other groups. Furthermore, the RSV (b+) group had higher C-reactive protein and Procalcitonin (PCT) levels and higher rates of High Flow Nasal Cannula-Oxygen Therapy (HFNC-OT) use. Multiple linear regression analysis revealed that PRISM-3 score, PCT levels, Pediatric Acute Respiratory Distress Syndrome diagnoses, inhaled steroid use, chronic illness status, and heart rate on admission were associated with the length of stay in the PICU. Conclusion: High flow nasal cannula-oxygen therapy continues to be the most frequently preferred respiratory support method in RSV infections. Viral infections accompanying RSV can increase the severity of the disease. [ABSTRACT FROM AUTHOR]

Obesity is a risk factor for increased morbidity and mortality in viral respiratory infection. Mucociliary clearance (MCC) in the airway is the primary host defense against viral infections. However, the impact of obesity on MCC is unclear, prompting this study. Using murine tracheal tissue culture and in vitro influenza A virus (IAV) infection models, we analyzed cilia-driven flow and ciliary beat frequency (CBF) in the airway epithelium to evaluate MCC. Short-term IAV infection increased cilia-driven flow and CBF in control mice, but not in high-fat diet-induced obese mice. Basal cilia-driven flow and CBF were also lower in obese mice than in control mice. Mechanistically, the increase of extracellular adenosine triphosphate (ATP) release during IAV infection, which was observed in the control mice, was abolished in the obese mice; however, the addition of ATP increased cilia-driven flow and CBF both in control and obese mice to a similar extent. In addition, RNA sequencing and reverse transcription-polymerase chain reaction revealed the downregulation of several cilia-related genes, including Dnah1, Dnal1, Armc4, and Ttc12 (the dynein-related genes); Ulk4 (the polychaete differentiation gene); Cep164 (the ciliogenesis and intraflagellar transport gene); Rsph4a, Cfap206, and Ppil6 (the radial spoke structure and assembly gene); and Drc3(the nexin-dynein regulatory complex genes) in obese murine tracheal tissues compared with their control levels. In conclusion, our studies demonstrate that obesity attenuates MCC under basal conditions and during IAV infection by downregulating the expression of cilia-related genes and suppressing the release of extracellular ATP, thereby increasing the susceptibility and severity of IAV infection. NEW & NOTEWORTHY: Our study shows that obesity impairs airway mucociliary clearance (MCC), an essential physical innate defense mechanism for viral infection. Mechanically, this is likely due to the obesity-induced downregulation of cilia-related genes and attenuation of extracellular ATP release. This study provides novel insights into the mechanisms driving the higher susceptibility and severity of viral respiratory infections in individuals with obesity. [ABSTRACT FROM AUTHOR]

Following the detection of highly pathogenic avian influenza (HPAI) virus in countries bordering Kenya to the west, we conducted surveillance among domestic and wild birds along the shores of Lake Victoria. In addition, between 2018 and 2020, we conducted surveillance among poultry and poultry workers in live bird markets and among wild migratory birds in various lakes that are resting sites during migration to assess introduction and circulation of avian influenza viruses in these populations. We tested 7464 specimens (oropharyngeal (OP) and cloacal specimens) from poultry and 6531 fresh fecal specimens from wild birds for influenza A viruses by real-time RT-PCR. Influenza was detected in 3.9% (n = 292) of specimens collected from poultry and 0.2% (n = 10) of fecal specimens from wild birds. On hemagglutinin subtyping, most of the influenza A positives from poultry (274/292, 93.8%) were H9. Of 34 H9 specimens randomly selected for further subtyping, all were H9N2. On phylogenetic analysis, these viruses were genetically similar to other H9 viruses detected in East Africa. Only two of the ten influenza A-positive specimens from the wild bird fecal specimens were successfully subtyped; sequencing analysis of one specimen collected in 2018 was identified as a low-pathogenicity avian influenza H5N2 virus of the Eurasian lineage, and the second specimen, collected in 2020, was subtyped as H11. A total of 18 OP and nasal specimens from poultry workers with acute respiratory illness (12%) were collected; none were positive for influenza A virus. We observed significant circulation of H9N2 influenza viruses in poultry in live bird markets in Kenya. During the same period, low-pathogenic H5N2 virus was detected in a fecal specimen collected in a site hosting a variety of migratory and resident birds. Although HPAI H5N8 was not detected in this survey, these results highlight the potential for the introduction and establishment of highly pathogenic avian influenza viruses in poultry populations and the associated risk of spillover to human populations. [ABSTRACT FROM AUTHOR]

Background: Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. Methods: In this study, the major antigen genes P1a of MP adhesion factor P1(3862‐4554 bases) and P30a of P30(49‐822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS‐P1a or NS‐P30a. The recombinant pHW2000 plasmids containing NS‐P1a or NS‐P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU‐P1a and rFLU‐P30a were rescued. RT‐PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU‐P1a and rFLU‐P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. Results: P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT‐PCR identification of the recombinant viruses rFLU‐P1a and rFLU‐P30a showed that P1a (693 bp), P30a (774 bp), NS‐P1a (1992bp) and NS‐P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. Conclusion: The recombinant viruses rFLU‐P1a and rFLU‐P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU‐P1a or rFLU‐P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human. [ABSTRACT FROM AUTHOR]

Novel bat H17N10 and H18N11 influenza A viruses (IAVs) are incapable of reassortment with conventional IAVs during co-infection. To date, the underlying mechanisms that inhibit bat and conventional IAV reassortment remain poorly understood. Herein, we used the bat influenza M gene in the PR8 H1N1 virus genetic background to determine the molecular basis that restricts reassortment of segment 7. Our results showed that NEP and M1 from bat H17N10 and H18N11 can interact with PR8 M1 and NEP, resulting in mediating PR8 viral ribonucleoprotein (vRNP) nuclear export and formation of virus-like particles with single vRNP. Further studies demonstrated that the incompatible packaging signals (PSs) of H17N10 or H18N11 M segment led to the failure to rescue recombinant viruses in the PR8 genetic background. Recombinant PR8 viruses (rPR8psH18M and rPR8psH17M) containing bat influenza M coding region flanked with the PR8 M PSs were rescued but displayed lower replication in contrast to the parental PR8 virus, which is due to a low efficiency of recombinant virus uncoating correlating with the functions of the bat M2. Our studies reveal molecular mechanisms of the M gene that hinder reassortment between bat and conventional IAVs, which will help to understand the biology of novel bat IAVs. IMPORTANCE Reassortment is one of the mechanisms in fast evolution of influenza A viruses (IAVs) and responsible for generating pandemic strains. To date, why novel bat IAVs are incapable of reassorting with conventional IAVs remains completely understood. Here, we attempted to rescue recombinant PR8 viruses with M segment from bat IAVs to understand the molecular mechanisms in hindering their reassortment. Results showed that bat influenza NEP and M1 have similar functions as respective counterparts of PR8 to medicating viral ribonucleoprotein nuclear export. Moreover, the incompatible packaging signals of M genes from bat and conventional IAVs and impaired bat M2 functions are the major reasons to hinder their reassortment. Recombinant PR8 viruses with bat influenza M open reading frames were generated but showed attenuation, which correlated with the functions of the bat M2 protein. Our studies provide novel insights into the molecular mechanisms that restrict reassortment between bat and conventional IAVs. [ABSTRACT FROM AUTHOR]

Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective. IMPORTANCE The innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses. [ABSTRACT FROM AUTHOR]

Production of the amyloidogenic prion protein, PrPSc, which forms infectious protein aggregates, or prions, is a key pathogenic event in prion diseases. Functional prion-like protein aggregations, such as the mitochondrial adaptor protein MAVS and the inflammasome component protein ASC, have been identified to play a protective role in viral infections in mammalian cells. In this study, to investigate if PrPSc could play a functional role against external stimuli, we infected prion-infected cells with a neurotropic influenza A virus strain, IAV/WSN. We found that prion-infected cells were highly resistant to IAV/WSN infection. In these cells, NF-κB nuclear translocation was disturbed; therefore, mitochondrial superoxide dismutase (mtSOD) expression was suppressed, and mitochondrial reactive oxygen species (mtROS) was increased. The elevated mtROS subsequently activated NLRP3 inflammasomes, leading to the suppression of IAV/ WSN-induced necroptosis. We also found that prion-infected cells accumulated a portion of PrP molecules in the cytosol, and that the N-terminal potential nuclear translocation signal of PrP impeded NF-κB nuclear translocation. These results suggest that PrPSc might play a functional role in protection against viral infections by stimulating the NLRP3 inflammasome-dependent antivirus mechanism through the cytosolic PrP-mediated disturbance of NF-κB nuclear translocation, which leads to suppression of mtSOD expression and consequently upregulation of the NLRP3 inflammasome activator mtROS. IMPORTANCE Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections. [ABSTRACT FROM AUTHOR]

Increased circulating tissue inhibitor of metalloproteinases‐1 (TIMP‐1) levels have been observed in patients with acute lung injury (ALI). However, the sex‐specific regulation of TIMP‐1 and the underlying molecular mechanisms have not been well elucidated. In this study, we found that plasma TIMP‐1 levels were significantly higher in COVID‐19 and H1N1 patients compared with those in healthy subjects (n = 25). TIMP‐1 concentrations were significantly different between males and females in each disease group. Among female but not male patients, TIMP‐1 levels significantly correlated with the PaO2/FiO2 ratio and hospital length of stay. Using the mouse model of ALI induced by the H1N1 virus, we found that TIMP‐1 is strikingly induced in PDGFRα‐positive cells in the murine lungs. Moreover, female mice showed a higher Timp‐1 expression in the lungs on day 3 postinfection. Mechanistically, we observed that estrogen can upregulate TIMP‐1 expression in lung fibroblasts, not epithelial cells. In addition, overexpression of estrogen receptor α (ERα) increased the TIMP‐1 promoter activity. In summary, TIMP‐1 is an estrogen‐responsive gene, and its promoter activity is regulated by ERα. Circulating TIMP‐1 may serve as a sex‐specific marker, reflecting the severity and worst outcomes in female patients with SARS‐CoV2‐ and IAV‐related ALI. [ABSTRACT FROM AUTHOR]