ETIDIOH - NEW

  Abstract Although seasonal influenza vaccination programs are effective at a population level, our data from inactivated influenza vaccine (IIV) cohorts in years 2015-2022 reveal that 50-60% of individuals do not seroconvert following immunization. The underlying mechanisms of vaccine non-responsiveness are far from understood. In this study, we sought to define key determinants of optimal B cell immune responses elicited by seasonal influenza vaccination, and to explore why some individuals fail to elicit humoral immunity following immunization. Immune responses associated with seroconversion and vaccine failure from individuals immunized with IIVs were compared at cellular and molecular levels using single-cell transcriptomics . We analyzed HA-specific B cell immunity across vaccine-responders, breakthrough infections and patients hospitalized with acute influenza. Droplet-based single-cell RNA sequencing and VDJ-sequencing of influenza-specific B cells from stored PBMCs was pe...

  Abstract Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool . However, IAV is at low abundance in wastewater , making it difficult to sequence . We benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods . Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes . However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes, but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches, potentially enabling broader surveillance and detection of emerging strains. However, probes were ...

  Abstract Background :  Continuous, non-invasive viral surveillance is essential to monitor emerging pathogens and guide public health responses. Most environmental surveillance studies use targeted qPCR approaches , and comparisons between wastewater and indoor air surveillance remain limited. We aimed to compare the utility of emergency department indoor air and urban wastewater for tracking circulating viruses and resolving genomic information.  Methods :  We conducted a matched-pair study comparing 19 weekly indoor air samples from the central ventilation exhaust shaft of an emergency department and 19 24-hour composite municipal wastewater samples in Leuven, Belgium , from December 2024 to April 2025. Both sample sets were processed using probe-based hybrid-capture viral metagenomics targeting over 3000 viral species , using influenza A as a clinically relevant test case.  Findings :  Wastewater captured higher overall viral diversity (233 versus 106 ...

  Abstract Currently, there is limited knowledge on the impact of immunity to hemagglutinin (HA) and/or neuraminidase (NA) on the transmission of influenza viruses . Therefore, using intramuscular vaccination , intranasal vaccination , or infection with reassortant viruses , we induced immunity to each antigen alone or both antigens combined in ferrets . We then assessed transmission of the 2009 pandemic H1N1 virus from these ferrets to naïve respiratory contacts . For all strategies used to induce immunity, combined immunity to HA and NA resulted in the largest reductions in transmission . Moreover, immunity to HA and NA conferred additive rather than synergistic reductions in transmission. No escape variants emerged in our transmission studies, and logistical regression showed that the probability of transmission was less than 50% when viral titers in donors were reduced to 101.5 and 102 median tissue culture infectious dose per ml on days 1 and 3 postinfection, respectively. The...

  Abstract Accumulating studies have identified the pivotal role of long non-coding RNAs (lncRNAs) in participating in host–virus interactions during virus infections . However, the regulatory roles of lncRNAs in influenza A virus (IAV) infection are still not fully elucidated . In this study, using high-throughput sequencing, we comprehensively compared the expression profiles of lncRNAs and mRNAs in mouse lungs infected either with the nonpathogenic parental (SDL124) H7N9 virus or its moderately pathogenic mouse-adapted (S8) variant . A total of 7636 significantly differentially expressed (SDE) lncRNAs were obtained in the S8-infected group compared to the mock group. As for the SDL124 group, 1042 SDE lncRNAs were identified. Subsequently, the mRNAs co-expressed with SDE lncRNAs were subjected to functional annotation and pathway enrichment analysis . The results indicated that the target mRNAs regulated by the S8 virus were mainly enriched in various immunological processes and ...

  Abstract While influenza A virus undergoes rapid antigenic drift in humans, at least some subtypes, such as H3, have relatively stable antigenicity in natural waterfowl reservoirs, despite the presence of immune pressure . However, the underlying mechanisms remain poorly understood. This study identified and characterized 187 antibodies to H3 hemagglutinin from experimentally infected mallard ducks , 18 of which were further analyzed by cryo-EM . Compared with human H3 antibodies , duck H3 antibodies exhibited higher glycan-binding propensity , more balanced immunodominance hierarchy , and targeted distinct epitopes . Other unique features of duck H3 antibodies included a convergent CDR H3-independent heavy chain-only binding mode and an N-glycosylated CDR H3 as decoy receptor . By annotating duck immunoglobulin germline genes , we also demonstrated the importance of gene conversion in duck H3 antibodies. Overall, our findings provide insights into how millennia of coevolution ha...

  Abstract Background :  The H1N1 influenza A virus evades host immunity through continuous antigenic drift, posing a significant challenge to broad-spectrum neutralizing antibody therapies. This study aims to systematically evaluate the neutralizing capacity of the broad-spectrum antibody C12H5 against H1N1 strains from different eras and identify key immune escape mutation sites .  Methods :  Three representative H1N1 virus strains from 2009, 2018, and 2023 were selected. An antigen–antibody binding prediction model based on the ESM-2 large language model was constructed by integrating 48,762 GISAID sequence data and deep mutation scanning data from the Bloom laboratory. Candidate escape sites were screened using SHAP (SHapley Additive exPlanations) value analysis. Mutant viruses were constructed via reverse genetics, and their neutralizing capacity and replication fitness were validated through hemagglutination inhibition assays, microneutralization assays, and vi...

  Abstract Influenza virus mRNAs are stable and competent for nuclear export and translation because they receive a 5′ cap(1) structure in a process called cap snatching 1. During cap snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that subsequently acts as a primer for the transcription of viral genes 4,5. Here we present the cryogenic electron microscopy structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF , positioning the FluPol endonuclease domain near the RNA exit channel of Pol II . These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure , trapped after cap snatching, shows that the cleaved capped RNA rearranges within FluPol, ...

  Abstract Influenza A virus is a highly contagious respiratory pathogen, and its rapid and continuous adaptive mutations for immune escape have limited the efficacy of existing vaccines and antiviral drugs. Here, we report a multimechanism anti-influenza platform based on the conjugation of zanamivir (ZMV) with amantadine (Aman). Aman acts as a hydrophobic tag that promotes the degradation of neuraminidase and concurrently enhances the physicochemical properties of ZMV , leading to improved membrane permeability and a significantly prolonged half-life. Meanwhile, the ZMV moiety counteracts Aman-induced cytotoxic autophagy . The resulting conjugate, compound 7j , exhibits potent activity against a wide range of neuraminidase and M2 ion channel mutations . Notably, a single intravenous dose of 7j fully protected mice from a lethal H1N1 challenge . Our work demonstrates that the rational fusion of ZMV and Aman achieves synergistic multimechanistic antiviral activity with enhanced eff...

  Abstract Newly emerging influenza virus strains 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 assist in mitigating disease symptoms via 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 vaccinatio...

  Abstract Background :  Genetic drift and host-associated adaptation in influenza A viruses threaten the long-term reliability of RT-qPCR-based diagnostics , particularly when nucleotide mismatches arise within primer and probe binding regions . Conventional assay evaluations often emphasize sequence conservation but rarely assess functional mismatch tolerance across divergent subtypes and hosts.  Methods :  We performed an in silico evaluation of a matrix (M) gene–targeted RT-qPCR assay by aligning primer and probe binding regions against 22 H1N1 isolates and representative H3N2 and H5N1 reference strains, including recent zoonotic isolates from avian and bovine hosts . Nucleotide mismatches were identified, quantified, and mapped relative to assay components and oligonucleotide termini. Mismatch burden was summarized by subtype and assay region.  Results :  H1N1 isolates exhibited complete conservation across primer and probe regions. In contrast, H3N2 a...

  ABSTRACT Swine influenza A virus (swIAV) is an important pathogen with regard to both the swine industry and public health . The pandemic A(H1N1) 2009 outbreak was caused by the swine-origin pandemic A(H1N1) 2009 [A(H1N1)pdm09] virus. Several reports have shown that several amino acid substitutions in the hemagglutinin (HA) antigenic sites can alter HA antigenicity. However, the impact of the amino acid deletion at position 155 on HA antigenicity remains unknown. In this study, we have isolated 11 samples of swIAVs from seven pig farms in Japan and found an amino acid deletion at position 155 of the HA region in one of the isolates of the H1N2 subtype . To examine the impact of this amino acid deletion on viral replication and HA antigenicity, we generated recombinant influenza A viruses possessing the H1 HA gene encoding either an artificial insertion or deletion of glycine at position 155. The growth kinetics of these recombinant viruses in two different cell lines demonstrated...

  Abstract The polymerase acidic (PA) protein is a subunit of the trimeric influenza A virus (IAV) RNA-dependent RNA polymerase and the target of the anti-influenza drug baloxavir marboxil (BXM). As with other direct-acting antivirals , treatment with BXM can lead to selection of viruses carrying resistance mutations . If these mutations have negligible fitness costs, resistant viruses can spread widely and render existing treatments obsolete . Multiple BXM resistance mutations in the nuclease domain of PA have been identified, with I38T and I38M amino acid substitutions occurring frequently. These mutations have minimal to no effects on viral polymerase activity , virus replication , or transmission . However, for reasons that are not well understood, viruses with BXM resistance substitutions have not been able to compete with parental wild-type strains . The IAV genome segment encoding PA also encodes the host shutoff nuclease PA-X, which shares the endonuclease domain with PA bu...

Abstract Key to the success of influenza A virus as a pathogen are its numerous tactics of immune evasion. To suppress anti-viral cellular and organismal responses , influenza A virus encodes several immunomodulatory proteins , including the endoribonuclease PA-X . PA-X decreases inflammation and immune responses in in vivo infections by limiting host gene expression . PA-X is conserved in 99% of all influenza A viral strains , pointing to its importance as a crucial immunomodulator . However, it is not yet known how PA-X activity alters the antiviral response in the human airway or how it benefits the virus. To define how influenza A virus uses this protein to evade immune responses , we characterized the impacts of PA-X on the host response to infection in the infected and bystander cells of the airway epithelium using a 3D ex vivo model. We discovered that PA-X exerts a dual action on immune responses , dampening aspects of both the innate and adaptive immune systems . Consistent wi...

  Abstract Influenza A virus (IAV) is a zoonotic pathogen with a broad host range, posing an ongoing threat to global public health . As the core subunit of the IAV polymerase , polymerase basic protein 1 (PB1) is essential for viral replication and transcription , and its mutations are key drivers of viral evolution . This review evaluates the impact of PB1 mutations on IAV evolution, with a focus on polymerase activity, host adaptation, transmissibility, and virulence. Additionally, it discusses the implications of these mutations for vaccine development. The review aims to provide insights that can inform influenza surveillance, identify novel antiviral targets, and guide vaccine design. Source:  Link:  https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2026.1768665/full ____

  Abstract Influenza-associated encephalitis/encephalopathy (IAE) is a severe neurological complication characterized by central nervous system dysfunction and structural damage following influenza virus infection . Predominantly affecting infants and young children , IAE exhibits its highest incidence in those under five years of age . Key clinical manifestations of IAE include acute seizures, sudden high fever, and impaired consciousness , frequently progressing to coma . Neuroimaging , particularly magnetic resonance imaging (MRI), often reveals multifocal brain lesions involving multiple brain regions, including the cerebellum, brainstem, and corpus callosum . The prognosis of IAE is poor , with a mortality rate reaching 30%. Current diagnosis relies heavily on clinical presentation and characteristic neuroimaging findings, as the precise pathogenesis of IAE remains elusive . While various research models, including cell lines, brain organoids, and animal models , have been dev...

  Abstract Selection of advantageous mutations drives the emergence of dominant variants during seasonal influenza epidemics . However, within-host detection of such variants remains rare , limiting our understanding of how selection operates at the scale of individual hosts. In this study, we used a controlled human infection model to examine the within-host evolutionary dynamics in thirteen participants intranasally infected with a seasonal H3N2 influenza A virus . Although this clinical trial is ongoing , our work represents a pre-planned, interim, exploratory analysis. Results in this system were contrasted with those observed in a ferret model of infection. The inoculum, used in both humans and ferrets, carried standing diversity that enabled evaluation of variant trajectories during infection. Although the dynamics were variable among participants, in humans , the minor variants in the PA and NP gene segments tended to increase in frequency as infection progressed. Variant dy...

  Abstract Hemagglutinins (HA) from different influenza A virus subtypes share as little as ~40% amino acid identity , yet their protein structure and cell entry function are highly conserved . Here we examine the extent that sequence constraints on HA differ across three subtypes . To do this, we first use pseudovirus deep mutational scanning to measure how all amino-acid mutations to an H7 HA affect its cell entry function. We then compare these new measurements to previously described measurements of how all mutations to H3 and H5 HAs affect cell entry function . We find that ~50% of HA sites display substantially diverged preferences for different amino acids across the HA subtypes. The sites with the most divergent amino-acid preferences tend to be buried and have biochemically distinct wildtype amino acids in the different HA subtypes. We provide an example of how rewiring the interactions among contacting residues has dramatically shifted which amino acids are tolerated at s...

  ABSTRACT 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 dive...

  ABSTRACT Multiple genes are involved in the pathogenicity of influenza A virus . Our previous study reported two naturally occurring amino acid mutations in the polymerase acidic (PA) protein as crucial determinants of the virulence of Eurasian avian-like H1N1 (EA H1N1) influenza viruses. PA-X, an accessory protein encoded by the PA gene , is thought to play a role in viral pathogenicity and regulation of host immune response , but its specific function remains unclear. In this study, we found that two genetically similar EA H1N1 influenza viruses , A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), induced significantly different suppression levels of host protein synthesis . The difference in host shutoff activity induced by PA-X protein was the key factor affecting the inhibition of host gene expression . Loss of PA-X expression significantly reduced its host shutoff activity , thereby enhancing host antiviral immune response . PA-X deficiency had no appa...