Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.     

Tolerance and Persistence of Ebola Virus in Primary Cells from Mops condylurus,a Potential Ebola Virus Reservoir

Although there have been documented Ebola virus disease outbreaks for more than 40 years, the natural reservoir host has not been identified. Recent studies provide evidence that the Angolan free-tailed bat (Mops condylurus), an insectivorous microbat, is a possible ebolavirus reservoir. To investigate the potential role of this bat species in the ecology of ebolaviruses, replication, tolerance, and persistence of Ebola virus (EBOV) were investigated in 10 different primary bat cell isolates from M. condylurus. Varying EBOV replication kinetics corresponded to the expression levels of the integral membrane protein NPC1. All primary cells were highly tolerant to EBOV infection without cytopathic effects. The observed persistent EBOV infection for 150 days in lung primary cells, without resultant selective pressure leading to virus mutation, indicate the intrinsic ability of EBOV to persist in this bat species. These results provide further evidence for this bat species to be a likely reservoir of ebolaviruses.     

Understanding Ebola Virus Transmission

An unprecedented number of Ebola virus infections among healthcare workers and patients have raised questions about our understanding of Ebola virus transmission. Here, we explore different routes of Ebola virus transmission between people, summarizing the known epidemiological and experimental data. From this data, we expose important gaps in Ebola virus research pertinent to outbreak situations. We further propose experiments and methods of data collection that will enable scientists to fill these voids in our knowledge about the transmission of Ebola virus.     

Use of the Syrian Hamster as a New Model of Ebola Virus Disease and Other Viral Hemorrhagic Fevers

Historically, mice and guinea pigs have been the rodent models of choice for therapeutic and prophylactic countermeasure testing against Ebola virus disease (EVD). Recently, hamsters have emerged as a novel animal model for the in vivo study of EVD. In this review, we discuss the history of the hamster as a research laboratory animal, as well as current benefits and challenges of this model. Availability of immunological reagents is addressed. Salient features of EVD in hamsters, including relevant pathology and coagulation parameters, are compared directly with the mouse, guinea pig and nonhuman primate models.     

Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.     

A Loop Region in the N-Terminal Domain of Ebola Virus VP40 Is Important in Viral Assembly,Budding, and Egress

Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys¹²⁷, Thr¹²⁹, and Asn¹³⁰) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress.     

埃博拉病毒病临床特征与治疗

埃博拉病毒病(Ebola virus disease,EVD)是由埃博拉病毒引起的烈性传染病,致死率高。EVD临床表现无特异性,确诊主要依靠病原学检查。目前以对症支持治疗为主,其他可能的治疗方案在不断研究探索中。本文对EVD的临床特征及治疗进行综述。     

Comparison of Aerosol Stability of Different Variants of Ebola Virus and Marburg Virus and Virulence of Aerosolised Ebola Virus in an Immune-Deficient Mouse

During outbreaks of virus diseases, many variants may appear, some of which may be of concern. Stability in an aerosol of several Ebola virus and Marburg virus variants was investigated. Studies were performed measuring aerosol survival using the Goldberg drum but no significant difference in biological decay rates between variants was observed. In addition, historic data on virulence in a murine model of different Ebola virus variants were compared to newly presented data for Ebola virus Kikwit in the A129 Interferon alpha/beta receptor-deficient mouse model. Ebola virus Kikwit was less virulent than Ebola virus Ecran in our mouse model. The mouse model may be a useful tool for studying differences in virulence associated with different variants whereas aerosol stability studies may not need to be conducted beyond the species level.     

A Characterization of Aerosolized Sudan Virus Infection in African Green Monkeys,Cynomolgus Macaques,and Rhesus Macaques

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and “Cuevavirus”. Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4–5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP.     

Balancing the Duty to Treat Patients with Ebola Virus Disease with the Risks to Dialysis Personnel

In 2014, the author was invited to present at the American Society for Nephrology’s annual conference in Philadelphia on the ethics of treating patients with Ebola virus disease. The argument was made that the status of health care workers, including nephrologists, was the dominant ethical standard that generated both the duty to treat and the conflicts between this commitment and other ethical commitments that arise in public health emergencies. Conflicts between duty to treat and personal safety, duty to community, and duty to colleagues were illustrated, and suggestions for designing ethics into medical practice were given. This article is a summary of that presentation.     

Potential Vaccines and Post-Exposure Treatments for Filovirus Infections

Viruses of the family Filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. While many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. Current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. Contemporary methods of supportive care and previous treatment approaches for human patients are also discussed.     

Structure of the Ebola VP35 interferon inhibitory domain

Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 Å and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.     

A Call to Action to Enhance Filovirus Disease Outbreak Preparedness and Response

The frequency and magnitude of recognized and declared filovirus-disease outbreaks have increased in recent years, while pathogenic filoviruses are potentially ubiquitous throughout sub-Saharan Africa. Meanwhile, the efficiency and effectiveness of filovirus-disease outbreak preparedness and response efforts are currently limited by inherent challenges and persistent shortcomings. This paper delineates some of these challenges and shortcomings and provides a proposal for enhancing future filovirus-disease outbreak preparedness and response. The proposal serves as a call for prompt action by the organizations that comprise filovirus-disease outbreak response teams, namely, Ministries of Health of outbreak-prone countries, the World Health Organization, Médecins Sans Frontières, the Centers for Disease Control and Prevention—Atlanta, and others.     

Experimental Inoculation of Egyptian Fruit Bats (Rousettus aegyptiacus) with Ebola Virus

Colonized Egyptian fruit bats (Rousettus aegyptiacus), originating in South Africa, were inoculated subcutaneously with Ebola virus (EBOV). No overt signs of morbidity, mortality, or gross lesions were noted. Bats seroconverted by Day 10–16 post inoculation (p.i.), with the highest mean anti-EBOV IgG level on Day 28 p.i. EBOV RNA was detected in blood from one bat. In 16 other tissues tested, viral RNA distribution was limited and at very low levels. No seroconversion could be demonstrated in any of the control bats up to 28 days after in-contact exposure to subcutaneously-inoculated bats. The control bats were subsequently inoculated intraperitoneally, and intramuscularly with the same dose of EBOV. No mortality, morbidity or gross pathology was observed in these bats. Kinetics of immune response was similar to that in subcutaneously-inoculated bats. Viral RNA was more widely disseminated to multiple tissues and detectable in a higher proportion of individuals, but consistently at very low levels. Irrespective of the route of inoculation, no virus was isolated from tissues which tested positive for EBOV RNA. Viral RNA was not detected in oral, nasal, ocular, vaginal, penile and rectal swabs from any of the experimental groups.     

The Baboon (Papio spp.) as a Model of Human Ebola Virus Infection

Baboons are susceptible to natural Ebola virus (EBOV) infection and share 96% genetic homology with humans. Despite these characteristics, baboons have rarely been utilized as experimental models of human EBOV infection to evaluate the efficacy of prophylactics and therapeutics in the United States. This review will summarize what is known about the pathogenesis of EBOV infection in baboons compared to EBOV infection in humans and other Old World nonhuman primates. In addition, we will discuss how closely the baboon model recapitulates human EBOV infection. We will also review some of the housing requirements and behavioral attributes of baboons compared to other Old World nonhuman primates. Due to the lack of data available on the pathogenesis of Marburg virus (MARV) infection in baboons, discussion of the pathogenesis of MARV infection in baboons will be limited.