Historical #Pandemic and Contemporary #Influenza A Viruses Reveal #PB2 M631L as a Convergent #Adaptation to #Human ANP32

 

Abstract

Understanding the genetic changes that allow avian influenza A viruses (IAVs) to switch their natural hosts and establish productive infection in humans is important for pandemic risk assessment. Adaptations in the IAV polymerase are required to overcome species-specific restrictions imposed by host ANP32 proteins. Notably, avian virus polymerase is generally only poorly supported by human ANP32 proteins due to species-specific differences. Consequently, efficient polymerase adaptation to the binding interface of human ANP32 requires distinct amino acid changes, such as PB2 E627K. A separate adaptation, PB2 M631L, has recently been reported in mammalian-adapted IAV; however, its functional role across divergent viral lineages and its relationship to host ANP32-dependent adaptation remain incompletely defined. Here, we examine PB2 M631L in the polymerases of a 1918 pandemic strain, a recombinant contemporary H1N1pdm09, and a recent clade 2.3.4.4b H5N1 virus. Using polymerase activity and protein-interaction assays, we show that PB2 M631L enhances polymerase activity and ANP32 binding in human—but not avian—contexts, and that this effect is conserved across multiple viral backgrounds. In H1N1pdm09, PB2 M631L also increased virus replication in mammalian cells. These findings indicate that PB2 M631L contributes to enhanced polymerase compatibility with human ANP32 proteins and are consistent with a role in adaptation across multiple influenza virus lineages. Our results highlight how analysis of historical pandemic strains can inform risk assessment for future emerging viruses.

Source: 

Link: https://www.mdpi.com/2076-2607/14/4/859

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The temporal #sequence of #influenza #H1N1 and #Mycoplasma pneumoniae co-infection causes disease severity in Syrian hamster models

 

Abstract

Introduction: 

Influenza H1N1 virus is one of the most prevalent subtypes among influenza viruses, and co-infection with Mycoplasma pneumoniae (Mp) is frequently documented in clinical respiratory infections. However, the pathological mechanisms underlying the temporal sequence of H1N1-Mp co-infection remain poorly characterized, and relevant animal models are lacking.

Methods: 

In this study, we established a model of influenza H1N1 and Mycoplasma pneumoniae co-infection in Syrian hamsters and infected two pathogens in interval of 72 hours. Clinical manifestations, body temperature, body weight, pathogen loads in nasal, pharyngeal, and anal swabs, as well as blood cytokine profiles were dynamically monitored over 14 days post-infection (dpi). Additionally, tissue pathogen loads, histopathological changes, routine blood parameters, and blood biochemistry indicators were evaluated at 7 and 14 dpi.

Results: 

The results demonstrated that hamsters first infected with H1N1 followed by Mp (F-M group) exhibited significantly more severe histopathological lesions (assessed by HE staining), higher pathogen loads, and dysregulated cytokine responses compared to other infection groups.

Conclusion: 

Our findings highlight the critical role of infection order in determining the severity of H1N1-Mp co-infection, providing novel insights into the temporal dynamics and pathogenic mechanisms of respiratory co-infections.

Source: 

Link: https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2026.1787294/full

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Porcine #influenza #mAbs to #H3, #H5, and #H7 hemagglutinins recognize H3 egg adapted site and target the HA stem

 

Abstract

Introduction

Monoclonal antibodies (mAbs) are critical tools for elucidating viral evolution, informing vaccine design, and developing antiviral therapeutics. Large-animal models, such as the pig, that closely mirror human immune responses are essential for understanding influenza immunity.

Methods

Pigs were either infected or sequentially immunized with influenza viruses and monoclonal antibodies directed against H3, H5, and H7 influenza virus haemagglutinins were isolated. Antibody specificity, breadth, epitope targeting (head versus stem), neutralizing capacity, and Fc-mediated activity were assessed across influenza subtypes.

Results

Pigs generated both strain-specific and broadly reactive mAbs targeting haemagglutinin head and stem epitopes. An H3-specific mAb (H3–57) selectively recognized the egg-adapted L194P mutation associated with reduced human vaccine effectiveness. H5 and H7 immunization induced neutralizing antibodies, including cross-group stem mAbs reactive with H1, H3, and H5 haemagglutinins. Fc-mediated activity correlated with antibody binding strength rather than epitope location.

Conclusions

These findings demonstrate that pigs mount antibody responses closely resembling those observed in humans, including recognition of conserved stem epitopes and adaptive head mutations. Porcine mAbs represent powerful new tools for dissecting influenza immunity, guiding vaccine design, and enhancing pandemic preparedness using a physiologically relevant large-animal model.

Source: 

Link: https://academic.oup.com/discovimmunology/article/5/1/kyag006/8503709

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#Oseltamivir aziridines are potent #influenza #neuraminidase #inhibitors and imaging agents

 

Significance

Influenza remains a major global health threat. We introduce oseltamivir-based aziridines that unite transition-state mimicry for tight binding with aziridine-enabled covalent capture of the catalytic tyrosine. This dual function yields potent, mechanism-based neuraminidase inhibition and enables activity-based quantification of active enzyme directly in complex samples. Across N1, N2, and influenza B enzymes, selected compounds show high potency against diverse viral neuraminidases and in live virus replication assays. By combining a clinically grounded scaffold with a reactivity handle, these molecules bridge therapeutic and diagnostic needs and offer a practical platform for neuraminidase imaging and antiviral development.

Abstract

Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by noncovalent binding and emerging resistance. We hypothesized that Oseltamivir aziridines would unite transition-state mimicry for tight binding, with aziridine-enabled covalent capture of the catalytic tyrosine, thereby supporting both therapy and activity-based quantification. Here, we present oseltamivir-based aziridines, inspired by cyclophellitol chemistry, that act as covalent inhibitors and activity-based probes via an N-acylaziridine warhead. Free-energy calculations, and NMR observations, indicate a 4H5 half-chair preference consistent with the NA transition state, and selected analogues inhibit multiple NA subtypes with low nanomolar binding constants. Diverse evidence establishes covalency: time-dependent inactivation, inhibitor washout, intact-mass shifts, MS/MS identification of a tyrosine adduct, and QM/MM reaction profiles, while cryoEM of N1 aligns with the proposed binding mode, revealing an elimination product. The inhibitors demonstrate formidable activity against diverse viral neuraminidases, including H5N1, and further enable imaging and quantification of active NA. With their dual therapeutic and diagnostic potential, these first-in-class inhibitors indeed benefit from transition state mimicry and covalency, and thus offer a powerful platform for antiviral development and neuraminidase imaging, addressing urgent global health needs in influenza treatment and prevention.

Source: 

Link: https://www.pnas.org/doi/10.1073/pnas.2504045123

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Defining #influenza-specific B cells in #vaccine #responders, non-responders and influenza breakthrough #infections

 

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 performed using 10x Genomics. Our results show that atypical B cells are the major subset of B cell responses in vaccine non-responders on day 28 post-vaccination. Conversely, individuals who seroconvert had diverse B cell phenotypes. The use of recombinant influenza-specific HA probes allowed us to dissect expression patterns on influenza HA-specific B cells. We found that HA-specific B cells of vaccine non-responders for A/H1N1 and A/H3N2 components displayed elevated atypical-like markers (CD11c, FcRL-5) at baseline, compared to responders. Analysis of differentially expressed genes (DEGs) between responders and non-responders identified differential expression of HLA-DR, CD74, CD83, and CXCR3 genes. We subsequently demonstrated reduced frequencies of HLA-DR-, CD74- and CD83-expressing B cells in patients hospitalized with influenza, compared to healthy participants. Hospitalized influenza patients also had significantly higher proportions of atypical CD21-CD27- B cells. Overall, our data demonstrate an association between elevated frequencies of atypical-like B cells with both lack of seroconversion following immunization and severe influenza infection. These findings broaden our understanding of humoral immunity in influenza vaccination and infection, providing novel insights for vaccination strategies and design.

Competing Interest Statement

Katherine Kedzierska has received paid honoraria from Pfizer. Hayley McQuilten has a consultancy role for Ena Therapeutics

Funder Information Declared

NHMRC

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.03.19.710321v1

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Developing and #Benchmarking #OneHealth Genomic #Surveillance #Tools for #Influenza A Virus in #Wastewater

 

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 costly, labor-intensive, and less sensitive than tiled-amplicon. When testing compatibility of sequencing methods with upstream virus concentration and extraction methods, ultrafiltration-based virus concentration outperformed large-volume direct extraction with all four sequencing methods. This set of benchmarking comparisons and custom panels provides needed information for the translation of IAV genomic sequencing into a routine component of wastewater surveillance.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

University of California, Berkeley, L22CR4507

NIH Common Fund, 4R00GM144747-03

Source: 

Link: https://www.biorxiv.org/content/10.1101/2025.09.19.676942v2

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A Tale of Two Lenses: #Emergency department indoor - #air hybrid-capture #metagenomics complements #wastewater by adding a human-focused respiratory #virus perspective

 

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 species) and more complete genomes compared to indoor air, showing a relatively stable composition, mainly of enteric and animal-associated viruses. Indoor air demonstrated lower overall diversity but was enriched for respiratory viruses, including influenza A, coronaviruses, metapneumovirus, and respiratory syncytial virus, and more frequently achieved high genome coverage for these pathogens. Although both sample types permitted influenza A subtype characterization, influenza A genomes from wastewater were often less well covered. When coverage thresholds were met, indoor air supported targeted antiviral resistance-site screening for influenza A and RSV-A. 

Interpretation: 

Wastewater and indoor air generate distinct but complementary viromes. Wastewater acts as a diverse, population-level monitor for One-Health applications, whereas indoor air serves as a targeted, human-centric sentinel system facilitating further genomic characterization for respiratory viruses.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

Mustafa Karatas is supported by a Research Foundation Flanders (FWO) fundamental research scholarship (number: 11P7I24N). C.G., L.C., E.H., S.G. and E.A. acknowledge support from the DURABLE project. The DURABLE project has been funded by the European Union, under the EU4Health Programme (EU4H), project no. 101102733. Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The computing power in this work was provided by the VSC (Flemish Supercomputer Centre), financed by the FWO and the Flemish government department EWI.

Source: MedRxIV, https://www.medrxiv.org/

Link: https://www.medrxiv.org/content/10.64898/2026.03.13.26348311v1

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#Immunity to #hemagglutinin and #neuraminidase results in additive reductions in #airborne #transmission of #influenza #H1N1 virus in #ferrets

 

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. These studies define the relationship between immunity to HA and NA on transmission and identify a threshold titer indicative of decreased transmission in ferrets.

Source: 

Link: https://www.science.org/doi/10.1126/sciadv.aea8719

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Systematic Identification of the Functional lncRNAs During #H7N9 Avian #Influenza Virus #Infection in Mice

 

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 exhibited a strong correlation with inflammatory-related signaling pathways. Moreover, 12 lncRNAs and 10 mRNAs co-expressed with SDE lncRNAs were selected and successfully verified by RT-qPCR. Among these lncRNAs, NONMMUG032982.2 and NONMMUG032328.2 exhibited strong antiviral activity against IAV. Additionally, these two lncRNAs were chosen for further in-depth bioinformatics analysis, including transcription factor prediction, coding capacity assessment, genomic location, construction of secondary structure, and prediction of potential interacting proteins. Taken together, these findings provide a cluster of lncRNAs probably associated with the virulence of IAV in mice and shed light on the anti-IAV effects of two functional lncRNAs, establishing a molecular foundation for further exploring the regulatory mechanisms of lncRNAs in IAV infection.

Source: Viruses, https://www.mdpi.com/journal/viruses

Link: https://www.mdpi.com/1999-4915/18/3/353

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Structural #insights into #antibody responses against #influenza A virus in its natural reservoir

 

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 have shaped the interplay between influenza A virus antigenic drift and antibody responses in the natural reservoir.

Competing Interest Statement

N.C.W. consults for HeliXon. The authors declare no other competing interests.

Funder Information Declared

National Institutes of Health, https://ror.org/01cwqze88, R01 AI165692

Carl R. Woese Institute for Genomic Biology, Carl R. Woese Institute for Genomic Biology Postdoctoral Fellowship

Vallee Foundation, https://ror.org/05nmp3276, Vallee Scholars Program

Foundation for Partnership Initiatives in the Niger Delta, https://ror.org/041nz5a71, Searle Scholars Program

Howard Hughes Medical Institute, https://ror.org/006w34k90, Emerging Pathogens Initiative

Source: BioRxIV, https://www.biorxiv.org/

Link: https://www.biorxiv.org/content/10.64898/2026.03.11.711171v1

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Identification of a Key #Hemagglutinin #Mutation Mediating #Antibody Escape in #Influenza #H1N1pdm09 Viruses

 

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 viral growth kinetics analysis. 

Results: 

Machine learning scoring identified five potential escape sites, with K147 exhibiting the highest overall score (0.92). SHAP analysis revealed that the K147 site within the HA protein’s 130-loop region received the highest importance score (0.28), significantly surpassing other candidate sites. Experimental validation revealed that the K147N mutation reduced neutralizing potency against C12H5 by 8-fold (from 1:1024 to 1:128) and approximately 6-fold in microneutralization assays (from 8.3 log2 to 5.7 log2), while exhibiting a replication advantage in MDCK cells. Microneutralization assays further confirmed an approximately 6-fold reduction in neutralization sensitivity. Structural analysis indicated that K147 is located at the periphery of the HA receptor-binding domain, immediately adjacent to the receptor-binding site. 

Conclusions: 

K147N is identified as the critical mutation mediating C12H5 immune escape, and this mutation has emerged in 2023 circulating strains. This study provides important molecular targets and early warning mechanisms for broad-spectrum antibody optimization and influenza vaccine updates.

Source: 

Link: https://www.mdpi.com/1999-4915/18/3/349

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Mechanism of co-transcriptional cap snatching by #influenza #polymerase

 

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 snatching1. 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 genes4,5. 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, directing the capped RNA 3′ end toward the FluPol polymerase active site for viral transcription initiation. Together, our results provide the molecular mechanisms of co-transcriptional cap snatching by FluPol.

Source: 

Link: https://www.nature.com/articles/s41586-026-10189-0

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#Zanamivir - #Amantadine Conjugate: A Dual-Action Agent with Broad-Spectrum Synergistic #Antiviral Efficacy

 

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 efficacy and safety, offering a new strategy for the development of next-generation anti-influenza drugs.

Source: 

Link: https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c03547

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Longitudinal #assessment of functional #antibodies to a novel #influenza virus strain across age groups

 

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 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 ADCC activity, both of which were notably lower in adolescents compared to adults and older adults. Without infection or vaccination, average amounts of H1N1pdm09-reactive antibodies remained relatively stable on 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 can potentially contribute to protection from severe disease. Accordingly, in case of a newly emerging virus, their further enhancement by vaccination could be beneficial as an immediate protective measure before a strain-specific vaccine becomes available.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

This study did not receive any funding

Source: 

Link: https://www.medrxiv.org/content/10.64898/2026.02.21.26346781v1

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Evaluating #primer and #probe #mismatch tolerance in an #Influenza A #matrix gene RT #qPCR using contemporary human and zoonotic strains

 

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 and H5N1 strains demonstrated subtype-specific sequence variability, with a total of eleven mismatches identified across seven non-H1N1 isolates (mean mismatch per isolate = 2.43). Probe mismatches predominated (63.6%), occurring primarily at internal positions, while primer mismatches were infrequent and largely avoided 3′ terminal nucleotides. Recent H5N1 isolates (2023–2024) shared conserved internal mismatches in the probe and forward primer, whereas a historical H5N1 isolate (2016) exhibited a distinct profile including a terminal probe mismatch. Despite this variability, mismatch patterns were consistent with preserved amplification potential. 

Conclusion: 

This study demonstrates that the evaluated influenza A M gene RT-qPCR assay exhibits inherent mismatch tolerance across human and zoonotic subtypes. By shifting diagnostic evaluation from strict sequence identity to functional resilience, our findings provide a framework for designing and maintaining robust molecular assays suitable for surveillance and pandemic preparedness amid ongoing viral evolution.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.02.23.707407v1

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Impact of an #aminoacid #deletion detected in the #hemagglutinin (HA) #antigenic site of swine #influenza A virus field strains on HA antigenicity

 

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 that the effect of amino acid deletion at position 155 of H1 HA on viral replication is limited. In contrast, microneutralization assay-based neutralization titers revealed that amino acid deletion significantly altered HA antigenicity. These results demonstrate that a naturally occurring amino acid deletion at position 155 in an H1 HA antigenic site can markedly alter HA antigenicity with only a limited impact on replication in vitro, highlighting the need to monitor such variants in swine populations and to assess their zoonotic potential.

Source: 

Link: https://journals.asm.org/doi/full/10.1128/jvi.01820-25?af=R

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Impaired #host shutoff is a fitness cost associated with #baloxavir marboxil #resistance #mutations in #influenza A virus PA/PA-X nuclease domain

 

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 but has a unique C-terminal domain generated by ribosomal frameshifting during translation. Unlike their effects on PA activity, the effects of BXM or the I38T/M substitutions on PA-X function remain uncharacterized. In our work, for the first time, we directly examine the effects of baloxavir and the I38T/M substitutions on PA-X activity and show that baloxavir inhibits PA-X activity in a dose dependent manner. Most importantly, we also demonstrate that the I38T/M mutations significantly impair the host shutoff activity of PA-X proteins from different IAV strains of H1N1, H3N2, and H5N1 subtypes. Our work reveals that the deleterious effects of I38T/M on PA-X function may represent an important barrier to the spread of BXM-resistant viruses.

Source: 

Link: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1013550

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Dual roles for #influenza A protein #PAX: limiting inflammatory response and disrupting #MHC I #antigen presentation in #human respiratory epithelium

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 with reports in model organisms, PA-X significantly decreases secretion of multiple cytokines from airway epithelium, including IFN-λ, which likely plays a role in its ability to reduce inflammation and lung damage. In addition, we revealed that PA-X decreases and delays MHC I antigen presentation from infected cells. This reduction likely aids influenza A virus in hiding from antigen-specific T cells and allows the virus to successfully replicate prior to immune detection. This new function for PA-X highlights how influenza A virus employs active mechanisms to block immune detection, in addition to tolerating high levels of antigen mutations to escape it. Moreover, as evasion of recognition by the adaptive immune system is particularly important during infections of animals and humans with pre-existing immunity, this immunomodulatory activity may be key to the longevity of influenza A viruses and their continued circulation.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69, R01AI137358, 75N93021C00017

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.01.30.702929v1

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#PB1 #mutations as key #drivers of #influenza A virus #evolution

 

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

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#Pathogenesis and #Research #Models of Acute #Influenza-Associated #Encephalitis / #Encephalopathy: An Update

 

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 developed to recapitulate IAE features, significant limitations persist in modeling the core clinical pathophysiology observed in pediatric patients, necessitating further model refinement. This review synthesizes the clinical spectrum of IAE, summarizes progress in understanding its pathogenesis, and critically evaluates existing research models. We aim to provide a foundation for utilizing experimental approaches to elucidate IAE mechanisms and identify potential therapeutic strategies.

Source: 

Link: https://www.mdpi.com/1999-4915/18/1/95

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