ETIDIOH - NEW

  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 Clade 2.2 H5N1 influenza viruses have caused an unusually high number of human infections , providing a unique opportunity to investigate early molecular steps associated with host adaptation . Although most work has focused on hemagglutinin (HA), the contribution of neuraminidase (NA) to these early adaptive events has remained unclear. By analyzing publicly available sequences from clade 2.2-infected patients , we identified 20 NA mutations and compared their phenotypes to 20 mutations acquired during diversification in primary human airway cells under drug-free conditions. Most patient-derived NA mutations resulted in modest reductions in sialidase activity , keeping activity within a functional range that supported improved replication in α2,6 sialylglycan (α2,6 Sia)-dominant environments , whereas excessive reduction impaired fitness . Notably, the phenotypes of culture-selected and patient-derived mutations were highly concordant , suggesting that these NA changes ...

  Abstract In November 2024, an adolescent female in British Columbia was hospitalized presenting with severe symptoms including respiratory failure due to infection with a novel H5N1 subtype influenza strain (BC24). Using cryogenic electron microscopy (cryo-EM), we show here that the N169 α2,3-linked auto-glycan that is found in the sialic acid binding site of previously studied H5 hemagglutinin (HA) proteins is absent in purified BC24 HA protein , suggesting greatly reduced affinity for α2,3-linked sialosides . Glycan microarray analysis shows that the BC24 HA protein displays reduced or no binding not just to most α2,3-linked sialosides, but also to α2,6-linked sialosides . Full-length BC24 HA expressed in A549 lung alveolar carcinoma cells drives membrane fusion, albeit at significantly lower levels than previous H5 HA proteins, and post-infection sera from the patient display strong binding to BC24 HA and HA proteins from other influenza subtypes. The high virulence of the BC2...

  Abstract Sialic acids (SAs) are abundantly expressed on vertebrate cell surfaces and are widely recognized as key viral attachment factors , particularly for influenza viruses . However, their role remains understudied in other orthomyxoviruses, such as thogoto and quaranja viruses , which are tick-borne viruses sporadically infecting humans. Enzymatic removal of SAs increased the infectivity of Thogoto and Dhori viruses , as well as pseudotypes carrying the glycoproteins of Oz, Sinu, and Wellfleet Bay viruses . A similar effect on pseudotype infectivity was observed following the binding of specific lectins to SAs. These findings indicate that, in contrast to influenza viruses, SAs act as a barrier to the entry of these orthomyxoviruses . Experimental evolution of the Sinu and Wellfleet Bay virus glycoproteins revealed point mutations that partially overcame this barrier . Given the abundance of sialic acids in mucosal tissues, we speculate that SAs may contribute to the inabili...

  Abstract Highly pathogenic H5Ny influenza A viruses are causing unprecedented, season-independent outbreaks across avian and mammalian species , including dairy cattle , a novel reservoir. The sialoside-binding properties of influenza A hemagglutinin (HA) are strongly related to its ability to infect and transmit between hosts . Mucin-like O-glycans , omnipresent in respiratory tracts, have been understudied as viral receptors due to their complexity. To address this, we synthesized 25 O-linked glycans with diverse sialosides , including modifications by fucosides and sulfates . Our findings reveal that H5Ny 2.3.4.4b viruses bind core 3 sialyl-Lewisx and Sia-Gal-β3GalNAc, O-linked glycans not recognized by classical H5 or other avian viruses . By determining crystal structures, we resolved the structural features of four glycans in an H5 hemagglutinin (HA) from a 2016 2.3.4.4b virus . While these viruses do not bind human-type receptors , their broad receptor specificity enhances...

  Abstract Highly pathogenic avian influenza H5N1 viruses of clade 2.3.4.4b have caused widespread avian mortality and sporadic mammalian infections , raising concerns about their potential for efficient replication in the human population . Efficient replication in the human upper respiratory tract is considered a key barrier to transmission . Here, we demonstrate that an H5N1 virus isolated from bovine milk in Texas in 2024 (H5N1Tex/24) replicates as efficiently as the 2009 pandemic H1N1 virus (H1N1HH4/09) in well-differentiated human nasal epithelial cells . These cells express both avian- and human-type influenza receptors , indicating receptor adaptation is unnecessary for entry . H5N1Tex/24 replicates effectively at 33 degrees Celsius , reflecting nasal cavity temperature , whereas earlier avian H5N1 strains require 37 degrees Celsius, suggesting that H5N1Tex/24 has acquired another key adaptive feature to the human upper respiratory tract. H5N1Tex/24 remains sensitive to int...

  Abstract In 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA resulting in spillbacks into poultry, wild birds and other mammals including humans . Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues , as well as cells of other mammals including humans . We find evidence of several mammalian adaptations in cattle including PB2 M631L , which is found in all cattle sequences, and PA K497R , which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface , an essential host factor for viral genome replication. We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins , particularly ANP32A, and thereby enhances virus replication in bovine mammary systems and primary human airway cultures . We show th...

  Abstract Since its emergence in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has posed a significant threat to human health . Recently, novel MERS-like coronaviruses with the potential for cross-species transmission have been identified. In this study, we focused on two newly isolated bat strains with putative health concern: BatCoV/Ii/GD/2014-422 (2014-422) and BtTp-BetaCoV/GX2012 (GX2012). We determined the cryo-EM structures of the spike glycoprotein trimer in the closed state for these two viruses. These structures display a more compact conformation compared to MERS-CoV spike . Biochemical characterization demonstrates that the spike receptor-binding domains (RBDs) of 2014-422 and GX2012 can bind to human dipeptidyl peptidase 4 (hDPP4). To investigate the structural determinants of pseudovirus infection, we solved the cryo-EM structures of 2014-422 RBD-hDPP4 and GX2012 RBD-hDPP4 complexes. The binding mode of the complex is conserved, but the angle of the RB...

  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 Enterovirus D68 (EV-D68), a neurotropic respiratory pathogen, poses a considerable clinical threat through its link to pediatric acute flaccid myelitis (AFM) and severe respiratory illness . The possibility of recurrent epidemics , evidenced since the 2014 outbreak, remains a major concern . Genomic determinants of virulence are central to this threat. Sequence variations that affect host–receptor interactions , immune evasion, and replication efficiency serve as critical modifiers of pathogenicity. This article systematically reviews the evidence for specific genomic sites that enhance EV-D68 virulence , focusing on three critical regions: the VP1 receptor-binding site , the 2Apro/TRAF3 cleavage site, and the 3Cpro immunoregulatory region . Mutations in the VP1 receptor-binding site can alter affinity for host receptors such as sialic acid, heparan sulfate, and MFSD6 , thereby shaping viral entry and tissue tropism . Alterations in the 2Apro/TRAF3 cleavage site may impair pro...

  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...

  ABSTRACT The 2009 pandemic H1N1 (pH1N1) influenza A virus (IAV) is a reassortant virus with two polymerase components, PA and PB2, originating from avian IAV . Avian IAV polymerase does not function efficiently in mammalian cells without host-adaptive mutations . The mechanism by which pH1N1 replicates in human hosts is not fully elucidated , as pH1N1 does not contain the host-adaptive PB2 E627K mutation required for species-specific interaction with ANP32 , which facilitates replicase (polymerase oligomer) formation. Our previous research revealed that mutations in PA played a key role in mammalian host adaptation of pH1N1. These mutations were found in two separate domains of PA, the C-terminal (CTD) and N-terminal domains (NTD). We reported that the NTD mutations increase the expression of NP through enhanced association of GRSF1 with the mRNA transcripts. However, the role of CTD mutations, which are located at the interface of the polymerase oligomers , has not been elucidat...

  Abstract Highly pathogenic avian influenza H5N1 poses an increasing public health risk, particularly following its spillover into dairy cows and associated human infections in the U.S. since March 2024. Here, we systematically identified critical PB2 mutations emerged during avian-to-cattle transmission and subsequent adaptation in cattle , notably PB2 M631L, which conferred pathogenicity in mice comparable to the well-characterized PB2 E627K mutation . Retrospective analysis reveals that PB2 631L also circulated in avian and human H5N1 strains during the 2013–2014 outbreaks in Cambodia and Vietnam . Additional adaptive mutations include established markers ( E627K, Q591R, D701N ), and novel variants ( I647V, G685R, K736R ). These mutations enhance polymerase activity by improving the utilization of both bovine and human ANP32A proteins , thereby increasing viral fitness and pathogenicity in mammals . The convergence of these adaptations highlights the elevated zoonotic risk of c...

  Summary Background Highly pathogenic avian influenza H5N1 viruses of the A/Goose/Guangdong/1/1996 lineage pose a global threat to wildlife, domestic animals, and humans . Cross-species transmission events to mammals, including humans , in the past 4 years highlight this threat. For influenza A viruses, crucial determinants of cross-species and intraspecies transmission to and among mammals include attachment to and replication in respiratory airway epithelial cells . Although these determinants have been studied for H5N1 viruses in the past, limited studies for clade 2.3.4.4b viruses exist. Therefore, the aim of this study was to determine the ability of recent clade 2.3.4.4b H5N1 viruses to attach to human respiratory tissues, to replicate in human airway epithelial cells and the associated immune response. Methods In this in-vitro study, we investigated three H5N1 clade 2.3.4.4b viruses (H5N1Gull2022, H5N1Polecat2022, and H5N1Bovine2024) in comparison with previously studied 2....

  Abstract Identifying animal species that are susceptible to the plethora of existing and emerging viruses is critical for predicting and containing disease outbreaks . Current efforts to assess viral tropism largely rely on experimental infection models , but such experiments are logistically and ethically infeasible for many wildlife species. To tackle this challenge, we developed a panel of airway organoids from ten taxonomically diverse wildlife and livestock species and evaluated their susceptibility to influenza viruses of mammalian (pH1N1) and avian (H5N1) origin . Our analyses revealed large species-specific differences in infection rate and cytopathogenicity that aligned with known in vivo data and field observations. Furthermore, we demonstrated that this organoid panel can serve as a powerful tool to elucidate receptor-binding mechanisms, viral dynamics, and early host adaptation in poorly characterized animal species. In summary, this work provides a robust and ethical...

  Abstract Efficient replication of influenza A viruses (IAVs) requires balanced activities of hemagglutinin (HA), neuraminidase (NA), and the RNA polymerase complex, whose functions are strongly influenced by PB2 mutations . We previously revealed three distinct evolutionary pathways for PB2 mutations, with two pathways leading to the emergence of viral strains responsible for human seasonal infections and the 2009 pandemic , and a third pathway giving rise to H5Nx highly pathogenic avian influenza viruses (HPAIVs) defined by a triad of mutations (I147T, K339T, and A588T) that occasionally spill over to humans . Here, we investigated the zoonotic risk posed by this triad and elucidated its evolutionary relationship with HA, NA, and vaccination . Recombinant PR8 and clade 2.3.2.1c H5N1 viruses carrying the triad replicated efficiently in embryonated chicken eggs and had moderate replication efficiency in mammalian cells ; moreover, mice infected with these viral strains exhibited m...

  Abstract Increase in human H5N1 spillover infections resulting from dissemination of highly pathogenic avian influenza (HPAI) virus into bird and mammal populations raises concerns about HPAI gaining human transmissibility . Studies identified hemagglutinin (HA) acid stability and receptor preference as essential traits that shape host tropism. Mutations that increase HA stability and affinity for α−2,6-linked sialic acids have been shown to confer airborne transmissibility in a ferret model , however mechanisms of activation of H5 subtype HA are poorly understood and the effect of adaptive mutations on HA function has been largely inferred from static structures. Here, we use hydrogen/deuterium-exchange mass spectrometry to dissect activation dynamics for two ancestral H5 HPAI HA , their transmission-adapted HA , and a contemporary HA . We identify variation in receptor binding site flexibility and demonstrate that adaptive mutations result in suppression of fusion peptide dynam...

  Editor’s summary Birds operate at body temperatures several degrees higher than those of mammals, and, like mammals, birds are infected by influenza viruses. Influenza viruses can move between animal hosts, often reassorting their gene segments as they transition. Knowing that the body temperature of humans often elevates when sick, Turnbull et al. investigated whether virus gene segments originating from hot-blooded birds may give the virus an advantage in feverish mammals. They found that a viral polymerase containing an avian origin PB1 subunit indeed allowed the virus to replicate at higher temperatures in vitro and in a hyperthermic mouse model. —Caroline Ash Structured Abstract INTRODUCTION Influenza A viruses circulate in diverse species of birds and periodically spill over to cause severe or fatal infections in humans . Avian influenza A viruses are adapted to replicate in the gastrointestinal tract of birds at ~40° to 42°C . By contrast, human-adapted seasonal influenza ...

  Abstract Three critically ill or fatal avian influenza A(H5N1) human infections have been reported in North America since November 2024 . Notably, all were infected with genotype D1.1 instead of B3.13, the dominant genotype before November 2024. Here, we demonstrated that D1.1 could replicate to higher titers in human nasal and airway organoid-derived transwell monolayers from 6 donors . D1.1 exhibited a better binding to α2,3- and α2,6-linked SA than B3.13. No significant differences in most inflammatory or antiviral cytokines/chemokines was observed. These observations suggest that D1.1 is better adapted to both the upper and lower human respiratory tract epithelium than B3.13. Source:  Link:  https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaf598/8341570#google_vignette ____

  Abstract SARS-CoV-2 Spike - the sole neutralization target, is highly resilient to the immune pressure driving genetic evolution . While potency and breadth of neutralization are widely studied, the incomplete neutralization - the mechanism of resistance without needing genetic change - remains unexplored . Several monoclonal antibodies , although potent, showed incomplete neutralization of genetically homogeneous pseudovirus suggesting the existence of distinct spike conformations . The residual infectivity at high antibody concentration indicates a viral fraction with intrinsic resistance to the antibody. Although the published studies on spike glycosylation, structure, and conformations provide evidence of spike heterogeneity the precise mechanism for the incomplete neutralization has not been established. In this study, we devise a method to separate the un-neutralized virion population , called as persistent fraction of infectivity (PF), and characterize the viral spike prot...