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Vaginal transmission from semen of male Ebola virus (EBOV) survivors has been implicated as a potential origin of Ebola virus disease (EVD) outbreaks. While EBOV in semen must traverse cervicovaginal mucus (CVM) to reach target cells, the behaviour of EBOV in CVM is poorly understood. CVM contains substantial quantities of IgG, and arrays of IgG bound to a virion can develop multiple Fc-mucin bonds, immobilizing the IgG/virion complex in mucus. Here, we measured the real-time mobility of fluorescent Ebola virus-like-particles (VLP) in 50 CVM specimens from 17 women, with and without ZMapp, a cocktail of 3 monoclonal IgGs against EBOV. ZMapp-mediated effective trapping of Ebola VLPs in CVM from a subset of women across the menstrual cycle, primarily those with Lactobacillus crispatus dominant microbiota. Our work underscores the influence of the vaginal microbiome on IgG-mucin crosslinking against EBOV and identifies bottlenecks in the sexual transmission of EBOV.

We present a model for virus-cell adhesion that can be used for quantitative extraction of adhesive properties from atomic force microscopy (AFM)-based force spectroscopy measurements. We extend a previously reported continuum model of viral cell interactions based on a single parameter representing adhesive energy density by using a cohesive zone model in which adhesion is represented by two parameters, a pull-off stress and associated characteristic displacement. This approach accounts for the deformability of the adhesive receptors, such as the Spike protein and transmembrane immunoglobulin and mucin domain (TIM) family that mediate adhesion of SARS-CoV-2 and Ebola viruses, and the omnipresent glycocalyx. Our model represents receptors as a Winkler foundation and aims to predict the pull-off force needed to break the adhesion between the virus and the cell. By comparing the force-separation curves simulated by the model and experimental data, we found that the model can effectively explain the AFM pull-off force trace, thus allowing quantification of the adhesion parameters. Our model provides a more refined understanding of viral cell adhesion and also establishes a framework for interpreting and predicting AFM force spectroscopy measurements.

Background: The inflammatory macrophage response contributes to severe Ebola virus disease, with liver and lung injury in humans., Objective: We sought to further define the activation status of hepatic and pulmonary macrophage populations in Ebola virus disease., Methods: We compared liver and lung tissue from terminal Ebola virus (EBOV)-infected and uninfected control cynomolgus macaques challenged via the conjunctival route. Gene and protein expression was quantified using the nCounter and GeoMx Digital Spatial Profiling platforms. Macrophage phenotypes were further quantified by digital pathology analysis., Results: Hepatic macrophages in the EBOV-infected group demonstrated a mixed inflammatory/non-inflammatory profile, with upregulation of CD163 protein expression, associated with macrophage activation syndrome. Hepatic macrophages also showed differential expression of gene sets related to monocyte/macrophage differentiation, antigen presentation, and T cell activation, which were associated with decreased MHC-II allele expression. Moreover, hepatic macrophages had enriched expression of genes and proteins targetable with known immunomodulatory therapeutics, including S100A9, IDO1, and CTLA-4. No statistically significant differences in M1/M2 gene expression were observed in hepatic macrophages compared to controls. The significant changes that occurred in both the liver and lung were more pronounced in the liver., Conclusion: These data demonstrate that hepatic macrophages in terminal conjunctivally challenged cynomolgus macaques may express a unique inflammatory profile compared to other macaque models and that macrophage-related pharmacologically druggable targets are expressed in both the liver and the lung in Ebola virus disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wanninger, Saldarriaga, Arroyave, Millian, Comer, Paessler and Stevenson.)

The Ebola virus is a deadly pathogen that has been threatening public health for decades. Recent studies have revealed alternative viral invasion routes where Ebola virus approaches cells via interactions among phosphatidylserine (PS), PS binding ligands such as Gas6, and TAM family receptors such as Axl. In this study, we investigate the interactions among phosphatidylserine on the Ebola viral-like particle (VLP) membrane, human Gas6, and human Axl using atomic force microscope-based single molecule force spectroscopy to compare their binding strength and affinity from a biomechanical perspective. The impact of calcium ions on their interactions is also studied and quantified to provide more details on the calcium-dependent phosphatidylserine-Gas6 binding mechanism. Our results indicate that, in the presence of calcium ions, the binding strengths of VLP-Gas6 and VLP-Gas6-Axl increase but are still weaker than that of Gas6-Axl, and the binding affinity of VLP-Gas6 and VLP-Gas6-Axl is largely improved. The binding strength and affinity of Gas6-Axl basically remain the same, indicating no impact in the presence of calcium ions. Together, our study suggests that, under physiological conditions with calcium present, the Ebola virus can utilize its membrane phosphatidylserine to dock on cell surface via Gas6-Axl bound complex.

Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus to illuminate assembly intermediates, as well as a 9 Å map of the complete intracellular assembly. This structure reveals a previously unresolved third and outer layer of NP complexed with VP35. The intrinsically disordered region, together with the C-terminal domain of this outer layer of NP, provides the constant width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in the virion. A comparison of intracellular nucleocapsids with prior in-virion nucleocapsid structures reveals that the nucleocapsid further condenses vertically in the virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nipah virus (NiV) and Ebola virus (EBOV) are highly pathogenic zoonotic viruses with case fatality rates of up to 90%. While the brain is a known target organ following NiV infection, involvement of the central nervous system in EBOV-infected patients only became more evident after the West African epidemic in 2013-2016. To gain a deeper comprehension of the neurotropism of NiV and EBOV with respect to target cells, affected brain regions and local inflammatory responses, murine organotypic brain slices (BS) were established and infected. Both NiV and EBOV demonstrated the capacity to infect BS from adult wt mice and mice lacking the receptor for type I IFNs (IFNAR -/- ) and targeted various cell types. NiV was observed to replicate in BS derived from both mouse strains, yet no release of infectious particles was detected. In contrast, EBOV replication was limited in both BS models. The release of several pro-inflammatory cytokines and chemokines, including eotaxin, IFN-γ, IL-1α, IL-9, IL-17a and keratinocyte-derived chemokine (KC), was observed in both virus-infected models, suggesting a potential role of the inflammatory response in NiV- or EBOV-induced neuropathology. It is noteworthy that the choroid plexus was identified as a highly susceptible target for EBOV and NiV infection, suggesting that the blood-cerebrospinal fluid barrier may serve as a potential entry point for these viruses.

The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses., (© 2024. The Author(s).)

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP 2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study's findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log 10 TCID 50 as the limit of detection.

The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013-2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Filovirus-host interactions play important roles in all stages of the virus lifecycle. Here, we identify LATS1/2 kinases and YAP, key components of the Hippo pathway, as critical regulators of EBOV transcription and egress. Specifically, we find that when YAP is phosphorylated by LATS1/2, it localizes to the cytoplasm (Hippo "ON") where it sequesters VP40 to prevent egress. In contrast, when the Hippo pathway is "OFF", unphosphorylated YAP translocates to the nucleus where it transcriptionally activates host genes and promotes viral egress. Our data reveal that LATS2 indirectly modulates filoviral VP40-mediated egress through phosphorylation of AMOTp130, a positive regulator of viral egress, but more surprisingly that LATS1/2 kinases directly modulate EBOV transcription by phosphorylating VP30, an essential regulator of viral transcription. In sum, our findings highlight the potential to exploit the Hippo pathway/filovirus axis for the development of host-oriented countermeasures targeting EBOV and related filoviruses., (© 2024. The Author(s).)

Filoviruses are negative-sense single-stranded RNA viruses often associated with severe and highly lethal hemorrhagic fever in humans and nonhuman primates, with case fatality rates as high as 90%. Of the known filoviruses, Ebola virus (EBOV), the prototype of the genus Orthoebolavirus , has been a major public health concern as it frequently causes outbreaks and was associated with an unprecedented outbreak in several Western African countries in 2013-2016, affecting 28,610 people, 11,308 of whom died. Thereafter, filovirus research mostly focused on EBOV, paying less attention to other equally deadly orthoebolaviruses (Sudan, Bundibugyo, and Taï Forest viruses) and orthomarburgviruses (Marburg and Ravn viruses). Some of these filoviruses have emerged in nonendemic areas, as exemplified by four Marburg disease outbreaks recorded in Guinea, Ghana, Tanzania, and Equatorial Guinea between 2021 and 2023. Similarly, the Sudan virus has reemerged in Uganda 10 years after the last recorded outbreak. Moreover, several novel bat-derived filoviruses have been discovered in the last 15 years (Lloviu virus, Bombali virus, Měnglà virus, and Dehong virus), most of which are poorly characterized but may display a wide host range. These novel viruses have the potential to cause outbreaks in humans. Several gaps are yet to be addressed regarding known and emerging filoviruses. These gaps include the virus ecology and pathogenicity, mechanisms of zoonotic transmission, host range and susceptibility, and the development of specific medical countermeasures. In this review, we summarize the current knowledge on non-Ebola filoviruses (Bombali virus, Bundibugyo virus, Reston virus, Sudan virus, Tai Forest virus, Marburg virus, Ravn virus, Lloviu virus, Měnglà virus, and Dehong virus) and suggest some strategies to accelerate specific countermeasure development.

The Ebola virus disease (EVD) has been endemic since 1976, and the case fatality rate is extremely high. EVD is spread by infected animals, symptomatic individuals, dead bodies, and contaminated environment. In this paper, we formulate an EVD model with four transmission modes and a time delay describing the incubation period. Through dynamical analysis, we verify the importance of blocking the infection source of infected animals. We get the basic reproduction number without considering the infection source of infected animals. And, it is proven that the model has a globally attractive disease-free equilibrium when the basic reproduction number is less than unity; the disease eventually becomes endemic when the basic reproduction number is greater than unity. Taking the EVD epidemic in Sierra Leone in 2014-2016 as an example, we complete the data fitting by combining the effect of the media to obtain the unknown parameters, the basic reproduction number and its time-varying reproduction number. It is shown by parameter sensitivity analysis that the contact rate and the removal rate of infected group have the greatest influence on the prevalence of the disease. And, the disease-controlling thresholds of these two parameters are obtained. In addition, according to the existing vaccination strategy, only the inoculation ratio in high-risk areas is greater than 0.4, the effective reproduction number can be less than unity. And, the earlier the vaccination time, the greater the inoculation ratio, and the faster the disease can be controlled., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs., Competing Interests: H.H.W. owns stocks in and is a consultant for and co-founder of EbuMab, ApS, Hemab, ApS, and GO-Therapeutics Inc. All other authors declare no conflicts of interest.

In tervening pro teins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCE In tervening pro teins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity., Competing Interests: The authors declare no conflict of interest.

Ebola virus and other orthoebolaviruses cause severe haemorrhagic fevers in humans, with very high case fatality rates. Their non-segmented single-stranded RNA genome encodes only seven structural proteins and a small number of non-structural proteins to facilitate the virus life cycle. The basics of this life cycle are well established, but recent advances have substantially increased our understanding of its molecular details, including the viral and host factors involved. Here we provide a comprehensive overview of our current knowledge of the molecular details of the orthoebolavirus life cycle, with a special focus on proviral host factors. We discuss the multistep entry process, viral RNA synthesis in specialized phase-separated intracellular compartments called inclusion bodies, the expression of viral proteins and ultimately the assembly of new virus particles and their release at the cell surface. In doing so, we integrate recent studies into the increasingly detailed model that has developed for these fundamental aspects of orthoebolavirus biology., (© 2024. Springer Nature Limited.)

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.

SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides' antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses., (© 2024. The Author(s).)

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Jain et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease., Competing Interests: Disclosures D.X.L., B.P., D.L.P., T.K.C., L.M.H., R.J.H., J.B., R.B., R.S.B., T.W., and M.R.H. are employed by Laulima Government Solutions, LLC, contracted by NIAID. A.M.W.H. is employed by Tunnell Government Services, Inc. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government. The authors declare no conflicts of interest., (Copyright © 2023 American Society for Investigative Pathology. All rights reserved.)

Introduction: Ebola virus (EBOV) is an RNA virus of the Filoviridae family that is responsible for outbreaks of hemorrhagic fevers in primates with a lethality rate as high as 90%. EBOV primarily targets host macrophages leading to cell activation and systemic cytokine storm, and fatal infection is associated with an inhibited interferon response, and lymphopenia. The EBOV surface glycoprotein (GP) has been shown to directly induce T cell depletion and can be secreted outside the virion via extracellular vesicles (EVs), though most studies are limited to epithelial cells and underlying mechanisms remain poorly elucidated., Methods: To assess the role of GP on EBOV-induced dysregulation of host immunity, we first utilized EBOV virus-like particles (VLPs) expressing VP40 and NP either alone (Bald-VLP) or in conjunction with GP (VLP-GP) to investigate early inflammatory responses in THP-1 macrophages and in a murine model. We then sought to decipher the role of non-classical inflammatory mediators such as EVs over the course of EBOV infection in two EBOV-infected rhesus macaques by isolating and characterizing circulatory EVs throughout disease progression using size exclusion chromatography, nanoparticle tracking-analysis, and LC-MS/MS., Results: While all VLPs could induce inflammatory mediators and recruit small peritoneal macrophages, pro-inflammatory cytokine and chemokine gene expression was exacerbated by the presence of GP. Further, quantification of EVs isolated from infected rhesus macaques revealed that the concentration of vesicles peaked in circulation at the terminal stage, at which time EBOV GP could be detected in host-derived exosomes. Moreover, comparative proteomics conducted across EV populations isolated from serum at various time points before and after infection revealed differences in host-derived protein content that were most significantly pronounced at the endpoint of infection, including significant expression of mediators of TLR4 signaling., Discussion: These results suggest a dynamic role for EVs in the modification of disease states in the context of EBOV. Overall, our work highlights the importance of viral factors, such as the GP, and host derived EVs in the inflammatory cascade and pathogenesis of EBOV, which can be collectively further exploited for novel antiviral development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Vucetic, Lafleur, Côté, Kobasa, Chan, Alvarez, Piccirillo, Dong and Olivier.)

Ebola virus (EBOV) induces cell death not only in infected permissive cells but also in nonpermissive, bystander cells by employing different mechanisms. Hydroxycarboxylic acid receptor 2 (HCAR2) has been reported to be involved in apoptotic cell death. We previously reported an increase in the expression of HCAR2-specific mRNA in EBOV-infected individuals with fatal outcomes. Here, we report that infection with an EBOV lacking the VP30 gene (EBOVΔVP30) results in the upregulation of HCAR2 mRNA expression in human hepatocyte Huh7.0 cells stably expressing VP30. Transient overexpression of HCAR2 reduced the viability of Huh7.0 cells and human embryonic kidney cells. Phosphatidylserine externalization and cell membrane permeabilization by HCAR2 overexpression was also observed. Interestingly, coexpression of HCAR2 with EBOV VP40 further reduced cell viability in transfected cells compared to HCAR2 coexpression with other viral proteins. Our data suggest that HCAR2 may contribute to EBOV-induced cell death., Competing Interests: Potential conflicts of interest. Y.K. has received unrelated funding support from FUJIFILM Toyama Chemical Co. LTD, Shionogi & Co. LTD, Daiichi Sankyo Pharmaceutical, Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Fuji Rebio, Tauns Laboratories, Inc., and FluGen., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Viral inclusion bodies (IBs) are potential sites of viral replication and assembly. How viral IBs form remains poorly defined. Here we describe a combined biophysical and cellular approach to identify the components necessary for IB formation during Ebola virus (EBOV) infection. We find that the eNP 0 VP35 complex containing Ebola nucleoprotein (eNP) and viral protein 35 (eVP35), the functional equivalents of nucleoprotein (N) and phosphoprotein (P) in non-segmented negative strand viruses (NNSVs), phase separates to form inclusion bodies. Phase separation of eNP 0 VP35 is reversible and modulated by ionic strength. The multivalency of eVP35, and not eNP, is also critical for phase separation. Furthermore, overexpression of an eVP35 peptide disrupts eNP 0 VP35 complex formation, leading to reduced frequency of IB formation and limited viral infection. Together, our results show that upon EBOV infection, the eNP 0 VP35 complex forms the minimum unit to drive IB formation and viral replication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I 3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

Ebola virus is a zoonotic pathogen with a geographic range covering diverse ecosystems that are home to many potential reservoir species. Although researchers have detected Ebola virus RNA and serological evidence of previous infection in different rodents and bats, the infectious virus has not been isolated. The field is missing critical knowledge about where the virus is maintained between outbreaks, either because the virus is rarely encountered, overlooked during sampling, and/or requires specific unknown conditions that regulate viral expression. This study assessed adipose tissue as a previously overlooked tissue capable of supporting Ebola virus infection. Adipose tissue is a dynamic endocrine organ helping to regulate and coordinate homeostasis, energy metabolism, and neuroendocrine and immune functions. Through in vitro infection of human and bat ( Eptesicus fuscus ) brown adipose tissue cultures using wild-type Ebola virus, this study showed high levels of viral replication for 28 days with no qualitative indicators of cytopathic effects. In addition, alterations in adipocyte metabolism following long-term infection were qualitatively observed through an increase in lipid droplet number while decreasing in size, a harbinger of lipolysis or adipocyte browning. The finding that bat and human adipocytes are susceptible to Ebola virus infection has important implications for potential tissue tropisms that have not yet been investigated. Additionally, the findings suggest how the metabolism of this tissue may play a role in pathogenesis, viral transmission, and/or zoonotic spillover events.

The liver is an early systemic target of Ebola virus (EBOV), but characterization beyond routine histopathology and viral antigen distribution is limited. We hypothesized Ebola virus disease (EVD) systemic proinflammatory responses would be reflected in temporally altered liver myeloid phenotypes. We utilized multiplex fluorescent immunohistochemistry (mfIHC), multispectral whole slide imaging, and image analysis to quantify molecular phenotypes of myeloid cells in the liver of rhesus macaques ( Macaca mulatta; n = 21) infected with EBOV Kikwit. Liver samples included uninfected controls ( n = 3), 3 days postinoculation (DPI; n = 3), 4 DPI ( n = 3), 5 DPI ( n = 3), 6 DPI ( n = 3), and terminal disease (6-8 DPI; n = 6). Alterations in hepatic macrophages occurred at ≥ 5 DPI characterized by a 1.4-fold increase in CD68+ immunoreactivity and a transition from primarily CD14 - CD16 + to CD14 + CD16 - macrophages, with a 2.1-fold decrease in CD163 expression in terminal animals compared with uninfected controls. An increase in the neutrophil chemoattractant and alarmin S100A9 occurred within hepatic myeloid cells at 5 DPI, followed by rapid neutrophil influx at ≥ 6 DPI. An acute rise in the antiviral myxovirus resistance protein 1 (MxA) occurred at ≥ 4 DPI, with a predilection for enhanced expression in uninfected cells. Distinctive expression of major histocompatibility complex (MHC) class II was observed in hepatocytes during terminal disease. Results illustrate that EBOV causes macrophage phenotype alterations as well as neutrophil influx and prominent activation of interferon host responses in the liver. Results offer insight into potential therapeutic strategies to prevent and/or modulate the host proinflammatory response to normalize hepatic myeloid functionality.

Zaïre ebolavirus (EBOV) causes Ebola virus disease (EVD), a devastating viral hemorrhagic fever in humans. Nonhuman primate (NHP) models of EVD traditionally use intramuscular infection with higher case fatality rates and reduced mean time-to-death compared to contact transmission typical of human cases of EVD. A cynomolgus macaque model of oral and conjunctival EBOV was used to further characterize the more clinically relevant contact transmission of EVD. NHPs challenged via the oral route had an overall 50% survival rate. NHPs challenged with a target dose of 1 × 10 2 PFU or 1 × 10 4 PFU of EBOV via the conjunctival route had 40% and 100% mortality, respectively. Classic signs of lethal EVD-like disease were observed in all NHPs that succumbed to EBOV infection including viremia, hematological abnormalities, clinical chemistries indicative of hepatic and renal disease, and histopathological findings. Evidence of EBOV viral persistence in the eye was observed in NHPs challenged via the conjunctival route. IMPORTANCE This study is the first to examine the Kikwit strain of EBOV, the most commonly used strain, in the gold-standard macaque model of infection. Additionally, this is the first description of the detection of virus in the vitreous fluid, an immune privileged site that has been proposed as a viral reservoir, following conjunctival challenge. The oral and conjunctival macaque challenge model of EVD described here more faithfully recapitulates the prodrome that has been reported for human EVD. This work paves the way for more advanced studies to model contact transmission of EVD, including early events in mucosal infection and immunity, as well as the establishment of persistent viral infection and the emergence from these reservoirs., Competing Interests: The authors declare no conflict of interest.

Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. KEY MESSAGES: • Ebola VLP can lead to metabolic alternations in endothelial cells and M1 and M2 macrophages. • Differential abundance of metabolites, mainly including fatty acids and sterol lipids, was observed after Ebola VLP exposure. • Multiple fatty acid-, steroid-, and amino acid-related metabolism pathways were observed perturbed., (© 2023. The Author(s).)

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-β), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans with high mortality. In Ebola virus disease (EVD) survivors, EBOV persistence in the eyes may break through the inner blood-retinal barrier (iBRB), leading to ocular complications and EVD recurrence. However, the mechanism by which EBOV affects the iBRB remains unclear. Here, we used the in vitro iBRB model to simulate EBOV in retinal tissue and found that Ebola virus-like particles (EBO-VLPs) could disrupt the iBRB. Cytokine screening revealed that EBO-VLPs stimulate pericytes to secrete vascular endothelial growth factor (VEGF) to cause iBRB breakdown. VEGF downregulates claudin-1 to disrupt the iBRB. Ebola glycoprotein is crucial for VEGF stimulation and iBRB breakdown. Furthermore, EBO-VLPs caused iBRB breakdown by stimulating VEGF in rats. This study provides a mechanistic insight into that EBOV disrupts the iBRB, which will assist in developing new strategies to treat EBOV persistence in EVD survivors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: In its earliest phases, Ebola virus disease's rapid-onset, high fever, and gastrointestinal symptoms are largely indistinguishable from other infectious illnesses. We aimed to characterise the clinical indicators associated with Ebola virus disease to improve outbreak response., Methods: In this retrospective analysis, we assessed routinely collected data from individuals with possible Ebola virus disease attending 30 Ebola health facilities in two provinces of the Democratic Republic of the Congo between Aug 1, 2018, and Aug 28, 2019. We used logistic regression analysis to model the probability of Ebola infection across 34 clinical variables and four types of possible Ebola virus disease exposures: contact with an individual known to have Ebola virus disease, attendance at any funeral, health facility consultation, or consultation with an informal health practitioner., Findings: Data for 24 666 individuals were included. If a patient presented to care in the early symptomatic phase (ie, days 0-2), Ebola virus disease positivity was most associated with previous exposure to an individual with Ebola virus disease (odds ratio [OR] 11·9, 95% CI 9·1-15·8), funeral attendance (2·1, 1·6-2·7), or health facility consultations (2·1, 1·6-2·8), rather than clinical parameters. If presentation occurred on day 3 or later (after symptom onset), bleeding at an injection site (OR 33·9, 95% CI 12·7-101·3), bleeding gums (7·5, 3·7-15·4), conjunctivitis (2·4, 1·7-3·4), asthenia (1·9, 1·5-2·3), sore throat (1·8, 1·3-2·4), dysphagia (1·8, 1·4-2·3), and diarrhoea (1·6, 1·3-1·9) were additional strong predictors of Ebola virus disease. Some Ebola virus disease-specific signs were less prevalent among vaccinated individuals who were positive for Ebola virus disease when compared with the unvaccinated, such as dysphagia (-47%, p=0·0024), haematemesis (-90%, p=0·0131), and bleeding gums (-100%, p=0·0035)., Interpretation: Establishing the exact time an individual first had symptoms is essential to assessing their infection risk. An individual's exposure history remains of paramount importance, especially in the early phase. Ebola virus disease vaccination reduces symptom severity and should also be considered when assessing the likelihood of infection. These findings about symptomatology should be translated into practice during triage and should inform testing and quarantine procedures., Funding: Médecins Sans Frontières and its research affiliate Epicentre., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We explore how animal host traits, phylogenetic identity and cell receptor sequences relate to infection status and mortality from ebolaviruses. We gathered exhaustive databases of mortality from Ebolavirus after exposure and infection status based on PCR and antibody tests. We performed ridge regressions predicting mortality and infection as a function of traits, phylogenetic eigenvectors and separately host receptor sequences. We found that mortality from Ebolavirus had a strong association to life history characteristics and phylogeny. In contrast, infection status related not just to life history and phylogeny, but also to fruit consumption which suggests that geographic overlap of frugivorous mammals can lead to spread of virus in the wild. Niemann Pick C1 (NPC1) receptor sequences predicted infection statuses of bats included in our study with very high accuracy, suggesting that characterizing NPC1 in additional species is a promising avenue for future work. We combine the predictions from our mortality and infection status models to differentiate between species that are infected and also die from Ebolavirus versus species that are infected but tolerate the virus (possible reservoirs of Ebolavirus). We therefore present the first comprehensive estimates of Ebolavirus reservoir statuses for all known terrestrial mammals in Africa., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Sundaram et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ebola virus disease (EVD) is a severe and frequently lethal disease caused by Ebola virus (EBOV). The latest occasional EVD outbreak (2013-2016) in Western African, which was accompanied by a high fatality rate, showed the great potential of epidemic and pandemic spread. Antiviral therapies against EBOV are very limited, strain-dependent (only antibody therapies are available) and mostly restricted to symptomatic treatment, illustrating the urgent need for novel antiviral strategies. Thus, we evaluated the effect of the clinically widely used antifungal itraconazole and the antidepressant fluoxetine for a repurposing against EBOV infection. While itraconazole, similar to U18666A, directly binds to and inhibits the endosomal membrane protein Niemann-Pick C1 (NPC1), fluoxetine, which belongs to the structurally unrelated group of weakly basic, amphiphile so-called "functional inhibitors of acid sphingomyelinase" (FIASMA) indirectly acts on the lysosome-residing acid sphingomyelinase via enzyme detachment leading to subsequent lysosomal degradation. Both, the drug-induced endolysosomal cholesterol accumulation and the altered endolysosomal pH, might interfere with the fusion of viral and endolysosomal membrane, preventing infection with EBOV. We further provide evidence that cholesterol imbalance is a conserved cross-species mechanism to hamper EBOV infection. Thus, exploring the endolysosomal host-pathogen interface as a suitable antiviral treatment may offer a general strategy to combat EBOV infection.

Despite increasing evidence that uveitis is common and consequential in survivors of Ebola virus disease (EVD), the host-pathogen determinants of the clinical phenotype are undefined, including the pathogenetic role of persistent viral antigen, ocular tissue-specific immune responses, and histopathologic characterization. Absent sampling of human intraocular fluids and tissues, these questions might be investigated in animal models of disease; however, challenges intrinsic to the nonhuman primate model and the animal biosafety level 4 setting have historically limited inquiry. In a rhesus monkey survivor of experimental Ebola virus (EBOV) infection, we observed and documented the clinical, virologic, immunologic, and histopathologic features of severe uveitis. Here we show the clinical natural history, resultant ocular pathology, intraocular antigen-specific antibody detection, and persistent intraocular EBOV RNA detected long after clinical resolution. The association of persistent EBOV RNA as a potential driver of severe immunopathology has pathophysiologic implications for understanding, preventing, and mitigating vision-threatening uveitis in EVD survivors., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Macrophages contribute to Ebola virus disease through their susceptibility to direct infection, their multi-faceted response to ebolaviruses, and their association with pathological findings in tissues throughout the body. Viral attachment and entry factors, as well as the more recently described influence of cell polarization, shape macrophage susceptibility to direct infection. Moreover, the study of Toll-like receptor 4 and the RIG-I-like receptor pathway in the macrophage response to ebolaviruses highlight important immune signaling pathways contributing to the breadth of macrophage responses. Lastly, the deep histopathological catalogue of macrophage involvement across numerous tissues during infection has been enriched by descriptions of tissues involved in sequelae following acute infection, including: the eye, joints, and the nervous system. Building upon this knowledge base, future opportunities include characterization of macrophage phenotypes beneficial or deleterious to survival, delineation of the specific roles macrophages play in pathological lesion development in affected tissues, and the creation of macrophage-specific therapeutics enhancing the beneficial activities and reducing the deleterious contributions of macrophages to the outcome of Ebola virus disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wanninger, Millian, Saldarriaga, Maruyama, Saito, Reyna, Taniguchi, Arroyave, Connolly, Stevenson and Paessler.)

Liver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Filoviruses enter cells through macropinocytosis and trafficking into the endosomes in which they bind to the receptor Niemann-Pick C1 protein (NPC1) for membrane fusion and entry into the cytoplasm. The endosomal receptor-binding is critical step for filovirus entry. Designing inhibitors to block receptor binding will prevent viral entry. Using available binding structural information from the co-crystal structures of the viral GP with the receptor NPC1 or with monoclonal antibodies, we have conducted structure-based design of peptide inhibitors to target the receptor binding site (RBS). The designed peptides were tested for their inhibition activity against pseudo-typed or replication-competent viruses in a cell-based assay. The results indicate that these peptides exhibited strong activities against both Ebola and Marburg virus infection. It is expected that these peptides can be further developed for therapeutic use to treat filovirus infection and combat the outbreaks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors involved in actin nucleation, such as Rac1 and Arp2/3, were shown important in both steps. However, downstream of nucleation, many other cell factors are needed to control actin dynamics. How these regulate EBOV infection remains largely unclear. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown of CAPG specifically inhibited viral infectivity and yield of infectious particles. Cell-based mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split-green fluorescent protein reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells.

Ebola virus (EBOV) infections continue to pose a global public health threat, with high mortality rates and sporadic outbreaks in Central and Western Africa. A quantitative understanding of the key processes driving EBOV assembly and budding could provide valuable insights to inform drug development. Here, we use a computational model to evaluate EBOV matrix assembly. Our model focuses on the assembly kinetics of VP40, the matrix protein in EBOV, and its interaction with phosphatidylserine (PS) in the host cell membrane. It has been shown that mammalian cells transfected with VP40-expressing plasmids are capable of producing virus-like particles (VLPs) that closely resemble EBOV virions. Previous studies have also shown that PS levels in the host cell membrane affects VP40 association with the plasma membrane inner leaflet and that lower membrane PS levels result in lower VLP production. Our computational findings indicate that PS may also have a direct influence on VP40 VLP assembly and budding, where a higher PS level will result in a higher VLP budding rate and filament dissociation rate. Our results further suggest that the assembly of VP40 filaments follow the nucleation-elongation theory, where initialization and oligomerization of VP40 are two distinct steps in the assembly process. Our findings advance the current understanding of VP40 VLP formation by identifying new possible mechanisms of PS influence on VP40 assembly. We propose that these mechanisms could inform treatment strategies targeting PS alone or in combination with other VP40 assembly steps., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: During the 2018-2020 Ebola virus disease (EVD) outbreak in the Democratic Republic of the Congo, risk communication and community engagement (RCCE) were prioritized in geographic areas in Uganda considered at high risk of introduction of EVD. To inform EVD preparedness in Uganda, we evaluated community perceptions and prevention practices related to EVD in 6 districts in Uganda., Methods: In March 2020, we conducted a population-based survey in 6 purposively selected districts in Uganda using multistage cluster sampling. We examined differences between districts classified as high- versus low risk for EVD in terms of their message exposure from RCCE; risk perception; and EVD knowledge, attitudes, and prevention practices., Results: A total of 3,485 respondents were interviewed (91% response rate). EVD message exposure was more common in the high- versus low-risk districts. EVD risk perceptions were low overall but greater in the high- versus low-risk districts. Comprehensive knowledge was significantly greater in the high- versus low-risk districts (adjusted prevalence ratio [aPR] 1.61, 95% confidence interval [CI]=1.35, 1.93). Respondents' engagement in all 3 EVD prevention practices (frequent handwashing with soap, avoiding physical contact with suspected Ebola patients, and avoiding burials involving contact with a corpse) was very low (4%). However, respondents with comprehensive knowledge were more likely to engage in all 3 EVD prevention practices compared to respondents without comprehensive knowledge (aPR 1.87, 95% CI=1.08, 3.25)., Conclusion: Our findings suggest that while RCCE efforts as part of EVD outbreak preparedness may have contributed to higher EVD knowledge in the targeted high-risk districts, uptake of prevention behaviors was similarly low across districts. In a non-outbreak setting, implementing targeted RCCE strategies may not be sufficient to motivate people to adopt protective behaviors in the absence of a high threshold of perceived threat such as in an active outbreak., (© Musaazi et al.)

Ebola virus (EBOV), one of the deadliest viruses, is the cause of fatal Ebola virus disease (EVD). The underlying mechanism of viral replication and EBOV-related hemorrhage is not fully understood. Here, we show that EBOV VP35, a cofactor of viral RNA-dependent RNA polymerase, binds human A kinase interacting protein (AKIP1), which consequently activates protein kinase A (PKA) and the PKA-downstream transcription factor CREB1. During EBOV infection, CREB1 is recruited into EBOV ribonucleoprotein complexes in viral inclusion bodies (VIBs) and employed for viral replication. AKIP1 depletion or PKA-CREB1 inhibition dramatically impairs EBOV replication. Meanwhile, the transcription of several coagulation-related genes, including THBD and SERPINB2, is substantially upregulated by VP35-dependent CREB1 activation, which may contribute to EBOV-related hemorrhage. The finding that EBOV VP35 hijacks the host PKA-CREB1 signal axis for viral replication and pathogenesis provides novel potential therapeutic approaches against EVD., (© 2022. The Author(s).)

Ebola virus (EBOV) and Marburg virus (MARV) continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW domain-containing host interactors via its conserved PPxY late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ, and WWOX. In addition to host WW domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 virus-like particles (VLPs) and recombinant vesicular stomatitis virus (VSV) M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOT's scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY-WW domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCE Filoviruses (Ebola virus [EBOV] and Marburg virus [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the filovirus matrix protein VP40 to regulate VP40-mediated egress of virus-like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus life cycle and the host cell.

Since the 2014-2016 epidemic, Ebola virus (EBOV) has spread to several countries and has become a major threat to global health. EBOV is a risk group 4 pathogen, which imposes significant obstacles for the development of countermeasures against the virus. Efforts have been made to develop anti-EBOV immunization and therapeutics, with three vaccines and two antibody-based therapeutics approved in recent years. Nonetheless, the high fatality of Ebola virus disease highlights the need to continuously develop antiviral strategies for the future management of EBOV outbreaks in conjunction with vaccination programs. This review aims to highlight potential EBOV therapeutics and their target(s) of inhibition, serving as a summary of the literature to inform readers of the novel candidates available in the continued search for EBOV antivirals.

Ebola virus (EBOV) targets immune cells and tries to inactivate dendritic cells and interferon molecules to continue its replication process. Since EBOV detailed mechanism has not been identified so far, it would be useful to understand the growth and spread of EBOV dynamics based on mathematical methods and simulation approaches. Computational approaches such as Cellular Automata (CA) have the advantage of simplicity over solving complicated differential equations. The spread of Ebola virus in lymph nodes is studied using a simplified Cellular Automata model with only four parameters. In addition to considering healthy and infected cells, this paper also considers T lymphocytes as well as cell movement ability during the simulation in order to investigate different scenarios in the dynamics of an EBOV system. It is shown that the value of the probability of death of T cells affects the number of infected cells significantly in the steady-state. For a special case of parameters set, the system shows oscillating dynamics. The results were in good agreement with an ordinary differential equation-based model which indicated CA method in combination with experimental discoveries could help biologists find out more about the EBOV mechanism and hopefully to control the disease., Competing Interests: The authors have declared that no competing interests exist.

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding., (© 2020. The Author(s).)

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5'-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-β production. Blocking VP35-dsRNA interaction and IFN-β suppression is a validated drug target. We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC 50 value of 8.5 μM. It reverts the EBOV VP35 inhibition of IFN-β production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a K D of 12 μM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC 50 value of 9.1 μM, showing no significant cytotoxicity. Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ebola virus disease (EVD) is a lethal disease caused by the highly pathogenic Ebola virus (EBOV), and its major symptoms in severe cases include vascular leakage and hemorrhage. These symptoms are caused by abnormal activation and disruption of endothelial cells (ECs) whose mediators include EBOV glycoprotein (GP) without the need for viral replication. However, the detailed molecular mechanisms underlying virus-host interactions remain largely unknown. Here, we show that EBOV-like particles (VLPs) formed by GP, VP40, and NP activate ECs in a GP-dependent manner, as demonstrated by the upregulation of intercellular adhesion molecules-1 (ICAM-1) expression. VLPs-mediated ECs activation showed a different kinetic pattern from that of TNF-α-mediated activation and was associated with apoptotic ECs disruption. In contrast to TNF-α, VLPs induced ICAM-1 overexpression at late time points. Furthermore, screening of host cytoskeletal signaling inhibitors revealed that focal adhesion kinase inhibitors were found to be potent inhibitors of ICAM-1 expression mediated by both TNF-α and VLPs. Our results suggest that EBOV GP stimulates ECs to induce endothelial activation and dysfunction with the involvement of host cytoskeletal signaling factors, which represent potential therapeutic targets for EVD.

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses., Competing Interests: JC has served as a consultant for Luna Biologics and Merck Sharp & Dohme Corp., is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Murin, Gilchuk, Crowe and Ward.)

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus., (© 2021 The Author(s) Published by S. Karger AG, Basel.)