Ebola Virus Infection Research Articles

A Pan-Ebolavirus Monoclonal Antibody Cocktail Provides Protection Against Ebola and Sudan Viruses.

Filoviruses, including ebolaviruses and marburgviruses, can cause severe and often fatal disease in humans. Over the past several years, antibody therapy has emerged as a promising strategy for the treatment of filovirus disease. Here, we describe 2 distinct cross-reactive monoclonal antibodies (mAbs) isolated from mice immunized with recombinant vesicular stomatitis virus-based filovirus vaccines. Both mAbs recognized the glycoproteins of multiple different ebolaviruses and exhibited broad but differential in vitro neutralization activities against these viruses. By themselves, each mAb provided partial to full protection against Ebola virus in mice, and in combination, the mAbs provided 100% protection against Sudan virus challenge in guinea pigs. This study identified novel mAbs that were elicited through immunization and able to provide protection from ebolavirus infection, thus enriching the pool of candidate therapeutics for treating Ebola disease.

A Recombinant Vesicular Stomatitis Virus-Based Vaccine Provides Postexposure Protection Against Bundibugyo Ebolavirus Infection.

The filovirus Bundibugyo virus (BDBV) causes severe disease with a mortality rate of approximately 20%-51%. The only licensed filovirus vaccine in the United States, Ervebo, consists of a recombinant vesicular stomatitis virus (rVSV) vector that expresses Ebola virus (EBOV) glycoprotein (GP). Ervebo was shown to rapidly protect against fatal Ebola disease in clinical trials; however, the vaccine is only indicated against EBOV. Recent outbreaks of other filoviruses underscore the need for additional vaccine candidates, particularly for BDBV infections. To examine whether the rVSV vaccine candidate rVSVΔG/BDBV-GP could provide therapeutic protection against BDBV, we inoculated seven cynomolgus macaques with 1000 plaque-forming units of BDBV, administering rVSVΔG/BDBV-GP vaccine to 6 of them 20-23 minutes after infection. Five of the treated animals survived infection (83%) compared to an expected natural survival rate of 21% in this macaque model. All treated animals showed an early circulating immune response, while the untreated animal did not. Surviving animals showed evidence of both GP-specific IgM and IgG production, while animals that succumbed did not produce significant IgG. This small, proof-of-concept study demonstrated early treatment with rVSVΔG/BDBV-GP provides a survival benefit in this nonhuman primate model of BDBV infection, perhaps through earlier initiation of adaptive immunity.

Magnetic Resonance Imaging for Monitoring of Hepatic Disease Induced by Ebola Virus: a Nonhuman Primate Proof-of-Concept Study.

Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD). However, the role of hepatic involvement in EVD progression is understudied. Medical imaging in established animal models of EVD (e.g., nonhuman primates [NHPs]) can be a strong complement to traditional assays to better investigate this pathophysiological process in vivo and noninvasively. In this proof-of-concept study, we used longitudinal multiparametric magnetic resonance imaging (MRI) to characterize liver morphology and function in nine rhesus monkeys after exposure to Ebola virus (EBOV). Starting 5 days postexposure, MRI assessments of liver appearance, morphology, and size were consistently compatible with the presence of hepatic edema, inflammation, and congestion, leading to significant hepatomegaly at necropsy. MRI performed after injection of a hepatobiliary contrast agent demonstrated decreased liver signal on the day of euthanasia, suggesting progressive hepatocellular dysfunction and hepatic secretory impairment associated with EBOV infection. Importantly, MRI-assessed deterioration of biliary function was acute and progressed faster than changes in serum bilirubin concentrations. These findings suggest that longitudinal quantitative in vivo imaging may be a useful addition to standard biological assays to gain additional knowledge about organ pathophysiology in animal models of EVD. IMPORTANCE Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD), but the contribution of hepatic pathophysiology to EVD progression is not fully understood. Noninvasive medical imaging of liver structure and function in well-established animal models of disease may shed light on this important aspect of EVD. In this proof-of-concept study, we used longitudinal magnetic resonance imaging (MRI) to characterize liver abnormalities and dysfunction in rhesus monkeys exposed to Ebola virus. The results indicate that in vivo MRI may be used as a noninvasive readout of organ pathophysiology in EVD and may be used in future animal studies to further characterize organ-specific damage of this condition, in addition to standard biological assays.

A Highly Attenuated Panfilovirus VesiculoVax Vaccine Rapidly Protects Nonhuman Primates Against Marburg Virus and 3 Species of Ebola Virus.

The family Filoviridae consists of several virus members known to cause significant mortality and disease in humans. Among these, Ebola virus (EBOV), Marburg virus (MARV), Sudan virus (SUDV), and Bundibugyo virus (BDBV) are considered the deadliest. The vaccine, Ervebo, was shown to rapidly protect humans against Ebola disease, but is indicated only for EBOV infections with limited cross-protection against other filoviruses. Whether multivalent formulations of similar recombinant vesicular stomatitis virus (rVSV)-based vaccines could likewise confer rapid protection is unclear. Here, we tested the ability of an attenuated, quadrivalent panfilovirus VesiculoVax vaccine (rVSV-Filo) to elicit fast-acting protection against MARV, EBOV, SUDV, and BDBV. Groups of cynomolgus monkeys were vaccinated 7 days before exposure to each of the 4 viral pathogens. All subjects (100%) immunized 1 week earlier survived MARV, SUDV, and BDBV challenge; 80% survived EBOV challenge. Survival correlated with lower viral load, higher glycoprotein-specific immunoglobulin G titers, and the expression of B-cell-, cytotoxic cell-, and antigen presentation-associated transcripts. These results demonstrate multivalent VesiculoVax vaccines are suitable for filovirus outbreak management. The highly attenuated nature of the rVSV-Filo vaccine may be preferable to the Ervebo "delta G" platform, which induced adverse events in a subset of recipients.

Pathogenesis of Aerosolized Ebola Virus Variant Makona in Nonhuman Primates.

Highly pathogenic filoviruses such as Ebola virus (EBOV) hold capacity for delivery by artificial aerosols, and thus potential for intentional misuse. Previous studies have shown that high doses of EBOV delivered by small-particle aerosol cause uniform lethality in nonhuman primates (NHPs), whereas only a few small studies have assessed lower doses in NHPs. To further characterize the pathogenesis of EBOV infection via small-particle aerosol, we challenged cohorts of cynomolgus monkeys with low doses of EBOV variant Makona, which may help define risks associated with small particle aerosol exposures. Despite using challenge doses orders of magnitude lower than previous studies, infection via this route was uniformly lethal across all cohorts. Time to death was delayed in a dose-dependent manner between aerosol-challenged cohorts, as well as in comparison to animals challenged via the intramuscular route. Here, we describe the observed clinical and pathological details including serum biomarkers, viral burden, and histopathological changes leading to death. Our observations in this model highlight the striking susceptibility of NHPs, and likely humans, via small-particle aerosol exposure to EBOV and emphasize the need for further development of diagnostics and postexposure prophylactics in the event of intentional release via deployment of an aerosol-producing device.

Single-cell profiling of lncRNA expression during Ebola virus infection in rhesus macaques

Single-cell profiling of lncRNA expression during Ebola virus infection in rhesus macaques

Single-cell profiling of lncRNA expression during Ebola virus infection in rhesus macaques

Single-cell profiling of lncRNA expression during Ebola virus infection in rhesus macaques

Survivors of Ebola virus disease exhibit elevated levels of autoantibodies

Abstract Survivors of Ebola virus disease (EVD) report a wide range of symptoms following recovery from acute viral infection, but underlying mechanisms are unclear. Many of these symptoms, collectively termed Post-Ebola Syndrome (PES), occur in the absence of detectable virus and resemble symptoms associated with autoimmune disease, such as arthralgia and myalgia, suggesting that EVD survivors may be experiencing a virus-induced autoimmune response. Studies in mice have shown that mice develop autoantibodies (autoAb) against HSP-60 and double stranded (ds) DNA that can bind to host tissues following Ebola virus (EBOV) infection. To determine if human EVD survivors also develop autoAb that may be associated with PES development, we profiled the humoral immune response of 354 survivors and 195 household contacts (HHC) from the 2014–2016 outbreak in Sierra Leone. Using Systems Serology, we measured the levels of antibodies against common autoantigens and found that EVD survivors had elevated levels of autoAb against dsDNA, HSP-60, IFNα, and citrullinated histone compared to HHC. As a subset of HHC were seropositive for EBOV glycoprotein (GP)-specific antibodies, separation of HHC into GP-seropositive and seronegative groups showed that the levels of autoAb in GP-seropositive HHC were elevated compared to GP-seronegative HHC, strongly suggesting that exposure/infection to EBOV tracks with presence of autoAb. As antibodies can activate innate immune cells via the antibody Fc domain, current studies are focused on analyzing the innate effector response mediated by these autoAb in EVD survivors and seropositive HHC. Together, our study highlights the potential role of autoAb in long-term sequelae associated with survival from EVD.

Establishing a Mouse Model for Sexual Transmission and Male Reproductive Tract Persistence of Ebola Virus.

Ebola virus disease (EVD) has resulted in the death of over 15 000 people since its discovery in 1976. At least 1 incident of re-emergence of EVD has been associated with persistent male reproductive tract infection in a patient surviving EVD greater than 500 days prior. To date, animal models of Ebola virus (EBOV) infection have failed to fully characterize the pathogenesis of reproductive tract infection. Furthermore, no animal model of sexual transmission of EBOV exists. In this study, we describe a roadmap to modeling sexual transmission of EBOV using a mouse-adapted EBOV isolate in immunocompetent male mice and female Ifnar-/- mice.

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

N'-Phenylacetohydrazide Derivatives as Potent Ebola Virus Entry Inhibitors with an Improved Pharmacokinetic Profile.

Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.

373 Identification of potential targets for immunotherapy in a cynomolgus macaque model of Ebola virus disease

OBJECTIVES/GOALS: Ebola virus infection causes severe disease and liver injury in humans. Macrophages contribute to inflammatory signaling and are prevalent in the liver. We assessed the activation status, including therapeutic target expression, of hepatic macrophages. METHODS/STUDY POPULATION: We compared formalin-fixed, paraffin-embedded liver tissue from terminal Ebola virus-infected and uninfected control cynomolgus macaques, a gold-standard model for human disease. We characterized region-specific protein and whole transcriptome expression in these tissues using GeoMx Digital Spatial Profiling. Macrophage (CD68+) and leukocyte (CD45+) accumulation in liver tissue was quantified by immunofluorescence image analysis using digital pathology software. RESULTS/ANTICIPATED RESULTS: Macrophage-specific (CD68+) regions in the liver of Ebola virus-infected macaques demonstrated a shift towards an inflammatory gene expression profile, as compared to those from healthy control tissue. These regions showed differential expression of monocyte/macrophage differentiation, antigen presentation, and T cell activation gene sets, which were associated with decreased MHC-II allele expression. Moreover, macrophage-specific regions in the infected macaques showed enriched expression of genes or proteins associated with known immunomodulatory therapeutics, including S100A9, IDO1, and CTLA-4. DISCUSSION/SIGNIFICANCE: These data demonstrate that hepatic macrophages express an inflammatory phenotype, that their ability to present antigens to the adaptive immune system may be impaired, and that they express therapeutically targetable markers for immunomodulation of these cells during Ebola virus infection.

Characterization of Ebola Virus Mucosal Challenge Routes in Cynomolgus Macaques.

Zaïre ebolavirus (EBOV) causes Ebola virus disease (EVD), a devastating viral hemorrhagic fever in humans. Nonhuman primate (NHP) models of EVD traditionally use intramuscular infection with higher case fatality rates and reduced mean time-to-death compared to contact transmission typical of human cases of EVD. A cynomolgus macaque model of oral and conjunctival EBOV was used to further characterize the more clinically relevant contact transmission of EVD. NHPs challenged via the oral route had an overall 50% survival rate. NHPs challenged with a target dose of 1 × 102 PFU or 1 × 104 PFU of EBOV via the conjunctival route had 40% and 100% mortality, respectively. Classic signs of lethal EVD-like disease were observed in all NHPs that succumbed to EBOV infection including viremia, hematological abnormalities, clinical chemistries indicative of hepatic and renal disease, and histopathological findings. Evidence of EBOV viral persistence in the eye was observed in NHPs challenged via the conjunctival route. IMPORTANCE This study is the first to examine the Kikwit strain of EBOV, the most commonly used strain, in the gold-standard macaque model of infection. Additionally, this is the first description of the detection of virus in the vitreous fluid, an immune privileged site that has been proposed as a viral reservoir, following conjunctival challenge. The oral and conjunctival macaque challenge model of EVD described here more faithfully recapitulates the prodrome that has been reported for human EVD. This work paves the way for more advanced studies to model contact transmission of EVD, including early events in mucosal infection and immunity, as well as the establishment of persistent viral infection and the emergence from these reservoirs.

Linked Mutations in the Ebola Virus Polymerase Are Associated with Organ Specific Phenotypes.

Ebola virus (EBOV) causes a severe infection called Ebola virus disease (EVD). The pathogenesis of EBOV infection is complex, and outcome has been associated with a variety of immunological and cellular factors. Disease can result from several mechanisms, including direct organ and endothelial cell damage as a result of viral replication. During the2013 to 2016 Western Africa EBOV outbreak, several mutants emerged, with changes in the genes of nucleoprotein (NP), glycoprotein (GP), and the large (L) protein. Reverse genetic analysis has been used to investigate whether these mutations played any role in pathogenesis with mixed results depending on the experimental system used. Previous studies investigated the impact of three single nonsynonymous mutations (GP-A82V, NP-R111C, and L-D759G) on the fatality rate of mouse and ferret models and suggested that the L-D759G mutation decreased the virulence of EBOV. In this study, the effect of these three mutations was further evaluated by deep sequencing to determine viral population genetics and the host response in longitudinal samples of blood, liver, kidney, spleen, and lung tissues taken from the previous ferret model. The data indicated that the mutations were maintained in the different tissues, but the frequency of minor genomic mutations were different. In addition, compared to wild-type virus, the recombinant mutants had different within host effects, where the D759G (and accompanying Q986H) substitution in the L protein resulted in an upregulation of the immune response in the kidney, liver, spleen, and lungs. Together these studies provide insights into the biology of EBOV mutants both between and within hosts. IMPORTANCE Ebola virus infection can have dramatic effects on the human body which manifest in Ebola virus disease. The outcome of infection is either survival or death and in the former group with the potential of longer-term health consequences and persistent infection. Disease severity is undoubtedly associated with the host response, often with overt inflammatory responses correlated with poorer outcomes. The scale of the2013 to 2016 Western African Ebola virus outbreak revealed new aspects of viral biology. This included the emergence of mutants with potentially altered virulence. Biobanked tissue from ferret models of EBOV infected with different mutants that emerged in the Western Africa outbreak was used to investigate the effect of EBOV genomic variation in different tissues. Overall, the work provided insights into the population genetics of EBOV and showed that different organs in an animal model can respond differently to variants of EBOV.

Ebanga™: The most recent FDA-approved drug for treating Ebola.

Ebolavirus (EBOV) is a virulent pathogen that causes Ebola virus disease (EVD), which is a life-threatening human condition with a fatality rate of up to 90%. Since the first outbreak in Africa in 1976, several outbreaks and epidemics of EBOV have occurred across the globe. While EVD is recognized as a serious threat to human health and outbreaks occur almost every year, the treatment options for the disease are limited. In designing therapeutic strategies against EBOV infection, viral structural proteins, such as glycoprotein (GP), could be an excellent target for neutralizing the virus. According to the latest research, GP-specific antibodies are the most efficient post-exposure treatments for EVD. Ansuvimab-zykl, i.e., mAb114 (Ebanga™), is a recent FDA-approved human immunoglobulin monoclonal antibody targeting EBOV GP. This review provides a brief overview of the pharmacological effects and safety profile of ansuvimab in clinical trials and provides insights into the precise mechanism of this new drug for treating EVD.

Epidemiological Analysis of Symmetry in Transmission of the Ebola Virus with Power Law Kernel

This study presents a mathematical model of non-integer order through the fractal fractional Caputo operator to determine the development of Ebola virus infections. To construct the model and conduct analysis, all Ebola virus cases are taken as incidence data. A symmetric approach is utilized for qualitative and quantitative analysis of the fractional order model. Additionally, stability is evaluated, along with the local and global effects of the virus that causes Ebola. Using the fractional order model of Ebola virus infections, the existence and uniqueness of solutions, as well the posedness and biological viability and disease free equilibrium points are confirmed. Many applications of fractional operators in modern mathematics exist, including the intricate and important study of symmetrical systems. Symmetry analysis is a powerful tool that enables the creation of numerical solutions for a given fractional differential equation very methodically. For this, we compare the results with the Caputo derivative operator to understand the dynamic behavior of the disease. The simulation demonstrates how all classes have convergent characteristics and maintain their places over time, reflecting the true behavior of Ebola virus infection. Power law kernel with the two step polynomial Newton method were used. This model seems to be quite strong and capable of reproducing the issue’s anticipated theoretical conditions.

Ebola viral factories are biomolecular condensates with specialized assembly dynamics for viral RNA synthesis.

A hallmark of Ebola virus (EBOV) infection is the formation of membrane-less organelles termed viral factories (VFs). Accumulation of viral proteins drives the formation of VFs, which offer a specialized compartment for viral replication. Multiple EBOV proteins are present inside VFs, but the involvement of individual viral components in VF assembly is unclear. Key to VF function is the recruitment of EBOV polymerase L, which, in complex with viral cofactor VP35, mediates replication and transcription of viral RNAs. How VFs spatially accommodate and control EBOV RNA synthesis remains elusive. Here, we detected both connected network-like and spherical Ebola VFs during infection, suggesting that spinodal decomposition and nucleation-and-growth both contribute to VF assembly. Using fluorescence recovery after photobleaching with live cell imaging, we measured highly dynamic molecular exchange within Ebola VFs reconstituted with mixtures of two viral proteins, NP and VP35. Co-expression of EBOV L partitions L into VFs but immobilizes a fraction of VP35, which likely results from L forming an arrested network pattern with interconnected foci inside VFs. We further introduced a surrogate viral genome into reconstituted VFs to allow L-directed viral RNA synthesis, from which we observed an intriguing link between the spacing of interconnected L-foci and a functional switch of viral RNA synthesis. We examined the same reconstituted VFs with electron microscopy and observed electron-dense materials in the cytoplasm, which resemble arrested L-network patterns observed with light microscopy. EBOV polymerase complex (L-VP35) localizes to select sites at the boundary of electron-dense materials, indicating viral RNA synthesis could initiate at multicomponent phase boundaries and benefit from the directional flux of viral proteins. In summary, our work provides an unprecedented view of EBOV propagation, which exploits fundamental rules of phase transition to regulate virus biogenesis.

Exploring the dimer-dimer association of the ebolavirus matrix protein VP40 at the human plasma membrane.

Ebolavirus (EBOV) infections cause the deadly Ebola Virus Disease (EVD) which has a high fatality rate in both human and non-human primates. The EBOV genome codes for only seven proteins that can change structures and have multiple functions during the viral assembly. The matrix protein VP40 is the primary component that makes up the cylindrical structure of the Ebola virus. In addition to its other functions during the viral life cycle, it forms the virion's viral matrix layer below the lipid envelope. Once the VP40 dimers are trafficked to the plasma membrane (PM), they assemble into linear filaments which further associate to form the viral matrix. The end-to-end association of the dimers via their C-terminal interfaces is a crucial step during the formation of the matrix filaments. A crystal structure of the CTD-CTD assembly is available and it provides insight into the nature of the interfacial interactions. In this study, we investigated the lipid interactions of the dimer-dimer filament at the inner leaflet of the PM. We used all-atom molecular dynamics simulations of the dimer-dimer structure of the VP40 associated with the PM and identified the major amino acid residues involved in the lipid interactions. We also compared the differences in the lipid contacts of VP40 of a single dimer versus the lipid contacts with the dimer-dimer conformation.

Resurgence of Ebola Virus: Transmission, Pathogenesis, Prevention and Cure

Ebola virus (EBOV) disease is a zoonotic disease that is caused by four species of genus Ebolavirus. The EBOV disease has outbreaks (1976-2022) in West and Central African countries, high mortality rate has made it a public health concern. EBOV is a filamentous virus having negative stranded and non-segmented RNA. The EBOV genome has the unique capability of forming soluble glycoprotein. Since its first emergence in human history, several effective therapeutics and vaccines are developed. These vaccines include replicative or non-replicative vectored vaccines, DNA vaccines and monovalent or polyepitopic vaccines. This article reviewed the structure of EBOV and pathophysiology as well as the immune response of EBOV infection. In addition to diagnosis and vaccinations available to cure EBOV at acute and chronic stages. Keywords: Zoonotic disease, Filovirus, Soluble glycoprotein, DNA vaccines, Polyepitopic, Vectored vaccines, Pathophysiology, Immune response

Characterization of an air-liquid interface primary human vaginal epithelium to study Ebola virus infection and testing of antivirals

Ebola virus (EBOV) is the causative agent of the often-fatal Ebola virus disease (EVD) characterized by hemorrhagic fever in humans and non-human primates. Sexual transmission from male survivors has been at the origin of multiple outbreak flare-ups between 2015 and 2021. However, this route is still poorly understood and the resulting EVD from it is also understudied. To support epidemiological studies documenting sexual transmission to women, and as a transition from previously using monolayer vaginal epithelial cells (VK2/E6E7), we first determined the biological relevance of two similar air-liquid interface models of the human vaginal epithelium (VEC and VLC Epivaginal™) and then characterized their susceptibility to EBOV and virus-induced inflammation. Finally, we evaluated toxicity of Polyphenylene Carboxymethylene (PPCM) microbicide in VLC and reassessed its antiviral effect.As expected, the VEC, but also VLC model showed stratified layers including a lamina propria under an epithelial structure similar to the full thickness of the human vaginal epithelium. However, we could not detect the immune cells featured in the most relevant model (VLC) of the vaginal epithelium using the dendritic cell CD1a and CD11c markers. Consistent with our previous work using the VK2/E6E7 cell line, infectious virus was detected from the apical side of both primary human cell systems, but only when using a high infective dose, with titers remaining at a constant level of 103−4 pfu/ml over 7 days suggesting lasting infectious virus shedding. In addition, infection caused disruption of the epithelium of both models and virus antigen was found from the apical superficial layers down to the lamina propria suggesting full virus penetration and overall confirming the susceptibility of the human vaginal tissue for EBOV. Just like previously seen in VK2/E6E7 cells, VLC infection also caused significant increase in inflammatory markers including IL-6, IL-8, and IP-10 suggesting vaginitis which is again consistent with tissue lesions seen in non-human primates. Finally, both virus infection and virus-induced inflammatory response in VLC could be prevented by a single 5-min PPCM microbicide treatment prior infection.