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Disrupting the conserved multivalent binding of hemagglutinin (HA) on influenza A virus (IAV) to sialic acids (SAs) on the host cell membrane offers a robust strategy to block viral attachment and infection, irrespective of antigenic evolution or drug resistance. In this study, we exploit red blood cell‐derived small extracellular vesicles (RBC sEVs) as nanodecoys by harnessing their high abundance of surface‐displayed SAs to interact with IAV through multivalent HA‐SA interactions. This high‐avidity binding inhibits viral adhesion to the cell surface, effectively preventing both attachment and infection in a dose‐dependent manner. Notably, enzymatic removal of SAs from RBC sEVs significantly diminishes their anti‐IAV efficacy. Our findings indicate that RBC sEVs possess intrinsic anti‐IAV properties due to their native multivalent SAs and hold considerable promise as antiviral therapeutics. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Methods Results Conclusions In December 2021, influenza A viruses (IAV) were detected in a population of farmed mink in British Columbia, Canada. Circulation of IAVs in farmed mink populations has raised public health concerns due to similarities between mustelid and human respiratory physiology, potentially facilitating spillover of zoonotic influenzas from livestock.Oropharyngeal specimens were collected from mink as part of a surveillance program for SARS‐CoV‐2. Diagnostic RT‐qPCR testing was performed using a multiplex assay targeting SARS‐CoV‐2, IAV, influenza B virus and respiratory syncytial virus. Whole viral genome sequencing was conducted on IAV‐positive specimens, followed by phylogenetic analysis with other animal and human IAV genome sequences from large global databases.IAVs were detected in 17 of 65 mink by RT‐qPCR. Based on genomic sequencing and phylogenetic analysis, these IAVs were subtyped as H3N2s that originated from reassortment of swine H3N2 (clade 1990.4 h), human seasonal H1N1 (pdm09) and swine H1N2 (clade 1A.1.1.3). This reassortant has been subsequently observed in swine in several Midwest American states, as well as in swine and turkeys in Ontario, suggesting its spillover into farmed mink in British Columbia was incidental to its broader dissemination in North American swine populations.These detections reaffirm the need for extensive genomic surveillance of IAVs in swine populations to monitor reassortments that might become public health concerns. They also highlight the need for closer surveillance of IAVs in mink to preserve animal health, protect agricultural interests, and monitor potential zoonotic threats. [ABSTRACT FROM AUTHOR]

Influenza A virus (H1N1) poses a significant threat to global human health that imperative demands the development of sensitive and accurate point‐of‐care testing (POCT) methods. Here, for the first time, Fe‐MoS2 nanosheets were employed as a multifunctional nanotag in the development of catalytic colorimetric‐photothermal dual‐mode lateral flow immunoassay (dLFIA) strips for the sensitive detection of H1N1 inactivated virus. The Fe‐MoS2 nanosheets featuring large size, high specific surface area, and ultrathin structure could flow smoothly on the strips and thus quickly produce an ideal colorimetric signal for qualitative analysis. Both the limit of detection (LOD) of catalytic colorimetric and photothermal signals reached 1000 copies/mL and the corresponding calculated LOD was 550 and 691 copies/mL, respectively, which were about 50–90‐fold more sensitive than traditional gold nanoparticles based‐LFIA (5 × 104 copies/mL). The developed assay could correctly identify eight positive clinical samples with Ct values less than 35 and 10 negative actual samples, proving significant promise for rapid, sensitive, and accurate detection of H1N1, especially in resource‐limited areas. [ABSTRACT FROM AUTHOR]

ABSTRACT Aims Methods and Results Conclusions The world experienced a huge number of outbreaks of highly pathogenic avian influenza (HPAI) in birds, which could represent one of the largest registered epidemics of infectious disease in food‐producing animals. Therefore, mammals, including humans, are continuously exposed to HPAI viruses leading to sporadic and sometimes unusual mammal infections. The aim of this paper is to assess the risk of crossing the avian/mammalian species barrier by the currently circulating HPAI viruses, focusing on the epidemiological situation of Belgium, a representative country for Western Europe.Information on transmission pathways and species susceptibility, based on the experimental and epidemiological data, was reviewed and weighted to assess the risk of mammal infection with HPAI A(H5N1) viruses of the circulating clade 2.3.4.4b. This risk is defined as the likelihood of mammal infection by birds crossed by the clinical consequences of this infection for this animal. From the Belgian perspective, it is concluded that this risk remains ‘low’ to ‘moderate’ for captive/domestic mammal species. However, this risk was categorised as ‘high’ for certain species, i.e. mammals that have the opportunity to have frequent direct or indirect close contacts with infected (dead) birds, such as wild felids, wild mustelids, foxes and wild marine carnivore mammals. For some mammal species, the uncertainty associated with the assessment remains high due to an ever‐changing situation.The longer the virus will continue to circulate in wildlife/the environment the stronger the probability of contact between infected birds and mammals will become. This will increase the related risk of viral adaptation for efficient transmission between mammal, posing concerns for public health. Regular reassessments based on the field and experimental data are therefore necessary to implement and adapt risk‐based mitigation measures. This will require continuous monitoring of avian influenza viruses in both birds and mammals as well as sharing of sequence data. [ABSTRACT FROM AUTHOR]

The SARS‐CoV‐2 pandemic has challenged more scientists to detect viruses and to visualize virus‐containing spots for diagnosis and infection control; however, detection principles of commercially available technologies are not optimal for visualization. Here, a convenient and universal homogeneous detection platform named proximity‐unlocked luminescence by sequential enzymatic reactions from antibody and antibody/aptamer (PULSERAA) is developed. This is designed so that the signal appears only when the donor and acceptor are in proximity on the viral surface. PULSERAA specifically detected in the range of 25–500 digital copies/mL of inactivated SARS‐CoV‐2 after simply mixing reagents; it is elucidated that the accumulation of chemical species in a limited space of the viral surface contributed to such high sensitivity. PULSERAA was quickly adapated to detect another virus variant, inactivated influenza A virus, and infectious SARS‐CoV‐2 in a clinical sample. Furthermore, on‐site (direct, rapid, and portable) visualization of the inactivated SARS‐CoV‐2‐containing spots by a conventional smartphone camera was achieved, demonstrating that PULSERAA can be a practical tool for preventing the next pandemic in the future. [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]

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

Exactly why human infection of avian influenza A virus H7N9 causes more severe disease in the elderly remains elusive. In this study, we found that H7N9 PB1‐F2 is a pathogenic factor in 15–18‐month‐old BALB/C mice (aged mice) but not in 6–8‐week‐old young adult mice (young mice). Recombinant influenza A virus with H7N9 PB1‐F2‐knockout was less pathogenic in aged mice as indicated with delayed weight loss. In contrast, survival of young mice infected with this virus was diminished. Furthermore, tissue damage, inflammation, proinflammatory cytokine and 2′3′‐cGAMP production in the lung were less pronounced in infected aged mice despite no change in viral titer. cGAS is known to produce 2′3′‐cGAMP to boost proinflammatory cytokine expression through STING‐NF‐κB signaling. We found that H7N9 PB1‐F2 promoted interferon β (IFNβ) and chemokine gene expression in cultured cells through the mitochondrial DNA‐cGAS‐STING‐NF‐κB pathway. H7N9 PB1‐F2 formed protein aggregate and caused mitochondrial cristae collapse, complex V‐dependent electron transport dysfunction, reverse electron transfer‐dependent oxidized mitochondrial DNA release to the cytoplasm and activation of cGAS‐STING‐NF‐κB signaling. PB1‐F2 N57 truncation, which is frequently observed in human circulating strains, mitigated H7N9 PB1‐F2‐mediated mitochondrial dysfunction and cGAS activation. In addition, we found that PB1‐F2 of pathogenic avian influenza viruses triggered more robust cGAS activation than their human‐adapted descendants. Our findings provide one explanation to age‐dependent pathogenesis of H7N9 infection. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) remains a significant global public health threat, causing substantial illness and economic burden. Despite existing antiviral drugs, the emergence of resistant strains necessitates alternative therapeutic strategies. This review explores the complex interplay between the ubiquitin proteasome system (UPS) and IAV pathogenesis. We discuss how IAV manipulates the UPS to promote its lifecycle, while also highlighting how host cells utilise the UPS to counteract viral infection. Recent research on deubiquitinases as potential regulators of IAV infection is also addressed. By elucidating the multifaceted role of the UPS in IAV pathogenesis, this review aims to identify potential targets for novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Abietane type diterpenic (dehydroabietic, 2,3‐dihydroquinopimaric and maleopimaric) acids were converted by the acid chloride method into a series of aliphatic and heterocyclic amine spacered conjugates. A number of structurally novel derivatives holding 1,2,3‐triazole moieties were designed and synthesized by treating of the propargylated amides and esters with a sugar azides using the Cu(I)‐catalyzed click chemistry approach. The synthesized N‐containing diterpene derivatives were tested for their potential inhibition of influenza A/PuertoRico/8/34 (H1N1) virus in MDCK cell culture and SARS‐CoV‐2 pseudovirus in BHK‐21‐hACE2 cells. Among tested forty‐five compounds ten derivatives were the most efficacious against influenza virus A with IC50 0.7–63.4 μM together with high selectivity index SI value from 11 from 94. Dihydroquinopimaric acid N‐ethylpiperazine‐amide and dehydroabietic acid 1,2,3‐triazoles spacered with glucose and lactose showed anti‐SARS‐CoV‐2 pseudovirus activity with EC50 values of 1.79–25.46 μM. Molecular docking and dynamics modeling investigated the binding mode of the lead compounds into the binding pocket of influenza A virus M2 protein and the RBD domain of SARS‐CoV‐2 spike glycoprotein. [ABSTRACT FROM AUTHOR]

Aim: 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. Methods and Results: 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. Conclusion: 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]

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

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

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]

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]

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]

Vaccination is the most effective strategy to combat influenza. Ideally, potent and persistent vaccine effects would be induced with a single vaccine dose. Here, we designed a virus‐like particle (VLP)‐based vaccine presenting multiple copies of the influenza hemagglutinin (HA) from A/Puerto Rico/8/1934 (PR8HA‐VLP) and examined its immunogenicity and protective efficacy in ferrets. Serum‐neutralizing antibodies were effectively induced against the homologous virus at 3‐week post‐vaccination with a single dose of PR8HA‐VLP with or without adjuvants. When the single‐immunized ferrets were challenged with the homologous virus, virus replication in the nasal mucosa was significantly reduced. Long‐term monitoring of serum titers revealed that after adjuvanted vaccination with PR8HA‐VLP, neutralizing antibodies were retained at similar levels 20‐ to 183‐week post‐vaccination, although a 4‐ to 8‐fold titer decline was observed from 3‐ to 20‐week post‐vaccination. Boost immunization at 183 weeks after the first immunization elicited higher neutralizing antibody titers than those at 3 weeks after the initial immunization in most of the animals. These results confirm that nanoparticle‐based vaccines are a promising approach to effectively elicit durable multiyear neutralizing antibody responses against influenza viruses. [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]

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]

The N121 site on the spike protein of SARS‐CoV‐2 is associated with heme and its metabolite, biliverdin, which can affect antibody binding. Both N121T and N121S substitutions have been observed in natural conditions and in a hamster model of dual infection with SARS‐CoV‐2 and Influenza A virus. Serum pseudotype neutralization assays against HIV‐1 particles carrying wild‐type, N121T, and N121S spikes with immune mouse and human sera revealed that N121T and N121S mutations had a greater impact on serum neutralization than biliverdin treatment. Although N121T and N121S substitutions are not currently major SARS‐CoV‐2 variants of concern, this study could provide fundamental information to prepare for potential future mutations at the N121 site of SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Influenza virus is a well‐known pathogen that can cause epidemics and pandemics. Several surveillance methods are being followed to monitor the transmission patterns and spread of influenza in the community. Wastewater‐based Epidemiology (WBE) can serve as an additional tool to detect the presence of influenza viruses. The current study primarily focuses on surveillance of Influenza A and Influenza B in wastewater treatment plant (WWTP) samples. A total of 100 wastewater samples were collected in July (n = 50) and August (n = 50) 2023 from four different WWTPs in Manipal and Udupi, district of Karnataka, India. The WWTP samples were processed and tested by Real‐Time reverse transcriptase PCR (RT‐PCR). The data generated was analysed in comparison with the clinical Influenza cases. Of the 100 samples, 18 (18%) tested positive for Influenza A virus and 2 (2%) tested positive for Influenza B virus, with a viral load ranging 1.4 x 102–2.2 x 103 gc/L for influenza A virus and 5.2 x 103–7.7 x 103gc/L for influenza B virus. On correlating the WWTP positivity with clinical case, it was found that influenza clinical cases and virus positivity in wastewater increased simultaneously, emphasizing WBE as a concurrent method for monitoring influenza virus activity. [ABSTRACT FROM AUTHOR]

The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA‐dependent RNA polymerase (RdRp) has emerged as a potential target for broad‐spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently‐used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring‐opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad‐spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high‐throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 μM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 μM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 μM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 μM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring‐opening" bases and suggests the "ring‐opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open‐loop bases to mimic Watson‐Crick base pairing better and improve antiviral activity. [ABSTRACT FROM AUTHOR]

Although rapid progression and a poor prognosis in influenza A virus (IAV) infection–induced acute exacerbation of chronic obstructive pulmonary disease (AECOPD) are frequently associated with metabolic energy disorders, the underlying mechanisms and rescue strategies remain unknown. We herein demonstrated that the level of resting energy expenditure increased significantly in IAV‐induced AECOPD patients and that cellular energy exhaustion emerged earlier and more significantly in IAV‐infected primary COPD bronchial epithelial (pDHBE) cells. The differentially expressed genes were enriched in the oxidative phosphorylation (OXPHOS) pathway; additionally, we consistently uncovered much earlier ATP exhaustion, more severe mitochondrial structural destruction and dysfunction, and OXPHOS impairment in IAV‐inoculated pDHBE cells, and these changes were rescued by melatonin. The level of OMA1‐dependent cleavage of OPA1 in the mitochondrial inner membrane and the shift in energy metabolism from OXPHOS to glycolysis were significantly increased in IAV‐infected pDHBE cells; however, these changes were rescued by OMA1‐siRNA or melatonin further treatment. Collectively, our data revealed that melatonin rescued IAV–induced cellular energy exhaustion via OMA1‐OPA1‐S to improve the clinical prognosis in COPD. This treatment may serve as a potential therapeutic agent for patients in which AECOPD is induced by IAV. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the pathogen epidemiology of community‐acquired pneumonia (CAP) among children in Southwest China before, during and after the COVID‐19 non‐pharmaceutical interventions (NPIs). Methods: Pathogen data of hospitalised children with CAP, including multiple direct immunofluorescence test for seven viruses, bacterial culture and polymerase chain reaction (PCR) for Mycoplasma pneumoniae, were analysed across three phases: Phase I (pre‐NPIs: 1 January 2019 to 31 December 2019), Phase II (NPI period: 1 January 2020 to 31 December 2020) and Phase III (post‐NPIs: 1 January 2023 to 31 December 2023). Results: A total of 7533 cases were enrolled, including 2444, 1642 and 3447 individuals in Phases I, II and III, respectively. M. pneumoniae predominated in Phases I and III (23.4% and 35.5%, respectively). In Phase II, respiratory syncytial virus (RSV) emerged as the primary pathogen (20.3%), whereas detection rates of influenza A virus (Flu A) and M. pneumoniae were at a low level (1.8% and 9.6%, respectively). In Phase III, both Flu A (15.8%) and M. pneumoniae epidemic rebounded, whereas RSV detection rate returned to Phase I level, and detection rates of Streptococcus pneumoniae and Haemophilus influenzae decreased significantly compared to those in Phase I. Detection rates of adenovirus and parainfluenza virus type 3 decreased phase by phase. Age‐stratified analysis and monthly variations supported the above findings. Seasonal patterns of multiple pathogens were disrupted during Phases II and III. Conclusions: COVID‐19 NPIs exhibited a distinct impact on CAP pathogen epidemic among children, with post‐NPIs increases observed in M. pneumoniae and Flu A prevalence. Continuous pathogen monitoring is crucial for effective prevention and control of paediatric CAP. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and influenza A virus (IAV) are two respiratory viruses and are similar concerning seasonal occurrence, transmission routes, clinical manifestations, and related immune responses. Recent studies showed that a substantial number of patients infected by SARS‐CoV‐2 were also coinfected with IAV. In this paper, we study the global properties of SARS‐CoV‐2 and IAV coinfection model in vivo. The role of humoral (antibody) immunity in controlling the coinfection is modeled. The model tracks uninfected epithelial cells, latent SARS‐CoV‐2‐infected epithelial cells, latent IAV‐infected epithelial cells, active SARS‐CoV‐2‐infected epithelial cells, active IAV‐infected epithelial cells, free SARS‐CoV‐2 particles, free IAV particles, SARS‐CoV‐2‐specific antibodies and IAV‐specific antibodies. The model includes six distributed‐time delays, two delays for the formation of latent SARS‐CoV‐2‐infected and latent IAV‐infected cells; two delays for the reactivation of latent SARS‐CoV‐2‐infected cells and latent IAV‐infected cells; and two delays for the maturation of new released SARS‐CoV‐2 and IAV virions. The regeneration and death of the uninfected epithelial cells are considered. We first examine the basic qualitative properties of the model, then we calculate all equilibria and prove their global stability. The global stability of equilibria is established using the Lyapunov method. The theoretical findings are demonstrated via numerical simulations. The importance of considering humoral immunity in the coinfection dynamics model is discussed. It is found that without modeling the humoral immunity, the case of IAV and SARS‐CoV‐2 coexistence will not occur. We discuss the effect of time delays on the dynamics of SARS‐CoV‐2 and IAV coinfection. It is noted that increasing the time delay length has similar impact as antiviral therapies. [ABSTRACT FROM AUTHOR]

Sialosides containing C8‐modified sialic acids are challenging synthetic targets but potentially useful probes for diagnostic substrate profiling of sialidases and elucidating the binding specificity of sialic acid‐interacting proteins. Here, we demonstrate efficient chemoenzymatic methods for synthesizing para‐nitrophenol‐tagged α2–3‐ and α2–6‐linked sialyl galactosides containing C8‐acetamido, C8‐azido, or C8‐amino derivatized N‐acetylneuraminic acid (Neu5Ac). High‐throughput substrate specificity studies showed that the C8‐modification of sialic acid significantly changes its recognition by sialidases from humans, various bacteria, and different influenza A and B viruses. Sialosides carrying Neu5Ac with a C8‐azido modification were generally well tolerated by all the sialidases we tested, whereas sialosides containing C8‐acetamido‐modified Neu5Ac were only cleaved by selective bacterial sialidases. In contrast, sialosides with C8‐amino‐modified Neu5Ac were cleaved by a combination of selective bacterial and influenza A virus sialidases. These results indicate that sialosides terminated with a C8‐amino or C8‐acetamido‐modified sialic acid can be used with other sialosides for diagnostic profiling of disease‐causing sialidase‐producing pathogens. [ABSTRACT FROM AUTHOR]

Background and Purpose: Severe influenza virus‐infected patients have high systemic levels of Th1 cytokines (including IFN‐γ). Intrapulmonary IFN‐γ increases pulmonary IFN‐γ‐producing T lymphocytes through the CXCR3 pathway. Virus‐infected mice lacking IP‐10/CXCR3 demonstrate lower pulmonary neutrophilic inflammation. AMG487, an IP‐10/CXCR3 antagonist, ameliorates virus‐induced lung injury in vivo through decreasing viral loads. This study examined whether AMG487 could treat H1N1 virus‐induced mouse illness through reducing viral loads or decreasing the number of lymphocytes or neutrophils. Experimental Approach: Here, we studied the above‐mentioned effects and underlying mechanisms in vivo. Key Results: H1N1 virus infection caused bad overall condition and pulmonary inflammation characterized by the infiltration of lymphocytes and neutrophils. From Day‐5 to Day‐10 post‐virus infection, bad overall condition, pulmonary lymphocytes, and IFN‐γ concentrations increased, while pulmonary H1N1 viral titres and neutrophils decreased. Both anti‐IFN‐γ and AMG487 alleviated virus infection‐induced bad overall condition and pulmonary lymphocytic inflammation. Pulmonary neutrophilic inflammation was mitigated by AMG487 on Day‐5 post‐infection, but was not mitigated by AMG487 on Day‐10 post‐infection. H1N1 virus induced increases of IFN‐γ, IP‐10, and IFN‐γ‐producing lymphocytes and activation of the Jak2‐Stat1 pathways in mouse lungs, which were inhibited by AMG487. Anti‐IFN‐γ decreased IFN‐γ and IFN‐γ‐producing lymphocytes on Day‐5 post‐infection. AMG487 but not anti‐IFN‐γ decreased viral titres in mouse lung homogenates or BALF. Higher virus load did not increase pulmonary inflammation and IFN‐γ concentrations when mice were treated with AMG487. Conclusion and Implications: AMG487 may ameliorate H1N1 virus‐induced pulmonary inflammation through decreasing IFN‐γ‐producing lymphocytes rather than reducing viral loads or neutrophils. [ABSTRACT FROM AUTHOR]

The vagus nerve circuit, operating through the alpha‐7 nicotinic acetylcholine receptor (α7 nAChR), regulates the inflammatory response by influencing immune cells. However, the role of vagal‐α7 nAChR signaling in influenza virus infection is unclear. In particular, does vagal‐α7 nAChR signaling impact the infection of alveolar epithelial cells (AECs), the primary target cells of influenza virus? Here, we demonstrated a distinct role of α7 nAChR in type II AECs compared to its role in immune cells during influenza infection. We found that deletion of Chrna7 (encoding gene of α7 nAChR) in type II AECs or disruption of vagal circuits reduced lung influenza infection and protected mice from influenza‐induced lung injury. We further unveiled that activation of α7 nAChR enhanced influenza infection through PTP1B‐NEDD4L‐ASK1‐p38MAPK pathway. Mechanistically, activation of α7 nAChR signaling decreased p38MAPK phosphorylation during infection, facilitating the nuclear export of influenza viral ribonucleoproteins and thereby promoting infection. Taken together, our findings reveal a mechanism mediated by vagal‐α7 nAChR signaling that promotes influenza viral infection and exacerbates disease severity. Targeting vagal‐α7 nAChR signaling may offer novel strategies for combating influenza virus infections. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) can rapidly disseminate among animals through various transmission routes, with emerging evidence suggesting the ocular surface as an important entrance. However, it remains unclear how the virus invades the respiratory tract after ocular exposure. Here, we demonstrated that H1N1 (A/swine/Guangdong/1/2011) utilizes the nasolacrimal system to rapidly spread from the ocular surface to the respiratory tract in the porcine model. In vivo and ex vivo, IAV could efficiently attach and replicate in conjunctiva epithelium, which has abundance of α‐2,6‐linked and α‐2,3‐linked sialic acid. After ocular inoculation, infectious virions swiftly migrate to the nasolacrimal duct of piglets and, via continual drainage, disseminate to the respiratory tract. Moreover, the detection of continual virus shedding as well as the successful isolation of virus from conjunctiva and respiratory tract tissue indicated the establishment of productive infection after the transocular route. This study presents evidence suggesting that IAVs could utilize the nasolacrimal system to swiftly spread to the respiratory tract following ocular exposure, which contributes to understanding the modes of transocular transmission of IAVs. [ABSTRACT FROM AUTHOR]

Sialyl‐Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty‐two isomeric asymmetrical tri‐antennary N‐glycans presenting SLex‐Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high‐affinity ligands for E‐selectin. The N‐glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5′‐diphospho‐N‐trifluoracetylglucosamine (UDP‐GlcNHTFA) that can be transferred by branching N‐acetylglucosaminyltransferases to give, after base treatment, GlcNH2‐containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E‐selectin bound equally well to linear glycans and tri‐antennary N‐glycans presenting SLex‐Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri‐antennary N‐glycans. Furthermore, several H5 HAs preferentially bound to N‐glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N‐glycans presenting SLex. [ABSTRACT FROM AUTHOR]

Background: Astragaloside IV (AS‐IV) is the most active monomer in the traditional Chinese herbal medicine Radix Astragali, which has a wide range of antiviral, anti‐inflammatory, and antifibrosis pharmacological effects, and shows protective effects in acute lung injury. Methods: This study utilized the immunofluorescence, flow cytometry, enzyme‐linked immunosorbent assay, quantitative reverse transcription‐polymerase chain reaction, western blot, and hematoxylin and eosin staining methods to investigate the mechanism of AS‐IV in reducing viral pneumonia caused by influenza A virus in A549 cells and BALB/c mice. Results: The results showed that AS‐IV suppressed reactive oxygen species production in influenza virus‐infected A549 cells in a dose‐dependent manner, and subsequently inhibited the activation of nucleotide‐binding oligomerization domain‐like receptor thermal protein domain associated protein 3 inflammasome and Caspase‐1, decreased interleukin (IL) ‐1β and IL‐18 secretion. In BALB/c mice infected with Poly (I:C), oral administration of AS‐IV can significantly reduce Poly (I:C)‐induced acute pneumonia and lung pathological injury. Conclusions: AS‐IV alleviates the inflammatory response induced by influenza virus in vitro and lung flammation and structural damage caused by poly (I:C) in vivo. [ABSTRACT FROM AUTHOR]

During the COVID‐19 pandemic, non‐pharmaceutical interventions were introduced to reduce exposure to respiratory viruses. However, these measures may have led to an "immunity debt" that could make the population more vulnerable. The goal of this study was to examine the transmission dynamics of seasonal influenza in the years 2023–2024. Respiratory samples from patients with influenza‐like illness were collected and tested for influenza A and B viruses. The electronic medical records of index cases from October 2023 to March 2024 were analyzed to determine their clinical and epidemiological characteristics. A total of 48984 positive cases were detected, with a pooled prevalence of 46.9% (95% CI 46.3–47.5). This season saw bimodal peaks of influenza activity, with influenza A peaked in week 48, 2023, and influenza B peaked in week 1, 2024. The pooled positive rates were 28.6% (95% CI 55.4–59.6) and 18.3% (95% CI 18.0–18.7) for influenza A and B viruses, respectively. The median values of instantaneous reproduction number were 5.5 (IQR 3.0–6.7) and 4.6 (IQR 2.4–5.5), respectively. The hospitalization rate for influenza A virus (2.2%, 95% CI 2.0–2.5) was significantly higher than that of influenza B virus (1.1%, 95% CI 0.9–1.4). Among the 17 clinical symptoms studied, odds ratios of 15 symptoms were below 1 when comparing influenza A and B positive inpatients, with headache, weakness, and myalgia showing significant differences. This study provides an overview of influenza dynamics and clinical symptoms, highlighting the importance for individuals to receive an annual influenza vaccine. [ABSTRACT FROM AUTHOR]

Pregnancy heightens susceptibility to influenza A virus (IAV) infection, thereby increasing the risk of severe pneumonia and maternal mortality. It also raises the chances of adverse outcomes in offspring, such as fetal growth restriction, preterm birth, miscarriage, and stillbirth in offsprings. However, the underlying mechanisms behind these effects remain largely unknown. Syncytiotrophoblast cells, crucial in forming the placental barrier, nutrient exchange and hormone secretion, have not been extensively studied for their responses to IAV. In our experiment, we used Forskolin‐treated BeWo cells to mimic syncytiotrophoblast cells in vitro, and infected them with H1N1, H5N1 and H7N9 virus stains. Our results showed that syncytiotrophoblast cells, with their higher intensity of sialic acid receptors, strongly support IAV infection and replication. Notably, high‐dose viral infection and prolonged exposure resulted in a significant decrease in fusion index, as well as gene and protein expression levels associated with trophoblast differentiation, β‐human chorionic gonadotropin secretion, estrogen and progesterone biosynthesis, and nutrient transport. In pregnant BALB/c mice infected with the H1N1 virus, we observed significant decreases in trophoblast differentiation and hormone secretion gene expression levels. IAV infection also resulted in preterm labor, fetal growth restriction, and increased maternal and fetal morbidity and mortality. Our findings indicate that IAV infection in syncytiotrophoblastic cells can result in adverse pregnancy outcomes by altering trophoblast differentiation, suppressing of β‐hCG secretion, and disrupting placental barrier function. [ABSTRACT FROM AUTHOR]

The nuclear export protein of the influenza A virus (NEP) is involved in many important processes of the virus life cycle. This makes it an attractive target for the treatment of a disease caused by a virus. Previously it has been shown, that recombinant variants of NEP are highly prone to aggregation in solution under various conditions with the formation of amyloid‐like aggregates. In the present work, the amyloid nature of NEP aggregates was evidenced by Congo red binding assays. Atomic force microscopy has shown that NEP can form two types of spherical nanoparticles, which provide an alternative pathway for the formation of amyloid‐like fibrils. Type I of these "fibrillogenic" spheres, formed under physiological conditions, represents the micelle‐like particles with height 10–60 nm, which can generate worm‐like flexible fibrils with the diameter 2.5–4.0 nm, length 20–500 nm and the Young's modulus ~73 MPa. Type II spherical aggregates with size of about 400–1000 nm, formed at elevated temperatures, includes fractions of drop‐like and vesicle‐like particles, generating more rigid amyloid‐like fibrils with height of ~8 nm, and length of up to 2 μm. The hypothetical mechanism of fibril formation via nanospherical structures was suggested. Research Highlights: AFM has revealed two types of the influenza A virus nuclear export protein spherical aggregates.They provide an alternative pathway for the formation of amyloid‐like fibrils.The mechanism of fibril formation via spherical structures is suggested. [ABSTRACT FROM AUTHOR]

Aims: Swine are a mixing vessel for the emergence of novel reassortant influenza A viruses (IAV). Interspecies transmission of swine‐origin IAV poses a public health and pandemic risk. In the United States, the majority of zoonotic IAV transmission events have occurred in association with swine exposure at agricultural fairs. Accordingly, this human‐animal interface necessitates mitigation strategies informed by understanding of interspecies transmission mechanisms in exhibition swine. Likewise, the diversity of IAV in swine can be a source for novel reassortant or mutated viruses that pose a risk to both swine and human health. Methods and Results: In an effort to better understand those risks, here we investigated the epidemiology of IAV in exhibition swine and subsequent transmission to humans by performing phylogenetic analyses using full genome sequences from 272 IAV isolates collected from exhibition swine and 23 A(H3N2)v viruses from human hosts during 2013–2015. Sixty‐seven fairs (24.2%) had at least one pig test positive for IAV with an overall estimated prevalence of 8.9% (95% CI: 8.3–9.6, Clopper‐Pearson). Of the 19 genotypes found in swine, 5 were also identified in humans. There was a positive correlation between the number of human cases of a genotype and its prevalence in exhibition swine. Additionally, we demonstrated that A(H3N2)v viruses clustered tightly with exhibition swine viruses that were prevalent in the same year. Conclusions: These data indicate that multiple genotypes of swine‐lineage IAV have infected humans, and highly prevalent IAV genotypes in exhibition swine during a given year are also the strains detected most frequently in human cases of variant IAV. Continued surveillance and rapid characterization of IAVs in exhibition swine can facilitate timely phenotypic evaluation and matching of candidate vaccine strains to those viruses present at the human‐animal interface which are most likely to spillover into humans. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to identify exposure to human, swine, and avian influenza A virus subtypes in rural companion and hunting dogs, backyard pigs, and feral pigs. Methods and Results: The study took place in a region of southeastern Mexico where the sampled individuals were part of backyard production systems in which different domestic and wild species coexist and interact with humans. We collected blood samples from pigs and dogs at each of the sites. We used a nucleoprotein enzyme‐linked immunosorbent assay to determine the exposure of individuals to influenza A virus. Haemagglutination inhibition was performed on the positive samples to determine the subtypes to which they were exposed. For data analysis, a binomial logistic regression model was generated to determine the predictor variables for the seropositivity of the individuals in the study. We identified 11 positive individuals: three backyard pigs, four companion dogs, and four hunting dogs. The pigs tested positive for H1N1 and H1N2. The dogs were positive for H1N1, H1N2, and H3N2. The model showed that dogs in contact with backyard chickens are more likely to be seropositive for influenza A viruses. Conclusions: We demonstrated the essential role hunting dogs could play as intermediate hosts and potential mixing vessel hosts when exposed to human and swine‐origin viral subtypes. These results are relevant because these dogs interact with domestic hosts and humans in backyard systems, which are risk scenarios in the transmission of influenza A viruses. Therefore, it is of utmost importance to implement epidemiological surveillance of influenza A viruses in backyard animals, particularly in key animals in the transmission of these viruses, such as dogs and pigs. [ABSTRACT FROM AUTHOR]

Considering the urgent demand for reliable and rapid detection of infectious respiratory viruses during unpredictable pandemics, an innovative ultrasensitive colorimetric immunoassay for influenza A (H1N1) virus detection is developed herein. The proposed approach leverages dual amplification by combining layer‐by‐layer interactions with the nanozyme effect of biotinylated gold nanoparticles (BGNPs). BGNPs assemble around the target via repeated incubation cycles under optimized conditions, resulting in a layered structure that increases optical density, producing a more intense signal proportional to the viral titer. Additionally, the nanozyme effect of the layered BGNPs induces oxidation of 3,3',5,5'‐tetramethylbenzidine, which further enhances the visible signal detectable by the naked eye. This synergetic nanoprobe‐based system demonstrates remarkable sensitivity, with a limit of detection of 101.29 EID50 mL−1, which is 2500‐fold higher than that of commercial rapid kits and conventional enzyme‐linked immunosorbent assays, within a rapid 55 min timeframe. Furthermore, the anti‐interference capability and portability of the developed system reinforce its practicality, making it a promising tool for field diagnostic tests that offers advanced, ultrasensitive, and early detection of respiratory viruses. [ABSTRACT FROM AUTHOR]

Pyrogallol, a natural polyphenol compound (1,2,3‐trihydroxybenzene), has shown efficacy in the therapeutic treatment of disorders associated with inflammation. Nevertheless, the mechanisms underlying the protective properties of pyrogallol against influenza A virus infection are not yet established. We established in this study that pyrogallol effectively alleviated H1N1 influenza A virus‐induced lung injury and reduced mortality. Treatment with pyrogallol was found to promote the expression and nuclear translocation of nuclear factor erythroid‐2‐related factor 2 (Nrf2) and peroxisome proliferator‐activated receptor gamma (PPAR‐γ). Notably, the activation of Nrf2 by pyrogallol was involved in elevating the expression of PPAR‐γ, both of which act synergistically to enhance heme oxygenase‐1 (HO‐1) synthesis. Blocking HO‐1 by zinc protoporphyrin (ZnPP) reduced the suppressive impact of pyrogallol on H1N1 virus‐mediated aberrant retinoic acid‐inducible gene‐I‐nuclear factor kappa B (RIG‐I–NF‐κB) signaling, which thus abolished the dampening effects of pyrogallol on excessive proinflammatory mediators and cell death (including apoptosis, necrosis, and ferroptosis). Furthermore, the HO‐1‐independent inactivation of janus kinase 1/signal transducers and activators of transcription (JAK1/STATs) and the HO‐1‐dependent RIG‐I‐augmented STAT1/2 activation were both abrogated by pyrogallol, resulting in suppression of the enhanced transcriptional activity of interferon‐stimulated gene factor 3 (ISGF3) complexes, thus prominently inhibiting the amplification of the H1N1 virus‐induced proinflammatory reaction and apoptosis in interferon‐beta (IFN‐β)‐sensitized cells. The study provides evidence that pyrogallol alleviates excessive proinflammatory responses and abnormal cell death via HO‐1 induction, suggesting it could be a potential agent for treating influenza. [ABSTRACT FROM AUTHOR]

To assess the positive rate of 11 respiratory pathogens in 2023, providing a comprehensive summary and analysis of the respiratory infection patterns after COVID‐19 pandemic. The study comprised 7544 inpatients suspected of respiratory infections who underwent respiratory pathogen multiplex polymerase chain reaction tests from July 2022 to December 31, 2023. We analyzed the positive rate of 11 pathogens over 18 months and the characterization of infection patterns among different age groups and immune states. Among 7544 patients (age range 4 months to 104 years, 44.99% female), the incidence of infected by at least one of the 11 pathogens was 26.07%. Children (55.18%, p < 0.05) experienced a significantly higher infection probability than adults (20.88%) and old (20.66%). Influenza A virus (8.63%), Mycoplasma pneumoniae (5.47%), and human rhinovirus (5.12%) were the most common pathogens. In children, M. pneumoniae (35.96%) replaced the predominant role of human respiratory syncytial virus (HRSV) (5.91%) in the pathogen spectrum. Age, immunosuppressed state, and respiratory chronic conditions were associated with a significantly higher risk of mixed infection. Immunosuppressed patients were more vulnerable to human coronavirus (4.64% vs. 1.65%, p < 0.05), human parainfluenza virus (3.46% vs. 1.69%, p < 0.05), and HRSV (2.27% vs. 0.55%, p < 0.05). Patterns in respiratory infections changed following regional epidemic control measures and the COVID‐19 pandemic. [ABSTRACT FROM AUTHOR]

Enveloped viruses carry one or multiple proteins with receptor‐binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor‐binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N‐ and C‐terminal, for influenza A virus hemagglutinins and SARS‐CoV‐2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties. [ABSTRACT FROM AUTHOR]

Antiviral therapy based on neuraminidase (oseltamivir) or polymerase (baloxavir marboxil) inhibitors plays an important role in the management of influenza infections. However, the emergence of drug resistance and the uncontrolled inflammatory response are major limitations in the treatment of severe influenza disease. Protectins D1 (PD1) and DX (PDX), part of a family of pro‐resolving mediators, have previously demonstrated anti‐influenza activity as well as anti‐inflammatory properties in various clinical contexts. Herein, we synthetized a series of simplified PDX analogs and assessed their in vitro antiviral activity against influenza A(H1N1) viruses, including oseltamivir‐ and baloxavir‐resistant variants. In ST6GalI‐MDCK cells, the PDX analog AN‐137B reduced viral replication in a dose‐dependent manner with IC50 values of 23.8 for A/Puerto Rico/8/1934 (H1N1) and between 32.6 and 36.7 µM for susceptible and resistant A(H1N1)pdm09 viruses. In MTS‐based cell viability experiments, AN‐137B showed a 50% cellular cytotoxicity (CC50) of 638.7 µM with a resulting selectivity index of 26.8. Of greater importance, the combination of AN‐137B with oseltamivir or baloxavir resulted in synergistic and additive in vitro effects, respectively. Treatment of lipopolysaccharide (LPS)‐stimulated macrophages with AN‐137B resulted in a decrease of iNOS activity as shown by the reduction of nitrite production, suggesting an anti‐inflammatory effect. In conclusion, our results indicate that the protectin analog AN‐137B constitutes an interesting therapeutic modality against influenza A virus, warranting further evaluation in animal models. [ABSTRACT FROM AUTHOR]

Infection with influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) poses a significant risk to human life, health, and the global economy. Vaccination is one of the most effective strategies in the fight against infectious viruses. In this study, we, for the first time, have evaluated the immunogenicity and protective effect of an influenza/SARS‐CoV‐2 Omicron subunit combined vaccine adjuvanted with MF59 and administered to BALB/c mice. Results showed that the combined vaccine induced high levels of IgG, IgG1, and IgG2a antibodies, as well as influenza A H1N1/California/2009 virus‐specific hemagglutination‐inhibiting antibodies in BALB/c mice. Moreover, this subunit combined vaccine induced high titers of neutralization antibodies against SARS‐CoV‐2 Omicron sublineage BA.5 pseudovirus and effectively reduced the viral load of authentic SARS‐CoV‐2 Omicron sublineage BA.5.2 in the cell culture supernatants. These results suggested that this subunit combined vaccine achieved protective effect against both H1N1 A/California/07/2009 strain and SARS‐CoV‐2 Omicron BA.5.2 variant. It is therefore expected that this study will establish the scientific foundation for the next‐step development of combined vaccines against other strains or variants of IAV and SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Objective. A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Methods. Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. Results. By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 μmol/L and 0.2 μmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 μmol/L; the final concentrations of FluA probe and primer were 0.2 μmol/L and 0.3 μmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 μmol/L and 0.25 μmol/L, respectively. Conclusion. A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes. [ABSTRACT FROM AUTHOR]

Introduction: Influenza A viruses cause global health concerns due to their high amino acid substitution rates. They are linked to yearly seasonal epidemics and occasional pandemics. This study focused on sequencing influenza A virus strains in Pakistan. Materials and Methods: We analyzed the genetic characteristics of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Pakistan from January 2020 to January 2023. Whole genome sequences from influenza A (n = 126) virus isolates were amplified and sequenced by the Oxford Nanopore (MinION) platform. Results: The HA genes of influenza A(H1N1)pdm09 underwent amino acid substitutions at positions K54Q, A186T, Q189E, E224A, R259K, and K308R in sequenced samples. The HA genes of influenza A(H3N2) had amino acid substitutions at G53D, E83K, D104G, I140M, S205F, A212T, and K276R in the sequenced samples. Furthermore, the HA gene sequences of influenza A(H1N1)pdm09 in this study belonged to subclade 6B.1A.5a.2a. Similarly, the HA gene sequences of influenza A(H3N2) were classified under six subclades (3C.3a.1 and 3C.2a1b.2a [2, 2a.1, 2b, 2c, and 2a.3b]). Notably, amino acid substitutions in other gene segments of influenza A(H1N1)pdm09 and A(H3N2) were also found. Conclusion: These findings indicate influenza A(H1N1)pdm09 and A(H3N2) viruses co‐circulated during the 2020–2023 influenza season in Pakistan. Continued surveillance is crucial for real‐time monitoring of possible high‐virulence variation and their relevance to existing vaccine strains. [ABSTRACT FROM AUTHOR]

Due to the extensive host range of influenza A viruses, it is difficult to determine the best diagnostic algorithm to efficiently screen samples from a variety of host species for influenza A viruses. While there are some influenza diagnostic algorithms that are specific to host species, to our knowledge, no single algorithm exists for the characterization of influenza A viruses across multiple host species. In this paper, we propose an algorithm that can serve as a guide for screening human, animal, and environmental samples for influenza A viruses of high human and animal health importance. [ABSTRACT FROM AUTHOR]

Influenza virus continually challenges both human and animal health. Moreover, influenza viruses are easy to mutate. In a certain degree, vaccines may not catch up with rapid mutant paces of viruses. Anti‐influenza drugs NIs (neuraminidase inhibitors) are one of the best choices. Therefore, based on ADMET properties, eight optimal natural multi‐targets NIs glycosides compounds (IC50 = 0.094–97.275 μM) are found from radix glycyrrhizae, flos sophorae, caulis spatholobi, radix astragali, radix glycyrrhizae, semen astragali complanati, and common fenugreek seed through network pharmacology, molecular docking, dynamics simulation, quantum chemistry, and in vitro experiment. Moreover, mechanism research illustrates these natural compounds treat influenza A virus through key targets TLR4, TNF, and IL6 (high fever, acute respiratory distress syndrome), MAPK1, and MAPK3 (MAPK signaling pathway, viral RNP export, and viral protein expression), IL1B (NOD‐like receptor signaling pathway, suppressed maturation of pro‐IL‐1β and pro‐IL‐18), CASP3 (apoptosis), AKT1 (inhibited premature apoptosis), and EP300 (viral myocarditis, chemoattraction of monocytes and macrophages, T‐cell activation antibody response). [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) infection causes respiratory disease. Recently, infection of IAV H5N1 among mammals are reported in farmed mink. Therefore, to discover antivirals against IAV, we screened a compound library by using the RNA‐dependent RNA polymerase (RdRp) assay system derived from H5N1 IAV including a drug‐resistant PA mutant (I38T) and a viral polymerase activity enhancing PB2 mutant (T271A). Upon screening, we found vidofludimus can be served as a potential inhibitor for IAV. Vidofludimus an orally active inhibitor for dihydroorotate dehydrogenase (DHODH), a key enzyme for the cellular de novo pyrimidine biosynthesis pathway. We found that vidofludimus exerted antiviral activity against wild‐type and drug‐resistant mutant IAV, with effective concentrations (EC50) of 2.10 and 2.11 μM, respectively. The anti‐IAV activity of vidofludimus was canceled by the treatment of uridine or cytidine through pyrimidine salvage synthesis pathway, or orotic acid through pyrimidine de novo synthesis pathway. This indicated that the main target of vidofludimus is DHODH in IAV RdRp expressing cells. We also produced recombinant seasonal IAV H1N1 virion and influenza B virus (IBV) RdRp assay system and confirmed vidofludimus also carried highly antiviral activity against seasonal IAV and IBV. Vidofludimus is a candidate drug for the future threat of IAV H5N1 infection among humans as well as seasonal influenza virus infection. [ABSTRACT FROM AUTHOR]

Airway epithelial cells contribute to a variety of lung diseases including allergic asthma, where IL‐4 and IL‐13 promote activation of the transcription factor STAT6. This leads to goblet cell hyperplasia and the secretion of effector molecules by epithelial cells. However, the specific effect of activated STAT6 in lung epithelial cells is only partially understood. Here, we created a mouse strain to selectively investigate the role of constitutively active STAT6 in Club cells, a subpopulation of airway epithelial cells. CCSP‐Cre_STAT6vt mice and bronchiolar organoids derived from these show an enhanced expression of the chitinase‐like protein Chil4 (Ym2) and resistin‐like molecules (Relm‐α, ‐β, ‐γ). In addition, goblet cells of these mice spontaneously secrete mucus into the bronchi. However, the activated epithelium resulted neither in impaired lung function nor conferred a protective effect against the migrating helminth Nippostrongylus brasiliensis. Moreover, CCSP‐Cre_STAT6vt mice showed similar allergic airway inflammation induced by live conidia of the fungus Aspergillus fumigatus and similar recovery after influenza A virus infection compared to control mice. Together these results highlight that STAT6 signaling in Club cells induces the secretion of Relm proteins and mucus without impairing lung function, but this is not sufficient to confer protection against helminth or viral infections. [ABSTRACT FROM AUTHOR]

With the determination of numerous viral and bacterial genome sequences, sequence‐trait relationships, such as the evolution of virulence and associations to geographic location or host, are now being studied. In these studies, phylogenetic trees were first reconstructed, and trait data were analysed based on the trees. However, in some cases, such as fast evolution sequences and gene‐sharing network data, reconstructing the phylogenetic tree is challenging. Even in such cases, it is possible to quantify the similarity between sequences and construct an similarity network.Here, we propose a novel approach, Network‐Trait association with Graph Fourier Transform (NeTaGFT), to analyse network‐trait associations. NeTaGFT is inspired by graph signalling process techniques. The graph in this study corresponds to a similarity network representing the similarities between virus samples, and the graph signal corresponds to trait data. By using graph Fourier transform, NeTaGFT aims to identify trait signals and associations of various traits from a similarity network.We validated that NeTaGFT can find signals associated with network structures and associations of traits with the simulation dataset. We applied NeTaGFT for influenza type A and virome gene‐sharing datasets. As a result, we identified several network structures and their associated traits.Our approach is expected to provide novel insights into network‐based approach not only for typical sequence‐trait relationships but also for various biological data, such as antibody evolution. [ABSTRACT FROM AUTHOR]

Biomimetic viral mineralization improves viral vaccine stability and immunogenicity using inorganic metals such as Ca, Al, or Fe. Mn is a metal found in high concentrations in mammalian tissues; however, under natural or laboratory conditions, Mn mineralization by medical viruses has yet to be established. Herein, a single IAV particle is successfully encapsulated with manganese phosphate (MnP) under specific conditions using the human influenza A virus (IAV). MnP‐mineralized IAVs (IAV@Mn) exhibited physiochemical and in vitro properties similar to Ca‐mineralized IAVs. In animal models, IAV@Mn shows limited replication in immune‐competent cells and a significant attenuation compared to naïve cells. Moreover, a single‐dose vaccination with IAV@Mn induced robust humoral and cellular immune responses and conferred significant protection against a wild‐type IAV challenge in mice. Thus, Mn mineralization in pathogenic viruses provides a rapid and universal strategy for generating an emergency vaccine in response to emerging viruses. [ABSTRACT FROM AUTHOR]