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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.

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.)

The Ebola virus (EBOV) is a member of the Orthoebolavirus genus, Filoviridae family, which causes severe hemorrhagic diseases in humans and non-human primates (NHPs), with a case fatality rate of up to 90%. The development of countermeasures against EBOV has been hindered by the lack of ideal animal models, as EBOV requires handling in biosafety level (BSL)-4 facilities. Therefore, accessible and convenient animal models are urgently needed to promote prophylactic and therapeutic approaches against EBOV. In this study, a recombinant vesicular stomatitis virus expressing Ebola virus glycoprotein (VSV-EBOV/GP) was constructed and applied as a surrogate virus, establishing a lethal infection in hamsters. Following infection with VSV-EBOV/GP, 3-week-old female Syrian hamsters exhibited disease signs such as weight loss, multi-organ failure, severe uveitis, high viral loads, and developed severe systemic diseases similar to those observed in human EBOV patients. All animals succumbed at 2-3 days post-infection (dpi). Histopathological changes indicated that VSV-EBOV/GP targeted liver cells, suggesting that the tissue tropism of VSV-EBOV/GP was comparable to wild-type EBOV (WT EBOV). Notably, the pathogenicity of the VSV-EBOV/GP was found to be species-specific, age-related, gender-associated, and challenge route-dependent. Subsequently, equine anti-EBOV immunoglobulins and a subunit vaccine were validated using this model. Overall, this surrogate model represents a safe, effective, and economical tool for rapid preclinical evaluation of medical countermeasures against EBOV under BSL-2 conditions, which would accelerate technological advances and breakthroughs in confronting Ebola virus disease., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Background: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain., Methods: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb)., Findings: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively., Interpretation: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration., Funding: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA., Competing Interests: Declaration of interests All authors have read and confirmed the manuscript and accepted the responsibility to submit it for publication. 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., (Published by Elsevier B.V.)

Ebola virus (EBOV), a major global health concern, causes severe, often fatal EBOV disease (EVD) in humans. Host genetic variation plays a critical role, yet the identity of host susceptibility loci in mammals remains unknown. Using genetic reference populations, we generate an F2 mapping cohort to identify host susceptibility loci that regulate EVD. While disease-resistant mice display minimal pathogenesis, susceptible mice display severe liver pathology consistent with EVD-like disease and transcriptional signatures associated with inflammatory and liver metabolic processes. A significant quantitative trait locus (QTL) for virus RNA load in blood is identified in chromosome (chr)8, and a severe clinical disease and mortality QTL is mapped to chr7, which includes the Trim5 locus. Using knockout mice, we validate the Trim5 locus as one potential driver of liver failure and mortality after infection. The identification of susceptibility loci provides insight into molecular genetic mechanisms regulating EVD progression and severity, potentially informing therapeutics and vaccination strategies., Competing Interests: Declaration of interests D.O.C. is employed by, has equity ownership in, and serves on the board of directors of TransViragen, the company that has been contracted by UNC-Chapel Hill to manage its Animal Models Core Facility. R.S.B. is a member of advisory boards for VaxArt, Takeda, and Invivyd, focused on unrelated projects., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ebola virus (EBOV) is a highly pathogenic virus that causes a severe illness called Ebola virus disease (EVD). EVD has a high mortality rate and remains a significant threat to public health. Research on EVD pathogenesis has traditionally focused on host transcriptional responses. Limited recent studies, however, have revealed some information on the significance of cellular microRNAs (miRNAs) in EBOV infection and pathogenic mechanisms, but further studies are needed. Thus, this study aimed to identify and validate additional known and novel human miRNAs in EBOV-infected adult retinal pigment epithelial (ARPE) cells and predict their potential roles in EBOV infection and pathogenic mechanisms. We analyzed previously available small RNA-Seq data obtained from ARPE cells and identified 23 upregulated and seven downregulated miRNAs in the EBOV-infected cells; these included two novel miRNAs and 17 additional known miRNAs not previously identified in ARPE cells. In addition to pathways previously identified by others, these miRNAs are associated with pathways and biological processes that include WNT, FoxO, and phosphatidylinositol signaling; these pathways were not identified in the original study. This study thus confirms and expands on the previous study using the same datasets and demonstrates further the importance of human miRNAs in the host response and EVD pathogenesis during infection.

Background: Ebola virus (EBOV) is a genus of negative-strand RNA viruses belonging to the family Filoviradae that was first described in 1976 in the present-day Democratic Republic of the Congo. It has intermittently affected substantial human populations in West Africa and presents itself as a global health menace due to the high mortality rate of patients, high transmission rate, difficult patient management, and the emergence of complicated autoimmune disease-like conditions post-infection., Objective: EBOV or other EBOV-like species as a biochemical weapon pose a significant risk; hence, the need to develop both prophylactic and therapeutic medications to combat the virus is unquestionable., Methods: In this review work, we have compiled the literature pertaining to transmission, pathogenesis, immune response, and diagnosis of EBOV infection. We included detailed structural details of EBOV along with all the available therapeutics against EBOV disease. We have also highlighted current developments and recent advances in therapeutic approaches against Ebola virus disease (EVD)., Discussion: The development of preventive vaccines against the virus is proving to be a successful effort as of now; however, problems concerning logistics, product stability, multi- dosing, and patient tracking are prominent in West Africa. Monoclonal antibodies that target EBOV proteins have also been developed and approved in the clinic; however, no small drug molecules that target these viral proteins have cleared clinical trials. An understanding of clinically approved vaccines and their shortcomings also serves an important purpose for researchers in vaccine design in choosing the right vector, antigen, and particular physicochemical properties that are critical for the vaccine's success against the virus across the world., Conclusion: Our work brings together a comprehensive review of all available prophylactic and therapeutic medications developed and under development against the EBOV, which will serve as a guide for researchers in pursuing the most promising drug discovery strategies against the EBOV and also explore novel mechanisms of fighting against EBOV infection., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia., (Copyright © 2021 the Author(s). Published by PNAS.)

Competing Interests: We declare no competing interests.

It has been shown that a very early cell-intrinsic response to infection is the upregulation of CD47 cell surface expression, a molecule known for delivering a "don't eat me signal" that inhibits macrophage-mediated phagocytosis and antigen presentation. Thus, blockade of CD47 signaling during lymphocytic choriomenigitis virus infections of mice has been shown to enhance the kinetics and potency of immune responses, thereby producing faster recovery. It seems counterintuitive that one of the earliest responses to infection would be immunoinhibitory, but it has been hypothesized that CD47 induction acts as an innate immune system checkpoint to prevent immune overactivation and immunopathogenic responses during certain infections. In the current study we examined the effect of CD47 blockade on lethal Ebola virus infection of mice. At 6 days post-infection, CD47 blockade was associated with significantly increased activation of B cells along with increases in recently cytolytic CD8 + T cells. However, the anti-CD47-treated mice exhibited increased weight loss, higher virus titers, and succumbed more rapidly. The anti-CD47-treated mice also had increased inflammatory cytokines in the plasma indicative of a "cytokine storm". Thus, in the context of this rapid hemorrhagic disease, CD47 blockade indeed exacerbated immunopathology and disease severity., (Copyright © 2021. Published by Elsevier B.V.)

Type I interferons (IFNs) are cytokines with both antiviral properties and protective roles in innate immune responses to viral infection. They induce an antiviral cellular state and link innate and adaptive immune responses. Yet, viruses have evolved different strategies to inhibit such host responses. One of them is the existence of viral proteins which subvert type I IFN responses to allow quick and successful viral replication, thus, sustaining the infection within a host. We propose mathematical models to characterise the intra-cellular mechanisms involved in viral protein antagonism of type I IFN responses, and compare three different molecular inhibition strategies. We study the Ebola viral protein, VP35, with this mathematical approach. Approximate Bayesian computation sequential Monte Carlo, together with experimental data and the mathematical models proposed, are used to perform model calibration, as well as model selection of the different hypotheses considered. Finally, we assess if model parameters are identifiable and discuss how such identifiability can be improved with new experimental data.

Ebola virus (EBOV), belonging to the species Zaire ebolavirus in the genus Ebolavirus , causes a severe febrile illness in humans with case fatality rates (CFRs) up to 90%. While there have been six virus species classified, which each have a single type virus in the genus Ebolavirus , CFRs of ebolavirus infections vary among viruses belonging to each distinct species. In this review, we aim to define the ebolavirus species-specific virulence on the basis of currently available laboratory and experimental findings. In addition, this review will also cover the variant-specific virulence of EBOV by referring to the unique biological and pathogenic characteristics of EBOV variant Makona, a new EBOV variant isolated from the 2013-2016 EBOV disease outbreak in West Africa. A better definition of species-specific and variant-specific virulence of ebolaviruses will facilitate our comprehensive knowledge on genus Ebolavirus biology, leading to the development of therapeutics against well-focused pathogenic mechanisms of each Ebola disease.

Ebola virus disease (EVD) is a serious global health concern because case fatality rates are approximately 50% due to recent widespread outbreaks in Africa. Well-defined nonhuman primate (NHP) models for different routes of Ebola virus exposure are needed to test the efficacy of candidate countermeasures. In this natural history study, four rhesus macaques were challenged via aerosol with a target titer of 1000 plaque-forming units per milliliter of Ebola virus. The course of disease was split into the following stages for descriptive purposes: subclinical, clinical, and decompensated. During the subclinical stage, high levels of venous partial pressure of carbon dioxide led to respiratory acidemia in three of four of the NHPs, and all developed lymphopenia. During the clinical stage, all animals had fever, viremia, and respiratory alkalosis. The decompensatory stage involved coagulopathy, cytokine storm, and liver and renal injury. These events were followed by hypotension, elevated lactate, metabolic acidemia, shock and mortality similar to historic intramuscular challenge studies. Viral loads in the lungs of aerosol-exposed animals were not distinctly different compared to previous intramuscularly challenged studies. Differences in the aerosol model, compared to intramuscular model, include an extended subclinical stage, shortened clinical stage, and general decompensated stage. Therefore, the shortened timeframe for clinical detection of the aerosol-induced disease can impair timely therapeutic administration. In summary, this nonhuman primate model of aerosol-induced EVD characterizes early disease markers and additional details to enable countermeasure development.

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses., Competing Interests: KC is a member of the scientific advisory boards of Integrum Scientific, LLC, Biovaxys Technology Corp, and the Pandemic Security Initiative of Celdara Medical, LLC, and he has consulted for Axon Advisors, LLC. JL is a consultant for Celdara Medical, LLC. Author JT was employed by company Mapp Biopharmaceutical. 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 © 2021 Wirchnianski, Wec, Nyakatura, Herbert, Slough, Kuehne, Mittler, Jangra, Teruya, Dye, Lai and Chandran.)

Biosafety, biosecurity, logistical, political, and technical considerations can delay or prevent the wide dissemination of source material containing viable virus from the geographic origin of an outbreak to laboratories involved in developing medical countermeasures (MCMs). However, once virus genome sequence information is available from clinical samples, reverse-genetics systems can be used to generate virus stocks de novo to initiate MCM development. In this study, we developed a reverse-genetics system for natural isolates of Ebola virus (EBOV) variants Makona, Tumba, and Ituri, which have been challenging to obtain. These systems were generated starting solely with in silico genome sequence information and have been used successfully to produce recombinant stocks of each of the viruses for use in MCM testing. The antiviral activity of MCMs targeting viral entry varied depending on the recombinant virus isolate used. Collectively, selecting and synthetically engineering emerging EBOV variants and demonstrating their efficacy against available MCMs will be crucial for answering pressing public health and biosecurity concerns during Ebola disease (EBOD) outbreaks.

Ebola virus (EBOV) attaches to target cells using two categories of cell surface receptors: C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic debris. Many enveloped viruses also contain exposed PS and can therefore exploit these receptors for cell entry. Viral infection can induce PS externalization in host cells, resulting in increased outer PS levels on budding virions. Scramblase enzymes carry out cellular PS externalization; thus, we targeted these proteins in order to manipulate viral envelope PS levels. We investigated two scramblases previously identified to be involved in EBOV PS levels, transmembrane protein 16F and Xk-related protein 8 (XKR8), as possible mediators of cellular and viral envelope surface PS levels during the replication of recombinant vesicular stomatitis virus containing its native glycoprotein (rVSV/G) or the EBOV glycoprotein (rVSV/EBOV-GP). We found that rVSV/G and rVSV/EBOV-GP virions produced in XKR8 knockout cells contain decreased levels of PS on their surfaces, and the PS-deficient rVSV/EBOV-GP virions are 70% less efficient at infecting cells through PS receptors. We also observed reduced rVSV and EBOV virus-like particle (VLP) budding in ΔXKR8 cells. Deletion of XKR8 in HAP1 cells reduced rVSV/G and rVSV/EBOV-GP budding by 60 and 65%, respectively, and reduced Ebola VLP budding more than 60%. We further demonstrated that caspase cleavage of XKR8 is required to promote budding. This suggests that XKR8, in addition to mediating virion PS levels, may also be critical for enveloped virus budding at the plasma membrane. IMPORTANCE Within the last decade, countries in western and central Africa have experienced the most widespread and deadly Ebola outbreaks since Ebola virus was identified in 1976. While outbreaks are primarily attributed to zoonotic transfer events, new evidence is emerging outbreaks may be caused by a combination of spillover events and viral latency or persistence in survivors. The possibility that Ebola virus can remain dormant and then reemerge in survivors highlights the critical need to prevent the virus from entering and establishing infection in human cells. Thus far, host cell scramblases TMEM16F and XKR8 have been implicated in Ebola envelope surface phosphatidylserine (PS) and cell entry using PS receptors. We assessed the contributions of these proteins using CRISPR knockout cells and two EBOV models: rVSV/EBOV-GP and EBOV VLPs. We observed that XKR8 is required for optimal EBOV envelope PS levels and infectivity and particle budding across all viral models.

Ebola virus (EBOV) expresses three different glycoproteins (GPs) from its GP gene. The primary product, soluble GP (sGP), is secreted in abundance during infection. EBOV sGP has been discussed as a potential pathogenicity factor, however, little is known regarding its functional role. Here, we analyzed the role of sGP in vitro and in vivo. We show that EBOV sGP has two different functions that contribute to infectivity in tissue culture. EBOV sGP increases the uptake of virus particles into late endosomes in HEK293 cells, and it activates the mitogen-activated protein kinase (MAPK) signaling pathway leading to increased viral replication in Huh7 cells. Furthermore, we analyzed the role of EBOV sGP on pathogenicity using a well-established mouse model. We found an sGP-dependent significant titer increase of EBOV in the liver of infected animals. These results provide new mechanistic insights into EBOV pathogenicity and highlight EBOV sGP as a possible therapeutic target., Competing Interests: The authors have declared that no competing interests exist.

Ebolaviruses Bundibugyo virus (BDBV) and Ebola virus (EBOV) cause fatal hemorrhagic disease in humans and nonhuman primates. While the host response to EBOV is well characterized, less is known about BDBV infection. Moreover, immune signatures that mediate natural protection against all ebolaviruses remain poorly defined. To explore these knowledge gaps, we transcriptionally profiled BDBV-infected rhesus macaques, a disease model that results in incomplete lethality. This approach enabled us to identify prognostic indicators. As expected, survival (∼60%) correlated with reduced clinical pathology and circulating infectious virus, although peak viral RNA loads were not significantly different between surviving and nonsurviving macaques. Survivors had higher anti-BDBV antibody titers and transcriptionally derived cytotoxic T cell-, memory B cell-, and plasma cell-type quantities, demonstrating activation of adaptive immunity. Conversely, a poor prognosis was associated with lack of an appropriate adaptive response, sustained innate immune signaling, and higher expression of myeloid-derived suppressor cell (MDSC)-related transcripts ( S100A8 , S100A9 , CEBPB , PTGS2 , CXCR1 , and LILRA3 ). MDSCs are potent immunosuppressors of cellular and humoral immunity, and therefore, they represent a potential therapeutic target. Circulating plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (tPA) levels were also elevated in nonsurvivors and in survivors exhibiting severe illness, emphasizing the importance of maintaining coagulation homeostasis to control disease progression. IMPORTANCE Bundibugyo virus (BDBV) and Ebola virus (EBOV) are ebolaviruses endemic to Africa that cause severe, often fatal hemorrhagic disease. BDBV is considered a less pathogenic ebolavirus due to its reduced lethality during human outbreaks, as well as in experimentally infected nonhuman primates. The reduced mortality of BDBV in NHP models, resulting in a pool of survivors, afforded us the unique opportunity of identifying immune correlates that confer protection against ebolaviruses. In this study, we discovered that the survival of BDBV-infected nonhuman primates (NHPs) was dependent on early development of adaptive (memory) immune responses and reduced myeloid-derived suppressor cell (MDSC)-related signaling. MDSCs are a heterogenous group of immune cells implicated in a number of diseases that are powerful immunosuppressors of cellular and humoral immunity. Thus, MDSCs represent a novel therapeutic target to prevent ebolavirus disease. To our knowledge, this is the first study to link increased morbidity with recruitment of these potent immunosuppressive cells.

Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication.

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014-2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 10 1 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Filoviruses (Family Filoviridae genera Ebolavirus and Marburgvirus) are negative-stranded RNA viruses that cause severe health effects in humans and non-human primates, including death. Except in outbreak settings, vaccines and other medical countermeasures against Ebola virus (EBOV) will require testing under the FDA Animal Rule. Multiple vaccine candidates have been evaluated using cynomolgus monkeys (CM) exposed to EBOV Kikwit strain. To the best of our knowledge, however, animal model development data supporting the use of CM in vaccine research have not been submitted to the FDA. This study describes a large CM database (122 CM, 62 female and 60 male, age 2 to 9 years) and demonstrates the consistency of the CM model through time to death models and descriptive statistics. CMs were exposed to EBOV doses of 0.1 to 100,000 PFU in 33 studies conducted at three Animal Biosafety Level 4 facilities, by three exposure routes. Time to death was modeled using Cox proportional hazards models with a frailty term that incorporated study-to-study variability. Despite significant differences attributed to exposure variables, all CMs exposed to the 100 to 1,000 pfu doses commonly used in vaccine studies died or met euthanasia criteria within 21 days of exposure, median 7 days, 93% between 5 and 12 days of exposure. Moderate clinical signs were observed 4 to 5 days after exposure and preceded death or euthanasia by approximately one day. Viremia was detected within a few days of infection. Hematology indices were indicative of viremia and the propensity for hemorrhage with progression of Ebola viremia. Changes associated with coagulation parameters and platelets were consistent with coagulation disruption. Changes in leukocyte profiles were indicative of an acute inflammatory response. Increased liver enzymes were observed shortly after exposure. Taken together, these factors suggest that the cynomolgus monkey is a reliable animal model for human disease., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Viral outbreaks present a particular challenge in countries in Africa where there is already a high incidence of other infectious diseases, including malaria which can alter immune responses to secondary infection. Ebola virus disease (EVD) is one such problem; understanding how Plasmodium spp. and Ebolavirus (EBOV) interact is important for future outbreaks., Methods: We conducted a systematic review in PubMed and Web of Science to find peer-reviewed papers with primary data literature to determine 1) prevalence of EBOV/Plasmodium spp. coinfection, 2) effect of EBOV/Plasmodium spp. coinfection on EVD pathology and the immune response, 3) impact of EBOV/Plasmodium spp. coinfection on the outcome of EVD-related mortality. Random effects meta-analyses were conducted with the R package meta to produce overall proportion and effect estimates as well as measure between-study heterogeneity., Results: From 322 peer-reviewed papers, 17 were included in the qualitative review and nine were included in a meta-analysis. Prevalence of coinfection was between 19% and 72%. One study reported significantly lower coagulatory response biomarkers in coinfected cases but no difference in inflammatory markers. Case fatality rates were similar between EBOV(+)/Pl(+) and EBOV(+)/Pl(-) cases (62.8%, 95% CI 49.3-74.6 and 56.7%, 95% CI 53.2-60.1, respectively), and there was no significant difference in risk of mortality (RR 1.09, 95% CI 0.90-1.31) although heterogeneity between studies was high. One in vivo mouse model laboratory study found no difference in mortality by infection status, but another found prior acute Plasmodium yoeli infection was protective against morbidity and mortality via the IFN-γ signalling pathway., Conclusion: The literature was inconclusive; studies varied widely and there was little attempt to adjust for confounding variables. Laboratory studies may present the best option to answer how pathogens interact within the body but improvement in data collection and analysis and in diagnostic methods would aid patient studies in the future., Competing Interests: The authors have declared that no competing interests exist.

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies., Competing Interests: Declaration of interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from and IDBiologics and AstraZeneca. Vanderbilt University has applied for a patent that is related to antibodies discussed in this work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The 2013-16 Ebola virus disease epidemic in west Africa caused international alarm due to its rapid and extensive spread resulting in a significant death toll and social unrest within the affected region. The large number of cases provided an opportunity to study the long-term kinetics of Zaire ebolavirus-specific immune response of survivors in addition to known contacts of those infected with the virus., Methods: In this observational cohort study, we worked with leaders of Ebola virus disease survivor associations in two regions of Guinea, Guéckédou and Coyah, to recruit survivors of Ebola virus disease, contacts from households of individuals known to have had Ebola virus disease, and individuals who were not knowingly associated with infected individuals or had not had Ebola virus disease symptoms to serve as negative controls. We did Zaire ebolavirus glycoprotein-specific T cell analysis on peripheral blood mononuclear cells (PBMCs) on location in Guinea and transported plasma and PBMCs back to Europe for antibody quantification by ELISA, functional neutralising antibody analysis using live Zaire ebolavirus, and T cell phenotype studies. We report on the longitudinal cellular and humoral response among Ebola virus disease survivors and highlight potentially paucisymptomatic infection., Findings: We recruited 117 survivors of Ebola virus disease, 66 contacts, and 23 negative controls. The mean neutralising antibody titre among the Ebola virus disease survivors 3-14 months after infection was 1/174 (95% CI 1/136-1/223). Individual results varied greatly from 1/10 to more than 1/1000 but were on average ten times greater than that induced after 1 month by single dose Ebola virus vaccines. Following reactivation with glycoprotein peptide, the mean T cell responses among 116 Ebola virus disease survivors as measured by ELISpot was 305 spot-forming units (95% CI 257-353). The dominant CD8+ polyfunctional T cell phenotype, as measured among 53 Ebola virus disease survivors, was interferon γ+, tumour necrosis factor+, interleukin-2-, and the mean response was 0·046% of total CD8+ T cells (95% CI 0·021-0·071). Additionally, both neutralising antibody and T cell responses were detected in six (9%) of 66 Ebola virus disease contacts. We also noted that four (3%) of 117 individuals with Ebola virus disease infections did not have circulating Ebola virus-specific antibodies 3 months after infection., Interpretation: The continuous high titre of neutralising antibodies and increased T cell response might support the concept of long-term protective immunity in survivors. The existence of antibody and T cell responses in contacts of individuals with Ebola virus disease adds further evidence to the existence of sub-clinical Ebola virus infection., Funding: US Food & Drug Administration, Horizon 2020 EU EVIDENT, Wellcome, UK Department for International Development., Translation: For the French translation of the abstract see Supplementary Materials section., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gamma irradiation (GI) is included in the CDC guidance on inactivation procedures to render a group of select agents and toxins nonviable. The Ebola virus falls within this group because it potentially poses a severe threat to public health and safety. To evaluate the impact of GI at a target dose of 50 kGy on neutralizing antibody titers induced by the rVSVΔG-ZEBOV-GP vaccine (V920), we constructed a panel of 48 paired human serum samples (GI-treated versus non-GI-treated) from healthy participants selected from a phase 3 study of V920 (study V920-012; NCT02503202). Neutralizing antibody titers were determined using a validated plaque-reduction neutralization test. GI of sera from V920 recipients was associated with approximately 20% reduction in postvaccination neutralizing antibody titers. GI was not associated with any change in pre-vaccination neutralizing antibody titers.

Heterologous prime-boost immunization regimens are a common strategy for many vaccines. DNA prime rAd5-GP boost immunization has been demonstrated to protect non-human primates against a lethal challenge of Ebola virus, a pathogen that causes fatal hemorrhagic disease in humans. This protection correlates with antibody responses and is also associated with IFNγ + TNFα + double positive CD8 + T-cells. In this study, we compared single DNA vs. multiple DNA prime immunizations, and short vs. long time intervals between the DNA prime and the rAd5 boost to evaluate the impact of these different prime-boost strategies on vaccine-induced humoral and cellular responses in non-human primates. We demonstrated that DNA/rAd5 prime-boost strategies can be tailored to induce either CD4 + T-cell or CD8 + T-cell dominant responses while maintaining a high magnitude antibody response. Additionally, a single DNA prime immunization generated a stable memory response that could be boosted by rAd5 3 years later. These results suggest DNA/rAd5 prime-boost provides a flexible platform that can be fine-tuned to generate desirable T-cell memory responses., 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 © 2021 Marcus, Thompson, Zhou, Bailey, Donaldson, Stanley, Asiedu, Foulds, Roederer, Moliva and Sullivan.)

Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 10 5 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 10 4 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge. IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV., (Copyright © 2021 American Society for Microbiology.)

Background: Sexual transmission chains of Ebola virus (EBOV) have been verified and linked to EBOV RNA persistence in semen, post-recovery. The rate of semen persistence over time, including the average duration of persistence among Ebola virus disease (EVD) survivors, is not well known. This cohort study aimed to analyze population estimates of EBOV RNA persistence rates in semen over time, and associated risk factors in a population of survivors from Sierra Leone., Methods and Findings: In this cohort study from May 2015 to April 2017 in Sierra Leone, recruitment was conducted in 2 phases; the first enrolled 100 male participants from the Western Area District in the capital of Freetown, and the second enrolled 120 men from the Western Area District and from Lungi, Port Loko District. Mean age of participants was 31 years. The men provided semen for testing, analyzed by quantitative reverse transcription PCR (qRT-PCR) for the presence of EBOV RNA. Follow-up occurred every 2 weeks until the endpoint, defined as 2 consecutive negative qRT-PCR results of semen specimen testing for EBOV RNA. Participants were matched with the Sierra Leone EVD case database to retrieve cycle threshold (Ct) values from the qRT-PCR analysis done in blood during acute disease. A purposive sampling strategy was used, and the included sample composition was compared to the national EVD survivor database to understand deviations from the general male survivor population. At 180 days (6 months) after Ebola treatment unit (ETU) discharge, the EBOV RNA semen positive rate was 75.4% (95% CI 66.9%-82.0%). The median persistence duration was 204 days, with 50% of men having cleared their semen of EBOV RNA after this time. At 270 days, persistence was 26.8% (95% CI 20.0%-34.2%), and at 360 days, 6.0% (95% CI 3.1%-10.2%). Longer persistence was significantly associated with severe acute disease, with probability of persistence in this population at 1 year at 10.1% (95% CI 4.6%-19.8%) compared to the probability approaching 0% for those with mild acute disease. Age showed a dose-response pattern, where the youngest men (≤25 years) were 3.17 (95% CI 1.60, 6.29) times more likely to be EBOV RNA negative in semen, and men aged 26-35 years were 1.85 (95% CI 1.04, 3.28) times more likely to be negative, than men aged >35 years. Among participants with both severe acute EVD and a higher age (>35 years), persistence remained above 20% (95% CI 6.0%-50.6%) at 1 year. Uptake of safe sex recommendations 3 months after ETU discharge was low among a third of survivors. The sample was largely representative of male survivors in Sierra Leone. A limitation of this study is the lack of knowledge about infectiousness., Conclusions: In this study we observed that EBOV RNA persistence in semen was a frequent phenomenon, with high population rates over time. This finding will inform forthcoming updated recommendations on risk reduction strategies relating to sexual transmission of EBOV. Our findings support implementation of a semen testing program as part of epidemic preparedness and response. Further, the results will enable planning of the magnitude of testing and targeted counseling needs over time., Competing Interests: The authors have declared that no competing interests exist.

Reston virus (RESTV), an ebolavirus, causes clinical disease in macaques but has yet only been associated with rare asymptomatic infections in humans. Its 2008 emergence in pigs in the Philippines raised concerns about food safety, pathogenicity, and zoonotic potential, questions that are still unanswered. Until today, the virulence of RESTV for pigs has remained elusive, with unclear pathogenicity in naturally infected animals and only one experimental study demonstrating susceptibility and evidence for shedding but no disease. Here we show that combined oropharyngeal and nasal infection of young (3- to 7-wk-old) Yorkshire cross pigs with RESTV resulted in severe respiratory disease, with most animals reaching humane endpoint within a week. RESTV-infected pigs developed severe cyanosis, tachypnea, and acute interstitial pneumonia, with RESTV shedding from oronasal mucosal membranes. Our studies indicate that RESTV should be considered a livestock pathogen with zoonotic potential., Competing Interests: The authors declare no competing interest.

COVID-19 is a highly contagious disease that has affected all African countries including the Democratic Republic of Congo (DRC). Formidable challenges limit precautionary measures which were instituted by the government to curb the pandemic. Insufficient COVID-19 testing laboratories, limited medical and personal protective equipment, and an inadequate number of health workers leave the country ill-equipped in the fight against the pandemic. Lack of assistance from the government to those who lost their jobs due to lockdown forced these individuals to go outside to find provisions, thus increasing the spread of the virus. Moreover, the fragile healthcare system is overburdened by civil conflicts and other epidemics and endemics amid the COVID-19 pandemic. The conflicts have led to thousands of deaths and hundreds of thousands of displacements and deprived many people of basic health services. The 11th outbreak of Ebola has been increasing at an alarming pace, and it is expected to soar because of a shortfall of funds and insufficient numbers of health workers. The DRC with the cooperation of regional powers needs to address these challenges in a manner similar to that used in the previous Ebola epidemics. Moreover, the government should have a balance in shifting the available resources between COVID-19 and other diseases. Until a vaccine is available, the DRC needs to be prudent when lifting restrictions to prevent explosion of new cases.

Background: The 2013-15 Ebola epidemic in West Africa was the largest so far recorded, and mainly affected three adjacent countries, Guinea, Liberia and Sierra Leone. The worst affected country (in terms of confirmed cases) was Sierra Leone. The present paper looks at the epidemic in Sierra Leone. The epidemic in this country was a concatenation of local outbreaks. These local outbreaks are not well characterized through analysis using standard numerical techniques. In part, this reflects difficulties in record collection at the height of the epidemic. This paper offers a different approach, based on application of field-based techniques of social investigation that provide a richer understanding of the epidemic., Methods: In a post-epidemic study (2016-18) of two districts (Bo and Moyamba) we use ethnographic data to reconstruct local infection pathways from evidence provided by affected communities, cross-referenced to records of the epidemic retained by the National Ebola Response Commission, now lodged in the Ebola Museum and Archive at Njala University. Our study documents and discusses local social and contextual factors largely missing from previously published studies., Results: Our major finding is that the epidemic in Sierra Leone was a series of local outbreaks, some of which were better contained than others. In those that were not well contained, a number of contingent factors helps explain loss of control. Several numerical studies have drawn attention to the importance of local heterogeneities in the Sierra Leone Ebola epidemic. Our qualitative study throws specific light on a number of elements that explain these heterogeneities: the role of externalities, health system deficiencies, cultural considerations and local coping capacities., Conclusions: Social issues and local contingencies explain the spread of Ebola in Sierra Leone and are key to understanding heterogeneities in epidemiological data. Integrating ethnographic research into epidemic-response is critical to properly understand the patterns of spread and the opportunities to intervene. This conclusion has significant implications for future interdisciplinary research and interpretation of standard numerical data, and consequently for control of epidemic outbreaks., Competing Interests: The authors have declared that no competing interests exist.

Mathematical modelling has successfully been used to provide quantitative descriptions of many viral infections, but for the Ebola virus, which requires biosafety level 4 facilities for experimentation, modelling can play a crucial role. Ebola virus modelling efforts have primarily focused on in vivo virus kinetics, e.g., in animal models, to aid the development of antivirals and vaccines. But, thus far, these studies have not yielded a detailed specification of the infection cycle, which could provide a foundational description of the virus kinetics and thus a deeper understanding of their clinical manifestation. Here, we obtain a diverse experimental data set of the Ebola virus infection in vitro, and then make use of Bayesian inference methods to fully identify parameters in a mathematical model of the infection. Our results provide insights into the distribution of time an infected cell spends in the eclipse phase (the period between infection and the start of virus production), as well as the rate at which infectious virions lose infectivity. We suggest how these results can be used in future models to describe co-infection with defective interfering particles, which are an emerging alternative therapeutic., Competing Interests: No authors have competing interests.

This study develops dose-response models for Ebolavirus using previously published data sets from the open literature. Two such articles were identified in which three different species of nonhuman primates were challenged by aerosolized Ebolavirus in order to study pathology and clinical disease progression. Dose groups were combined and pooled across each study in order to facilitate modeling. The endpoint of each experiment was death. The exponential and exact beta-Poisson models were fit to the data using maximum likelihood estimation. The exact beta-Poisson was deemed the recommended model because it more closely approximated the probability of response at low doses though both models provided a good fit. Although transmission is generally considered to be dominated by person-to-person contact, aerosolization is a possible route of exposure. If possible, this route of exposure could be particularly concerning for persons in occupational roles managing contaminated liquid wastes from patients being treated for Ebola infection and the wastewater community responsible for disinfection. Therefore, this study produces a necessary mathematical relationship between exposure dose and risk of death for the inhalation route of exposure that can support quantitative microbial risk assessment aimed at informing risk mitigation strategies including personal protection policies against occupational exposures., (© 2020 Society for Risk Analysis.)

The fusion of the viral and target cell membranes is a key step in the life cycle of all enveloped viruses. Here, a range of structural data is used to generate an evidence-based model of the active conformation of an archetypical type-I fusion protein, the Ebola glycoprotein 2 (GP2). The stability of the trimeric complex is demonstrated using molecular dynamics and validated by simulating the interaction of the complex with a lipid bilayer. In this model, the fusion peptides project away from the central helix bundle parallel to the target membrane. This maximizes contact with the host membrane, enhances lateral stability, and would explain why, when activated, viral fusion proteins are trimeric.

Patients and physicians worldwide are facing tremendous health care hazards that are caused by the ongoing severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) pandemic. Remdesivir (GS-5734) is the first approved treatment for severe coronavirus disease 2019 (COVID-19). It is a novel nucleoside analog with a broad antiviral activity spectrum among RNA viruses, including ebolavirus (EBOV) and the respiratory pathogens Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and SARS-CoV-2. First described in 2016, the drug was derived from an antiviral library of small molecules intended to target emerging pathogenic RNA viruses. In vivo , remdesivir showed therapeutic and prophylactic effects in animal models of EBOV, MERS-CoV, SARS-CoV, and SARS-CoV-2 infection. However, the substance failed in a clinical trial on ebolavirus disease (EVD), where it was inferior to investigational monoclonal antibodies in an interim analysis. As there was no placebo control in this study, no conclusions on its efficacy in EVD can be made. In contrast, data from a placebo-controlled trial show beneficial effects for patients with COVID-19. Remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. Although this is an important milestone in the fight against COVID-19, approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. Further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable., (Copyright © 2020 American Society for Microbiology.)

Multiple cell surface molecules including TAM receptors (TYRO3, AXL, and MERTK), a family of tyrosine kinase receptors, can serve as attachment receptors for Ebola virus (EBOV) entry into cells. The interaction of these receptors with EBOV particles is believed to trigger the initial internalization events that lead to macropinocytosis. However, the details of how these interactions lead to EBOV internalization have yet to be elucidated. Here, we screened receptor tyrosine kinase (RTK) inhibitors for anti-EBOV activity by using our previously established biologically contained Ebola virus that lacks the VP30 gene (EBOVΔVP30) and identified several RTKs, including human epidermal growth factor receptor 2 (HER2), as potential targets of anti-EBOV inhibitors and as novel host factors that have a role in EBOV infection. Of these identified RTKs, it was only HER2 whose knockdown by siRNAs impaired EBOVΔVP30-induced AKT1 phosphorylation, an event that is required for AKT1 activation and subsequent macropinocytosis. Stable expression of HER2 resulted in constitutive activation of AKT1, resulting in the enhancement of EBOVΔVP30 growth, EBOV GP-mediated entry, and macropinocytosis. Moreover, we found that HER2 interacts with the TAM receptors, and in particular forms a complex with TYRO3 and EBOVΔVP30 particles on the cell surface. Interestingly, HER2 was required for EBOVΔVP30-induced TYRO3 and AKT1 activation, but the other TAM receptors (TYRO3 and MERTK) were not essential for EBOVΔVP30-induced HER2 and AKT1 activation. Our findings demonstrate that HER2 plays an important role in EBOV entry and provide novel insights for the development of therapeutics against the virus., Competing Interests: The authors have declared that no competing interests exist.

The conduct of clinical trials during the West Africa Ebola outbreak in 2014 highlighted many ethical challenges. How these challenges were addressed, what clinical studies were conducted during that outbreak, and the lessons learned for dealing with future outbreaks were the subject of a National Academy of Medicine committee report titled Integrating Clinical Research into Epidemic Response: The Ebola Experience . This report suggested improvements for research during subsequent emerging or re-emerging outbreaks and is summarized in this review. We also discuss the current Ebola outbreak in the Democratic Republic of the Congo and highlight how the dialogue has changed and how successful clinical trials have been implemented. We conclude with a description of productive efforts to include pregnant women and children in therapeutic and vaccine trials during outbreaks that are currently ongoing.

Interaction between filovirus glycoprotein (GP) and the Niemann-Pick C1 (NPC1) protein is essential for membrane fusion during virus entry. Some single-nucleotide polymorphism (SNPs) in two surface-exposed loops of NPC1 are known to reduce viral infectivity. However, the dependence of differences in entry efficiency on SNPs remains unclear. Using vesicular stomatitis virus pseudotyped with Ebola and Marburg virus GPs, we investigated the cell-to-cell spread of viruses in cultured cells expressing NPC1 or SNP derivatives. Eclipse and virus-producing phases were assessed by in vitro infection experiments, and we developed a mathematical model describing spatial-temporal virus spread. This mathematical model fit the plaque radius data well from day 2 to day 6. Based on the estimated parameters, we found that SNPs causing the P424A and D508N substitutions in NPC1 most effectively reduced the entry efficiency of Ebola and Marburg viruses, respectively. Our novel approach could be broadly applied to other virus plaque assays., Competing Interests: The authors have declared that no competing interests exist.

Microbicides play critical roles in infection prevention and control of Ebola virus by decontaminating high-touch environmental surfaces (HITES), interrupting the virus-HITES-hands nexus. We evaluated the efficacy of formulations containing different microbicidal actives for inactivating Ebola virus-Makona strain (EBOV/Mak) on stainless-steel carriers per ASTM E2197-11. Formulations of sodium hypochlorite (NaOCl) (0.05-1%), ethanol (70%), chloroxylenol (PCMX) (0.12-0.48% by weight) in hard water, and a ready-to-use disinfectant spray with 58% ethanol (EDS), were tested at contact times of 0, or 0.5 to 10 min at ambient temperature. EBOV/Mak was inactivated (> 6 log 10 ) by 70% ethanol after contact times ≥ 2.5 min, by 0.5% and 1% NaOCl or EDS (> 4 log 10 ) at contact times ≥ 5 min, and by 0.12-0.48% PCMX (> 4.2 log 10 ) at contact times ≥ 5 min. Residual infectious virus in neutralized samples was assessed by passage on cells and evaluation for viral cytopathic effect. No infectious virus was detected in cells inoculated with EBOV/Mak exposed to NaOCl (0.5% or 1%), PCMX (0.12% to 0.48%), or EDS for ≥ 5 min. These results demonstrate ≥ 6 log 10 inactivation of EBOV/Mak dried on prototypic surfaces by EDS or formulations of NaOCl (≥ 0.5%), PCMX (≥ 0.12%), or 70% ethanol at contact times ≥ 5 min.

Besides the common Fc receptor (FcR)-mediated mechanism of antibody-dependent enhancement (ADE), Ebola virus (EBOV) is known to utilize the complement component C1q for ADE of infection. This mechanism is FcR-independent and mediated by cross-linking of virus-antibody-C1q complexes to cell surface C1q receptors, leading to enhanced viral entry into cells. Using confocal microscopy, we found that virus-like particles (VLPs) consisting of EBOV glycoprotein, nucleoprotein, and matrix protein attached to the surface of human kidney 293 cells more efficiently in the presence of an ADE monoclonal antibody and C1q than with the antibody or C1q alone, and that there was no significant difference in the efficiency of VLP uptake into endosomes between the C1q-mediated ADE and non-ADE entry. Accordingly, both ADE and non-ADE infection were similarly decreased by inhibitors of the signaling pathways known to be required for endocytosis. These results suggest that C1q-mediated ADE of EBOV infection is simply caused by increased attachment of virus particles to the cell surface, which is distinct from the mechanism of FcR-mediated ADE requiring intracellular signaling to promote phagocytosis/macropinocytosis., Competing Interests: The authors have declared that no competing interests exist.

Background: Within the past decade, Africa has faced several recurrent outbreaks of Ebola virus disease (EVD), including the 2014-2016 outbreak in West Africa and the recent 2018-2020 Kivu outbreak in the Democratic Republic of Congo. The study thus aimed at quantifying and mapping the scientific output of EVD research published within 2010-2020 though a bibliometric perspective., Methods: EVD-related publications from 2010 to 2020 were retrieved from the Web of Science (WoS) and Scopus databases by using the keywords 'Ebola', 'Ebola Virus Disease', 'Ebolas', and 'ebolavirus'. Biblioshiny software (using R-studio cloud) was used to categorise and evaluate authors', countries' and journals' contribution. VOSviewer was used for network visualisation., Results: According to the used search strategy, a total of 3865 and 3848 EVD documents were published in WoS and Scopus, respectively. The average citation per document was 16.1 (WoS) and 16.3 (Scopus). The results show an overall increase in the publication trend within the study period. The leading countries in EVD research were the USA and UK, with over 100 papers in both databases, including Nigeria and South Africa. NIAID and CDC-USA were the most influential institutions, while "Infectious Diseases" and "Medicine" were the most decisive research fields. The most contributing authors included Feldmann H and Qiu XG with over 60 papers in each database, while Journal of Infectious Diseases was the most crucial journal. The most cited article was from Aylward et al. published in 2014, while recent years displayed a keyword focus on "double-blind", "efficacy", "ring vaccination" and "drug effect"., Conclusion: This bibliometric analysis provides an updated historical perspective of progress in EVD research and has highlighted the role played by various stakeholders. However, the contribution of African countries and institutions is not sufficiently reflected, implying a need for increased funding and focus on EVD research for effective prevention and control., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Joseph Kawuki et al.)

Vaccine solutions rarely reach the public until after an outbreak abates; an Ebola vaccine was approved 5 years after peak outbreak and SARS, MERS, and Zika vaccines are still in clinical development. Despite massive leaps forward in rapid science, other regulatory bottlenecks are hamstringing the global effort for pandemic vaccines., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK's use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks.

Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1 + cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry. IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics., (Copyright © 2020 Bo et al.)

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain. IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA., (Copyright © 2020 American Society for Microbiology.)

Ongoing Ebola virus disease outbreaks in the Democratic Republic of the Congo follow the largest recorded outbreak in Western Africa (2013-2016). To combat outbreaks, testing of medical countermeasures (therapeutics or vaccines) requires a well-defined, reproducible, animal model. Here we present Ebola virus disease kinetics in 24 Chinese-origin rhesus monkeys exposed intramuscularly to a highly characterized, commercially available Kikwit Ebola virus Filovirus Animal Non-Clinical Group (FANG) stock. Until reaching predetermined clinical disease endpoint criteria, six animals underwent anesthesia for repeated clinical sampling and were compared to six that did not. Groups of three animals were euthanized and necropsied on days 3, 4, 5, and 6 post-exposure, respectively. In addition, three uninfected animals served as controls. Here, we present detailed characterization of clinical and laboratory disease kinetics and complete blood counts, serum chemistries, Ebola virus titers, and disease kinetics for future medical countermeasure (MCM) study design and control data. We measured no statistical difference in hematology, chemistry values, or time to clinical endpoint in animals that were anesthetized for clinical sampling during the acute disease compared to those that were not.

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus.

Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels. We find that transfer functions are robust across a wide range of stimulation paradigms and animals, and reveal a second vascular component of neurovascular coupling upon very strong stimulation. We propose that transfer functions can be considered as reliable quantitative reporters to follow neurovascular coupling dynamics.

Introduction: Ebola Virus Disease (EVD) is the international health emergency paradigm due to its epidemiological presentation pattern, impact on public health, resources necessary for its control, and need for a national and international response., Study Objective: The objective of this work is to study the evolution and progression of the epidemiological presentation profile of Ebola disease outbreaks since its discovery in 1976 to the present, and to explore the possible reasons for this evolution from different perspectives., Methods: Retrospective observational study of 38 outbreaks of Ebola disease occurred from 1976 through 2019, excluding laboratory accidents. United Nations agencies and programs; Ministries of Health; the US Centers for Disease Control and Prevention (CDC); ReliefWeb; emergency nongovernmental organizations; and publications indexed in PubMed, EmBase, and Clinical Key have been used as sources of data. Information on the year of the outbreak, date of beginning and end, duration of the outbreak in days, number of cases, number of deaths, population at risk, geographic extension affected in Km2, and time of notification of the first cases to the World Health Organization (WHO) have been searched and analyzed., Results: Populations at risk have increased (P = .024) and the geographical extent of Ebola outbreaks has grown (P = .004). Reporting time of the first cases of Ebola to WHO has been reduced (P = .017) and case fatality (P = .028) has gone from 88% to 62% in the period studied. There have been differences (P = .04) between the outbreaks produced by the Sudan and Zaire strains of the virus, both in terms of duration and case fatality ratio (Sudan strain 74.5 days on average and 62.7% of case fatality ratio versus Zaire strain with 150 days on average and 55.4% case fatality ratio)., Conclusion: There has been a change in the epidemiological profile of the Ebola outbreaks from 1976 through 2019 with an increase in the geographical extent of the outbreaks and the population at risk, as well as a significant decrease in the outbreaks case fatality rate. There have been advances in the detection and management capacity of outbreaks, and the notification time to the WHO has been reduced. However, there are social, economic, cultural, and political obstacles that continue to greatly hinder a more efficient epidemiological approach to Ebola disease, mainly in Central Africa.