Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

Medical countermeasures against henipaviruses: a review and public health perspective.

Henipaviruses, including Nipah virus, are regarded as pathogens of notable epidemic potential because of their high pathogenicity and the paucity of specific medical countermeasures to control infections in humans. We review the evidence of medical countermeasures against henipaviruses and project their cost in a post-COVID-19 era. Given the sporadic and unpredictable nature of henipavirus outbreaks, innovative strategies will be needed to circumvent the infeasibility of traditional phase 3 clinical trial regulatory pathways. Stronger partnerships with scientific institutions and regulatory authorities in low-income and middle-income countries can inform coordination of appropriate investments and development of strategies and normative guidelines for the deployment and equitable use of multiple medical countermeasures. Accessible measures should include global, regional, and endemic in-country stockpiles of reasonably priced small molecules, monoclonal antibodies, and vaccines as part of a combined collection of products that could help to control henipavirus outbreaks and prevent future pandemics.

A STAT-1 knockout mouse model for Machupo virus pathogenesis.

BACKGROUND: Machupo virus (MACV), a member of the Arenaviridae, causes Bolivian hemorrhagic fever, with ~20% lethality in humans. The pathogenesis of MACV infection is poorly understood, and there are no clinically proven treatments for disease. This is due, in part, to a paucity of small animal models for MACV infection in which to discover and explore candidate therapeutics. METHODS: Mice lacking signal transducer and activator of transcription 1 (STAT-1) were infected with MACV. Lethality, viral replication, metabolic changes, hematology, histopathology, and systemic cytokine expression were analyzed throughout the course of infection. RESULTS: We report here that STAT-1 knockout mice succumbed to MACV infection within 7-8 days, and presented some relevant clinical and histopathological manifestations of disease. Furthermore, the model was used to validate the efficacy of ribavirin in protection against infection. CONCLUSIONS: The STAT-1 knockout mouse model can be a useful small animal model for drug testing and preliminary immunological analysis of lethal MACV infection.

Opportunities for Refinement of Non-Human Primate Vaccine Studies.

Non-human primates (NHPs) are used extensively in the development of vaccines and therapeutics for human disease. High standards in the design, conduct, and reporting of NHP vaccine studies are crucial for maximizing their scientific value and translation, and for making efficient use of precious resources. A key aspect is consideration of the 3Rs principles of replacement, reduction, and refinement. Funders of NHP research are placing increasing emphasis on the 3Rs, helping to ensure such studies are legitimate, ethical, and high-quality. The UK's National Centre for the 3Rs (NC3Rs) and the Coalition for Epidemic Preparedness Innovations (CEPI) have collaborated on a range of initiatives to support vaccine developers to implement the 3Rs, including hosting an international workshop in 2019. The workshop identified opportunities to refine NHP vaccine studies to minimize harm and improve welfare, which can yield better quality, more reproducible data. Careful animal selection, social housing, extensive environmental enrichment, training for cooperation with husbandry and procedures, provision of supportive care, and implementation of early humane endpoints are features of contemporary good practice that should and can be adopted more widely. The requirement for high-level biocontainment for some pathogens imposes challenges to implementing refinement but these are not insurmountable.

Natural history of disease in cynomolgus monkeys exposed to Ebola virus Kikwit strain demonstrates the reliability of this non-human primate model for Ebola virus disease.

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.

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.

High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test.

Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials.

Assessment of the potential for host-targeted iminosugars UV-4 and UV-5 activity against filovirus infections in vitro and in vivo.

Iminosugars are host-directed antivirals with broad-spectrum activity. The iminosugar, N-butyl-deoxynojirimycin (NB-DNJ or Miglustat®), is used in humans for treatment of Gaucher's disease and has mild antiviral properties. More potent analogs of NB-DNJ have been generated and have demonstrated activity against a variety of viruses including flaviviruses, influenza, herpesviruses and filoviruses. In the current study, a panel of analogs based on NB-DNJ was analyzed for activity against Ebola (EBOV) and Marburg viruses (MARV). The antiviral activity of NB-DNJ (UV-1), UV-2, UV-3, UV-4 and UV-5 against both EBOV and MARV was demonstrated in Vero cells. Subsequent studies to examine the activity of UV-4 and UV-5 using rodent models of EBOV and MARV were performed. In vivo efficacy studies provided inconsistent data following treatment with iminosugars using filovirus mouse models. A tolerability study in nonhuman primates demonstrated that UV-4 could be administered at much higher dose levels than rodents. Since UV-4 was active in vitro, had been demonstrated to be active against influenza and dengue in vivo, and was being tested in a Phase 1 clinical trial, a small proof-of-concept nonhuman primate trial was performed to determine whether this antiviral candidate could provide clinical benefit to EBOV-infected individuals. Administration of UV-4B did not provide a clinical or survival benefit to macaques infected with EBOV-Makona; however, dosing of animals was not optimal in this study. Efficacy may be improved by thrice daily dosing (e.g. by nasogastric tube feeding) to match the efficacious dosing regimens demonstrated against dengue and influenza viruses.

Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses.

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.

Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.

A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4) studies. After standardization studies were completed, Good Laboratory Practices (GLP)-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay's precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP) serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.

Development of a new vaccine for the prevention of Lassa fever.

BACKGROUND: Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge. METHODS AND FINDINGS: We developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection. CONCLUSION: Our data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease.

Animal models for ebolavirus countermeasures discovery: what defines a useful model?

INTRODUCTION: Ebolaviruses are highly pathogenic filoviruses, which cause disease in humans and nonhuman primates (NHP) in Africa. The Zaire ebolavirus outbreak in 2014, which continues to greatly affect Western Africa and other countries to which the hemorrhagic fever was exported due to travel of unsymptomatic yet infected individuals, was complicated by the lack of available licensed vaccines or therapeutics to combat infection. After almost a year of research at an increased pace to find and test vaccines and therapeutics, there is now a deeper understanding of the available disease models for ebolavirus infection. Demonstration of vaccine or therapeutic efficacy in NHP models of ebolavirus infection is crucial to the development and eventual licensure of ebolavirus medical countermeasures, so that safe and effective countermeasures can be accelerated into human clinical trials. AREAS COVERED: The authors describe ebolavirus hemorrhagic fever (EHF) disease in various animal species: mice, guinea pigs, hamsters, pigs and NHP, to include baboons, marmosets, rhesus and cynomolgus macaques, as well as African green monkeys. Because the NHP models are supremely useful for therapeutics and vaccine testing, emphasis is placed on comparison of these models, and their use as gold-standard models of EHF. EXPERT OPINION: Animal models of EHF varying from rodents to NHP species are currently under evaluation for their reproducibility and utility for modeling infection in humans. Complete development and licensure of therapeutic agents and vaccines will require demonstration that mechanisms conferring protection in NHP models of infection are predictive of protective responses in humans, for a given countermeasure.

Pre-symptomatic diagnosis and treatment of filovirus diseases.

Filoviruses are virulent human pathogens which cause severe illness with high case fatality rates and for which there are no available FDA-approved vaccines or therapeutics. Diagnostic tools including antibody- and molecular-based assays, mass spectrometry, and next-generation sequencing are continually under development. Assays using the polymerase chain reaction (PCR) have become the mainstay for the detection of filoviruses in outbreak settings. In many cases, real-time reverse transcriptase-PCR allows for the detection of filoviruses to be carried out with minimal manipulation and equipment and can provide results in less than 2 h. In cases of novel, highly diverse filoviruses, random-primed pyrosequencing approaches have proved useful. Ideally, diagnostic tests would allow for diagnosis of filovirus infection as early as possible after infection, either before symptoms begin, in the event of a known exposure or epidemiologic outbreak, or post-symptomatically. If tests could provide an early definitive diagnosis, then this information may be used to inform the choice of possible therapeutics. Several exciting new candidate therapeutics have been described recently; molecules that have therapeutic activity when administered to animal models of infection several days post-exposure, once signs of disease have begun. The latest data for candidate nucleoside analogs, small interfering RNA (siRNA) molecules, phosphorodiamidate (PMO) molecules, as well as antibody and blood-product therapeutics and therapeutic vaccines are discussed. For filovirus researchers and government agencies interested in making treatments available for a nation's defense as well as its general public, having the right diagnostic tools to identify filovirus infections, as well as a panel of available therapeutics for treatment when needed, is a high priority. Additional research in both areas is required for ultimate success, but significant progress is being made to reach these goals.

Evaluation of Ebola Virus Inhibitors for Drug Repurposing.

A systematic screen of FDA-approved drugs was performed to identify compounds with in vitro antiviral activities against Ebola virus (EBOV). Compounds active (>50% viral inhibition and <30% cellular toxicity) at a single concentration were tested in dose-response assays to quantitate the antiviral activities in replication and viral entry assays as well as cytotoxicity in the Vero cell line used to conduct these assays. On the basis of the approved human dosing, toxicity/tolerability, and pharmacokinetic data, seven of these in vitro hits from different pharmacological classes (chloroquine (CQ), amiodarone, prochlorperazine, benztropine, azithromycin, chlortetracycline, and clomiphene) were evaluated for their in vivo efficacy at a single dose and were administered via either intraperitoneal (ip) or oral route. Initially, azithromycin (100 mg/kg, twice daily, ip), CQ (90 mg/kg, twice daily, ip), and amiodarone (60 mg/kg, twice daily, ip) demonstrated significant increases in survival in the mouse model. After repeat evaluation, only CQ was found to reproducibly give significant efficacy in the mouse model with this dosing regimen. Azithromycin and CQ were also tested in a guinea pig model of EBOV infection over a range of doses, but none of the doses increased survival, and drug-related toxicity was observed at lower doses than in the mouse. These results show the benefits and specific challenges associated with drug repurposing and highlight the need for careful evaluation of approved drugs as rapidly deployable countermeasures against future pandemics.

Cross-protection conferred by filovirus virus-like particles containing trimeric hybrid glycoprotein.

Filoviruses are causative agents of hemorrhagic fever, and to date no effective vaccine or therapeutic has been approved to combat infection. Filovirus glycoprotein (GP) is the critical immunogenic component of filovirus vaccines, eliciting high levels of antibody after successful vaccination. Previous work has shown that protection against both Ebola virus (EBOV) and Marburg virus (MARV) can be achieved by vaccinating with a mixture of virus-like particles (VLPs) expressing either EBOV GP or MARV GP. In this study, the potential for eliciting effective immune responses against EBOV, Sudan virus, and MARV with a single GP construct was tested. Trimeric hybrid GPs were produced that expressed the sequence of Marburg GP2 in conjunction with a hybrid GP1 composed EBOV and Sudan virus GP sequences. VLPs expressing these constructs, along with EBOV VP40, provided comparable protection against MARV challenge, resulting in 75 or 100% protection. Protection from EBOV challenge differed depending upon the hybrid used, however, with one conferring 75% protection and one conferring no protection. By comparing the overall antibody titers and the neutralizing antibody titers specific for each virus, it is shown that higher antibody responses were elicited by the C terminal region of GP1 than by the N terminal region, and this correlated with protection. These data collectively suggest that GP2 and the C terminal region of GP1 are highly immunogenic, and they advance progress toward the development of a pan-filovirus vaccine.

TMC-114 (Tibotec).

Tibotec (formerly Tibotec-Virco) is developing TMC-114 as a potential treatment for HIV-1 infection. In February 2001, TMC-126 was revealed as the series prototype, from which TMC-114 was developed. Because of its improved antiviral and superior pharmacokinetic properties, TMC-114 was selected for clinical development. Phase II trials of TMC-114 are underway.

Bioterrorism and emerging infectious disease - antimicrobials, therapeutics and immune-modulators. SARS coronavirus.

The purpose of this meeting was to provide a forum for expert presentations and discussion about the threats of bioterrorism and emerging infectious diseases, and to address the issues relating to epidemics, prevention of infection and treatment of some of these emerging infectious diseases classified as potential agents of bioterror. Included in the talks were state-of-the-art presentations about infectious clone technology and recombinant viruses, pathogen and receptor interactions at the cellular and molecular level, genomic responses to infection, and new information on antiviral mechanisms of action. Severe acute respiratory syndrome (SARS) and progress toward understanding the epidemic was addressed, and other sessions were presented concerning immune therapy and immunopotentiation of disease, siRNA and gene silencing, host responses to pathogen infections, as well as the use of genetic engineering to circumvent and direct the immune response. Many discussions were held and data were presented about possible compounds and new drugs that may have antiviral properties, yet there were few discussions of any available new drugs. This report addresses reverse genetics of SARS virus, as well as its epidemiology, and a host of different recent approaches to developing antivirals effective against SARS, including some potential vaccine candidates. Also presented are hypotheses about the human immune response to SARS infection, as well as immune therapies against botulinum and anthrax toxins. This report also addresses antiviral approaches exploiting siRNAs, and different aspects of the host immune response to many of the different dangerous pathogens discussed at this meeting. Finally, approaches to circumventing and directing the immune response using genetic engineering will be reported.

HSPA5 is an essential host factor for Ebola virus infection.

Development of novel strategies targeting the highly virulent ebolaviruses is urgently required. A proteomic study identified the ER chaperone HSPA5 as an ebolavirus-associated host protein. Here, we show using the HSPA5 inhibitor (-)- epigallocatechin gallate (EGCG) that the chaperone is essential for virus infection, thereby demonstrating a functional significance for the association. Furthermore, in vitro and in vivo gene targeting impaired viral replication and protected animals in a lethal infection model. These findings demonstrate that HSPA5 is vital for replication and can serve as a viable target for the design of host-based countermeasures.

Marburgvirus hijacks nrf2-dependent pathway by targeting nrf2-negative regulator keap1.

Marburg virus (MARV) has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2). Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses.