ETIDIOH - NEW

  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 Marburgviruses (MBVs) cause severe haemorrhagic fever with higher fatality rates than Ebola virus (EBOV). Here we show that the MBV glycoprotein (GP) mediates viral entry more efficiently than EBOV GP . Using cryo-EM , we determined structures of MBV GP in three states : (1) unbound; (2) bound to its endosomal receptor NPC1; and (3) complexed with a neutralizing nanobody . The glycan cap shields the receptor-binding site from NPC1 but only partially from the nanobody , enabling limited immune evasion . After glycan cap cleavage , NPC1 binds to MBV GP in a distinct orientation compared with EBOV GP, providing an additional anchor and enhancing receptor affinity . NPC1 engagement also induces substantial conformational changes in MBV GP, probably facilitating membrane fusion . Furthermore, MBV GP is susceptible to the neutralizing nanobody , which mimics NPC1 at the receptor-binding site. Together, our findings reveal MBV GP as a highly efficient entry mediator and suggest...

  Abstract A human infection with clade 2.3.4.4b H5N1 influenza A virus in Canada revealed minority variants E190D and Q226H in the hemagglutinin (HA) receptor-binding site (RBS). Because mutations at positions 190 and 226 have been associated with altered receptor specificity in other influenza subtypes, we investigated their impact on receptor binding in H5 HA . Using a recombinant protein approach and an ELISA-based glycan-binding assay , we assessed binding to representative avian- and human-type sialylated glycans . Both single mutations and their combination resulted in a complete loss of detectable binding to the tested glycans . To evaluate whether this phenotype was background-dependent, Q226H was additionally introduced into two other H5 HA proteins , each representing a distinct clade . In both cases, the mutation similarly abolished receptor binding . These findings independently validate recent glycan microarray observations and demonstrate that the patient-derived E19...

  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 In this study, the infection dynamics, replication, and pathogenicity of a recombinant virus containing a deletion of ORF6 (rWA1ΔORF6) on the backbone of the highly virulent SARS-CoV-2 WA1 virus (rWA1) were investigated and compared to the parental rWA1 virus. While both rWA1 and rWA1ΔORF6 viruses replicated efficiently in cultured cells , the rWA1ΔORF6 virus produced smaller plaques, suggesting reduced cell-to-cell spread. Luciferase reporter assays revealed immune-suppressing effects of ORF6 on interferon (IFN) and nuclear factor kappa B (NF-κB) signaling pathways. Pathogenesis assessment in cats revealed that animals inoculated with rWA1 were lethargic and presented with fever on days 2 and 4 post-infection (pi), whereas rWA1ΔORF6-inoculated animals developed subclinical infection . Additionally, animals inoculated with rWA1ΔORF6 presented reduced infectious virus shedding in nasal and oral secretions and broncho-alveolar lavage fluid when compared with the rWA1-inocu...

  Abstract The H7N9 avian influenza virus (AIV) has posed a major global public health concern since its first detection in China in 2013 . Transmitted among wild birds and poultry , this virus has crossed the species barrier to infect humans , causing severe respiratory disease and high mortality . Although the widespread use of H7 vaccines has markedly reduced human infections , the ongoing circulation and adaptive evolution of the virus in poultry remain a serious threat . In this study, we analyzed three highly pathogenic H7N9 isolates collected in China in 2022, representing two hemagglutinin (HA) gene evolutionary lineages : Group.y.2.3 (isolate 229-4, chicken origin; isolate 782-2, quail origin) and Group.y.2.4 (isolate 621, quail origin). Pathogenicity was compared through phylogenetic analysis , molecular characterization, and infection experiments in both avian and mammalian models . Group.y.2.3 isolates displayed stronger replication and pathogenicity in chickens and mic...

  ABSTRACT An outbreak of H3N1 low-pathogenic avian influenza virus (LPAIV) in Belgium in 2019 caused unexpected levels of mortality and morbidity in poultry . These viruses possess an NA polymorphism associated with plasminogen (PLG) binding , as well as an atypical sequence around the HA cleavage site ; accordingly, HA cleavage mediated by NA-driven PLG recruitment has been proposed to underlie their systemic spread and pathogenicity. To test this, we established a reverse genetics system for A/chicken/Belgium/460/2019 and created single mutations in HA (K345R) and NA (S122N) that restored the viruses to normal consensus, as well as an HA/NA double mutant. Confirming previous work, trypsin-independent spread and HA cleavage of wild-type Ck/Belgium were observed in the presence of fetal bovine serum containing PLG in vitro. Dose-dependent HA cleavage and trypsin-independent spread were also observed in the presence of purified chicken PLG . Compared to the wild-type virus, both HA...

  ABSTRACT The recent emergence of highly pathogenic avian influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) in dairy cattle presents substantial challenges to the agricultural sector and public health. Mechanistic studies of infection and transmission in cattle have proven difficult due to animal handling restrictions and the limited availability of established cell culture models. Primary bovine embryonic fibroblasts (BeEFs) were isolated and investigated here as a model to study influenza A virus (IAV) infection dynamics. We compared sialylation profiles , infectious virus production, viral replication, and plaque morphology in BeEFs following infection with the bovine HPAI H5N1 and an earlier 2.3.4.4b genotype (B1.1) isolated in 2022. The data presented here demonstrate increased expression of α-2,3 sialic acids compared to α-2,6 sialic acids in BeEFs , similar to sialylation profiles previously reported in bovine mammary tissue . These data also display increased viral fi...

  Abstract The spillover of highly pathogenic avian influenza (HPAI) A H5N1 virus to mammalian hosts raises major concerns due to its pandemic potential . Cats are frequently affected mammals, often succumbing to systemic and neurological disease . Here, we characterized the pathogenesis and transmissibility of two H5N1 genotypes, B3.13 and D1.1, in cats . Infected cats exhibited high-level viremia and virus shedding in nasal, oral, and fecal secretions were consistently detected. The virus replicated initially in the upper respiratory tract and lungs , followed by systemic dissemination and neuroinvasion . Notably, the virus crossed the blood-brain-barrier by infecting endothelial cells , spreading to astrocytes and neurons , causing multifocal encephalitis . D1.1-virus infection caused protracted disease with lower shedding and no transmissibility , whereas B3.13 virus caused rapid onset with efficient shedding and transmission. These findings reveal critical H5N1 neuropathogenes...

  ABSTRACT Dogs are considered mixing vessels for influenza viruses , posing a pandemic potential via viral reassortment . Our previous studies indicated that the avian-origin H3N2 canine influenza virus (A/canine/Zhejiang/1/2010, abbreviated C1) is virulent in canine and mice . Furthermore, we found that the HA and NA genes of C1 share a close genetic relationship with an H3N2 avian influenza virus (A/duck/Shanghai/06/2009, abbreviated D6), but they exhibit distinct pathogenicity . However, the understanding mechanisms remain unclear. In the present study, we explored the genetic determinants that contribute to the different pathogenicity between the C1 and D6. By using the reverse genetics approaches, we rescued several single-gene and position-substituted reassortant viruses based on the C1. The replication in Madin–Darby canine kidney cells and pathogenic trial in mice showed that the neuraminidase (NA) gene played a critical role in C1 virulence. Further analysis demonstrated ...

  Abstract Nipah virus is a highly pathogenic virus in the family Paramyxoviridae that utilizes two distinct surface glycoproteins to infect cells . The receptor-binding protein (RBP) binds host receptors whereas the fusion protein (F) merges viral and host membranes . Here, we use nonreplicative pseudoviruses to safely measure the effects of all F single amino acid residue mutations on its cell entry function and neutralization by monoclonal antibodies . We compare mutational tolerance in F with previous experimental measurements for RBP and show that F is much more functionally constrained than the RBP . We also identify mutationally intolerant sites on the F trimer surface and core that are critical for proper function, and describe mutations that are candidates for stabilizing F in the prefusion conformation for vaccine design . We quantify how F mutations affect neutralization by six monoclonal antibodies, and show that the magnitude of mutational effects on neutralization var...

  Abstract Influenza D virus (IDV), primarily found in livestock species, has demonstrated cross-species transmission potential , yet its threat to humans remains poorly understood . Here, we curated a panel of IDV isolates collected during field surveillance from 2011 to 2020 from swine and cattle to assess their ability to infect human airway cells as a proxy for zoonotic threat assessment. Using lung epithelial cell lines , primary well-differentiated airway epithelial cultures, and precision-cut lung slices , we demonstrated that IDV efficiently propagates in cells and tissues from the human respiratory tract , reaching titers comparable to human influenza A virus (IAV). Infection kinetics in primary porcine airway cultures and respiratory tissues mirrored those from human , suggesting similar infectivity across species. To define host responses to IDV infection, we evaluated innate immune sensing and downstream interferon signaling in human respiratory cells. IDV infection res...

  ABSTRACT Oropouche virus (OROV), a neglected arbovirus, has historically been considered a self-limiting infection associated with febrile illness . However, the recent surge in cases since late 2023 has been marked by atypical outcomes , highlighting its underestimated clinical impact . Gastrointestinal symptoms such as diarrhea have also been reported, but the prevalence and mechanistic insight remain largely elusive. Here, through a meta-analysis of 12 identified clinical studies , we revealed a pooled prevalence of diarrhea as 15% (95% CI, 10%–20%) among the Oropouche patient population. In primary human intestinal organoid-based experimental models , we demonstrated productive infection by both a recent patient isolate (OROV-2024) and a historical strain (Be An19991). This is shown by the accumulation of intracellular OROV RNA, release of infectious particles, and immunostaining of OROV glycoprotein Gc. Interestingly, OROV infection mildly triggered the expression of type II...

  Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus that can cause zoonotic disease in humans with lethal severe viral pneumonia . Dromedary camels are the source of zoonotic infection. As of November 2025, MERS-CoV has resulted in a total of 2630 reported cases, 37% of these being fatal . The number of reported human cases has been on a decreasing trend since 2016 and reached a nadir during the COVID-19 pandemic . The reason for the reduction of cases is unclear and may be multifactorial. We hypothesized that mutations accumulating in the virus spike protein may have reduced zoonotic potential . Here, we investigate the impact of recently emerged virus spike-protein mutations on virus replication competence using pseudoviruses and replication-competent recombinant viruses. We found that virus spike variants detected in 2019 and some from 2023 show a reduced cell entry, lower viral replication and reduced fitness in human primary alveolar e...

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 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...