ETIDIOH - NEW

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...

Abstract   Background :  The current avian influenza A(H5N1) epizootic poses a significant threat to public health , with sporadic infections in humans raising concerns about potential adaptation for efficient human transmission . Laboratory studies have provided evidence that the polymerase basic protein 2 (PB2) E627K mutation facilitates more efficient replication in mammals and humans. This mutation has been detected in Canadian poultry, wild birds and mammals .  Objective :  Our objective was to summarize the current state of evidence on the impact of the avian influenza PB2 E627K mutation on human adaptation, transmission, epidemiology and clinical outcomes in natural human infections.  Methods :  We employed a search strategy across MEDLINE, Embase, Scopus, Global Health and CAB Abstracts for articles published from each database’s inception until mid-May 2023.  Results :  We identified nine eligible articles for review that addressed human ...

{Excerpt} Highly pathogenic H5N1 avian influenza (HPAI) viruses started circulating in lactating dairy cattle in the USA at the end of 2023 (ref. 1) and these viruses are now rapidly spreading between cows2. Eisfeld et al.3 found that a clade 2.3.4.4b H5N1 virus from this cattle outbreak can bind to α2,6-linked sialyl-glycopolymers on microtitre plates . Here we show that the haemagglutinin from a clade 2.3.4.4b H5N1 virus binds poorly to glycans that terminate with α2-6 sialic acids. This is an important finding, as α2,6 sialic acid is abundant in the upper respiratory tract of humans , and acquisition of α2,6 sialic acid receptor specificity is believed to be required for efficient transmission of influenza virus in humans and is considered a risk factor for the emergence of a new pandemic virus4. (...) Source: Nature,  https://www.nature.com/articles/s41586-025-08821-6 ____

Significance We explored how H5Nx influenza viruses , which can infect many different birds and mammals, could adapt to infect humans by altering the hemagglutinin (HA). HA must change to bind human-type receptors for transmission between people. We compared two strains from viruses isolated in 2016 and found that one ( 2.3.4.4e ) can switch to human receptor binding with a single mutation , while another ( 2.3.4.4b ) might require more complex changes to bind simple human-type receptors. These findings highlight the potential for specific strains to evolve and become a pandemic threat, underscoring the importance of monitoring mutations that could lead to human-type receptor adaptation. Abstract H5Nx viruses continue to wreak havoc in avian and mammalian species worldwide . The virus distinguishes itself by the ability to replicate to high titers and transmit efficiently in a wide variety of hosts in diverse climatic environments. Fortunately, transmission to and between humans is sca...

Abstract Rationale and Objectives :  Iron availability and metabolism are important in the pathogenesis of bacterial infections . More recently, links have been reported between iron and the severity of viral infections . In this study, we characterize a crucial relationship between iron metabolism and IAV infection and disease.  Methods :  Iron-related gene expression was assessed in human airway epithelial cells (AEC) infected with IAV. AECs were cultured with ferric iron, iron-loaded transferrin, or iron chelator, deferoxamine (DFO), prior to infection with IAV. Mice were placed on a high iron diet for 8 weeks prior to infection with IAV or treated with anti-transferrin receptor-1 (TFR1) antibody during IAV infection. The effects of iron modulation and depletion of TFR1-mediated responses on IAV infection were assessed.  Measurements and main results :  Iron-related gene expression and metabolism are altered systemically and in lung tissues and AECs during IA...

Abstract High pathogenicity (HP) avian influenza viruses (AIV) generally evolve from low pathogenicity (LP) precursors after transmission from wild birds to chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo), causing severe economic losses worldwide. Turkeys are more susceptible to AIV infection than chickens and are considered potential bridging hosts that facilitate the emergence of HPAIV . Beyond the polybasic cleavage site (pCS) in hemagglutinin (HA), little is known about other virulence determinants of HPAIV in these species. In 2015, HPAIV H7N7 and its LP ancestor were isolated from the same chicken farm, which differed by 16 nonsynonymous mutations across all eight gene segments, in addition to the pCS. Here we identify the genetic determinants, including the pCS, that contributed to the HPAIV H7N7 virulence, transmission, replication, and tissue distribution in chickens and turkeys. Notably, the non-structural (NS1) or matrix (M) proteins ’ encoding segments...

Abstract The current outbreak of highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype clade 2.3.4.4b in dairy cattle in the United States has affected nearly 900 dairy farms and resulted in at least 39 human infections, putting health authorities and the scientific community on high alert. Here we characterize the virus growth properties and host-pathogen interactions of an isolate obtained from a sick dairy cow in Texas in vitro and in vivo and compare it to an older HPAI isolate. Despite so far being associated with mild disease in human patients, the cattle H5N1 virus showed superior growth capability and rapid replication kinetics in a panel of human lung cell lines in vitro . In vivo, cattle H5N1 exhibited more intense pathogenicity in mice , with rapid lung pathology and high virus titers in the brain , accompanied by high mortality after challenge via different inoculation routes. Additionally, the cattle H5N1 demonstrated efficient antagonism of overexpressed RI...

Abstract The novel H10N3 avian influenza virus (AIV) has infected four individuals since 2021 and caused severe respiratory damage , posing a significant threat to public health . However, its pathogenic mechanisms remain poorly understood. Our findings revealed that H10N3 infection induces severe lung damage and causes death in mice , even at low doses. The elevated levels of multiple pro-inflammatory factors in the bronchoalveolar lavage fluid were significantly increased during infection, displaying hallmarks of a cytokine storm . Transcriptome sequencing further revealed systematic activation of inflammation-related pathways, predicting that viral infection induces multiple forms of programmed cell death , including apoptosis, pyroptosis, and necroptosis . Protein-level validation showed that the activation of key cell death markers, including Caspase-3, GSDMD, and MLKL , significantly increased as the infection progressed, with their dynamic changes correlating strongly with the e...

Significance Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic coronavirus that continues to cause periodic outbreaks in humans with a case-fatality rate of approximately 35%. MERS-CoV generally transmits poorly, but superspreading events are well documented. Efficient human-to-human transmission of respiratory viruses generally correlates with a tropism for the upper respiratory tract, but this tropism for MERS-CoV remains poorly understood. Characterizing the MERS-CoV tropism in the human upper respiratory tract is of critical importance to understand its epidemiology and pandemic potential of future MERS-CoV variants and other dipeptidyl peptidase 4 (DPP4)-utilizing coronaviruses present in animal reservoirs. Abstract Transmissibility of respiratory viruses is a complex viral trait that is intricately linked to tropism. Several highly transmissible viruses, including severe acute respiratory syndrome coronavirus 2 and Influenza viruses, specifically targ...

Abstract Highly pathogenic avian influenza viruses (HPAIV) pose a serious public health concern . In March 2024, a first-time outbreak of HPAIV H5N1 in dairy cattle herds was reported in the United States (US). Since then, the virus has continued to spread in cattle herds and spilt over into humans . We recently showed that the first human isolate reported in the US in Texas (HPhTX) from a dairy worker in an affected cattle farm has enhanced replication kinetics and pathogenicity in mice compared to a closely related bovine isolate (HPbTX). However, the molecular determinants of differential pathogenicity have not yet been identified. Herein, we show that HPhTX has enhanced polymerase activity , compared with HPbTX, in human cells and that the polymerase basic 2 (PB2) protein is the main factor responsible for this difference. Through single and combined site-directed mutagenesis and swapping the three amino acids different between HPhTX and HPbTX, we found that PB2 mutation E627K is t...