Conserved #influenza A #epitope candidate regions and a benchmark of ESM-2 #sequence features

 

Abstract

Influenza A virus antigenic drift forces annual vaccine reformulation, motivating the search for conserved epitope candidates that could support broadly protective vaccines. We systematically screened influenza A virus sequences (H1N1, H3N2, H5N1; nine viral proteins) to define 98 conserved candidate regions, 38 of which were identical across the H1N1, H3N2, and H5N1 consensus sequences, all in the polymerase complex and nucleoprotein (PB2, PB1, PA, NP), whereas the ten surface-glycoprotein (HA/NA) candidates were subtype-specific. We then benchmarked two protein-language-model (ESM-2) features against alignment conservation. Group-masked log-probability correlated moderately with MSA conservation (Spearman rho = 0.25 to 0.39 for HA) but provided no incremental value for T-cell epitope discrimination (change in AUROC +0.004, p = 0.46); attention-derived contact-density was not a valid solvent-accessibility proxy. A curated antibody-epitope benchmark (22 clusters, 5 neutralization-supported) was underpowered for a high-confidence B-cell test. We document data-quality and reproducibility pitfalls (length heterogeneity, coordinate mapping, and pseudoreplication) and release the auditable benchmark. These results provide an auditable candidate resource and show that, in the evaluated benchmarks, ESM-2 sequence scores did not improve epitope prioritization beyond alignment-derived conservation.

Competing Interest Statement

The authors have declared no competing interest.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.08.16.745106v1

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Assessment of Quantitative #Genetic #Distances Supports the Separation of #H17N10 and #H18N11 Subtypes of #Influenza a Virus into a Distinct Species

 

Abstract

The taxonomic status of the H17N10 and H18N11 influenza A viruses isolated from bats remains unclear due to the absence of quantitative classification criteria at this taxonomic level. A total of 3328 representative IAV genomes, encompassing all eight protein-coding segments, were analysed. Various genetic distance-based metrics were assessed at the pairwise level, including intra- and intergroup nucleotide distances, dN/dS ratios, and transition/transversion ratios, to facilitate the differentiation of the Alphainfluenzavirus genus into distinct taxa. Pairwise distances for seven of the eight segments (PB2, PB1, PA, NP, M, NA, NS) consistently differentiated the H17–H18 group from H1–H16. Across segments, intergroup nucleotide divergence was consistently above a lower bound of ~25%, with segment-specific values extending to higher levels (up to ~40% in PB2 and PA), while intragroup divergence remained substantially lower. The HA segment did not conform to this pattern, which is consistent with the hypothesis of ancient reassortment. The distribution of pairwise dN/dS values for the PB2, PB1, PA, and NP segments is evidently bimodal. Intergroup comparisons were consistently higher across all segments, whereas intragroup values remained lower. A similar lower boundary of approximately 0.12 was observed across segments, while the upper range of intergroup values varied by gene. Overall, the results support a consistent gene-specific separation pattern. Previously demonstrated absence of reassortment compatibility between bat viruses (H17–H18) and canonical influenza A (H1–H16) viruses indicates that these lineages have evolved independently over an extended period. These consistent genomic patterns provide support for the hypothesis that H17N10 and H18N11 viruses may represent a separate species within the genus Alphainfluenzavirus.

Source: 

Link: https://www.mdpi.com/1999-4915/18/8/838

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#Development and Characterization of a Recombinant #Bundibugyo Virus Expressing a Fluorescent Reporter Protein

 

Abstract

Bundibugyo virus disease (BVD), caused by Bundibugyo virus (BDBV), is associated with substantial morbidity and mortality, with previous outbreaks reporting case fatality rates of 30%–50%. The ongoing BDBV outbreak in the Democratic Republic of the Congo and Uganda highlights the urgent need for virus-specific research tools and medical countermeasures. Unlike Ebola virus disease caused by Zaire ebolavirus, no licensed vaccines or specific therapeutics are currently available for BVD. The lack of research tools has limited studies with BDBV. To facilitate antiviral testing and neutralization studies, we developed the first recombinant BDBV expressing the fluorescent reporter protein ZsGreen (rBDBV-ZsG). As a proof of concept, we tested a set of previously characterized antiviral compounds and demonstrated comparable inhibitory profiles between rBDBV-ZsG and the wild-type BDBV parental strain. Furthermore, the utility of rBDBV-ZsG was successfully evaluated in neutralization assays, demonstrating robust sensitivity and specificity using monoclonal antibodies and convalescent serum samples.

Source: 

Link: https://www.tandfonline.com/doi/full/10.1080/22221751.2026.2709847

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Characterization of #bovine-derived #H5N1 viruses expressing fluorescent and luminescent reporter #proteins

ABSTRACT

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle reported in the USA. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication, facilitating high-throughput screens. In this study, we engineered three recombinant H5N1 clade 2.3.4.4b reporter viruses expressing nanoluciferase (NLuc) and two fluorescent reporter proteins, miniGFP2 and UnaG within the open reading frame of the nonstructural gene of the bovine A/Cattle/Texas/063224-24-1/2024 (TX2/24) virus. All reporter viruses replicated efficiently in vitro, presenting replication kinetics comparable to the parental rTX2/24 virus, but exhibited smaller plaque sizes, suggesting reduced cell-to-cell spread. In vivo infection studies in mice showed comparable pathogenicity among all four viruses, although rTX2/24-miniGFP2 and rTX2/24-UnaG exhibited decreased virus shedding relative to rTX2/24 and rTX2/24-NLuc. Virus titrations and in situ localization of virus replication sites demonstrated robust replication in respiratory tissues, with slightly attenuated systemic dissemination of all three reporter viruses. Fluorescent virus neutralization assays using miniGFP2 and UnaG reporter viruses accurately quantified neutralizing antibody titres in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Additionally, the utility of the NLuc reporter virus for antiviral screening was validated against oseltamivir in vitro. Collectively, these results establish the H5N1 TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis and for use in serological and antiviral drug screens.

Source: 

Link: https://www.microbiologyresearch.org/content/journal/jgv/10.1099/jgv.0.002298

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Single #influenza A viruses induce #nanoscale cellular #reprogramming at the virus-cell interface

Abstract

During infection, individual virions trigger specific cellular signaling at the virus-cell interface, a nanoscale region of the plasma membrane in direct contact with the virus. However, virus-induced receptor recruitment and cellular activation are transient processes that occur within minutes at the nanoscale. Hence, the temporal and spatial kinetics of such early events often remain poorly understood due to technical limitations. To address this challenge, we develop a protocol to covalently immobilize labelled influenza A viruses on glass surfaces before exposing them to live epithelial cells. Our method extends the observation time for virus-plasma membrane association while minimizing viral modifications, facilitating live imaging of virus-cell interactions. Using single-molecule super-resolution microscopy, we investigate virus-receptor interaction showing that viral receptors exhibit reduced mobility at the virus-binding site, which leads to a specific local receptor accumulation and turnover. We further follow the dynamics of clathrin-mediated endocytosis at the single-virus level and demonstrate the recruitment of adaptor protein 2 (AP-2), previously thought to be uninvolved in influenza A virus infection. Finally, we examine the nanoscale organization of the actin cytoskeleton at the virus-binding site, showing a local and dynamic response of the cellular actin cortex to the infecting virus.

Source: Nature Communications, https://www.nature.com/articles/s41467-025-58935-8

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