Multiplex serological profiling reveals diverse avian and mammalian influenza A virus exposure in swine.

INAAC: An affinity chromatography strategy enabling characterization and quantification of influenza neuraminidase antigens in vaccines.

H5N1 highly pathogenic avian influenza viruses have spread globally and pose a pandemic risk. Prior studies suggest that early life exposures to group 1 influenza viruses (H1N1 and H2N2) prime antibodies that cross-react to the hemagglutinin of H5N1, which is also a group 1 virus. However, less is known about how immune history affects antibody responses against the H5N1 neuraminidase (NA). We measured NA inhibition antibodies against multiple H5N1 viruses using sera from 155 individuals born between 1927 and 2016. Individuals likely primed in childhood with H1N1 viruses possessed higher levels of antibodies that cross-react with the NA of H5N1 viruses compared to those primed with H2N2 or H3N2 viruses. While young children rarely possessed cross-reactive N1 antibodies, childhood infections with contemporary H1N1, but not H3N2, viruses elicited them. We also measured antibodies against an H5N5 virus (A6 genotype) that recently caused a fatal infection in the United States. Consistent with the lack of circulation of N5 viruses in humans, we found low levels of antibodies against the N5 NA. Our data suggest that immune history greatly impacts the generation of cross-reactive NA antibodies, and that reassortment with other NAs may increase the risk of H5 infection of humans.

Avian influenza H9N2 viruses cause economic losses to the poultry industry and pose a public health risk. Two major H9N2 lineages dominate globally: the G1 lineage (genotype G1-B), prevalent in the Middle East, Africa and South Asia, and the BJ/94 lineage (predominantly genotype G57), dominant in China, Vietnam, South Korea, Indonesia and the Far East. We investigated replication, transmission and pathogenicity of prototype strains from these two lineages to link genotype to phenotype. The G57 virus A/Ck/Vietnam/H7F-14-BN4-315/2014 (Vietnam/315) was more lethal and showed greater tissue dissemination in chicken embryos than the G1-B virus A/chicken/Pakistan/UDL-01/2008 (Pakistan/UDL-01). Vietnam/315 exhibited higher replication in directly infected and contact chickens, with increased oropharyngeal and cloacal shedding and tissue dissemination. In contrast, the Pakistan/UDL-01 virus was shed mainly from the oropharynx at lower levels and remained localized to nasal tissues and trachea, highlighting differences in replication, tissue tropism and transmission. Gene analysis showed that the matrix (M) gene of Vietnam/315 enhanced replication in primary chicken kidney cells, while polymerase basic 2 (PB2), haemagglutinin (HA), neuraminidase (NA) and M genes promoted increased replication in Madin-Darby canine kidney cells. Both viruses preferentially bound to sulphated avian-like receptors. However, Vietnam/315 showed higher NA activity and a more acid-stable HA (pH fusion 5.2) than Pakistan/UDL-01. These findings indicate that the G57 genotype strain examined exhibits greater replication and transmission fitness than the G1-B lineage strain in vivo, ex vivo and in vitro. Although based on prototype viruses, the results suggest that reassortment involving G57 genotype genes could enhance viral fitness and increase animal and human infection risk.

Influenza viruses undergo antigenic drift, the gradual accumulation of mutations that cause antigenic changes in the viral surface proteins hemagglutinin (HA) and neuraminidase (NA). Although selection for antigenic variants is detectable on the global scale, the processes by which antigenic variants are generated and selected in individual hosts remain unclear. It has been hypothesized that selection for antigenic variants may occur during the establishment of a new infection, rather than over time in a single host. Here, we leveraged a large household cohort study to assess whether selection was detectable between acutely infected hosts. We investigated influenza A virus evolution using specimens from 384 children and household contacts with RT-PCR-confirmed influenza A infection, representing infections with A(H1N1)pdm09 and A(H3N2) viruses from 2017-19. In agreement with prior studies, we found that acute infections involved weak purifying selection across the viral genome. In addition, we identified 40 transmission events occurring in 31 households. During transmission, evolution between hosts was characterized by tight transmission bottlenecks and weak purifying selection. We found variability in the strength and direction of selection on antigenic regions of HA, but no clear evidence for selection of antigenic variants during transmission. Together, our results indicate that stochastic processes and weak natural selection dominate most acute influenza A virus infections and transmission events, and that selection of antigenic variants during transmission between acutely infected hosts is likely to be exceedingly rare.

On the brink of emergence: an evolutionary approach to Influenza A virus H5N1 isolated from humans.

Phylogenetic patterns and spatiotemporal evolution of H1N1pdm09 in Anhui Province, China, 2009-2023.

BackgroundPediatric influenza remains a significant global public health challenge, driving continuous advancements in vaccines, antiviral therapies, and infection control strategies. As the volume of related literature expands rapidly, systematically synthesizing these developments is essential. Therefore, this study conducts a comprehensive bibliometric analysis of pediatric influenza prevention and treatment research. By examining structural and temporal trends in the literature, it aims to identify historical evolution, research hotspots, and emerging trends.MethodsData were extracted from the Web of Science Core Collection (WoSCC) for studies on pediatric influenza prevention and treatment (1994–2024). Bibliometric tools such as CiteSpace, HistCite and Alluvial Generator were used to conduct multidimensional visual analyses of publication trends, highly cited literature, academic collaboration networks, keyword clustering, and emergent keywords in the field.ResultsResearch on pediatric influenza has witnessed extensive international scientific collaboration. Active topics emerged at different time points, with 108 disciplines, 720 keywords, and 1,885 papers with citation bursts. Keyword clustering revealed six emerging research subfields: #1 influenza a, #2 immunization, #3 oseltamivir, and so on. Keyword alluvial maps revealed that the most persistent research themes were live attenuated influenza vaccine, neuraminidase (NA), and response, and the emerging keywords are syncytial virus, acute respiratory illness, and immunization. Recent reference clustering further delineated five cutting-edge subfields: #2 baloxavir, #3 vaccine effectiveness, and so forth.ConclusionsResearch on pediatric influenza prevention and control has undergone three distinct phases, with current studies focusing on interactions with coronavirus disease 2019 (COVID-19), vaccine hesitancy, influenza vaccine efficacy, and antiviral drug resistance mechanisms. Future research should prioritize elucidating influenza mixed infection pathways, accelerating broad-spectrum vaccine development, and innovating antiviral treatment regimens. Concurrently, strengthening public health interventions and health communication mechanisms is crucial for enhancing global influenza prevention and control efficacy among children.

Background:Although the hemagglutination inhibition (HAI) titer remains the gold standard correlate of protection against influenza, it does not fully capture the broader antibody responses that contribute to immunity.Methods:We analyzed immune responses in paired pre-infection and convalescent sera from 306 RT-PCR–confirmed A/H3N2 infections from two household studies (2014–18) in Managua, Nicaragua. Antibody responses were measured by HAI and enzyme-linked immunosorbent assays (ELISAs) against full-length hemagglutinin (HA), the HA stalk, and neuraminidase (NA). Participants were classified as HAI responders (≥4-fold HAI rise), alternate responders (no HAI rise but ≥4-fold boost in ≥1 ELISA), or no-response individuals (no ≥4-fold rise in any assay). We compared demographic, clinical, and pre-infection antibody characteristics across these groups. We also analyzed predictors of an NA response.Results:Overall, 77% of participants had HAI seroconversion or a 4-fold rise. Among the 23% HAI non-responders, 62% had alternate antibody responses. No-response individuals had the highest pre-infection HAI and full-length HA titers (p < 0.0001), the lowest viral loads, and the fewest fever or influenza like illness (ILI) symptoms (p < 0.01). An NA response was more common among symptomatic individuals (p = 0.0483) and those with low or high baseline NA titers.Conclusions:High baseline HAI titers can limit detectable 4-fold rises and are associated with milder illness. Evaluating additional immune responses may capture a more complete picture of the host response to infection, thereby improving surveillance and informing vaccine development.

ABSTRACT Antibodies against neuraminidase (NA) are an independent correlate of protection and the antigenic relatedness between H1N1pdm and H5N1 NAs suggests that seasonal influenza infection may provide cross-reactive immunity. In this study, recent H1N1pdm infection elicited modest NA-inhibiting (NAI) antibody responses against contemporary A/Victoria/4897/2022 (H1N1pdm) but strong responses against older A/California/7/2009 (H1N1pdm). Convalescent sera exhibited significantly higher cross-reactive NAI against clade 2.3.4.4b H5N1 (A/Texas/37/2024) than non-flu A patients, whereas H3N2 infection did not elicit such cross-reactivity. NAI titres were comparable between N1-California and N1-Texas but lower against N1-Victoria, indicating greater antigenic similarity between N1-California and N1-Texas. This was supported by stronger inhibition of N1-Texas by N1-California sheep antiserum compared with N1-Victoria sheep antiserum. Surprisingly, H1N1pdm-infected patients exhibited very low NAI against clade 2.3.2.1a H5N1 (N1-Bangladesh; GMT of 171 vs GMT 1159 for N1-Texas). Mouse antisera demonstrated reduced reciprocal inhibition between N1-Texas and N1-Bangladesh, consistent with their antigenic divergence. When four residues in N1-Bangladesh were substituted with their corresponding residues in N1-Texas, N1-Bangladesh antiserum showed reduced NAI, while N1-Texas antisera showed increased inhibition compared with wild-type N1-Bangladesh. This suggests that these amino acid differences are partially responsible for their antigenic divergence. Importantly, longitudinal analysis revealed that boosted cross-reactive NAI responses waned by day 90 post-infection, highlighting their limited durability. Together, these findings demonstrate that seasonal H1N1pdm infection can transiently boost cross-reactive NAI antibodies against clade 2.3.4.4b H5N1, but antigenic divergence in clade 2.3.2.1a limits cross-reactivity. As such, the impact of pre-existing antibody during an H5N1 outbreak is dependent on the infecting clade.

Background: Influenza A(H3N2) continues to evolve rapidly, frequently eroding population immunity and challenging seasonal vaccine strain selection. During the 2025/26 season, the A(H3N2) subclade K (J.2.4.1) expanded quickly across multiple regions and showed evidence of antigenic divergence in standard assays. Methods: In this study, we combined phylogenetic analyses of hemagglutinin (HA) and neuraminidase (NA) sequences with a systematic synthesis of recent peer-reviewed studies and official surveillance reports to comprehensively define the molecular profile and early epidemiological dynamics of subclade K. Results: Our phylogenetic reconstructions of HA and NA genes confirmed the emergence of a coherent and recently diversified lineage characterized by coordinated evolution of surface glycoproteins and broad geographic representation during 2025. Integration of molecular, temporal, and surveillance evidence further supported rapid expansion with limited early regional structuring. Antigenic analyses reported in peer-reviewed studies described reduced haemagglutination inhibition reactivity to vaccine reference antisera for many subclade K viruses, whereas vaccine effectiveness (VE) estimates from multiple settings remained moderate. Conclusions: Overall, the available genetic, antigenic, and epidemiological evidence indicates that subclade K represents a recently diversified A(H3N2) lineage associated with rapid international spread during the 2025/26 season, highlighting the importance of integrated HA/NA genomic surveillance and timely antigenic characterization to support evidence-based vaccine strain selection.

Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses continue to expand geographically and across mammalian hosts, raising concern about pandemic potential. The degree and specificity of pre-existing immunity in humans are key determinants of this risk. We analyzed hemagglutinin (HA)- and neuraminidase (NA)-specific antibody responses in 300 sera collected from adults in New York City. While HA directed binding antibodies to clade 2.3.4.4b H5 were low and hemagglutination-inhibiting antibodies were absent, we detected widespread binding and functional NA antibodies against N1 neuraminidases from clade 2.3.4.4b H5N1 viruses. Neuraminidase inhibition (NI) titers were highest against North American D1.1 genotype N1 viruses and correlated strongly with neutralizing activity, whereas HA-binding antibodies did not. An additional N-linked glycosylation site, as found in the NA of a human D1.1 isolate from British Columbia, reduced susceptibility to NI antibodies. Antibodies titer to N5 from H5N5 were low to minimal. These findings indicate that population-level immunity to clade 2.3.4.4b H5 viruses is dominated by NA-directed antibodies, with important implications for pandemic risk assessment.IMPORTANCEUnderstanding how pre-existing human immunity shapes susceptibility to emerging influenza viruses is central to pandemic preparedness. Here, we determined that human sera contain widespread, functional antibodies targeting H5N1 neuraminidase, which correlate with virus neutralization, whereas HA-directed responses are limited. We further show that acquisition of an NA glycosylation site reduces antibody inhibition, highlighting a potential pathway for immune evasion. These results identify neuraminidase-specific immunity as a major immunological barrier to severe H5N1 disease in humans and emphasize the need to incorporate NA antigenicity into influenza surveillance, risk assessment, and next-generation vaccine design.

Maternal-infant immunity against influenza is improved through vaccination during pregnancy. We conducted an in-depth analysis of antibody (Ab) responses in sera from pregnant and non-pregnant women immunized with an unadjuvanted inactivated influenza A (H1N1) monovalent vaccine during the 2009 pandemic (NCT00992719). Pregnant women received either the standard 15 µg dose or the increased 30 µg dose, while non-pregnant women received the 15 µg dose. Ab specific for influenza hemagglutinin (HA), HA stalk, and neuraminidase (NA), as well as canonical functions of hemagglutination inhibition (HAI), microneutralization, and neuraminidase inhibition, was examined at baseline, 21 days post-vaccination, at delivery, and in cord blood. Ab subclasses, Fc receptor binding, and Fc-mediated immune functions, including cellular cytotoxicity, phagocytosis, and complement deposition, were also assessed. The vaccine was well tolerated and highly immunogenic in recipients; most participants had a ≥4-fold increase in Ab titers post-vaccination for HAI (HAI > 70%) and HA-specific IgG (IgG > 50%). Pregnant women who received the 15 µg dose had lower vaccine responses, as measured by NA-specific IgG, Fc receptor binding, and cell-mediated activity, compared with the other groups. Immunization of pregnant women with the 30 µg dose resulted in more robust humoral immunity, including a larger number of HA Ab features reaching 4-fold increases compared to the other groups, a more durable antiviral function, and increased NA-specific Ab features that were transferred to the infant as compared to pregnant women who received the standard 15 µg dose. Increasing the antigen content of seasonal vaccines could enhance immunity against influenza in mothers and infants and warrants further study.IMPORTANCEPregnant women and infants are at-risk groups for influenza infection. Vaccination is recommended during pregnancy to stimulate adaptive immunity and protect both the mother and infant early in life. We characterized the immune responses of pregnant and non-pregnant women to an unadjuvanted inactivated 2009 pandemic influenza A (H1N1). Pregnant women immunized with the 15 µg standard seasonal influenza vaccine dose developed lower NA-specific responses compared to non-pregnant women immunized with the same vaccine dose. Vaccination of pregnant women with a 30 µg dose resulted in more robust and durable post-vaccination responses, particularly longer-lasting functional antibodies and NA-specific antibodies in maternal and cord blood. A deeper analysis of antibody responses beyond the traditional hemagglutination inhibition (HAI) assay suggests that a higher-dose influenza vaccine, already recommended for the elderly, could be beneficial for pregnant women and warrants further investigation.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT00992719.

Avian influenza infections cause substantial economic losses in the poultry industry and raise public health concerns due to viral adaptation and cross-species transmission. The frequent antigenic drift of influenza viruses further complicates the prevention and treatment of avian respiratory infections. In this study, we generated high-affinity heavy-chain variable domain (VHH) nanobodies from naïve alpaca/camelid VHH libraries using phage display combined with H9N2 influenza A virus (IAV)-infected Madin-Darby Canine Kidney (MDCK) cells. Based on binding affinity and neutralization potential, we identified seven hemagglutinin (HA)-specific and two neuraminidase (NA)-specific VHHs. Molecular docking predicted the interaction sites of HA-specific VHHs (L1-2, L1-4, A5) and NA-specific VHHs (L1-3, L2-2), providing mechanistic insights. Notably, the three HA-specific VHHs (L1-2, L1-4, A5) showed cross-reactivity to representative HA subtypes (H1, H3, and influenza B), indicating recognition of conserved epitopes across divergent influenza strains. For the first time, these camelid nanobodies were fused to the chicken IgY Fc domain, and the expression cassette was integrated into the Saccharomyces cerevisiae genome, achieving a secretion yield of 15-20 mg/L of VHH-Fc antibodies. Experimental validation confirmed that the three HA-specific VHHs-Fc constructs effectively blocked viral infection, while the two NA-specific VHH-Fc constructs (L1-3, L2-2) inhibited NA activity, demonstrating the functional efficacy of the yeast-secreted VHH-IgY Fc platform. This novel IgY Fc fusion approach offers a scalable platform with enhanced stability, extended circulation potential, and applicability in poultry.

Avian influenza viruses (AIVs) of the H6 subtype demonstrate broad host tropism and represent a persistent concern for both poultry and public health due to their documented potential for cross-species transmission. This study aimed to isolate and characterize two novel H6 subtype AIVs from duck farms in China, focusing on their genetic evolution, molecular characterization, and antigenic properties. Virus isolation was performed from AIV-positive cloacal swabs. Whole genomes were sequenced and phylogenetically analyzed. Bioinformatic tools were employed to identify critical amino acid motifs, glycosylation patterns, and reassortment events. Antigenic cross-reactivity was evaluated through hemagglutination inhibition (HI) assays. Two strains, designated as H6N6 (SHT) and H6N2 (GXG), were successfully isolated. Phylogenetic analysis classified their hemagglutinin (HA) genes into the ST/2853-like and ST/339-like lineages, respectively. The GXG strain was identified as a triple-reassortant virus, with its matrix (M) gene derived from an H9N2 AIV, and contained a unique arginine insertion at residue 169 of the HA. In contrast, the SHT strain possessed a deletion in the neuraminidase (NA) stalk region (residues 58-68). Asymmetric antigenic cross-reactivity was observed: antiserum against GXG partially inhibited SHT, while SHT antiserum showed no inhibition of GXG. Epidemiological data confirmed the dominance of the ST/2853-like lineage in China, with N6 as the predominant NA subtype from 2015 to 2022. In conclusion, the two H6 AIV isolates exhibit distinct genetic and antigenic characteristics, carry mammalian adaptation markers, and highlight a potential cross-species transmission risk. These findings underscore the necessity for continued surveillance and the development of appropriately matched vaccine candidates.

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that mediates virus attachment to host sialic acid-containing receptors, exhibits neuraminidase activity, and promotes membrane fusion. Notably, purified HN protein also targets sialic acids on malaria-parasitised red blood cells (PRBCs), thereby reducing parasite viability, and is thus a potential therapeutic molecule against the malaria parasite. To gain further insights into the structural and functional basis of its sialic acid recognition, we performed comparative analyses of the sialic acid-binding pocket across different viral lectins. It revealed a Type-VI sialic acid-binding module in HN, characterised by fewer interactions with the functional groups of sialic acid compared to Type-I influenza neuraminidases (NA). Introduction of a rationale-based specific mutation (I175Y) in the central binding pocket of HN transformed its sialic acid-binding module from Type-VI to Type-I. It resulted in significantly higher affinity for sialic acid, accompanied by a substantial loss in its neuraminidase activity. This disruption in the delicate functional balance between receptor-binding and enzymatic cleavage highlights a trade-off between optimising one activity at the cost of another. Biologically, the mutant HN exhibited enhanced hemadsorption, or cell-binding activity, and thus showed greater ability to block NDV replication in both cell monolayers and in-ovo systems. The mutant protein also demonstrated an increased capacity to target PRBCs and showed potent anti-plasmodial activity, compared to the wild-type. This enhanced targeting capability highlights the mutant as a promising candidate for developing precision drug delivery systems for malaria.

Intranasal prime-boost immunization with trivalent influenza virus neuraminidase proteins and conserved HCA2 sequences fused to a circularly permuted E. coli heat-labile enterotoxin B subunit.

Characterisation of immune responses targeting highly pathogenic avian influenza A(H5) viruses in health-care workers in the Netherlands: an observational, cross-sectional analysis.

Antioxidant-antiinflammatory dual-pathway synergistic therapy: An intelligent polysialic acid nano-delivery platform against Glaesserella parasuis.

Influenza viruses present an ongoing global health risk because they are always changing, which in turn results in the ineffectiveness of current strain-specific vaccines and leaves the world vulnerable to potential pandemics. The need for a universal influenza vaccine, designed to develop lasting broadly protective immunity against volatile influenza virus strains has led to advances in immunogen design. Nanotechnology, specifically self-assembled nanovaccines, offers a truly revolutionary "bottom-up" strategy to address this issue. Nanovaccines that spontaneously self-assemble into easily discernable pathogen-like nanoparticles, including protein cages (e.g., ferritin) and virus-like particles, provide densely displayed conserved influenza epitopes-such as hemagglutinin (HA) stalk, neuraminidase (NA), and M2 ectodomain (M2e)-in a multivalent array, greatly enhancing B-cell activation, initiated by extensive receptor crosslinking, and generating immune responses to a magnitude and breadth that is unattainable with soluble antigens. Moreover, self-assembled nanovaccines, often in adjuvant-free or self-adjuvanting formulations, not only induce durable and broad cross-protective humoral and cellular immunity but also offer protection from numerous heterosubtypic viral challenges. While significant hurdles remain in scaling the process to a manufacturing level and subsequently translating it into the clinic, self-assembling nanovaccines represent a paradigm shift in influenza prevention, providing a rational and promising pathway toward the development of a universal vaccine and a rapid response platform for future pandemics.