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      A New Approach for Monitoring Ebolavirus in Wild Great Apes

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          Abstract

          Background

          Central Africa is a “hotspot” for emerging infectious diseases (EIDs) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. Ebolavirus is suspected to have caused recent declines in resident great apes. While ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm Ebola virus disease (EVD) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure.

          Methodology/Principal findings

          Here we report the first successful noninvasive detection of antibodies against Ebola virus (EBOV) from wild ape feces. Using this method, we have been able to identify gorillas with antibodies to EBOV with an overall prevalence rate reaching 10% on average, demonstrating that EBOV exposure or infection is not uniformly lethal in this species. Furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to EBOV (protected from exposure by rivers as topological barriers of transmission).

          Conclusions/Significance

          Our new approach will contribute to a strategy to protect apes from future EBOV infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. Finally, since human EVD is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where EBOV causes case-fatality rates of up to 88%.

          Author Summary

          Ebolavirus causes deadly outbreaks in wild great apes, and has been reported as a significant threat to the survival of wild lowland gorillas in Central Africa. Improved knowledge of basic information regarding geographic distribution of ebolavirus in great ape populations, including the identification of immunologically naïve populations and the determination of whether apes survive virus exposure, will be needed in order for protective interventions such as immunization to be effective. However, monitoring ebolavirus infection in wild gorillas by current methods is challenging because of the difficulty in obtaining diagnostic samples from these elusive primates. Additionally, there are limitations associated with the available laboratory assays used to document ebolavirus infection. Here we report the first successful noninvasive detection of EBOV immunity in wild great apes, demonstrating survival in this species. This tool will be useful in a comprehensive strategy aimed at the protection of this endangered species and improved prevention of EVD outbreaks in human populations.

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          Most cited references37

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          Origin of the human malaria parasite Plasmodium falciparum in gorillas.

          Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here we develop a single-genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in faecal samples from wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed and almost always made up of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas comprised parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla origin and not of chimpanzee, bonobo or ancient human origin.
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            Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients.

            Ebola virus is very pathogenic in humans. It induces an acute hemorrhagic fever that leads to death in about 70% of patients. We compared the immune responses of patients who died from Ebola virus disease with those who survived during two large outbreaks in 1996 in Gabon. In survivors, early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells, which was indicated by the upregulation of FasL, perforin, CD28 and gamma interferon mRNA in peripheral blood mononuclear cells. In contrast, fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM. Early activation of T cells, indicated by mRNA patterns in peripheral blood mononuclear cells and considerable release of gamma interferon in plasma, was followed in the days preceding death by the disappearance of T cell-related mRNA (including CD3 and CD8). DNA fragmentation in blood leukocytes and release of 41/7 nuclear matrix protein in plasma indicated that massive intravascular apoptosis proceeded relentlessly during the last 5 days of life. Thus, events very early in Ebola virus infection determine the control of viral replication and recovery or catastrophic illness and death.
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              Development of a preventive vaccine for Ebola virus infection in primates.

              Outbreaks of haemorrhagic fever caused by the Ebola virus are associated with high mortality rates that are a distinguishing feature of this human pathogen. The highest lethality is associated with the Zaire subtype, one of four strains identified to date. Its rapid progression allows little opportunity to develop natural immunity, and there is currently no effective anti-viral therapy. Therefore, vaccination offers a promising intervention to prevent infection and limit spread. Here we describe a highly effective vaccine strategy for Ebola virus infection in non-human primates. A combination of DNA immunization and boosting with adenoviral vectors that encode viral proteins generated cellular and humoral immunity in cynomolgus macaques. Challenge with a lethal dose of the highly pathogenic, wild-type, 1976 Mayinga strain of Ebola Zaire virus resulted in uniform infection in controls, who progressed to a moribund state and death in less than one week. In contrast, all vaccinated animals were asymptomatic for more than six months, with no detectable virus after the initial challenge. These findings demonstrate that it is possible to develop a preventive vaccine against Ebola virus infection in primates.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                September 2014
                18 September 2014
                : 8
                : 9
                : e3143
                Affiliations
                [1 ]Wildlife Conservation Society, Bronx, New York, New York, United States of America
                [2 ]Vaccine Research Center, National Institute for Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America
                [3 ]Departamento Biologia Animal (Vertebrados), Facultad de Biologia, Universidad de Barcelona, Barcelona, Spain
                [4 ]ECOFAC, Libreville, Gabon
                [5 ]AIDS and Cancer Virus Program, Frederick National Laboratory, Frederick, Maryland, United States of America
                [6 ]University of Alabama at Birmingham, Birmingham, Alabama, United States of America
                Centers for Disease Control and Prevention, United States of America
                Author notes

                I have read the journal's policy and the authors of this manuscript have the following competing interests: DJ, one of the co-authors, is currently employed by Metabiota, Inc. This affiliation does not alter the authors adherence to all PLOS NTDs policies on sharing data and materials.

                Conceived and designed the experiments: NJS PER SM KNC. Performed the experiments: SM MB GF. Analyzed the data: NJS PER SM DJ GF. Contributed reagents/materials/analysis tools: DJ PER NJS ZS BFK BH AUO MB PR WBK. Contributed to the writing of the manuscript: PER SM NJS.

                [¤a]

                Current address: Metabiota Inc., Nanaimo, British Columbia, Canada

                [¤b]

                Current address: Departments of Medicine and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America

                [¤c]

                Current address: Ecohealth Alliance, New York, New York, United States of America

                Article
                PNTD-D-14-00630
                10.1371/journal.pntd.0003143
                4169258
                25232832
                4e130222-4e10-404d-8456-b9933adc43ec
                Copyright @ 2014

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 15 April 2014
                : 22 July 2014
                Page count
                Pages: 10
                Funding
                This work was supported by the United States Fish and Wildlife Service, the Arcus Foundation, the Paul G. Allen Family Foundation, Mr. and Mrs. Bradley L. Goldberg, the United States Agency for International Development's Emerging Pandemic Threats PREDICT program, CSR Tullow Oil Society, the Réseau des Aires Protégées d'Afrique Centrale (RAPAC), the extramural grants (R01 AI50529 and R01 AI58715) and the Intramural Research Program of the U.S. National Institutes of Health, National Institute of Allergy and Infectious Diseases, Vaccine Research Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript and the findings do not represent the opinions of the US government.
                Categories
                Research Article
                Biology and Life Sciences
                Medicine and Health Sciences
                Research and Analysis Methods
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are contained within the manuscript.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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