This study aimed to investigate the immunopharmacological mechanism of Saposhnikoviae Radix(SR) against respiratory viruses and bacteria infections based on immune signal activation. Mouse models of pneumonia induced by intranasal influenza A virus(H1N1) infection and intratracheal lipopolysaccharide(LPS) instillation were established. The effects of the water extract of SR on pulmonary CT imaging, histological damage, and inflammatory cytokine expression in lung tissues were evaluated. Using bone marrow-derived macrophages, this study analyzed the regulatory effects of the extract on macrophage proliferation and phagocytic function. RNA-seq and bioinformatics analysis, combined with molecular biology experiments, were employed to explore the regulatory role of the water extract of SR on the type Ⅰ interferon(IFN-Ⅰ) signaling pathway. The findings revealed that the water extract of SR significantly alleviated H1N1 infection-induced lung imaging and pathological damage in mice, reduced lung viral gene copies, and downregulated the expression of inflammatory cytokines such as interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), and CXC motif chemokine ligand 10(CXCL10). Concurrently, drug treatment demonstrated similar lung tissue protective functions in the LPS-induced mouse pneumonia model. Cell experiments showed that the extract promoted proliferation of macrophages and enhanced their phagocytic capacity toward neutral red. Transcriptomic analysis revealed that genes upregulated by the extract of SR were significantly enriched in pathways including H1N1 infection, Toll-like receptor signaling, NOD-like receptor signaling, and IFN signaling. Finally, qRT-PCR and Western blot confirmed that the extract activated the interferon signaling pathway. In conclusion, the extract of SR activates innate immunity centered on IFN-Ⅰ signaling and enhances macrophage function, thereby improving the host's defense against viral and bacterial infections. It possesses potential preventive and therapeutic value for respiratory tract infection-associated pneumonia and lung injury.

Influenza A(H1N1)pdm09 continues to evolve through antigenic drift, posing challenges to vaccine effectiveness. The present study investigated the evolutionary dynamics, glycosylation patterns, and immunological features of the hemagglutinin (HA) gene of Influenza A(H1N1)pdm09 from the region. A total of 120 positive samples collected between 2022 and 2024 were sequenced using the Illumina MiSeq platform alongside previously generated HA sequences from the laboratory during 2009-2019. Mutational analysis was performed against the reference strain (A/California/07/2009) and the vaccine strain (A/Wisconsin/67/2022), followed by phylogenetic, structural, N-glycosylation, and epitope analysis using bioinformatics tools. Several persistent mutations (P100S, S220T, R240Q, I338V, E391K) were observed across study years, while substitutions such as S101N, D114N, A273T, K300E, S468N, E516K, and K180Q were consistently detected after 2015. Recent circulating strains (2022-2024) acquired additional mutations (K71Q, N146D, K147N, L178I, N173K, and A203T) and predominantly exhibited S202I rather than the earlier S202T substitution. Phylogenetically, these strains clustered within clade 6B.1 A.5a.2a. The presence of an additional N-glycosylation site at residue 179 after 2017 may suggest immune-driven selection. Notably, T137A, K186Q, and E277D substitutions identified in 2024 Influenza A(H1N1)pdm09 strains were associated with the loss of predicted epitope-binding regions relative to the vaccine strain A/Wisconsin/67/2022. These amino acid changes suggest potential alterations in antigenic structure that may influence immune recognition. No differences in mutation distribution were observed between the indoor and outdoor cases. Overall, the study represents the first long-term genomic surveillance of Influenza A(H1N1)pdm09 from Northeast India and demonstrates continuous genetic diversification and evolutionary dynamics of circulating Influenza A(H1N1)pdm09 strains.

Influenza is a highly contagious virus with most individuals showing recovery within a week after symptom onset. However, children, the elderly, and those with chronic health conditions are at high risk of developing serious complications. Extensive immune cell infiltration of the lung and massive production of pro-inflammatory cytokines can result in lung tissue damage and disruption, resulting in hypoxia and death. During the 2024-2025 influenza season in the USA, influenza caused an estimated 27,000-130,000 deaths. Our previous studies have shown that agents known to drive polarization of macrophages into the M2a phenotype mitigated inflammatory responses to influenza H1N1 A/PR/8/34 (PR8). Since IRS2 dampens the IL-4-induced differentiation to the M2a subtype, we sought to determine the impact of IRS2 on PR8 infection. Contrary to expectations, IRS2-/- mice exhibited enhanced susceptibility to PR8 when compared to IRS2+/+ mice, which was associated with a significant drop in blood oxygen saturation. Additionally, PR8 infection induced significantly greater inflammation in the lungs of IRS2-/- mice than IRS2+/+ mice. Conversely, viral replication was not significantly different. Significantly increased levels of the inflammatory mediator HMGB1 and airway epithelial cell denuding were observed in the lungs of IRS2-/- mice, with a concomitant decrease in M2a gene expression. Mice with myeloid-specific knockout of IRS2 showed no enhanced sensitivity to PR8, supporting the hypothesis that the major impact of IRS2-deficiency on host response to PR8 infection is not myeloid cell-intrinsic. Overall, these results suggest that in the absence of IRS2, airway epithelial cells are especially sensitive to influenza-induced damage through dysregulated inflammation, resulting in increased susceptibility during infection.

Influenza virus outbreaks remain a persistent public health concern, yet traditional metabolomics methods are inadequate for addressing key analytical challenges of "dark matter" in influenza research. By integrating quantitative MS[1] data, MS[2]-derived fragmentation trees and molecular fingerprints, structure-based comparative metabolomics enhances predictive capability for chemical structures, and enables the discovery of candidate metabolic markers without the need for database spectra. In this study, we established a C57BL/6J mouse model of H1N1 infection (with PBS as control) and performed structure-based comparative metabolomics on fecal samples using liquid chromatography-mass spectrometry (LC-MS). Quantitative analysis of MS[1] data identified 40 differential metabolites, while qualitative analysis of MS[2] data enabled their structural annotation. A candidate metabolite marker, LysoPE 15:0, along with other potential metabolic markers, was annotated and validated using Mirror plot, CFM-ID, and sim-Rank-Network. Our findings demonstrate that structure-based comparative metabolomics enables library spectra-free annotation of metabolomic "dark matter" and provides a methodological workflow for discovering candidate metabolite markers in other diseases.

To address the limitations of egg-based influenza vaccine production, serum-free suspension culture of Madin-Darby Canine Kidney cells (MDCK) has emerged as a promising alternative. In this study, a quadrivalent influenza virus subunit vaccine was developed using this platform by culturing four viral strains, followed by purification, inactivation, and formulation. Preclinical safety evaluations in rats, rabbits, and guinea pigs demonstrated no systemic toxicity. In mice, immunization with the vaccine elicited significant hemagglutination inhibition and microneutralization antibodies against H1N1, H3N2, BY, and BV strains at 28 and 42 days post-vaccination. These results indicate that the vaccine candidate possesses a favorable safety profile and induces robust immunogenicity.

Viral infections trigger cellular stress responses, and host stress proteins play key roles in antiviral defense. Here we identify stress-responsive protein arachidonate lipoxygenase-15 (ALOX15) is a critical component of mitochondrial antiviral innate immunity. Loss of Alox15 impairs mitochondrial antiviral signaling (MAVS)-mediated type I interferon production, resulting in increased susceptibility to influenza virus, an effect reversed by adeno-associated virus-mediated lung delivery of Alox15. ALOX15 translocates to mitochondria in response to H1N1 and other RNA viruses (H3N2 and human coronavirus-229E), independent of its enzymatic activity. This mitochondrial localization is also strikingly observed in peripheral blood mononuclear cells from influenza-infected individuals. Mechanistically, ALOX15 is recruited to mitochondria by polymerized MAVS, displacing the deubiquitinase USP19 and sustaining MAVS K63-linked ubiquitination and aggregation. Leveraging these insights, we developed a synergistic ALOX15 regulation-based therapeutic strategy for influenza infection by combining the ALOX15 transcriptional activator songorine with its enzymatic inhibitor PD146176. Together, our findings establish ALOX15 as an essential component of mitochondrial antiviral immunity and a promising host-directed target for antiviral therapy.

Detecting natural selection operating at the amino acid sequence level of proteins is essential for understanding the functional importance of amino acid sites and substitutions. Relatively large number of sequences are required for detecting natural selection using statistical methods including the parsimony method. However, applicability of the parsimony method declines as the number of sequences increases, partly due to the difficulty in inferring ancestral sequences at interior nodes of the phylogenetic tree. Here, an attempt was demonstrated to detect natural selection by the parsimony method without inferring ancestral sequences, through estimating the numbers of synonymous and nonsynonymous substitutions utilizing the minimum numbers of nucleotide and amino acid substitutions along the phylogenetic tree. In the analysis of 19,949 sequences for hemagglutinin of influenza A virus A(H1N1)pdm09, recurrent positive selection was detected at the amino acid sites in B cell and T cell epitopes. In addition, episodic positive selection was detected for the amino acid substitution giving rise to N-linked glycosylation at position 162. These results suggested a possibility that the parsimony method may remain applicable to large-scale sequence analyses for detecting natural selection.

Influenza A viruses (IAVs) have recently expanded their host range to bovine species, raising critical questions about tissue tropism, viral replication capacity, and impacts on dairy production and zoonotic risk. Extensive shedding of highly pathogenic avian influenza virus (HPAIV) H5N1 in milk from naturally infected cattle in the USA highlights the bovine mammary gland as a relevant site of viral replication. To investigate these dynamics, we established and characterized bovine mammary gland organoids (MGOs) derived from primary epithelial cells and maintained in chemically defined media. This efficient, scalable model reconstructs structural and functional hallmarks of the bovine udder, including luminal-basal polarity, lineage-specific cytokeratin patterns, and lactation-associated differentiation. We then used this system to study low and highly pathogenic avian, as well as swine and human IAVs. Viruses from diverse host species productively infected MGOs, largely independent of culturing conditions. Mammalian origin H1N1 strains matched bovine H5N1 in efficiency, but low pathogenic avian H9N2 showed restricted replication, indicating subtype-specific differences in mammary tropism. This suitability study develops a MGO platform that provides a tractable basis for detailed molecular investigation of mammary infection biology and influenza pathogenesis in cattle, to inform strategies that protect both animal and public health.

We evaluated the magnitude and function of antibody responses to seasonal influenza vaccination in individuals with HIV receiving ART compared with individuals without HIV. In a prospective cohort of 78 adults, influenza strain-specific IgG levels were measured up to one-year post-vaccination. Linear mixed-effects models assessed longitudinal trends. IgG levels peaked at two weeks and declined gradually in both groups. HIV status did not significantly influence antibody magnitude or neutralizing activity, and H1N1 antibody levels correlated with neutralization titers. These findings demonstrate robust, durable, and functional humoral response in well-controlled HIV, supporting the effectiveness of annual influenza vaccination in this population.

Influenza A viruses of divergent pathogenicity elicit distinct host immune responses, yet the underlying cellular and spatial dynamics remain poorly defined. Here, we integrate single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to systematically compare the immune landscapes in murine lungs following infection with seasonal H1N1 or a highly pathogenic avian influenza H5N1 virus belonging to clade 2.3.2.1. Analysis of 84,162 immune cells identifies 12 distinct cell types, suggesting that H1N1 infection is associated with robust interferon and inflammatory responses, characterized by alveolar macrophage depletion and extensive infiltration of monocytes and neutrophils. In contrast, H5N1 clade 2.3.2.1 infection is characterized by attenuated interferon signaling, delayed monocyte activation, dysregulated neutrophil maturation, and impaired intercellular communication-features that may be distinct from previously characterized H5N1 strains. Spatial mapping indicates distinct distribution and interaction patterns of immune cells between the two infections. Functional validation through myeloid cell depletion supports the critical role of the monocyte-macrophage axis in disease progression. These findings provide a high-resolution atlas of pulmonary immune responses to influenza viruses of differing pathogenicity and highlight cell-type-specific mechanisms of immune modulation.IMPORTANCESeasonal H1N1 influenza virus causes annual epidemics, while highly pathogenic avian H5N1 virus has a high mortality rate and pandemic potential. Understanding why H5N1 causes more severe disease is critical for developing better treatments. In this study, we used advanced single-cell and spatial technologies to create a detailed map of the immune response in the lungs of mice infected with either H1N1 or a clade 2.3.2.1 H5N1 virus. We discovered that H1N1 triggers a strong, well-organized immune response that controls the virus. In contrast, H5N1 infection leads to a disorganized and weakened response where key immune cells fail to activate and communicate properly. These findings suggest that the H5N1 virus may evade and suppress the host's immune system. Our study provides a high-resolution immune atlas and identifies potential targets for new therapies against severe influenza.

An Igλ light chain bias (IgL) has been identified in ferrets after a primary influenza virus infections. To further study if this phenotype is consistent between species, serum immunoglobulin Igλ and Igκ light chain (IgL) prevalence was evaluated in humans, mice, and pigs following a primary respiratory virus infection. Each IgL population was tested for its binding capacity to the immunodominant viral glycoprotein of the infecting virus. Humans infected with their initial SARS-CoV-2 virus infection had a marginal Igκ bias against the spike (S) glycoprotein, but had no observable IgL bias toward the receptor binding domain of the S protein. Mice had an Igκ bias against the influenza virus hemagglutinin (HA) after a primary infection with an H1N1 or H3N2 influenza virus. Conversely, pigs had an Igλ bias to the HA after a primary infection with either influenza virus subtype. These results highlight that IgL biases vary in different species after a primary viral respiratory infection. Overall, these results have implications for how immunological data should be interpreted between species and the selection of animal models for various applications.