Porcine γδ T cells express cytotoxic cell-associated markers and display killing activity but are not selectively cytotoxic against PRRSV- or swIAV-infected macrophages.

Background: Gamma-delta (γδ) T cells are a major immune cell subset in pigs. Approximately 50% of circulating T cells are γδ T cells in young pigs and up to 30% in adult sows. Despite this abundance, the functions of porcine γδ T cells are mostly unidentified. In humans and mice, activated γδ T cells exhibit broad innate cytotoxic activity against a wide variety of stressed, infected, and cancerous cells through death receptor/ligand-dependent and perforin/granzyme-dependent pathways. However, so far, it is unknown whether porcine γδ T cells have the ability to perform cytotoxic functions. Methods: In this study, we conducted a comprehensive phenotypic characterization of porcine γδ T cells isolated from blood, lung, and nasal mucosa. To further analyze the cytolytic potential of γδ T cells, in vitro cytotoxicity assays were performed using purified γδ T cells as effector cells and virus-exposed or mock-treated primary porcine alveolar macrophages as target cells. Results: Our results show that only CD2+ γδ T cells express cytotoxic markers (CD16, NKp46, perforin) with higher perforin and NKp46 expression in γδ T cells isolated from lung and nasal mucosa. Moreover, we found that γδ T cells can exhibit cytotoxic functions in a cell-cell contact and degranulation-dependent manner. However, porcine γδ T cells did not seem to specifically target Porcine Reproductive and Respiratory Syndrome Virus or swine Influenza A Virus-infected macrophages, which may be due to viral escape mechanisms. Conclusion: Porcine γδ T cells express cytotoxic markers and can exhibit cytotoxic activity in vitro. The specific mechanisms by which porcine γδ T cells recognize target cells are not fully understood but may involve the detection of cellular stress signals.

Revealing Different Pathways for Influenza A Virus To Reach Microtubules after Endocytosis by Quantum Dot-Based Single-Virus Tracking.

Actin- and microtubule (MT)-based transport systems are essential for intracellular transport. During influenza A virus (IAV) infection, MTs provide long tracks for virus trafficking toward the nucleus. However, the role of the actin cytoskeleton in IAV entry and especially the transit process is still ambiguous. Here, by using quantum dot-based single-virus tracking, it was revealed that the actin cytoskeleton was crucial for the virus entry via clathrin-mediated endocytosis (CME). After entry via CME, the virus reached MTs through three different pathways: the virus (1) was driven by myosin VI to move along actin filaments to reach MTs (AF); (2) was propelled by actin tails assembled by an Arp2/3-dependent mechanism to reach MTs (AT); and (3) directly reached MTs without experiencing actin-related movement (NA). Therefore, the NA pathway was the main one and the fastest for the virus to reach MTs. The AT pathway was activated only when plenty of viruses entered the cell. The viruses transported by the AF and AT pathways shared similar moving velocities, durations, and displacements. This study comprehensively visualized the role of the actin cytoskeleton in IAV entry and transport, revealing different pathways for IAV to reach MTs after entry. The results are of great significance for globally understanding IAV infection and the cellular endocytic transport pathway.

Inactivation of highly pathogenic avian influenza virus with high temperature short time continuous flow pasteurization and virus detection in bulk milk tanks.

Infections of dairy cattle with clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) were reported in March 2024 in the U.S. and viable virus was detected at high levels in raw milk from infected cows. This study aimed to determine the potential quantities of infectious HPAIV in raw milk in affected states where herds were confirmed positive by USDA for HPAIV (and therefore were not representative of the entire population), and to confirm that the commonly used continuous flow pasteurization using the FDA approved 72°C (161°F) for 15 s conditions for high temperature short time (HTST) processing, will inactivate the virus. Double-blinded raw milk samples from bulk storage tanks from farms (n=275) were collected in four affected states. Samples were screened for influenza A using quantitative real-time RT-PCR (qrRT-PCR) of which 158 (57.5%) were positive and were subsequently quantified in embryonating chicken eggs. Thirty-nine qrRT-PCR positive samples (24.8%) were positive for infectious virus with a mean titer of 3.5 log10 50% egg infectious doses (EID50) per mL. To closely simulate commercial milk pasteurization processing systems, a pilot-scale continuous flow pasteurizer was used to evaluate HPAIV inactivation in artificially contaminated raw milk using the most common legal conditions in the US: 72°C (161°F) for 15s. Among all replicates at two flow rates (n=5 at 0.5L/min; n=4 at 1L/min), no viable virus was detected. A mean reduction of ≥5.8 ± 0.2 log10 EID50/mL occurred during the heating phase where the milk is brought to 72.5°C before the holding tube. Estimates from heat-transfer analysis support that standard U.S. continuous flow HTST pasteurization parameters will inactivate >12 log10 EID50/mL of HPAIV, which is ∼9 log10 EID50/mL greater than the mean quantity of infectious virus detected in raw milk from bulk storage tank samples. These findings demonstrate that the US milk supply is safe when pasteurized.

Signals from intestinal microbiota mediate the crosstalk between the lung-gut axis in an influenza infection mouse model.

Introduction: Introduction: The influenza virus primarily targets the respiratory tract, yet both the respiratory and intestinal systems suffer damage during infection. The connection between lung and intestinal damage remains unclear. Methods: Our experiment employs 16S rRNA technology and Liquid Chromatography-Mass Spectrometry (LC-MS) to detect the impact of influenza virus infection on the fecal content and metabolites in mice. Additionally, it investigates the effect of influenza virus infection on intestinal damage and its underlying mechanisms through HE staining, Western blot, Q-PCR, and flow cytometry. Results: Our study found that influenza virus infection caused significant damage to both the lungs and intestines, with the virus detected exclusively in the lungs. Antibiotic treatment worsened the severity of lung and intestinal damage. Moreover, mRNA levels of Toll-like receptor 7 (TLR7) and Interferon-b (IFN-b) significantly increased in the lungs post-infection. Analysis of intestinal microbiota revealed notable shifts in composition after influenza infection, including increased Enterobacteriaceae and decreased Lactobacillaceae. Conversely, antibiotic treatment reduced microbial diversity, notably affecting Firmicutes, Proteobacteria, and Bacteroidetes. Metabolomics showed altered amino acid metabolism pathways due to influenza infection and antibiotics. Abnormal expression of indoleamine 2,3-dioxygenase 1 (IDO1) in the colon disrupted the balance between helper T17 cells (Th17) and regulatory T cells (Treg cells) in the intestine. Mice infected with the influenza virus and supplemented with tryptophan and Lactobacillus showed reduced lung and intestinal damage, decreased Enterobacteriaceae levels in the intestine, and decreased IDO1 activity. Discussion: Overall, influenza infection caused damage to lung and intestinal tissues, disrupted intestinal microbiota and metabolites, and affected Th17/Treg balance. Antibiotic treatment exacerbated these effects. Supplementation with tryptophan and Lactobacillus improved lung and intestinal health, highlighting a new understanding of the lung-intestine connection in influenza-induced intestinal disease.

Leveraging virucidal potential of an anti-microbial coating agent to mitigate fomite transmission of respiratory viruses.

In the wake of the COVID-19 pandemic, respiratory tract infections have emerged as a significant global threat, yet their impact on public health was previously underappreciated. This study investigated the antiviral efficacy of the nano-coating agent BARRIER90, composed of silicon-quaternary ammonium compound and a naturally derived biopolymer, against three distinct respiratory viruses: Influenza A (H1N1), Adenovirus Type 1, and Enterovirus-Coxsackie B1. BARRIER90 exhibited robust and sustained virucidal activity, persisting up to 90 days post-coating, against the enveloped virus, Influenza A, with significant reduction in viral plaques. Contrastingly, its efficacy against non-enveloped viruses revealed transient activity against Enterovirus-Coxsackie B1, with almost no antiviral activity observed against Adenovirus Type 1. These findings indicate the potential of antimicrobial coatings in mitigating viral transmission through contaminated surfaces (fomites), which harbour pathogenic viruses for longer periods. Antimicrobial coatings may facilitate infection control in various settings, including healthcare facilities and shared workspaces.

Preventive and therapeutic effects of a super-multivalent sialylated filamentous bacteriophage against the influenza virus.

The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.

Pediatric plastic bronchitis associated with smoke inhalation and influenza A: case report and literature review.

Plastic bronchitis is a relatively uncommon illness that has been reported in all age groups. This case report describes a specific manifestation of plastic bronchitis in two pediatric brothers influenced by both smoke inhalation and influenza A virus infection. The therapeutic approach mainly involved symptomatic supportive care, antiviral therapy, repeated bronchoscopic alveolar lavage, and bronchial cast removal. Eventually, both patients went into remission. Bronchoscopy proved to be helpful in diagnosing and treating these cases.

Indole-core inhibitors of influenza a neuraminidase: iterative medicinal chemistry and molecular modeling.

Influenza viruses that cause seasonal and pandemic flu are a permanent health threat. The surface glycoprotein, neuraminidase, is crucial for the infectivity of the virus and therefore an attractive target for flu drug discovery campaigns. We have designed and synthesized more than 40 3-indolinone derivatives. We mainly investigated the role of substituents at the 2 position of the core as well as the introduction of substituents or a nitrogen atom in the fused phenyl ring of the core for inhibition of influenza virus neuraminidase activity and replication in vitro and in vivo. After evaluating the compounds for their ability to inhibit the viral neuraminidase, six potent inhibitors 3c, 3e, 7c, 12o, 12v, 18d were progressed to evaluate for cytotoxicity and inhibition of influenza virus A/PR/8/34 replication in in MDCK cells. Two hit compounds 3e and 12o were tested in an animal model of influenza virus infection. Molecular mechanism of the 3-indolinone derivatives interactions with the neuraminidase was revealed in molecular dynamic simulations. Proposed inhibitors bind to the 430-cavity that is different from the conventional binding site of commercial compounds. The most promising 3-indolinone inhibitors demonstrate stronger interactions with the neuraminidase in molecular models that supports proposed binding site.

Avian and Human Influenza A Virus Receptors in Bovine Mammary Gland.

An outbreak of influenza A (H5N1) virus was detected in dairy cows in the United States. We detected influenza A virus sialic acid -α2,3/α2,6-galactose host receptors in bovine mammary glands by lectin histochemistry. Our results provide a rationale for the high levels of H5N1 virus in milk from infected cows.

Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination.

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

Clinical effects of basic nursing combined with psychological intervention on treatment compliance of patients with Influenza-A(H1N1).

Objective: To investigate the effects of basic nursing combined with psychological intervention on treatment compliance, self-care ability, clinical efficacy, lung function and nursing satisfaction of patients with Influenza-A(H1N1). Method: This was application research. Eighty patients with influenza-A (H1N1) admitted to The First Affiliated Hospital of Hebei North University from January 2020 to December 2022 were included as subjects and randomly divided into the observation group(n=40) and the control group(n=40). Patients in the control group were given routine basic nursing intervention, while those in the observation group were treated with combined psychological intervention in addition to basic nursing. The differences in treatment compliance, self-care ability, clinical efficacy, lung function and nursing satisfaction were compared between the two groups. Results: After the intervention, the treatment compliance score and the total self-care ability score of the observation group were higher than those of the control group, with statistically significant differences(P<0.05). After treatment, the clinical efficacy of the observation group was significantly higher than that of the control group(P<0.05). Before treatment, no significant difference was observed between the two groups in terms of various indexes of lung function, which were better in the observation group than in the control group after treatment(P<0.05). Conclusion: Basic nursing combined with psychological intervention results in a variety of benefits in the treatment of patients with Influenza-A(H1N1), such as improved treatment compliance and self-care ability, ameliorated lung function, as well as enhanced treatment outcomes and nursing satisfaction, which needs to be promoted in clinical practice.

Unraveling Novel Strategies: Targeting Miz1 for Degradation to Enhance Antiviral Defense against Influenza A Virus.

The ubiquitin system has been shown to play an important role in regulation of immune responses during viral infection. In a recent article published in Science Signaling, Wu and colleagues revealed that transcriptional factor Miz1 plays a pro-viral role in influenza A virus (IAV) infection by suppressing type I interferons (IFNs) production through recruiting HDAC1 to ifnb1 promoter. They show that a series of E3 ligases combinatorially regulates Miz1 ubiquitination and degradation and modulates IFNs production and viral replication.

Obesity impairs ciliary function and mucociliary clearance in the murine airway epithelium.

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, although the addition of ATP increased cilia-driven flow and CBF both in control and obese mice to a similar extent. Additionally, 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 to 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.

Lipid nanoparticle-encapsulated DNA vaccine confers protection against swine and human-origin H1N1 influenza viruses.

In 2009, a novel swine-origin H1N1 virus emerged, causing a pandemic. The virus, known as H1N1pdm09, quickly displaced the circulating H1 lineage and became the dominant seasonal influenza A virus subtype infecting humans. Human-to-swine spillovers of the H1N1pdm09 have occurred frequently, and each occurrence has led to sustained transmission of the human-origin H1N1pdm09 within swine populations. In the present study, we developed a lipid nanoparticle-based DNA vaccine (LNP-DNA) containing the hemagglutinin gene of a swine-origin H1N1pdm09. In pigs, this LNP-DNA vaccine induced a robust antibody response after a single intramuscular immunization and protected the pigs against challenge infection with the homologous swine-origin H1N1pdm09 virus. In a mouse model, the LNP-DNA vaccine induced antibody and T-cell responses and protected mice against lethal challenge with a mouse-adapted human-origin H1N1pdm09 virus. These findings demonstrate the potential of the LNP-DNA vaccine to protect against both swine- and human-origin H1N1pdm09 viruses. IMPORTANCE: Swine influenza A virus (IAV) is widespread and causes significant economic losses to the swine industry. Moreover, bidirectional transmission of IAV between swine and humans commonly occurs. Once introduced into the swine population, human-origin IAV often reassorts with endemic swine IAV, resulting in reassortant viruses. Thus, it is imperative to develop a vaccine that is not only effective against IAV strains endemic in swine but also capable of preventing the spillover of human-origin IAV. In this study, we developed a lipid nanoparticle-encapsulated DNA plasmid vaccine (LNP-DNA) that demonstrates efficacy against both swine- and human-origin H1N1 viruses. The LNP-DNA vaccines are non-infectious and non-viable, meeting the criteria to serve as a vaccine platform for rapidly updating vaccines. Collectively, this LNP-DNA vaccine approach holds great potential for alleviating the impact of IAV on the swine industry and preventing the emergence of reassortant IAV strains.

Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen.

BACKGROUND: Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management. Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention. Quantum dot-encoded microspheres have been widely used in immunodetection. The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis. Thus, establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis. AIM: To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology, which forms the foundation for the assays of multiple respiratory virus biomarkers. METHODS: Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B. The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer, and the detection conditions were optimized to establish the influenza A and B antigen codetection method, which was utilized for their detection in clinical samples. The results were compared with the fluorescence quantitative polymerase chain reaction (PCR) method to validate the clinical performance of this method. RESULTS: The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens, respectively, which both ranged from 15.6 to 250000 pg/mL. In the clinical sample evaluation, the proposed method well correlated with the fluorescent quantitative PCR method, with positive, negative, and overall compliance rates of 57.4%, 100%, and 71.6%, respectively. CONCLUSION: A multiplex assay for quantitative detection of influenza A and B virus antigens has been established, which is characterized by high sensitivity, good specificity, and a wide detection range and is promising for clinical applications.

Shark fin sign: A rare EKG finding in a patient with Takotsubo cardiomyopathy from influenza A infection.

This case demonstrated the rare "shark fin" ECG pattern, an ST-segment elevation typically seen in acute myocardial infarction. We reported a case of takotsubo cardiomyopathy secondary to influenza A infection with multiple organ failure, showing the shark fin sign and resulting in in-patient mortality and various complication.

The Hemagglutinin of Influenza A Virus Induces Ferroptosis to Facilitate Viral Replication.

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.

Time-resolved scRNA-seq reveals transcription dynamics of polarized macrophages with influenza A virus infection and antigen presentation to T cells.

AbstractThroughout history, the influenza A virus has caused numerous devastating global pandemics. Macrophages, as pivotal innate immune cells, exhibit a wide range of immune functions characterized by distinct polarization states, reflecting their intricate heterogeneity. In this study, we employed the time-resolved single-cell sequencing technique coupled with metabolic RNA labelling to elucidate the dynamic transcriptional changes in distinct polarized states of bone marrow-derived macrophages (BMDMs) upon infection with the influenza A virus. Our approach not only captures the temporal dimension of transcriptional activity, which is lacking in conventional scRNA-seq methods, but also reveals that M2-polarized Arg1_macrophages is the sole state supporting successful replication of influenza A virus. Furthermore, we identified distinct antigen presentation capabilities to CD4+ T and CD8+ T cells across diverse polarized states of macrophages. Notably, the M1 phenotype, exhibited by both bone marrow-derived macrophages (BMDMs) and murine alveolar macrophages (AMs), demonstrated superior conventional and cross-presentation abilities for exogenous antigens, with a particular emphasis on cross-presentation capacity. Additionally, as CD8+ T cell differentiation progressed, M1 polarization exhibited an enhanced capacity for cross-presentation. All three phenotypes of BMDMs, including M1, demonstrated robust presentation of CD4+ regulatory T cells, while displaying limited ability to present naive CD4+ T cells. These findings offer novel insights into the immunological regulatory mechanisms governing distinct polarized states of macrophages, particularly their roles in restricting the replication of influenza A virus and modulating antigen-specific T cell responses through innate immunity.

Inhibition of RAN attenuates influenza a virus replication and nucleoprotein nuclear export.

Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.

Highly pathogenic avian influenza A virus subtype H5N1 (clade 2.3.4.4b) isolated from a natural protected area in Peru.

The panzootic caused by H5N1 avian influenza viruses is a high concern for wild birds' conservation and the study of spillover events into mammals. The near coding-complete genome of H5N1 clade 2.3.3.4b sequencing in the Miseq Illumina platform was performed from a bird located in Pantanos of Villa National Wildlife Refuge.