Influenza A virus (IAV) zoonotic transmission and continuous evolution in multiple host species heighten the risk of emerging novel strains at the human-animal interface. Digitally immune-optimized hemagglutinin (HA), neuraminidase (NA), and matrix-2 (M2) vaccine antigens were engineered and combined as a single DNA expression construct (DVX-H1N1) to maximize immune responses to human seasonal, pandemic, and zoonotic H1N1 IAVs. Immunization of mice with DVX antigens induced broad neutralizing antibody responses to human- and swine-origin H1N1 isolates. The DVX-H1N1 candidate was then evaluated in the well-recognized swine challenge model for human influenza in comparison to two benchmark controls representing conventional whole inactivated virus (WIV) vaccines that were either homologous or heterologous to the H1N1 pandemic 2009 subclade 1A.3.3.2 challenge strain, A/swine/England/1353/2009 (H1N1). Serology demonstrated that DVX-H1N1 immunization induced immune responses across a broad spectrum of human and swine H1N1 strains representing antigenic drift and reassortment. Following intranasal virus challenge, nasal shedding of viral RNA was significantly suppressed in the DVX-H1N1 and the homologous control WIV vaccinated groups in comparison to naive controls and the heterologous WIV vaccinated groups. These findings demonstrate that in both swine and mouse models, the DVX-H1N1 vaccine induced broad immune responses and, in pigs, controlled viral infection comparable to conventional strain-matched, but unlike mismatched WIV vaccines.

The Orthomyxoviridae family includes influenza D virus (IDV), an emerging pathogen primarily affecting cattle and swine; there is evidence of cross-species transmission and potential zoonotic risk. Although active human infections have yet to be confirmed, high seroprevalence in cattle-exposed populations highlights the need for continued surveillance. We developed and validated a rapid, field-deployable reverse transcription loop-mediated isothermal amplification assay for IDV detection; specificity was 99.2% and sensitivity ranged from 95.6% (cycle quantification <30) to 81.8% (cycle quantification <40). This method offers a cost-effective, accessible alternative to quantitative reverse transcription PCR, enabling improved monitoring of IDV and reinforcing preparedness for emerging influenza threats.

Swine influenza A viruses (IAV-S) caused the 2009 H1N1 pandemic and pose a future zoonotic and pandemic threat. Vietnam represents a critical hotspot for IAV-S emergence within East and Southeast Asia, with dense swine and human populations and intensive livestock trade. We conducted genomic surveillance of IAV-S in Vietnam during 2020-2024, extending previous surveillance from 2013-2019. We identified multiple co-circulating H1 and H3 clades, including pandemic H1N1, Eurasian avian-like, and European lineages, by conducting phylogenetic analysis of 56 IAV-S isolates (21 H1N1, 31 H1N2, and 4 H3N2). Three H1 clades persisted exclusively in Vietnam, circulating up to 12 years. Phylogeographic analysis revealed multiple independent introduction events from North America, Europe, China, Thailand, and Cambodia. We detected extensive reassortment that frequently involved pandemic H1N1 virus internal genes. We identified several lineage-specific mutations associated with mammalian adaptation. Our findings underscore the ongoing IAV-S evolution and need for sustained surveillance in Vietnam.

Domestic pigs are susceptible to avian, swine, and human influenza A viruses (IAVs). This facilitates co-infections of different IAVs that result in genetic reassortment. In combination with husbandry practices such as high-density farming and frequent human-pig interactions, pigs serve as efficient zoonotic hosts for IAV. Despite significance in zoonotic transmission, factors influencing IAV pathogenesis, evolution, and transmission in pigs remain poorly studied. One factor that may alter IAV dynamics in pigs is co-infection with endemic swine viruses such as porcine reproductive and respiratory syndrome virus (PRRSV; Betaarterivirus; Nidovirales) that causes widespread disease in pigs on farms globally. During initial outbreaks, PRRSV triggers transplacental infections and massive fetal loss in naïve herds. Once herd immunity or vaccination is established, PRRSV may persist in affected herds, resulting in transplacental infections and the birth of piglets that appear normal or weak. These piglets are concerning from a zoonotic perspective because they may be immunocompromised and potentially serve as more efficient hosts for IAV, characterized by prolonged IAV shedding, enhanced viral replication, impaired seroconversion, and consequently extended co-infection windows that favor reassortment. Here, I summarize studies supporting the hypothesis that fetal exposure to PRRSV can alter IAV pathogenesis in surviving piglets, potentially enhancing IAV pathogenesis and transmission. I also highlight key experiments that need to be conducted to validate or invalidate a causal relationship between fetal PRRSV infection, persistent immunopathology in surviving piglets, and the resulting permissive environment for enhanced IAV evolution and transmission. Addressing this knowledge gap is essential for advancing our understanding of PRRSV and IAV biology in its zoonotic pig host and for developing effective strategies to mitigate the risk of IAV spillovers to humans.

Vaccines are a safe and highly effective tool for protecting both humans and animals against infectious diseases. Although vaccines currently in use in humans have robust safety records, historical examples show that some vaccines have resulted in adverse outcomes, including vaccine-associated enhanced respiratory disease (VAERD). VAERD occurs when preexisting vaccine-induced immunity worsens respiratory disease, rather than conferring protection during infection. Enhanced disease in the lower respiratory tract characteristic of VAERD includes worsened clinical manifestations such as pneumonia and bronchiolitis, as well as increased pulmonary pathology in vaccinated, infected individuals compared with their unvaccinated, infected counterparts. Mechanistically, VAERD has been associated with TH2-mediated immunopathology or antibody-dependent enhancement and is primarily associated with specific virus families, including Pneumoviridae, Orthomyxoviridae and Coronaviridae. Notable cases of VAERD have been associated with a respiratory syncytial virus vaccine candidate evaluated in clinical trials in the 1960s, experimental coronavirus vaccines in animal models, and in veterinary vaccines for influenza virus in swine. Existing approved vaccines are safe and highly effective tools for protecting both humans and animals against respiratory pathogens. However, as new respiratory vaccines are developed, they must also undergo careful testing for VAERD or other adverse outcomes. This review aims to provide the field with an overview of VAERD as a resource to support vaccine development and safety studies.

Swine are a crucial species in influenza A virus (IAV) ecology, capable of supporting replication and consequently evolution of human, avian, and swine strains. They are also the source of the 2009 pandemic H1N1 virus (H1N1pdm09). Understanding how swine IAV breach the species barrier is essential for detection of potentially pandemic viruses, but is often overlooked compared to avian IAV. We hypothesize that the HA-NA balance of (pre)pandemic swine viruses is a key feature that needs adjustment as swine and humans differ in their sialoglycome. We focused on a set of (pre)pandemic swine viruses collected in Mexico following the H1N1pdm09 emergence. Phylogenetic analysis showed that the emergence was preceded by substitutions in HA and NA predicted to affect their function. Thus, HA acquired substitution A227E, previously shown to reduce receptor binding in H1N1pdm09, while NA obtained substitution S369N in the 2nd sialic acid binding site (2SBS) predicted to reduce NA cleavage. The substitutions' effect on the HA-NA balance, was confirmed with biolayer interferometry using recombinant proteins attached to nanoparticles. Engineering recombinant attenuated influenza viruses with swine HA and NA with/without the mentioned substitutions, showed that the 2SBS substitution negatively affected replication in primary human and swine cultures. Interestingly, replication was restored by the HA substitution. Overall, we show that the emergence of H1N1pdm09 in humans was preceded by substitutions in HA and NA that were acquired in swine and adjusted the HA-NA balance. We hypothesize that adjustment of the HA-NA balance was a critical step for crossing the host-species barrier.

The lateral patch epitope of the H1 hemagglutinin (HA) was a dominant target of antibodies following exposure to the 2009 pandemic H1N1 virus. However, the conservation and mechanisms of antigenic drift in the lateral patch remain unresolved. Here, we used lateral patch-specific monoclonal antibodies (mAbs) to understand the antigenicity of the lateral patch of human, avian, and swine H1Nx viruses spanning from 1918 to 2022. We found that lateral patch-specific mAbs demonstrated broad binding to pre-2009 H1N1 viruses and to the pandemic 2009 H1N1. However, we identified discrete mutations that drove multiple antibody evasion pathways for lateral patch-targeting mAbs in post-2009 H1N1 viruses. Despite this, we observed that the lateral patch remains well conserved across enzootic sources, suggesting that existing lateral patch antibodies could protect against a future zoonotic H1Nx pandemic. Together, these data support that the lateral patch remains an attractive target for pandemic preparedness against emergent H1Nx viruses.IMPORTANCESeasonal influenza viruses continually undergo antigenic drift to evade antibody-mediated immunity, yet how individual antibody specificities shape this evolution remains poorly understood. We investigated the conservation and antigenic evolution of the H1 hemagglutinin (HA) lateral patch, an epitope targeted by broadly neutralizing antibodies. Using human monoclonal antibodies, we found that the lateral patch had remained highly conserved across nearly a century of human and animal influenza virus circulation but has undergone antigenic drift multiple times in pre- and post-2009 seasonal H1N1 viruses. Structural and mutational analyses revealed how these substitutions alter antibody recognition and shape the genetic features of lateral patch-targeting antibodies across individuals born in different eras. The lateral patch is also conserved among swine and avian H1Nx viruses, highlighting its potential as a target of protective immunity against pandemic H1Nx viruses. These findings provide a comprehensive framework for understanding antibody-driven evolution within a broadly conserved influenza virus epitope.

Narcolepsy is a rare neurological disorder caused by the loss of function in neurons producing orexin or hypocretins, disrupting the sleep-wake cycle. In Sweden and Finland approximately 800 patients developed narcolepsy after swine flu (H1N1) vaccination between 2009 and 2010, probably as an autoimmune side effect. The vaccinated cohorts are of great scientific interest owing to their specific exposomes. We aimed to identify new biomarkers by analyzing serum from 39 narcolepsy type 1 (NT1) patients and 48 controls in Sweden, with subsequent validation in 38 NT1 patients and 44 controls from Finland. Three pre-defined Olink panels, each comprising 92 proteins, were used to quantify 276 serum proteins in Swedish participants, with two panels validated in the Finnish cohort. Additionally, transcriptome analyses explored gene expression of overlapping significant proteins using the only publicly available narcolepsy transcriptome cohort. Our findings identified six overlapping proteins that showed nominally significant differential expression in both cohorts. UNC5C, VWC2, CPA2, GFR-α-1, and CLEC10A were consistently downregulated in NT1 patients, whereas HAGH showed opposite directions of effect between cohorts. UNC5C was also replicated at the transcriptomic level. In addition, ADAM23 in the Swedish cohort and SCARB2 in the Finnish cohort were significantly downregulated. Furthermore, ILKAP levels were nominally lower in vaccinated patients compared with controls. Overall, this study identifies candidate serum biomarkers that may provide insight into the pathogenesis of NT1. However, these findings are exploratory and hypothesis-generating and should not be considered established biomarkers. Independent validation in larger and well-characterized cohorts is required to confirm their potential clinical and biological relevance.

Newly emerging influenza viruses pose a constant threat as they encounter a population lacking neutralizing antibodies against the new strain. However, cross-reactive non-neutralizing antibodies (nnABs) may be present and help mitigate disease symptoms through various effector mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Although nnABs to influenza virus have received more attention lately, little information is available on their age-related prevalence, steady-state levels, functional properties, and changes in these parameters over time. Using longitudinal samples from adolescents, adults, and older adults, collected before and after the 2009 swine flu pandemic, we comprehensively characterized the specificity and functionality of nnAB responses against H1N1 pandemic 2009 (H1N1pdm09) virus. Remarkably, all participants exhibited cross-reactive antibodies to this virus before having encountered it through infection or vaccination, with the highest baseline levels observed in older adults. The levels of these IgG antibodies showed a strong correlation with engagement of fragment crystallizable γ receptor IIIa (FcγRIIIa) and NK cell activation, both of which were notably lower in adolescents than in adults and older adults. Without infection or vaccination, average amounts of H1N1pdm09-reactive antibodies remained relatively stable on the population level over the 5-year study period. However, on an individual level, substantial increases and decreases occurred. H1N1pdm09 infection or vaccination significantly enhanced specific antibody levels and the FcγRIIIa-engaging capacity of these antibodies in all age groups. ADCC-mediating antibodies increased, however, only in adolescents, reaching the same level as observed in the adult groups. Taken together, our results demonstrate the presence of cross-reactive, non-neutralizing, functional, and boostable antibodies against a never-encountered influenza virus strain across all age groups. These antibodies might contribute to protection from severe disease. Accordingly, in case of a newly emerging influenza virus, their further enhancement by vaccination could be beneficial as an immediate protective measure before a strain-specific vaccine becomes available.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus (IAV) is an important respiratory pathogen in pigs and poses a zoonotic risk to humans in close contact. While IAV epidemiology has been extensively studied in large-scale production systems, data from Switzerland - characterized by small herds and limited live pig imports - remain scarce. This exploratory nationwide cross-sectional study aimed to assess the association between herd-level IAV detection and reported respiratory disease in pig herds, and to explore associations with husbandry-, animal-, and human health-related factors. Between November 2023 and April 2025, 25 Swiss pig herds with caretaker-suspected respiratory symptoms were investigated. In each herd, five nasal swabs were collected and analyzed by quantitative PCR. Herd managers completed an interview, and clinical examinations were performed. Overall, 56 % (95 % CI: 37,1 - 73,3) of herds tested positive for IAV, comparable to reports from other European countries. The estimated intra-herd detection rate was 49,6 % (95 % CI: 31,2 - 68,0). Respiratory disease outbreaks associated with IAV detection showed indications of seasonal variation, with no positive herds identified during summer. Across age groups, pigs aged 11-14 weeks had a higher likelihood of IAV detection, with 15,79-fold increased odds (95 % CI: 1,50 - 860,4), although with considerable uncertainty. The interpretation is limited by the small sample size, heterogeneous data, and reliance on single time-point qPCR detection. The results suggest that IAV detection in clinically apparent respiratory outbreaks may follow seasonal patterns in Swiss pig herds. Weaners and newly introduced fattening pigs may play a role in such respiratory outbreaks and could represent relevant targets for IAV surveillance in Switzerland. Continued monitoring and the implemen tation of appropriate control measures remain important given the virus's zoonotic potential and impact on pig health.

Cell culture-based manufacturing platforms, already well-established for human influenza vaccines, are increasingly being adopted in the veterinary field as a powerful alternative to traditional embryonated hen's eggs, providing improved scalability and safety. In this study, we established a serum-free suspension culture process using Madin-Darby canine kidney (MDCK) cells for the efficient production of swine influenza virus (SIV). To optimize SIV production, we investigated multiple customized serum-free media in shaking flask cultures and selected an optimal formulation that supported efficient viral production. When this optimized medium was applied to a bioreactor culture, it supported robust cell growth, achieving a maximum viable cell density of 3.8 × 10[6] cells/mL prior to infection. Subsequent direct SIV infection experiments, conducted without media exchange in both shaking flask and bioreactor cultures, consistently yielded high and stable virus titers reaching a maximum log2 titer of 8.0, thus confirming the excellent scalability and reproducibility of the process. Moreover, the test trivalent vaccine developed in this study using the bioreactor-produced SIV antigen triggered high hemagglutination inhibition (HI) antibody titers and robust geometric mean titers (GMT) in animal models, thereby demonstrating superior immunogenicity to egg-based commercial vaccines. Taken together, these findings suggest that MDCK cell-based suspension culture platform may provide a scalable, high-yield, and reliable framework for the modern manufacturing of cell-based SIV vaccines.

Cross-species transmission of influenza A virus (IAV) represents a major risk for emergence of new pandemics, however, the host molecular responses invoked by cross-species IAV infection remain incompletely defined. MicroRNAs (miRNAs) are important post-transcriptional regulators of innate immunity, but their role during infection with IAV strains of the same subtype but of different host origin has not been investigated. Here, we investigated pulmonary miRNA responses in upper and lower lung sites from pigs inoculated with either a swine-adapted (swH1N1) or a human-adapted (huH1N1) IAV originating from the 2009H1N1 pandemic strain. High-throughput microfluidic RT-qPCR profiling of 115 miRNAs and selected antiviral genes revealed strain-dependent differences, with 33 miRNAs being differentially expressed in pig lungs (upper and lower lung) after human-adapted IAV infection, while only eight miRNAs were altered after swine-adapted IAV infection. Expression of several miRNAs correlated inversely with predicted target pattern recognition receptor (PRR) and interferon-stimulated gene (ISG) transcripts after huH1N1 infection, including miR-19a/b, miR-21-5p, and miR-93-3p, suggesting miRNA-mediated fine-tuning of antiviral signaling. Notably, extensive downregulation of miRNAs in the lower lungs after huH1N1 infection coincided with robust ISG induction, despite huH1N1 viral lung load being comparable to swH1N1 viral load. The swine-adapted IAV, in contrast, triggered stronger RIG-I-like receptor and cGAS transcription without substantial miRNA changes. Together, these findings demonstrate that pulmonary miRNA responses to IAV are both strain- and lung site-specific and may contribute to shaping the outcome of infection depending on the degree of viral host adaptation.

Superoxide dismutase 2 (SOD2), a key mitochondrial antioxidant enzyme, is essential for maintaining cellular redox homeostasis by scavenging superoxide radicals. While viruses often induce oxidative stress, the specific role of SOD2 in antiviral defense remains unclear. Here, we report that vaccinia virus (VACV) infection triggers mitochondrial and cellular reactive oxygen species (ROS) and selectively upregulates SOD2, but not SOD1. Genetic knockout of SOD2 exacerbated mitochondrial ROS (mtROS) accumulation and significantly enhanced VACV replication and spread, resulting in larger viral plaques. Conversely, SOD2 overexpression constrained plaque formation and suppressed viral dissemination. Mechanistically, the antiviral function of SOD2 does not strictly rely on its enzymatic activity or mitochondrial targeting, as neither the deacetylation-mimicking mutant nor the mutant lacking the mitochondrial localization signal peptide appreciably impaired its antiviral potency. Furthermore, in a rabbit model, local overexpression of human SOD2 attenuated the poxvirus lesion formation. Our findings unveil an important yet easily overlooked role of SOD2 in antiviral defense and posit it as a promising candidate for the development of host-directed therapeutics against poxviruses.