ETIDIOH - NEW

Abstract Influenza A virus (IAV) poses a significant global health risk, with highly pathogenic strains like H5N1 (CFR ~52%) causing severe disease compared to less lethal but more transmissible strains like H1N1 (CFR 0.01-0.03%). Although IAV primarily infects lung epithelial cells , causing cell death and tissue damage , the molecular basis of strain-specific pathogenesis remains poorly understood. Here we show that in cell culture , H5N1 induced more rapid and extensive cell death than H1N1. Since Interferon (IFN) signaling is key to innate immunity, we examined its role in virus-induced cell death using STAT1-knockout A549 cells and JAK/STAT pathway inhibitors like Baricitinib . Both approaches reduced cell death across various IAV strains, including H1N1, H5N1, H7N9 , and H3N2 . However, inhibition increased viral titers , raising concerns about its clinical use in isolation. To overcome this, we tested a combination of Oseltamivir (antiviral) and Baricitinib (anti-inflammatory). ...

ABSTRACT The ongoing outbreak of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b has affected at least 989 dairy herds across 17 states in the United States (U.S.) and resulted in 70 confirmed human infections , underscoring the urgent need to understand the pathogenesis and therapeutic interventions of emerging H5N1 viruses. In this study, we modelled infection with a highly pathogenic recombinant human A/Texas/37/2024 H5N1 (rHPh-TX H5N1) strain using human airway organoids (HAO) to investigate viral replication, innate immune response , infection-induced fibrogenesis, and potential therapeutic interventions . rHPh-TX H5N1 replicated efficiently in HAO, eliciting a robust interferon (IFN) response and pro-inflammatory cytokine production . Prolonged infection led to the accumulation of fibroblast-like cells surrounding infected regions, marked by increased alpha-smooth muscle actin (α-SMA) expression and upregulation of transforming growth factor-beta (TGF-β), indicative ...

Abstract The structural instability of influenza hemagglutinin (HA) is related to its function in low pH-mediated membrane fusion , which requires prior cleavage of the premature HA0 by a host protease . The precise determinants underlying the stability and cleavability of HA remain to be fully understood and have implications for risk assessment of zoonotic influenza A viruses (IAV), viral transmissibility and vaccine production. To address this, we conducted random mutagenesis on early 2009 pandemic H1 HA, followed by selection of acid-stable viruses and detailed profiling of the mutant HAs. This resulted in identification of four mutations , which increase the acid-stability and decrease the fusion-promoting activity of H1 HA, without compromising viral entry and replication in cells. The newly recognized mutations are situated in the globular head , vestigial esterase and membrane-proximal part of H1 HA, in regions involved in the refolding of HA at low pH. A fifth mutation, D346N,...

Abstract Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness . While Middle East respiratory syndrome coronavirus ( MERS-CoV ) uses DPP4 for entry , the receptors of many MERS-related betacoronaviruses remain unknown . The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells . Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2 , revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use...

Abstract The H1N1 swine influenza viruses CQ91 and CQ445, isolated from pigs in China, exhibited distinct virulence in mice despite sharing similar genomic constellations. CQ91 demonstrated higher pathogenicity (MLD50: 5.4 log10 EID50) and replication efficiency in mice compared to CQ445 (MLD50: 6.6 log10 EID50). Through reverse genetics, we found that the attenuation of CQ445 was due to a single substitution of glutamic acid (E) with lysine (K) at position 343 in the PB2 protein . Introducing the CQ445-PB2 (343K) into CQ91 significantly reduced viral replication and pathogenicity in mice, while replacing CQ445-PB2 with CQ91-PB2 (343E) restored virulence. In vitro studies showed that the K343E mutation impaired viral replication in MDCK and A549 cells and reduced polymerase activity in minigenome assays. Mechanistically, the amino acid at position 343 in the PB2 affects the transcription stage of the viral replication process . Structural modeling indicated that the charge reversal cau...

ABSTRACT The Henipavirus genus comprises five viral species, of which the prototype members, Hendra virus (HeV) and Nipah virus (NiV), are reported to infect humans. In humans and other spill-over hosts , HeV/NiV can cause severe respiratory and/or encephalitic disease , with mortality rates exceeding 50%; currently, there are no approved human vaccines and only limited therapeutic options . As members of the family Paramyxoviridae , henipaviruses have six “core” structural proteins and typically three additional accessory proteins that are expressed from the P gene. Several of these proteins are multifunctional, with roles in forming intracellular interfaces with the host (in particular, M, P, V, W, and C proteins), to modulate processes including antiviral responses, supporting viral replication. Understanding the molecular basis of these interfaces and their functions is critical to delineate the mechanisms of pathogenesis and may inform new strategies to combat infection and diseas...

ABSTRACT In recent years, high pathogenicity avian influenza viruses (HPAIVs) have spread among wild, captive, and domestic birds, as well as mammals . Beyond the resulting economic and ecological losses , spillover into mammals has raised concerns about a potential pandemic . Viral tropism refers to the spectrum of host species, organs, and cells susceptible and permissive to viral infection . It is a potent driver of infection dynamics and shedding patterns, which presents important variations both between and within hosts: in poultry, HPAIV leads to systemic endothelial infection in domestic chickens , whereas neurological and selective epithelial infections are observed in domestic ducks . In mammals , infection can result in respiratory and neurological disease , but the recent outbreaks in domestic dairy cows highlighted a unique and remarkable adaptation to the mammary gland prone to viral shedding in milk. The present review explores viral tropism of HPAIV across recent spillov...

Abstract In 2022-23, the world witnessed the largest recorded outbreak of monkeypox virus (MPXV). Neurological manifestations were reported alongside the detection of MPXV DNA and MPXV-specific antibodies in the cerebrospinal fluid of patients. Here, we analyze the susceptibility of neural tissue to MPXV using human neural organoids (hNOs) exposed to a clade IIb isolate . We report susceptibility of several cell types to the virus, including neural progenitor cells and neurons . The virus efficiently replicates in hNOs, as indicated by the exponential increase of infectious viral titers and establishment of viral factories . Our findings reveal focal enrichment of viral antigen alongside accumulation of cell-associated infectious virus , suggesting viral cell-to-cell spread . Using an mNeonGreen-expressing recombinant MPXV, we confirm cell-associated virus transmission . We furthermore show the formation of beads in infected neurites, a phenomenon associated with neurodegenerative diso...

Abstract Although influenza A viruses (IAV) are notorious respiratory pathogens, some IAV strains can reach and replicate in the central nervous system (CNS) in vivo. In particular, highly pathogenic avian influenza viruses (HPAIV) pose a threat for future pandemics and are linked to greater neurotropic potential . Moreover, neurotropic IAV strains have shown a more significant impact on the onset and pathogenesis of neurodegenerative diseases (NDD). However, despite its clinical relevance , the dynamics and cellular tropism of IAV infection in the CNS are not well understood. In this study, we analyzed the replication of HPAIV H7N7 in vitro using a primary murine triple co-culture system comprising neurons, astrocytes, and microglia . We found that microglia become highly infected early on and induce a strong pro-inflammatory response before undergoing apoptosis . Using fluorescence microscopy with automated single-cell profiling, we found that, in contrast to non-neurotropic H3N2, th...

Abstract The influenza A virus polymerase, consisting of a heterotrimer of three viral proteins, carries out both transcription and replication of the viral RNA genome . These distinct activities are regulated by viral proteins that vary in abundance during infection, and by various co-opted host cell proteins, which serve as targets for the development of novel antiviral interventions . However, little is known about which host proteins direct transcription and which replication. In this report, we performed a differential interactome screen to identify host proteins co-opted as either transcription- or replication-specific factors. We found that distinct sets of host proteins interact with the influenza polymerase as it carries out the different activities. We functionally characterised HMGB2 and RUVBL2 as replication-specific cofactors and RPAP2 as a transcription-specific cofactor. Our data demonstrate that comparative proteomics can be used as a targeted approach to uncover virus-...

Abstract The global spread of the A/goose/Guangdong/1/96-lineage H5N1 highly pathogenic avian influenza (HPAI) viruses is accompanied by an expanded host range and the establishment of sustained viral transmission among dairy cattle . To evaluate if the evolving H5N1 viruses have changed tissue tropism over time, we compared the binding patterns of recombinant hemagglutinin (HA) proteins derived from clade 1 (A/Vietnam/1203/04, H5VN) and circulating clade 2.3.4.4b viruses detected from a wild bird (A/Eurasian Teal/Hong Kong/AFCD-HKU-23-14009-01020/2023, H5HK) and dairy cattle (A/bovine/Ohio/B24OSU-439/2024, H5OH). The HA protein of A(H1N1)pdm09 virus was included for comparison. Using bio-layer interferometry, H1 protein preferentially bound to the 2,6-linked sialoside 6'SLNLN while H5 proteins preferentially bound to the 2,3-linked sialoside 3'SLN. H5OH showed higher binding affinity to 3'SLN than H5HK and H5VN. The attachment pattern of H1 and H5 proteins to the respirato...

Abstract The 2024 outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in U.S. dairy cattle presented an unprecedented scenario where the virus infected bovine mammary glands and was detected in milk , raising serious concerns for public health and the dairy industry. Unlike previously described subclinical influenza A virus (IAV) infections in cattle, H5N1 infection induced severe clinical symptoms , including respiratory distress, mastitis, and abnormal milk production . To understand the host immune responses and changes, particularly in the mammary gland, we performed single-cell RNA sequencing analysis on bovine milk somatic cells (bMSCs) in vitro exposed to an H5N1 isolate from an infected dairy farm. We identified ten distinct cell clusters and observed a shift toward type-2 immune responses , characterized by T cells expressing IL13 and GATA3 , and three different subtypes of epithelial cells based on the expression of genes associated with milk production. Our stud...

ABSTRACT The eight-segmented RNA genome of influenza A virus (IAV) is transcribed and spliced into 10 major viral mRNAs in the nucleus of infected cells. Both transcription and splicing are facilitated by the host RNA polymerase II (Pol II) machinery via interactions between the viral ribonucleoprotein (vRNP) complex and various host factors. In this study, we demonstrate that IAV vRNPs recruit species-specific heterogeneous nuclear ribonucleoprotein M (hnRNPM) to support their replication in human and avian cells through distinct mechanisms. In A549 cells, human hnRNPM specifically facilitates the efficient transcription of HA, NA, M, and NS segments of WSN virus in a gene coding sequence-dependent manner. In contrast, in DF-1 cells, chicken hnRNPM restricts excessive splicing of M segment mRNA to ensure proper M2 protein production. Notably, human hnRNPM, with 34 additional amino acids compared with its chicken counterpart, fails to inhibit the M2 expression in DF-1 cells, whereas bo...

Abstract Fever during influenza A virus (IAV) infection is triggered by the innate immune response . Various factors contribute to this response, including IAV mini viral RNAs (mvRNA), which trigger RIG-I signaling when their replication and transcription are dysregulated by template loops (t-loop). It is presently not well understood whether the fever response to IAV infection impacts subsequent viral replication and innate immune activation . Here we show that IAV infection at temperatures that simulate fever leads to increased mvRNA synthesis and antiviral signaling . Mathematical modeling and experimental analyses reveal that differential IAV nucleoprotein and RNA polymerase production underlies the increased mvRNA level. Moreover, at the higher infection temperature mvRNAs with dysregulating t-loops contribute most to the innate immune activation. We propose that fever during IAV infection can establish a positive feedback loop in which elevated aberrant RNA synthesis and innate i...

Abstract Influenza viruses replicate and transcribe their genome in the context of a conserved ribonucleoprotein (RNP) complex . By integrating cryo–electron microscopy single-particle analysis and cryo–electron tomography , we define the influenza RNP as a right-handed, antiparallel double helix with the viral RNA encapsidated in the minor groove . Individual nucleoprotein subunits are connected by a flexible tail loop that inserts into a conserved pocket in its neighbor. We visualize the viral polymerase in RNP at different functional states , revealing how it accesses the RNA template while maintaining the double-helical architecture of RNP by strand sliding. Targeting the tail loop binding interface, we identify lead compounds as potential anti-influenza inhibitors . These findings elucidate the molecular determinants underpinning influenza virus replication and highlight a promising target for antiviral development. Source: Science,  https://www.science.org/doi/10.1126/science...

Abstract Highly pathogenic avian influenza (HPAI) A(H5N1) virus emerged in lactating dairy cattle in March 2024, causing mastitis-related disease and infections in other farm animals and workers . Recent work identified α2,6 and α2,3-linked sialic acids (SA), which serve as influenza virus receptors, in the lactating bovine mammary gland ; however, their distribution across stages of mammary growth and development remains unknown. We compared the distribution of tissue sialylation in mammary glands of prepubertal dairy calves , pregnant dairy heifers , and lactating cows . Mammary glands at all physiological stages expressed both α2,6 SA, the preferred receptor linkage for human influenza viruses, and α2,3 SA, the preferred receptor linkage for avian influenza viruses. Importantly, mammary glands of pregnant dairy heifers exhibited the highest overall expression of α2,3 SA, observed in both tissue and alveolar lumens. Our results suggest that pregnant dairy heifers, like lactating dair...

ABSTRACT Sporadic human infections of avian influenza virus (AIV) raise significant public health concerns . A critical factor limiting the transmission of AIVs is the shift in receptor-binding preference from Siaα2,3 to Siaα2,6. To reveal the adaptive selection dynamics during the host adaptation process of AIVs, this study generated a viral library with random mutations in the HA gene of the H9N2 strain . Upon passaging the viral library in chickens and mice , the predominantly selected variants exhibited a preference for Siaα2,3 receptors . Notably, the wild-type strain remained dominant in both inoculated and direct-contact chickens, while variants with the ΔL226/R229I substitutions were preferentially selected in mice. Ferrets have a predominance of Siaα2,6 in their respiratory tract. As expected, the variant harboring the N289D mutation, which prefers Siaα2,6 binding, was enriched during in vivo passaging in ferrets . The mice-adapted variant with the ΔL226/R229I mutations causes...

Abstract Prior to the discovery of bat influenza A virus (IAV) subtypes H17N10 and H18N11 , all IAVs were thought to bind sialic acid residues via hemagglutinin (HA) to mediate attachment and subsequent viral entry. However, H17 and H18 engage a proteinaceous receptor : the major histocompatibility complex class II (MHCII). The mechanistic details of this hitherto unknown protein-mediated entry are not understood. Given that conventional IAVs rely on multivalent binding to sialylated glycans , we hypothesized that bat HA similarly interacts with multiple MHCII molecules. Using photoactivated localization microscopy (PALM) on fixed and live cells, we demonstrate that bat IAV particles attach to pre-existing MHCII clusters and induce a further increase in cluster size upon binding. To measure the impact of viral attachment on the dynamics of MHCII, we employ an “inverse attachment” approach, immobilizing viral particles on coverslips before seeding live MHCII-expressing cells on top. Sin...

Abstract Highly pathogenic H5Nx 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 H5Nx 2.3.4.4b viruses uniquely bind core 3 sialyl-Lewisx and Sia-Gal-β3GalNAc, glycans not recognized by classical H5 or other avian viruses. By determining its crystal structure, we resolved the structural features of both structures in an H5 hemagglutinin (HA) from a 2016 2.3.4.4b virus. While these viruses do not bind human-type receptors , their promiscuous receptor specificity enhances ...

Abstract Lassa mammarenavirus (LASV), a member of the family Arenaviridae , is a highly pathogenic virus capable of causing severe systemic infections in humans . The primary host reservoir is the Natal multimammate mouse (Mastomys natalensis), with human infections typically occurring through mucosal exposure to virus-containing aerosols from rodent excretions . To better understand the molecular mechanisms underlying LASV replication in the respiratory tract, we utilized differentiated primary human airway epithelial cells (HAECs) grown under air–liquid interface conditions, closely mimicking the bronchial epithelium in vivo. Our findings demonstrate that HAECs are permissive to LASV infection and support productive virus replication . While LASV entry into polarized HAECs occurred through both apical and basolateral surfaces , progeny virus particles were predominantly released from the apical surface , consistent with an intrinsic apical localization of the envelope glycoprotein GP...