Depletion of Bone Marrow Hematopoietic Cells in Ebolavirus-Infected Rhesus Macaques: A Possible Cause of Hematologic Abnormalities in Ebolavirus Disease.

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease.

Ebola Virus Disease Features Hemophagocytic Lymphohistiocytosis/Macrophage Activation Syndrome in the Rhesus Macaque Model.

BACKGROUND: Ebola virus (EBOV) disease (EVD) is one of the most severe and fatal viral hemorrhagic fevers and appears to mimic many clinical and laboratory manifestations of hemophagocytic lymphohistiocytosis syndrome (HLS), also known as macrophage activation syndrome. However, a clear association is yet to be firmly established for effective host-targeted, immunomodulatory therapeutic approaches to improve outcomes in patients with severe EVD. METHODS: Twenty-four rhesus monkeys were exposed intramuscularly to the EBOV Kikwit isolate and euthanized at prescheduled time points or when they reached the end-stage disease criteria. Three additional monkeys were mock-exposed and used as uninfected controls. RESULTS: EBOV-exposed monkeys presented with clinicopathologic features of HLS, including fever, multiple organomegaly, pancytopenia, hemophagocytosis, hyperfibrinogenemia with disseminated intravascular coagulation, hypertriglyceridemia, hypercytokinemia, increased concentrations of soluble CD163 and CD25 in serum, and the loss of activated natural killer cells. CONCLUSIONS: Our data suggest that EVD in the rhesus macaque model mimics pathophysiologic features of HLS/macrophage activation syndrome. Hence, regulating inflammation and immune function might provide an effective treatment for controlling the pathogenesis of acute EVD.

Expanded profiling of Remdesivir as a broad-spectrum antiviral and low potential for interaction with other medications in vitro.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness.

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants.

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2.

Remdesivir is efficacious in rhesus monkeys exposed to aerosolized Ebola virus.

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.

Remdesivir (GS-5734) Is Efficacious in Cynomolgus Macaques Infected With Marburg Virus.

Marburg virus (MARV) is a filovirus with documented human case-fatality rates of up to 90%. Here, we evaluated the therapeutic efficacy of remdesivir (GS-5734) in nonhuman primates experimentally infected with MARV. Beginning 4 or 5 days post inoculation, cynomolgus macaques were treated once daily for 12 days with vehicle, 5 mg/kg remdesivir, or a 10-mg/kg loading dose followed by 5 mg/kg remdesivir. All vehicle-control animals died, whereas 83% of animals receiving a 10-mg/kg loading dose of remdesivir survived, as did 50% of animals receiving a 5-mg/kg remdesivir regimen. Remdesivir-treated animals exhibited improved clinical scores, lower plasma viral RNA, and improved markers of kidney function, liver function, and coagulopathy versus vehicle-control animals. The small molecule remdesivir showed therapeutic efficacy in this Marburg virus disease model with treatment initiation 5 days post inoculation, supporting further assessment of remdesivir for the treatment of Marburg virus disease in humans.

Recent successes in therapeutics for Ebola virus disease: no time for complacency.

The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.

Characterization of Ebola Virus Disease (EVD) in Rhesus Monkeys for Development of EVD Therapeutics.

Recent Ebola virus (EBOV) outbreaks in West Africa and the Democratic Republic of the Congo have highlighted the urgent need for approval of medical countermeasures for treatment and prevention of EBOV disease (EVD). Until recently, when successes were achieved in characterizing the efficacy of multiple experimental EVD therapeutics in humans, the only feasible way to obtain data regarding potential clinical benefits of candidate therapeutics was by conducting well-controlled animal studies. Nonclinical studies are likely to continue to be important tools for screening and development of new candidates with improved pharmacological properties. Here, we describe a natural history study to characterize the time course and order of progression of the disease manifestations of EVD in rhesus monkeys. In 12 rhesus monkeys exposed by the intramuscular route to 1000 plaque-forming units of EBOV, multiple endpoints were monitored for 28 days following exposure. The disease progressed rapidly with mortality events occurring 7-10 days after exposure. Key disease manifestations observed consistently across the infected animals included, but were not limited to, viremia, fever, systemic inflammation, coagulopathy, lymphocytolysis, renal tubular necrosis with mineralization, and hepatocellular degeneration and necrosis.

Cholesterol-conjugated stapled peptides inhibit Ebola and Marburg viruses in vitro and in vivo.

Filoviridae currently includes five official and one proposed genera. Genus Ebolavirus includes five established and one proposed ebolavirus species for Bombali virus (BOMV), Bundibugyo virus (BDBV), Ebola virus (EBOV), Reston virus (RESTV), Sudan virus (SUDV) and Taï Forest virus (TAFV), and genus Marburgvirus includes a single species for Marburg virus (MARV) and Ravn virus (RAVV). Ebola virus (EBOV) has emerged as a significant public health concern since the 2013-2016 Ebola Virus Disease outbreak in Western Africa. Currently, there are no therapeutics approved and the need for Ebola-specific therapeutics remains a gap. In search for anti-Ebola therapies we tested the idea of using inhibitory properties of peptides corresponding to the C-terminal heptad-repeat (HR2) domains of class I fusion proteins against EBOV infection. The fusion protein GP2 of EBOV belongs to class I, suggesting that a similar strategy to HIV may be applied to inhibit EBOV infection. The serum half-life of peptides was expanded by cholesterol conjugation to allow daily dosing. The peptides were further constrained to stabilize a helical structure to increase the potency of inhibition. The EC50s of lead peptides were in low micromolar range, as determined by a high-content imaging test of EBOV-infected cells. Lead peptides were tested in an EBOV lethal mouse model and efficacy of the peptides were determined following twice-daily administration of peptides for 9 days. The most potent peptide was able to protect mice from lethal challenge of mouse-adapted Ebola virus. These data show that engineered peptides coupled with cholesterol can inhibit viral production, protect mice against lethal EBOV infection, and may be used to build novel therapeutics against EBOV.

T-705 induces lethal mutagenesis in Ebola and Marburg populations in macaques.

Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.

Characterization of the plasma proteome of nonhuman primates during Ebola virus disease or melioidosis: a host response comparison.

BACKGROUND: In-depth examination of the plasma proteomic response to infection with a wide variety of pathogens can assist in the development of new diagnostic paradigms, while providing insight into the interdependent pathogenic processes which encompass a host's immunological and physiological responses. Ebola virus (EBOV) causes a highly lethal infection termed Ebola virus disease (EVD) in primates and humans. The Gram negative non-spore forming bacillus Burkholderia pseudomallei (Bp) causes melioidosis in primates and humans, characterized by severe pneumonia with high mortality. We sought to examine the host response to infection with these two bio-threat pathogens using established animal models to provide information on the feasibility of pre-symptomatic diagnosis, since the induction of host molecular signaling networks can occur before clinical presentation and pathogen detection. METHODS: Herein we report the quantitative proteomic analysis of plasma collected at various times of disease progression from 10 EBOV-infected and 5 Bp-infected nonhuman primates (NHP). Our strategy employed high resolution LC-MS/MS and a peptide-tagging approach for relative protein quantitation. In each infection type, for all proteins with > 1.3 fold abundance change at any post-infection time point, a direct comparison was made with levels obtained from plasma collected daily from 5 naïve rhesus macaques, to determine the fold changes that were significant, and establish the natural variability of abundance for endogenous plasma proteins. RESULTS: A total of 41 plasma proteins displayed significant alterations in abundance during EBOV infection, and 28 proteins had altered levels during Bp infection, when compared to naïve NHPs. Many major acute phase proteins quantitated displayed similar fold-changes between the two infection types but exhibited different temporal dynamics. Proteins related to the clotting cascade, immune signaling and complement system exhibited significant differential abundance during infection with EBOV or Bp, indicating a specificity of the response. CONCLUSIONS: These results advance our understanding of the global plasma proteomic response to EBOV and Bp infection in relevant primate models for human disease and provide insight into potential innate immune response differences between viral and bacterial infections.

Virulence of Marburg Virus Angola Compared to Mt. Elgon (Musoke) in Macaques: A Pooled Survival Analysis.

Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model.

Ebola Virus Causes Intestinal Tract Architectural Disruption and Bacterial Invasion in Non-Human Primates.

In the 2014⁻2016 West Africa Ebola Virus (EBOV) outbreak, there was a significant concern raised about the potential for secondary bacterial infection originating from the gastrointestinal tract, which led to the empiric treatment of many patients with antibiotics. This retrospective pathology case series summarizes the gastrointestinal pathology observed in control animals in the rhesus EBOV-Kikwit intramuscular 1000 plaque forming unit infection model. All 31 Non-human primates (NHPs) exhibited lymphoid depletion of gut-associated lymphoid tissue (GALT) but the severity and the specific location of the depletion varied. Mesenteric lymphoid depletion and necrosis were present in 87% (27/31) of NHPs. There was mucosal barrier disruption of the intestinal tract with mucosal necrosis and/or ulceration most notably in the duodenum (16%), cecum (16%), and colon (29%). In the intestinal tract, hemorrhage was noted most frequently in the duodenum (52%) and colon (45%). There were focal areas of bacterial submucosal invasion in the gastrointestinal (GI) tract in 9/31 (29%) of NHPs. Only 2/31 (6%) had evidence of pancreatic necrosis. One NHP (3%) experienced jejunal intussusception which may have been directly related to EBOV. Immunofluorescence assays demonstrated EBOV antigen in CD68+ macrophage/monocytes and endothelial cells in areas of GI vascular injury or necrosis.

Persistent Marburg Virus Infection in the Testes of Nonhuman Primate Survivors.

Sexual transmission of filoviruses was first reported in 1968 after an outbreak of Marburg virus (MARV) disease and recently caused flare-ups of Ebola virus disease in the 2013-2016 outbreak. How filoviruses establish testicular persistence and are shed in semen remain unknown. We discovered that persistent MARV infection of seminiferous tubules, an immune-privileged site that harbors sperm production, is a relatively common event in crab-eating macaques that survived infection after antiviral treatment. Persistence triggers severe testicular damage, including spermatogenic cell depletion and inflammatory cell invasion. MARV mainly persists in Sertoli cells, leading to breakdown of the blood-testis barrier formed by inter-Sertoli cell tight junctions. This disruption is accompanied by local infiltration of immunosuppressive CD4+Foxp3+ regulatory T cells. Our study elucidates cellular events associated with testicular persistence that may promote sexual transmission of filoviruses and suggests that targeting immunosuppression may be warranted to clear filovirus persistence in damaged immune-privileged sites.

Intracellular conversion and in vivo dose response of favipiravir (T-705) in rodents infected with Ebola virus.

During the 2013-2016 Ebola virus (EBOV) outbreak in West Africa, our team at USAMRIID evaluated the antiviral activity of a number of compounds, including favipiravir (T-705), in vitro and in mouse and nonhuman primate (NHP) models of Ebola virus disease. In this short communication, we present our findings for favipiravir in cell culture and in mice, while an accompanying paper presents the results of NHP studies. We confirmed previous reports that favipiravir has anti-EBOV activity in mice. Additionally, we found that the active form of favipiravir is generated in mice in tissues relevant for the pathogenesis of EBOV infection. Finally, we observed that protection can be achieved in mice down to 8 mg/kg/day, which is lower than the dosing regimens previously reported. An accompanying paper reports the results of treating nonhuman primates infected with EBOV or with Marburg virus with oral or intravenous favipiravir.

Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus.

Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV). While no survival benefit was observed in two studies employing once- or twice-daily oral dosing of favipiravir during EBOV infection of NHPs, an antiviral effect was observed in terms of extended time-to-death and reduced levels of viral RNA. However, oral dosing in biosafety level-4 (BSL-4) presents logistical and technical challenges, and repeated anesthesia events may potentially worsen survival outcome in animals. For the third study of treatment of MARV infection, we therefore made use of catheters, jackets, and tethers for intravenous (IV) dosing and blood collection, which minimized the requirement for repeated anesthesia events. When MARV infection was treated with IV favipiravir, five of six animals (83%) survived infection, while all untreated NHPs succumbed. An accompanying report presents the results of favipiravir treatment of EBOV infection in mice.

Enhancing the antiviral potency of ER α-glucosidase inhibitor IHVR-19029 against hemorrhagic fever viruses in vitro and in vivo.

Targeting host functions essential for viral replication has been considered as a broad spectrum and resistance-refractory antiviral approach. However, only a few host functions have, thus far, been validated as broad-spectrum antiviral targets in vivo. ER α-glucosidases I and II have been demonstrated to be essential for the morphogenesis of many enveloped viruses, including members from four families of viruses causing hemorrhagic fever. In vivo antiviral efficacy of various iminosugar-based ER α-glucosidase inhibitors has been reported in animals infected with Dengue, Japanese encephalitis, Ebola, Marburg and influenza viruses. Herein, we established Huh7.5-derived cell lines with ER α-glucosidase I or II knockout using CRISPR/Cas9 and demonstrated that the replication of Dengue, Yellow fever and Zika viruses was reduced by only 1-2 logs in the knockout cell lines. The results clearly indicate that only a partial suppression of viral replication can possibly be achieved with a complete inhibition of ER-α-glucosidases I or II by their inhibitors. We therefore explore to improve the antiviral efficacy of a lead iminosugar IHVR-19029 through combination with another broad-spectrum antiviral agent, favipiravir (T-705). Indeed, combination of IHVR-19029 and T-705 synergistically inhibited the replication of Yellow fever and Ebola viruses in cultured cells. Moreover, in a mouse model of Ebola virus infection, combination of sub-optimal doses of IHVR-19029 and T-705 significantly increased the survival rate of infected animals. We have thus proved the concept of combinational therapeutic strategy for the treatment of viral hemorrhagic fevers with broad spectrum host- and viral- targeting antiviral agents.

Clinical Laboratory Values as Early Indicators of Ebola Virus Infection in Nonhuman Primates.

The Ebola virus (EBOV) outbreak in West Africa during 2013-2016 demonstrated the need to improve Ebola virus disease (EVD) diagnostics and standards of care. This retrospective study compared laboratory values and clinical features of 3 nonhuman primate models of lethal EVD to assess associations with improved survival time. In addition, the study identified laboratory values useful as predictors of survival, surrogates for EBOV viral loads, and triggers for initiation of therapeutic interventions in these nonhuman primate models. Furthermore, the data support that, in nonhuman primates, the Makona strain of EBOV may be less virulent than the Kikwit strain of EBOV. The applicability of these findings as potential diagnostic and management tools for EVD in humans warrants further investigation.

Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys.

Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013-2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68+ cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence.

Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.