ETIDIOH - NEW

  Abstract Human H3N2 influenza viruses, introduced during the 1968 pandemic, have evolved to recognize human-type sialic acid-containing receptors (Neu5Acα2-6Gal) extended with at least three LacNAc (Galβ1-4GlcNAc) repeats. To investigate this restriction in the context of virus attachment to the airway epithelium , we comprehensively analyzed the glycome of human nasal and tracheal epithelial cells . Using a synthetic N-glycan library that reflects the structural diversity of the human airway glycome, we found that only bi-antennary N-glycans with extended human-type receptors on at least one branch serve as receptors for the recent H3 hemagglutinins (HAs). Such receptors are found on tracheal epithelium but are deficient in nasal epithelium. Immunofluorescence analysis on human trachea reveals that recent H3 HAs preferentially attach to ciliated cells , consistent with single-cell RNA sequencing analysis indicating that these cells express glycosyltransferases that produce exten...

  ABSTRACT Ebola virus (EBOV), the causative agent of Ebola virus disease, remains one of the World Health Organization’s top 10 threats to global health . Infectious EBOV virions can be found on the surface of skin late in infection and may be transmitted to others through skin-to-skin contact . We investigate in vivo EBOV tropism and the kinetics of virus movement to and from the skin. Increasing viral loads were detected over time in the skin of EBOV-infected non-human primates and mice , with antigen detected in dermal stromal and immune cells . Epidermal cells within and surrounding hair follicles also harbored viral antigen , suggesting a novel mechanism of virus egress to the epidermal surface. During late infection, proinflammatory responses were elevated in infected visceral organs but minimal in the skin despite significant viral loads. We observed similar viral trafficking and cell tropism in the skin of mice intraperitoneally infected with a low containment EBOV model v...

  ABSTRACT Influenza viruses utilize host proteases to activate the viral fusion protein, hemagglutinin (HA), into its fusion-competent form. Although proteolytic activation of HA is essential for virus replication, the cell-type dependence of HA activation within the airway epithelium and the subcellular location(s) in which it occurs are not well established. To address these questions, we investigated the proteolytic activation of HA in differentiated human airway epithelial cells using contemporary and historical H1N1 and H3N2 strains . We find that activation is efficient across viral strains and subtypes but depends on cellular tropism , with ciliated cells activating HA more effectively than non-ciliated cells. Similar to prior observations in immortalized cell lines, we find that HA activation occurs intracellularly, constraining the antiviral activity of host-directed protease inhibitors . These results establish that HA activation within the airway epithelium depends on c...

  ABSTRACT Alterations in the PB2-627 domain of avian influenza virus (AIV) can potentially increase the risk of cross-host species infections in humans and mammals . Recently, there has been a rise in human cases of AIV infections without the presence of the known mammalian determinant PB2-E627K . Here, we identified a variant, PB2-627V , which has evolved in poultry and has contributed to the increase in human AIV infections . By screening global PB2 sequences , we discovered a new independent cluster of PB2-627V that emerged in the 2010s , prevalent in avian, mammalian, and human AIV isolates , including those of H9N2, H7N9, H3N8, 2.3.4.4b H5N1 , and other subtypes. We functionally assessed its host adaptation , fitness , and transmissibility across three subtypes of AIVs (H9N2, H7N9, and H3N8) in different host models . PB2-627V combines the viral properties of avian-like PB2-627E and human-like PB2-627K , facilitating AIVs to efficiently infect and replicate in chickens and mi...

  Abstract Mpox poses a heightened risk of severe disease and mortality among individuals with HIV , yet the molecular mechanisms and immunopathology underlying multi-organ damage caused by the mpox virus (MPXV), particularly in the context of HIV co-infection, remain poorly understood. Here, we observe increased MPXV replication, more extensive skin lesions, and impaired humoral and cellular immune responses in SIV-MPXV co-infected rhesus macaques compared to those infected with MPXV alone. Multi-organ proteomic and phosphoproteomic analyses reveals upregulation of proteins involved in immune and inflammatory pathways in skin lesions and across multiple organs, especially in immune-related tissues. Abnormal activation of DNA replication and cell cycle signaling pathways , which may contribute to enhanced viral replication, is evident in both MPXV and SIV-MPXV co-infected groups. CDK4/6 may present a potential therapeutic target to suppress MPXV replication. These comprehensive pro...

  Abstract Importance :  Highly pathogenic avian influenza (HPAI) A H5N1 has been recognized for nearly three decades as a threat to avian species and as a virus with pandemic potential if spillover into human populations occurs. Recently the virus has evolved capacity to infect many mammalian species , including dairy cattle , increasing the risk for human exposure and the pandemic threat. Sialic acids (SA) serve as binding sites for influenza viruses. The distribution of SA determines infectivity of specific influenza viruses across species and tissue tropism . Hemagglutinin (HA) of human and swine adapted influenza viruses bind primarily to SA with α2,6-galactose linkages and avian influenza viruses preferentially bind to SA with α2,3-galactose linkages . Recently, the bovine udder was found to contain SA with α2,3 linkages which allow the H5N1 virus to bind to bovine udder epithelium and to infect milk. The distribution of SA receptors in the human mammary gland is unknown...

Highlights •  MjHKU4r-CoV-1 with high fusogenicity induces inflammatory responses in human cells •  6-HB structure determination unveils MjHKU4r-S-mediated membrane fusion mechanism •  MjHKU4r-CoV-1 HR2 peptides exhibit potent activity by targeting viral HR1 domain •  Stapled peptide MjHKU4r-HR2P10 shows potent and broad-spectrum anti-CoV activity Summary Unlike preceding MERS-related coronaviruses, the recently identified MjHKU4r-CoV-1 strain can directly infect human cells . Nonetheless, its potential pathogenic attributes and underlying molecular mechanisms remain unclear. We find that MjHKU4r-CoV-1 induces significant inflammation , including interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), and exhibits pronounced fusogenicity mediated by its spike (S) protein, leading to extensive syncytium formation . This suggests the possibility that MjHKU4r-CoV-1 possesses strong pathogenic potential in humans . Further, we successfully reveal the molecular mechan...

Abstract Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infections have caused substantial mortality events in marine mammals in recent years. We hypothesized that the high number of infections and disease severity could be related to cell tropism in respiratory tracts . Therefore, we examined the attachment pattern of an H5N1 clade 2.3.4.4b virus (H52022) as a measure for cell tropism in the respiratory tracts of harbor seals, gray seals, harbor porpoises, and bottlenose dolphins and compared it with an H5N1 clade 2.1.3.2 virus (H52005) and a human seasonal H3N2 virus using virus histochemistry. Both H5 viruses attached abundantly to olfactory and respiratory mucosa in the upper respiratory tract of both seal species. H52022 attached more abundantly than H52005 to epithelial cells in the lower respiratory tract of all species. The observed attachment possibly explains the susceptibility of marine mammal species for recent H5N1 viruses and the observed development of se...

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...