Impact of Ebola epidemics on the daily operation of existing systems in Eastern Democratic Republic of the Congo: a brief review.

AIMS: to provide insights into the recent Ebola virus disease (EVD) outbreaks on different aspects of daily life in the Democratic Republic of the Congo and propose possible solutions. METHODS: We collected information regarding the effects of EVD outbreaks on existing systems in the eastern part of the Democratic Republic of the Congo (DRC). We searched the PubMed database using the terms "impact effect Ebola outbreak system", "Management Ebola Poor Resources Settings", "Health Economic Challenges Ebola" and "Economic impact Ebola systems." Only studies focusing on epidemiology, diagnostics, sequencing, vaccination, therapeutics, ecology, work force, governance, healthcare provision and health system, and social, political, and economic aspects were considered. The search included the electronic archives of EVD outbreak reports from government and partners. RESULTS: EVD outbreaks negatively impacts the functions of countries. The disruption in activities is proportional to the magnitude of the epidemic and slows down the transport of goods, decreases the region's tourist appeal, and increases 'brain drain'. Most low- and medium-income countries, such as the DRC, do not have a long-term holistic emergency plan for unexpected situations or sufficient resources to adequately implement countermeasures against EVD outbreaks. Although the DRC has acquired sufficient expertise in diagnostics, genomic sequencing, administration of vaccines and therapeutics, clinical trials, and research activities, deployment, operation, and maintenance of these expertise and associated tools remains a concern. LIMITATIONS: Despite the data search extension, additional reports addressing issues related to social aspects of EVD outbreaks in DRC were not retrieved. CONCLUSION: National leadership has not yet taken the lead in strategic, operational, or financial aspects. Therefore, national leaders should double their efforts and awareness to encourage local fundraising, sufficient budget al.location, infrastructure construction, equipment provision, and staff training, to effectively support a holistic approach in response to outbreaks, providing effective results, and all types of research activities.

Cheminformatics-Based Study Identifies Potential Ebola VP40 Inhibitors.

The Ebola virus (EBOV) is still highly infectious and causes severe hemorrhagic fevers in primates. However, there are no regulatorily approved drugs against the Ebola virus disease (EVD). The highly virulent and lethal nature of EVD highlights the need to develop therapeutic agents. Viral protein 40 kDa (VP40), the most abundantly expressed protein during infection, coordinates the assembly, budding, and release of viral particles into the host cell. It also regulates viral transcription and RNA replication. This study sought to identify small molecules that could potentially inhibit the VP40 protein by targeting the N-terminal domain using an in silico approach. The statistical quality of AutoDock Vina's capacity to discriminate between inhibitors and decoys was determined, and an area under the curve of the receiver operating characteristic (AUC-ROC) curve of 0.791 was obtained. A total of 29,519 natural-product-derived compounds from Chinese and African sources as well as 2738 approved drugs were successfully screened against VP40. Using a threshold of -8 kcal/mol, a total of 7, 11, 163, and 30 compounds from the AfroDb, Northern African Natural Products Database (NANPDB), traditional Chinese medicine (TCM), and approved drugs libraries, respectively, were obtained after molecular docking. A biological activity prediction of the lead compounds suggested their potential antiviral properties. In addition, random-forest- and support-vector-machine-based algorithms predicted the compounds to be anti-Ebola with IC50 values in the micromolar range (less than 25 µM). A total of 42 natural-product-derived compounds were identified as potential EBOV inhibitors with desirable ADMET profiles, comprising 1, 2, and 39 compounds from NANPDB (2-hydroxyseneganolide), AfroDb (ZINC000034518176 and ZINC000095485942), and TCM, respectively. A total of 23 approved drugs, including doramectin, glecaprevir, velpatasvir, ledipasvir, avermectin B1, nafarelin acetate, danoprevir, eltrombopag, lanatoside C, and glycyrrhizin, among others, were also predicted to have potential anti-EBOV activity and can be further explored so that they may be repurposed for EVD treatment. Molecular dynamics simulations coupled with molecular mechanics Poisson-Boltzmann surface area calculations corroborated the stability and good binding affinities of the complexes (-46.97 to -118.9 kJ/mol). The potential lead compounds may have the potential to be developed as anti-EBOV drugs after experimental testing.

Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding.

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.

Ebola Entry Inhibitors Discovered from Maesa perlarius.

Ebola virus disease (EVD), a disease caused by infection with Ebola virus (EBOV), is characterized by hemorrhagic fever and a high case fatality rate. With limited options for the treatment of EVD, anti-Ebola viral therapeutics need to be urgently developed. In this study, over 500 extracts of medicinal plants collected in the Lingnan region were tested against infection with Ebola-virus-pseudotyped particles (EBOVpp), leading to the discovery of Maesa perlarius as an anti-EBOV plant lead. The methanol extract (MPBE) of the stems of this plant showed an inhibitory effect against EBOVpp, with an IC50 value of 0.52 µg/mL, which was confirmed by testing the extract against infectious EBOV in a biosafety level 4 laboratory. The bioassay-guided fractionation of MPBE resulted in three proanthocyanidins (procyanidin B2 (1), procyanidin C1 (2), and epicatechin-(4ß→8)-epicatechin-(4ß→8)-epicatechin-(4ß→8)-epicatechin (3)), along with two flavan-3-ols ((+)-catechin (4) and (-)-epicatechin (5)). The IC50 values of the compounds against pseudovirion-bearing EBOV-GP ranged from 0.83 to 36.0 µM, with 1 as the most potent inhibitor. The anti-EBOV activities of five synthetic derivatives together with six commercially available analogues, including EGCG ((-)-epigallocatechin-3-O-gallate (8)), were further investigated. Molecular docking analysis and binding affinity measurement suggested the EBOV glycoprotein could be a potential molecular target for 1 and its related compounds.

The Methanolic Extract of Perilla frutescens Robustly Restricts Ebola Virus Glycoprotein-Mediated Entry.

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBOV infection.

Review: Insights on Current FDA-Approved Monoclonal Antibodies Against Ebola Virus Infection.

The unprecedented 2013-2016 West Africa Ebola outbreak accelerated several medical countermeasures (MCMs) against Ebola virus disease (EVD). Several investigational products (IPs) were used throughout the outbreak but were not conclusive for efficacy results. Only the Randomized Controlled Trial (RCT) on ZMapp was promising but inconclusive. More recently, during the second-largest Ebola outbreak in North Kivu and Ituri provinces, Democratic Republic of the Congo (DRC), four IPs, including one small molecule (Remdesivir), two monoclonal antibody (mAb) cocktails (ZMapp and REGN-EB3) and a single mAb (mAb114), were evaluated in an RCT, the Pamoja Tulinde Maisha (PALM) study. Two products (REGN-EB3 and mAb114) demonstrated efficacy as compared to the control arm, ZMapp. There were remarkably few side effects recorded in the trial. The FDA approved both medications in this scientifically sound study, marking a watershed moment in the field of EVD therapy. These products can be produced relatively inexpensively and can be stockpiled. The administration of mAbs in EVD patients appears to be safe and effective, while several critical knowledge gaps remain; the impact of early administration of Ebola-specific mAbs on developing a robust immune response for future Ebola virus exposure is unknown. The viral mutation escape, leading to resistance, presents a potential limitation for single mAb therapy; further improvements need to be explored. Understanding the contribution of Fc-mediated antibody functions such as antibody-dependent cellular cytotoxicity (ADCC) of those approved mAbs is still critical. The potential merit of combination therapy and post-exposure prophylaxis (PEP) need to be demonstrated. Furthermore, the PALM trial has accounted for 30% of mortality despite the administration of specific treatments. The putative role of EBOV soluble Glycoprotein (sGP) as a decoy to the immune system, the virus persistence, and relapses might be investigated for treatment failure. The development of pan-filovirus or pan-species mAbs remains essential for protection. The interaction between FDA-approved mAbs and vaccines remains unclear and needs to be investigated. In this review, we summarize the efficacy and safety results of the PALM study and review current research questions for the further development of mAbs in pre-exposure or emergency post-exposure use.

Assessing the determinants of Ebola virus disease transmission in Baka Community of the Tropical Rainforest of Cameroon.

BACKGROUND: Ebola virus disease (EVD) is a severe, often fatal illness in humans and nonhuman primates caused by the Ebola virus. The recently approved rVSV-EBOV vaccine is not available in many high-risk countries hence prevention is paramount. The design of effective prevention interventions requires an understanding of the factors that expose communities at risk. It was based on this that we investigated the Baka community of Abong-Mbang Health District in tropical rain forest of Cameroon. METHODS: A cross-sectional study was conducted with participants randomly selected from 13 villages in Abong-Mbang by multi-stage cluster sampling. A questionnaire was administered to them to collect demographic information, data on knowledge of EVD, their feeding and health-seeking behaviour. Data was analyzed using the chi-square test. Knowledge of EVD was assessed using an 8 item Morisky Scale. An adapted Threat Capability Basic Risk Assessment Guide was used to determine their risk of exposure to infection. RESULTS: A total of 510 participants, most of whom were hunters (31.4%), farmers (29.8%), and had primary education (62.7%), were included in this study. Although 83.3% participants had heard of EVD, most (71%) did not know its cause. Their source of information was mainly informal discussions in the community (49%). Misconceptions were identified with regards to the cause and mode of transmission. Only 43.1% accepted EVD could be transmitted from human-to-human. Generally, participants' knowledge of EVD was poor. Demographic factors such as level of education, occupation and ethnic group significantly affected knowledge of EVD. The majority of participants were at a very high risk of exposure to infection as they consumed various forms of bush meat and were involved in other risky practices such as scarification and touching of corpses. Although over half of participants seek medical care, most of them preferred traditional medicine. Socio-cultural and service-related factors were deterrent factors to medical care. CONCLUSION: Participants generally had poor knowledge of EVD and were at high risk of infection. We recommend rigorous sensitization campaigns in the study area to educate the population on EVD and clarify the misconceptions identified. EVD surveillance is recommended particularly as outbreaks have often been reported in the Congo Basin.

A Surrogate Animal Model for Screening of Ebola and Marburg Glycoprotein-Targeting Drugs Using Pseudotyped Vesicular Stomatitis Viruses.

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.

Flavaglines as natural products targeting eIF4A and prohibitins: From traditional Chinese medicine to antiviral activity against coronaviruses.

Flavaglines are cyclopenta[b]benzofurans found in plants of the genus Aglaia, several species of which are used in traditional Chinese medicine. These compounds target the initiation factor of translation eIF4A and the scaffold proteins prohibitins-1 and 2 (PHB1/2) to exert various pharmacological activities, including antiviral effects against several types of viruses, including coronaviruses. This review is focused on the antiviral effects of flavaglines and their therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Association between multivitamin supplementation and mortality among patients with Ebola virus disease: An international multisite cohort study.

INTRODUCTION: Micronutrient supplementation is recommended in Ebola Virus Disease (EVD) care; however, there is limited data on its therapeutic effects. METHODS: This retrospective cohort study included patients with EVD admitted to five Ebola Treatment Units (ETU) in Sierra Leone and Liberia during September 2014 to December 2015. A uniform protocol was used to guide ETU care, however, due to supply limitations, only a subset of patients received multivitamins. Data on demographics, clinical characteristics, and laboratory testing was collected. The outcome of interest was facility-based mortality and the primary predictor was multivitamin supplementation initiated within 48 h of admission. The multivitamin formulations included: thiamine, riboflavin, niacin and vitamins A, C, and D3. Propensity score models (PSM) were used to match patients based on covariates associated with multivitamin administration and mortality. Mortality between cases treated and untreated within 48 h of admission were compared using generalized estimating equations to calculate relative risk with bootstrap methods employed to assess statistical significance. RESULTS: There were 424 patients with EVD who had sufficient treatment data for analysis, of which 261 (61.6%) had daily multivitamins initiated within 48 h of admission. The mean age of the cohort was 30.5 years and 59.4% were female. In the propensity score matched analysis, mortality was 53.5% among patients receiving multivitamins and 66.2% among patients not receiving multivitamins, resulting in a relative risk for mortality of 0.81 (p = 0.03) for patients receiving multivitamins. CONCLUSION: Early multivitamin supplementation was associated with lower overall mortality. Further research on the impact of micronutrient supplementation in EVD is warranted.

Ebola in the Eastern Democratic Republic of Congo: One Health approach to infectious disease control.

The Democratic Republic of Congo (DRC) is facing its tenth outbreak of Ebola virus disease (EVD), in North-Kivu and Ituri provinces. This is the second most deadly EVD outbreak in history, after the one that occurred in West Africa in 2014. The DRC Ministry of Health (MoH), supported by the World Health Organization (WHO) and a range of regional and international partners, are implementing EVD response plans in these affected areas such as screening of suspect cases at points of entry, case detection, contact tracing, laboratory testing, case management and infection prevention and control, safe and dignified burials, ring vaccination (this involves vaccination of infected individuals, direct contacts of infected individuals and contacts of their contacts), and therapeutics, community mobilization and free access to healthcare services. Despite these efforts, there has been a sharp rise in the number of confirmed cases within the identified affected areas, and due to a number of challenges unique to DRC, there has been an expansion in the geographical extent of transmission. The significance of the proximity of these regions to wildlife and the Virunga National Park is questionable in the EVD transmission dynamics. The close interaction between human, animal, and environmental factors, in combination with high population movement due to regular rebel attacks in these regions, suggest the need for the integration of the One Health approach in the holistic response plans for control and prevention of EVD. This paper seeks to highlight the implications and importance of a One Health-based approach into the infectious diseases control program implementation in DRC.

Nutritional Care for Patients with Ebola Virus Disease.

During the Ebola virus disease (EVD) outbreak of 2014-2016 in West Africa, practitioners faced challenges providing nutritional care for patients in Ebola treatment units (ETUs). The current EVD outbreak in the Democratic Republic of the Congo demonstrates the need to understand lessons learned from previous outbreaks and to update nutritional guidelines. We conducted a literature review to identify articles that included nutrition as an integral part of supportive care. We found little information on the specific nutritional care or practical challenges within an ETU. This review showed that nutritional care for EVD patients is poorly described, and therefore the optimal composition and implementation of nutritional care remain unknown. We recommend that researchers and practitioners share specific and practical details of their experiences in providing nutritional support within ETUs to expand the knowledge base and ultimately improve the nutritional care for an increasingly prevalent patient population.

A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures.

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.

Vitamin A Supplementation Was Associated with Reduced Mortality in Patients with Ebola Virus Disease during the West African Outbreak.

BACKGROUND: Micronutrient supplementation is recommended in Ebola virus disease (EVD); however, there are limited data on therapeutic impacts of specific micronutrients. OBJECTIVE: To evaluate the association between vitamin A supplementation and mortality in EVD. METHODS: This retrospective cohort included patients with EVD admitted to 5 International Medical Corps Ebola Treatment Units (ETUs) in 2 countries during 2014-2015. Protocolized treatments with micronutrients were used at all ETUs: however, because of resource constraints, only a subset of patients received vitamin A. Standardized data on demographics, clinical characteristics, malaria status, and Ebola viral loads (cycle threshold values) were collected. The outcome of interest was mortality between cases treated with 200,000 IU of vitamin A on care days 1 and/or 2, and those not. Propensity scores based on the first 48 h of care were derived using covariates of age, ETU duration, malaria status, cycle threshold values, and clinical symptoms. Patients were matched 1:1 using nearest neighbors with replacement. Mortality between cases treated and not treated with vitamin A was compared using generalized estimating equations to calculate RR with associated 95% CI. RESULTS: There were 424 cases analyzed, of which 330 (77.8%) were treated with vitamin A. The mean age was 30.5 y and 40.3% were men. The most common symptoms were diarrhea (85.6%), anorexia (80.7%), and abdominal pain (76.9%). Mortality proportions among cases treated and not treated with vitamin A were 55.0% and 71.9%, respectively. In the propensity-matched analysis, mortality was significantly lower among cases receiving vitamin A (RR = 0.77, 95% CI: 0.59, 0.99; P = 0.041). In a subgroup analysis of patients treated with multivitamins already containing vitamin A, additional vitamin A supplementation did not impact mortality. CONCLUSION: Early vitamin A supplementation was associated with reduced mortality in patients with EVD, and should be further studied and considered for use in future epidemics.

Contemporary Anti-Ebola Drug Discovery Approaches and Platforms.

The Ebola virus has a grave potential to destabilize civil society as we know it. The past few deadly Ebola outbreaks were unprecedented in size: The 2014-15 Ebola West Africa outbreak saw the virus spread from the epicenter through to Guinea, Sierra Leone, Nigeria, Congo, and Liberia. The 2014-15 Ebola West Africa outbreak was associated with almost 30,000 suspected or confirmed cases and over 11,000 documented deaths. The more recent 2018 outbreak in the Democratic Republic of Congo has so far resulted in 216 suspected or confirmed cases and 139 deaths. There is a general acceptance within the World Health Organization (WHO) and the Ebola outbreak response community that future outbreaks will become increasingly more frequent and more likely to involve intercontinental transmission. The magnitude of the recent outbreaks demonstrated in dramatic fashion the shortcomings of our mass casualty disease response capabilities and lack of therapeutic modalities for supporting Ebola outbreak prevention and control. Currently, there are no approved drugs although vaccines for human Ebola virus infection are in the trial phases and some potential treatments have been field tested most recently in the Congo Ebola outbreak. Treatment is limited to pain management and supportive care to counter dehydration and lack of oxygen. This underscores the critical need for effective antiviral drugs that specifically target this deadly disease. This review examines the current approaches for the discovery of anti-Ebola small molecule or biological therapeutics, their viral targets, mode of action, and contemporary platforms, which collectively form the backbone of the anti-Ebola drug discovery pipeline.

Intranasal vaccination with ebola virus GP amino acids 258-601 protects mice against lethal challenge.

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.

Ebola: Lessons on Vaccine Development.

The West African Ebola virus (EBOV) epidemic has fast-tracked countermeasures for this rare, emerging zoonotic pathogen. Until 2013-2014, most EBOV vaccine candidates were stalled between the preclinical and clinical milestones on the path to licensure, because of funding problems, lack of interest from pharmaceutical companies, and competing priorities in public health. The unprecedented and devastating epidemic propelled vaccine candidates toward clinical trials that were initiated near the end of the active response to the outbreak. Those trials did not have a major impact on the epidemic but provided invaluable data on vaccine safety, immunogenicity, and, to a limited degree, even efficacy in humans. There are plenty of lessons to learn from these trials, some of which are addressed in this review. Better preparation is essential to executing an effective response to EBOV in the future; yet, the first indications of waning interest are already noticeable.

High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system.

The massive epidemic of Ebola virus disease (EVD) in West Africa, followed in recent months by two outbreaks in the Democratic Republic of the Congo, underline the importance of this severe disease. Because Ebola virus (EBOV) must be manipulated under biosafety level 4 (BSL4) containment, the discovery and development of virus-specific therapies have been hampered. Recently, a transient transfection-based transcription- and replication competent virus-like particle (trVLP) system was described, enabling modeling of the entire EBOV life cycle under BSL2 conditions. Using this system, we optimized the condition for bulk co-transfection of multiple plasmids, developed a luciferase reporter-based assay in 384-well microtiter plates, and performed a high-throughput screening (HTS) campaign of an 8,354-compound collection consisting of U.S. Food & Drug Administration (FDA) -approved drugs, bioactives, kinase inhibitors, and natural products in duplicates. The HTS achieved a good signal-to-background ratio with a low percent coefficient of variation resulting in Z' = 0.7, and data points were reproducible with R2 = 0.89, indicative of a robust assay. After applying stringent hit selection criteria of ≥70% EBOV trVLP inhibition and ≥70% cell viability, 381 hits were selected targeting early, entry, and replication steps and 49 hits targeting late, maturation, and secretion steps in the viral life cycle. Of the total 430 hits, 220 were confirmed by dose-response analysis in the primary HTS assay. They were subsequently triaged by time-of-addition assays, then clustered and ranked according to their chemical structures, biological functions, therapeutic index, and maximum inhibition. Several novel drugs have been identified to very efficiently inhibit EBOV. Interestingly, most showed pharmacological activity in treatments for central nervous system-related diseases. We developed and screened an HTS assay using the novel EBOV trVLP system. Newly identified inhibitors are useful tools to study the poorly understood EBOV life cycle. In addition, they also provide opportunities to either repurpose FDA-approved drugs or develop novel viral interventions to combat EVD.

Identification of a small molecule inhibitor of Ebola virus genome replication and transcription using in silico screening.

Ebola virus (EBOV) causes a severe haemorrhagic fever in humans and has a mortality rate over 50%. With no licensed drug treatments available, EBOV poses a significant threat. Investigations into possible therapeutics have been severely hampered by the classification of EBOV as a BSL4 pathogen. Here, we describe a drug discovery pathway combining in silico screening of compounds predicted to bind to a hydrophobic pocket on the nucleoprotein (NP); with a robust and rapid EBOV minigenome assay for inhibitor validation at BSL2. One compound (MCCB4) was efficacious (EC50 4.8 µM), exhibited low cytotoxicity (CC50 > 100 µM) and was specific, with no effect on either a T7 RNA polymerase driven firefly luciferase or a Bunyamwera virus minigenome. Further investigations revealed that this small molecule inhibitor was able to outcompete established replication complexes, an essential aspect for a potential EBOV treatment.