An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a.

Generation of a shelf-stable, broadly-reactive influenza vaccine formulated with TLR4, TLR7/8, or TLR9 stimulating adjuvants.

Background/Objectives: While new vaccines are in development; one strategy to increase influenza vaccine coverage is to repurpose current influenza vaccines for intranasal delivery. Methods: To address this goal; mice were vaccinated intranasally with either a split inactivated virus vaccine (Fluzone) or a recombinant HA vaccine (Flublok) at one of two doses (1 μg high dose or 0.1 μg low dose). Both vaccines were adjuvanted with either a STING agonist; c-di-AMP (CDA); or a combination of a synthetic toll-like receptor (TLR) 4 and TLR7/8 agonist (TRAC478). Results: Mice vaccinated with either vaccine plus adjuvant had higher hemagglutination-inhibition titers than mice administered unadjuvanted vaccines. Mice vaccinated with either vaccine plus CDA had on average higher numbers of H3 and influenza B hemagglutinin (HA)-specific antibody-secreting cells (ASCs); whereas mice vaccinated with vaccine plus TRAC478 had on average higher number of H1 HA-specific ASCs. All vaccinated mice challenged with the H1N1 influenza virus were protected against both morbidity and mortality with no detectable virus in their lungs. Mice challenged with the H3N2 influenza virus all lost weight over the first 5 days of infection. Adding TRAC478 with either a high or low dose vaccine resulted in 80–100% survival following challenge. Almost all mice vaccinated with Flublok plus CDA died from H3N2 influenza virus challenged with ~2 logs higher viral lung titers than mice administered Flublok only or Flublok plus TRAC478. Conclusions: Overall; Fluzone and Flublok can effectively be used for intranasal vaccination.

Influenza viruses are highly prevalent and continuously evolving respiratory pathogens that pose a significant threat to global public health and the poultry industry. Immunization remains the primary approach to preventing infection and reducing the risk of an influenza pandemic. Nanoparticles not only serve as delivery vehicles for antigens but also enhance antigen-specific immune responses. Polysaccharide nanoparticles not only amplify the immunostimulatory capacity of polysaccharides but also exhibit superior adjuvant effects. In this study, we investigated the effect of cationic Lentinan (CLNT) as an adjuvant for influenza vaccines. CLNT significantly enhanced the production of specific antibodies against influenza antigens (H1N1 and H9N2) and promoted a strong humoral immune response. Compared with the control group, the CLNT group showed 8.49- and 5.23-fold increases in H1N1- and H9N2-specific IgG antibody levels, respectively. CLNT also activated dendritic cells, T cells, and B cells, thus triggering the cellular immune response. In addition, CLNT effectively inhibited the replication of H1N1 and H9N2 viruses in the trachea and lungs, indicating its potent protective immune effect against influenza virus challenge. This study suggests that CLNT enhances both the potency and breadth of immune responses against influenza viruses, presenting it as a promising adjuvant and offering new options for designing next-generation influenza vaccines.

The emergence and broad circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in wild birds and its spillover into dairy cows with sustained transmission in this species pose a major risk to felines, which are highly susceptible and often succumb to the infection. Here, we developed a novel recombinant hemagglutinin H5-based vaccine and evaluated its safety, immunogenicity, and protective efficacy against HPAI H5N1 virus in domestic cats. Immunization of cats with H5-vaccine candidate elicited robust levels of neutralizing antibodies against H5N1 virus and protection against disease, mortality, and infection upon H5N1 virus challenge. The vaccine-elicited immunity significantly reduced virus shedding and viremia, limiting systemic spread and disease severity in immunized animals. Importantly, beyond protecting susceptible felids, vaccinating cats against the H5N1 virus could also reduce the risk of human exposure - underscoring the One Health impact of implementing such a vaccination strategy in feline populations.

Avian influenza H5N1 has pandemic potential and historically has caused more severe disease in pregnant women than the general population. With increasing transmission of H5N1 detected among placental mammals, animal models are necessary for testing countermeasures, including during pregnancy. Pregnant outbred mice were infected with a contemporary strain of bovine H5N1 during the second or third trimester equivalent. Second trimester infection causedin uterotransmission, with infectious virus detected in the uterus, placenta, and fetus. Birth following third trimester infection resulted in offspring with decreased size, neurodevelopmental delays, and adolescent behavioral impairments, with infectious virus detected in the neonatal milk ring and lungs, as well as mammary tissues. H5N1 viral protein colocalized with trophoblast cells in the placenta and epithelial cells in mammary tissue that spatially overlapped with lectins for α2,3-linked SA. With the pandemic potential of H5N1, our vertical transmission model in placental mammals is essential for understanding viral spread and evaluating treatments during pregnancy.

Avian influenza virus cross-species infection in humans poses a major threat to global public health. Species-specific differences between avian ANP32A and mammalian ANP32 proteins create a natural barrier against viral cross-species infection by directly impairing the functional interaction between the avian-origin viral RNA polymerase and mammalian ANP32 proteins, thereby restricting viral genome replication. The key to overcoming this barrier lies in the adaptation of viral RNA polymerase to host ANP32 family proteins. This mini-review summarizes the mechanisms and variations in influenza virus adaptation to ANP32 proteins across different species. Influenza viruses adapt to species-specific ANP32 proteins through various mutations and display distinct preferences for specific ANP32 family members within the same host. Additionally, alternative splicing variants of ANP32A within a single species further modulate viral RNA polymerase adaptability. Despite this diversity, the underlying interaction mechanism remains conserved: ANP32-polymerase binding is necessary but not sufficient for optimal polymerase activity. This interaction facilitates the formation of asymmetric polymerase dimers and specifically supports viral genome replication, while the step from cRNA to vRNA remains subject to species-specific restrictions. This explains the classic adaptive mechanism of the PB2 E627K mutation, which restores efficient viral genome replication through acid-base pairing with ANP32A. Furthermore, adaptive mutations in emerging strains such as H3N2 canine influenza virus and recent cases of H5N1 in dairy cows underscore the need for continuous viral surveillance and deeper mechanistic studies on virus-ANP32 interactions. Such research is strategically critical for advancing the One Health approach and mitigating future influenza pandemics.

Neuromedin B and its receptor NMBR inhibit H9N2 infection.

Since early-2024 unprecedented outbreaks of highly pathogenic avian influenza H5N1 clade 2.3.4.4b have been ongoing in dairy cattle in the United States with significant consequences for the dairy industry and public health. Estimation of key epidemiological parameters is required to support outbreak response, including predicting the likely effectiveness of interventions and testing strategies. Here, we pool limited publicly available data from four studies of naturally and experimentally infected dairy cattle. We quantify Ct value trajectories of infected dairy cattle and the relationship between Ct value and the log-titer of infectious virus, a proxy for infectiousness. We estimate that following infection minimum Ct values are rapidly reached within 1-2 days with a population mean Ct value of 15.7 (12.9, 18.4). We identify a threshold Ct value of 21.8 (19.9, 24.6), with values of Ct value above this threshold representing little-to-no infectious viral load. Finally, assuming a direct relationship between Ct value and infectiousness, we estimate the distribution of the duration of infectiousness for dairy cattle (i.e., the duration their Ct value remains below the critical threshold) with a population median of 7.8 (4.1, 13.9) days. Our estimates will be critical inputs to the development of outbreak management guidelines and modeling analyses informing response strategies.

Genetic and biological features of avian influenza virus subtype H10N5 in environmental samples related to a case of dual infection by H10N5 and H3N2.

Influenza A virus demonstrates significant potential as an oncolytic agent. Nevertheless, being replication-competent pathogens, they may induce adverse effects in cancer patients who have compromised immune systems. Here, the results show that the 4-hydroxytamoxifen (4-HT)-dependent H1N1 influenza virus S218 can inhibit the proliferation and lung metastasis of melanoma in vivo, and 4-HT enhances the anti-tumor effect of S218 virus. To activate the S218 virus in the tumors, tumor-targeted nanostructured lipid carriers (NLCs) modified with folate (FA) are developed to encapsulate 4-HT. This approach improves the tumor-targeting capability and anti-tumor immunity of the S218 virus. Furthermore, the combination of the S218 virus with the small molecule STING agonist SR717 further enhances the regression of lung metastases. The findings demonstrate a novel oncolytic strategy utilizing nanocarriers to deliver small-molecule drugs for activating viruses in the tumors, offering insights for oncolytic virus development.

An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification.

Chaihu Guizhi decoction alleviates H1N1 influenza virus and Staphylococcus aureus co-infection in mice by reducing inflammatory cytokines and regulating Th17/Treg balance.

To investigate the molecular characteristics of H3N2 canine influenza viruses circulating in Jiangsu, China, we isolated a H3N2 strain (A/Canine/Nanjing/CnNj01-2025) from a dog presenting with respiratory signs at the Veterinary Teaching Hospital of Nanjing Agricultural University. All eight gene segments were sequenced and compared with those of two human H3N2 strains and five avian H3N2 strains. Antigenicity and receptor-binding properties were also assessed. Phylogenetic analysis revealed that the canine isolate descended from the avian lineage and formed an independent evolutionary clade, while the human strains were more distantly related to the avian lineage. Glycosylation analysis of the HA protein revealed that the canine strain carried seven N-glycosylation sites, including a unique site at residue 97/81 (HA/H3 numbering), which serves as a molecular signature of the canine strain. Several amino-acid substitutions were identified in major antigenic sites, including D97/81N, A176/160T, N204/188D, V212/196I, and W237/222L. Analysis of internal genes showed that the canine strain harbored PB2 292T and 590S mammalian adaptation mutations, which are also present in human strains. Hemagglutination inhibition (HI) assays of the canine strain indicated moderate serologic cross-reactivity with a human H3N2 antiserum (16-fold reduction), whereas avian strains showed no cross-reactivity. Receptor-binding assays demonstrated that the virus retained predominant α-2,3 sialic acid binding, comparable to that of avian influenza viruses, and gained a modest affinity for human-type α-2,6 sialic acid receptors. Therefore, the canine H3N2 virus has undergone significant antigenic drift, developed partial serological cross-reactivity with human strains, and acquired detectable but limited binding affinity for human-type receptors. Overall, our findings suggest that the current canine H3N2 influenza virus exhibits distinct genetic and antigenic variations from human and avian strains. Continuous molecular and serological surveillance of canine influenza viruses is therefore warranted to monitor their evolutionary trends and assess the potential for cross-species transmission.

H5N1 is a highly pathogenic avian influenza virus of major global concern. Since 2023, it has circulated widely among wild and farmed birds, with increasing spillover into mammals, including minks, seals, and cattle, and sporadic infections in humans in Chile, the UK, and the USA. The risk of a future pandemic is considered high because ongoing viral evolution could enable efficient human-to-human transmission. The hemagglutinin (HA) glycoprotein is the principal determinant of host range, mediating viral attachment and entry through interactions with sialylated glycans and potentially additional host surface proteins. Here, we developed an artificial intelligence (AI)-based pipeline integrating structural modeling, protein–protein interaction prediction, and biological filtering to identify human cell surface proteins with high likelihood of interacting with H5N1 HA. These interactions may contribute to viral entry and tropism and therefore represent promising candidates for experimental validation and therapeutic targeting. Our findings highlight the utility of AI-driven pipelines in accelerating the discovery of host factors relevant to pandemic influenza viruses.

Phellinus linteus, a medicinal mushroom with an ancient history in traditional medicine, serves as a valuable raw material for functional foods. While previous studies have shown promising antiviral effects, the present work is the most comprehensive investigation of P. linteus mycelial extracts and active compounds thereof against the H1N1 influenza virus infection in in vitro and animal models. Antiviral activity was evaluated using three treatment approaches (preventive, co-treatment, and therapeutic) in MDCK cells infected with H1N1 virus. In vivo studies employed male BALB/c mice exposed to aerosolized H1N1 virus (1.6 × 106 PFU/mL). In the animal experiment, mice received either P. linteus mycelia powder (PL) at 1000 mg/kg/day or PLw/PLe at 350 mg/kg/day for 7 days before and after infection. Ethanol extracts (PLe) demonstrated superior antiviral properties compared to water extracts (PLw) in cell survival and viral inhibition tests. Animal studies revealed that both PL and PLe significantly improved recovery after H1N1 infection, with survival rates of 60% and 40%, respectively, versus 25% in controls. The treatments effectively restored depleted immune cell populations, indicating broad immunomodulatory effects. These findings highlight the potential of P. linteus as a functional food ingredient with promising therapeutic applications against viral infections through its action on both innate and adaptive immune responses.

Highly pathogenic avian influenzas (HPAIs) is a continuing public health threat. Here, we describe the development of plasmid-encoded H5N1 hemagglutinin antigens representing clades that have caused human zoonoses. Electroporation (EP) delivery of clade 2.3.2.1c HA (pCamb) DNA was immunogenic in mice but only partially protective against clade 2.3.4.b challenge. Homologous challenge resulted in complete protection, suggesting that matched clade antigens are important for protection. Contemporary clade 2.3.4.4b HA (pMich) DNA plasmids supported robust cellular and humoral responses when delivered via EP. Co-immunization with plasmids representing both H5 clades supported high titers of binding and neutralizing antibodies against both antigens and complete protection from clade 2.3.4.4b challenge. We formulated the pMich plasmid DNA in lipid nanoparticles (LNPs). A single dose of pMich DNA-LNP supported long-lived immunity in mice that was protective against challenge at both acute and memory timepoints. These data demonstrate that DNA can support robust anti-HPAI immunity and protection.

Abstract: This article presents a case of liver cirrhosis complicated by H1N1 influenza virus infection, and analyzes it in the context of medical literature since 2020. The patient, a 73-year-old male with decompensated cirrhosis of unknown origin, was admitted to the hospital due to fever, cough, and breathing difficulties. After receiving antiviral treatment (oseltamivir), liver protection, and management of complications, his condition improved. The discussion section focuses on the mechanisms of influenza infection in patients with liver cirrhosis, treatment strategies, and the prevention and management of complications, emphasizing the importance of early diagnosis and comprehensive treatment.

Influenza viruses cause hundreds of thousands of infections globally every year. In the past century, seasonal influenza viruses have included H1N1, H2N2 and H3N2 strains. H2N2 influenza viruses circulated in the human population between 1957–1968. Previously, our group demonstrated a lack of H2N2 influenza virus immunity in individuals born after 1968, as well as the effectiveness of hemagglutinin (HA) based vaccines for multiple influenza virus subtypes. In this study, H2 antigenic maps and radial graphs were generated using previously published data from H2 HA vaccinations of ferrets and seasonal influenza vaccinations of humans. The antigenic maps revealed a stark difference in clustering of HA antigens between the ferrets and humans, and the radial graphs showed specific antigen recognition varies greatly between different influenza preimmune ferrets. These maps also revealed the significant impact that different pre-existing immunities have on antigenic recognition and clustering of antigens after vaccine boost. From these data, we predicted two possible antigenically significant sites containing various mutations that have not been previously reported and showed that one of these sites is relevant using mouse anti-sera.

In developing countries particularly in field conditions unfavorable environmental conditions, lack of availability of appropriate transport media (TM) and maintenance of cold chain during transport; sample collection, storage, and transportation is more challenging. Considering these facts, five TM out of which three laboratory-based media named phosphate-buffered saline (PBS), 50% glycerol + PBS, and normal saline (NS) and two commercially available media including viral transport medium (VTM), charcoal based viral transport medium (CVTM) were compared to protect infectivity of the H5N1 influenza virus. Spiked fecal sample and allantoic fluid with and without these TM were placed in field simulatory storage and transportation conditions and in every 12 h time interval these samples were tested for virus isolation in embryonated chicken egg inoculation and identification by HA test and RT PCR upto 7 days. Survivability of the virus was detected by calculating the percent infectivity and analysed by logistic regression analysis. NS, PBS, and CVTM, were most effective media in maintaining the integrity of the test virus. These media are easily available and economical and less complex to prepare. A significant difference was found in survivability of the virus only in VTM in between allantoic fluid and fecal sample.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-15987-6.