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BackgroundThe 2013-16 Ebola virus disease epidemic in west Africa caused international alarm due to its rapid and extensive spread resulting in a significant death toll and social unrest within the affected region. The large number of cases provided an opportunity to study the long-term kinetics of Zaire ebolavirus-specific immune response of survivors in addition to known contacts of those infected with the virus.MethodsIn this observational cohort study, we worked with leaders of Ebola virus disease survivor associations in two regions of Guinea, Guéckédou and Coyah, to recruit survivors of Ebola virus disease, contacts from households of individuals known to have had Ebola virus disease, and individuals who were not knowingly associated with infected individuals or had not had Ebola virus disease symptoms to serve as negative controls. We did Zaire ebolavirus glycoprotein-specific T cell analysis on peripheral blood mononuclear cells (PBMCs) on location in Guinea and transported plasma and PBMCs back to Europe for antibody quantification by ELISA, functional neutralising antibody analysis using live Zaire ebolavirus, and T cell phenotype studies. We report on the longitudinal cellular and humoral response among Ebola virus disease survivors and highlight potentially paucisymptomatic infection.FindingsWe recruited 117 survivors of Ebola virus disease, 66 contacts, and 23 negative controls. The mean neutralising antibody titre among the Ebola virus disease survivors 3-14 months after infection was 1/174 (95% CI 1/136-1/223). Individual results varied greatly from 1/10 to more than 1/1000 but were on average ten times greater than that induced after 1 month by single dose Ebola virus vaccines. Following reactivation with glycoprotein peptide, the mean T cell responses among 116 Ebola virus disease survivors as measured by ELISpot was 305 spot-forming units (95% CI 257-353). The dominant CD8+ polyfunctional T cell phenotype, as measured among 53 Ebola virus disease survivors, was interferon γ+, tumour necrosis factor+, interleukin-2-, and the mean response was 0·046% of total CD8+ T cells (95% CI 0·021-0·071). Additionally, both neutralising antibody and T cell responses were detected in six (9%) of 66 Ebola virus disease contacts. We also noted that four (3%) of 117 individuals with Ebola virus disease infections did not have circulating Ebola virus-specific antibodies 3 months after infection.InterpretationThe continuous high titre of neutralising antibodies and increased T cell response might support the concept of long-term protective immunity in survivors. The existence of antibody and T cell responses in contacts of individuals with Ebola virus disease adds further evidence to the existence of sub-clinical Ebola virus infection.FundingUS Food & Drug Administration, Horizon 2020 EU EVIDENT, Wellcome, UK Department for International Development.TranslationFor the French translation of the abstract see Supplementary Materials section.

Ebola virus (EBOV) haemorrhagic fever remains a threat to global public health with an urgent need for an effective treatment. In order to achieve these goals, access to non-human primate (NHP) laboratory models is an essential requirement. Here, we present the first NHP-EBOV laboratory model readily available to the European scientific community, based on infection of Mauritian cynomolgus macaques using a Central-African EBOV strain and increasing virus challenge dose (10, 100, or 1000 focus forming units per animal). The outcome of these experiments was assessed using clinical, hematological, and biochemical criteria. All challenge doses resulted in fatal infections within 8–11 days. Symptoms appeared from day 5 after infection onwards and disease progression was slower than in previous reports based on Asian cynomolgus macaques. Thus, our model resembled human disease more closely than previous models (onset of symptoms estimated 2–21 days after infection) extending the period of time available for therapeutic intervention. To establish the dynamics of virus genome variation, the study included the first detailed analysis of major and minor genomic EBOV variants during the course of the disease. Major variants were scarce and the population of minor variants was shaped by selective pressure similar to genomic mutations observed in Nature. This primate model provides a robust baseline for future genomic studies in the context of therapeutic methods for treating Ebola virus-infected patients.

Lassa fever is a hemorrhagic fever endemic to West Africa and caused by Lassa virus, an Old-World arenavirus. It may be fatal, but most patients recover from acute disease and some experience asymptomatic infection. The immune mechanisms associated with these different outcomes have not yet been fully elucidated, but considerable progress has recently been made, through the use of in vitro human models and non-human primates, the only relevant animal model that mimics the pathophysiology and immune responses induced in patients. We discuss here the roles of the various components of the innate and adaptive immune systems in Lassa virus infection and in the control of viral replication and pathogenesis.

Hemorrhagic fever outbreaks are difficult to diagnose and control in part because of a lack of low-cost and easily accessible diagnostic structures in countries where etiologic agents are present. Furthermore, initial clinical symptoms are common and shared with other endemic diseases such as malaria or typhoid fever. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need, particularly in outbreak settings. Therefore, rapid diagnostic tests such as lateral flow can be broadly deployed and are typically well-suited to rapidly diagnose hemorrhagic fever viruses, such as Ebola virus. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect very low amount of virus in blood. Here, we developed and characterized an immunoassay test based on immunochromatography coupled to silver amplification technology to detect the secreted glycoprotein of EBOV. The glycoprotein is among the first viral proteins to be detected in blood. This strategy aims at identifying infected patients early following onset of symptoms by detecting low amount of sGP protein in blood samples. The limit of detection achieved by this sGP-targeted kit is 2.2 x 104 genome copies/ml in plasma as assayed in a monkey analytical cohort. Clinical performance evaluation showed a specificity of 100% and a sensitivity of 85.7% when evaluated with plasma samples from healthy controls and patients infected with Zaire Ebola virus from Macenta, Guinea. This rapid and accurate diagnostic test could therefore be used in endemic countries for early detection of infected individuals in point of care settings. Moreover, it could also support efficient clinical triage in hospitals or clinical centers and thus reducing transmission rates to prevent and better manage future severe outbreaks., Author summary Ebola virus disease is a severe disease caused by Ebola virus, a member of the filovirus family, which occurs in humans and other primates. Ebola is believed to be zoonotic, however the natural reservoir is unknown. Overlapping symptoms with other endemic diseases, such as malaria and cholera, make accurate diagnostic challenging. Outbreaks of Ebola have been widespread as the consequence of the absence of available rapid, sensitive, specific, robust, and affordable licensed diagnostic test in remote areas, where outbreaks usually start. Here we have established and validated a rapid diagnostic test, which is fast, sensitive, specific, efficient, affordable, and user-friendly. Its analytical characteristics make it suitable for clinical management during Ebola virus outbreaks in remote areas. Of interest, this rapid diagnostic test detects the presence of an early viral antigen, the secreted glycoprotein, found in blood of patients shortly after infection, suggesting that it could be used to identify infected patients shortly after onset of symptoms.

In March 2014, the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea. Virologic investigation identified Zaire ebolavirus (EBOV) as the causative agent. Full-length genome sequencing and phylogenetic analysis showed that EBOV from Guinea forms a separate clade in relationship to the known EBOV strains from the Democratic Republic of Congo and Gabon. Epidemiologic investigation linked the laboratory-confirmed cases with the presumed first fatality of the outbreak in December 2013. This study demonstrates the emergence of a new EBOV strain in Guinea.

Background Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. Methods and Findings Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in “cycle threshold” [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A “target value” of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%–32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%–91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within, In the context the recent Ebola outbreak, Xavier Anglaret and colleagues test an experimental treatment, favipiravir, for Ebola virus disease in a multicenter non-randomized trial., Editors' Summary Background In 2014 and 2015, an Ebola virus outbreak larger than any known before occurred in West Africa. Ebola virus disease (EVD) is highly contagious, and many infected people die. Central to the emergency response to the recent outbreak were local Ebola treatment centers where patients were diagnosed, were isolated, and received supportive care. With thousands of patients dying and many health workers contracting the disease, fear was ubiquitous and distrust abundant. While conducting research in this environment was extremely challenging, the urgent need for treatments and the opportunity to conduct studies that could bring such treatments closer to reality was also recognized. In September 2014, WHO released a short list of existing drugs that were candidates for clinical trials among patients infected in the outbreak. Favipiravir, an antiviral drug developed in Japan for patients with severe influenza, was on the list. Why Was This Study Done? Because of the urgent need to find drugs that could reduce deaths caused by Ebola, the researchers decided to conduct a clinical trial using favipiravir in patients with EVD in Guinea. In view of the circumstances, they decided against a randomized controlled trial and instead designed a study where all participants would receive the same treatment. In randomized controlled trials only some participants receive the treatment in addition to standard care, while others serve as a control group and receive standard care only, or standard care plus a placebo. Such studies allow stronger conclusions to be drawn about whether a treatment is safe and whether it works or not. The researchers had two main reasons for this decision. First, patients from the same family or village often sought EVD treatment at the same time, and the researchers felt that it was ethically unacceptable to randomize such groups, with only some of them receiving the experimental drug. Second, the strict isolation procedures imposed to interrupt virus transmission had intensified fear in affected communities and fueled rumors of illicit drug experimentation and organ theft at the treatment centers. In this context, the researchers worried that a randomized study might increase distrust among the community and the reluctance of patients to seek care. Rather than seeking definitive answers about the safety and efficacy of favipiravir in patients with EVD, the objectives of the study as it was designed were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak and to learn lessons from the experience. In addition, the researchers planned to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD in the hope that their preliminary findings could improve the design of subsequent trials and the chance to provide conclusive answers. What Did the Researchers Do and Find? After 13 weeks of preparation, the trial took place from December 2014 to April 2015 at four separate Ebola treatment centers, three in rural areas and one in an urban setting. In addition to standard care (which included rehydration, antimalarial and antibacterial therapies, and medication to reduce fever, pain, and nausea), all participants were given favipiravir by mouth for ten days, at doses substantially higher than those recommended for patients with influenza. Outcomes measured were mortality, viral load changes over time (based on blood samples), and adverse events. EVD was confirmed with an assay that used patient blood and provided an estimate of the viral load, that is, of how much virus the blood contained. Because viral load was known to influence the course of EVD, the researchers analyzed the participants in two groups, namely, those with a viral load estimate above a certain threshold and those with viral load estimate below the threshold. They also used existing data from Guinean patients diagnosed with Ebola earlier in the outbreak who had received only standard care and calculated an expected mortality rate for patients above and below the viral load threshold. The researchers were able to enroll 126 participants in the trial. Of these, 111 were included in the final analysis. Of 99 adult and adolescent participants 13 years and older, 55 were in the lower viral load group and 44 in the higher viral load group. Mortality was 20% in the former and 91% in the latter. Neither mortality rate was significantly different from that of earlier patients who had received only standard care. The researchers also found that favipiravir was well tolerated. None of the patients stopped the course of treatment, vomiting following drug intake was rare, and no severe adverse events were attributed to the drug. The researchers did not see a difference in mortality between patients who reported onset of symptoms less than three days before the start of treatment and those whose symptoms had started more than three days the start of treatment. What Do these Findings Mean? The report shows that it is possible to conduct an emergency trial during an outbreak in a low-resource setting. In fact, at the time of its acceptance, this paper reported on an Ebola treatment trial larger than any other yet published. The experience described should be useful for similar undertakings in the future. The following conditions contributed to the success of the trial: close collaboration between researchers, local health officials, and affected communities on one hand, and flexibility in design, conduct, and analysis based on close monitoring and interim assessments on the other. Besides using interim results to influence the conduct and analysis of their own trial, the researchers also shared these results with the scientific community in real time, and this feedback influenced other research during the outbreak. The trial could not answer definitively whether favipiravir treatment was safe or reduced mortality in patients with EVD. The results suggest that the drug is unlikely to be beneficial for patients with very high viral loads, at least when given by itself. They also suggest that favipiravir is safe in patients with lower viral loads, and that in such patients additional efficacy studies are warranted. Intermediate analysis of various measurements in trial participants showed that the estimate of viral load from the field EVD diagnosis test is a good proxy for the actual viral load (determined after the samples were shipped to and analyzed in a reference laboratory in France) and suitable as a surrogate marker. The results also confirm that viral load is a strong predictor of mortality. Additional Information This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001967. The World Health Organization has pages on Ebola virus disease, trials of Ebola treatments and vaccines, and the current update of the list of suitable drugs for testing or use in patients infected with Ebola (originally compiled in September 2014) US Centers for Disease Control and Prevention has information on the Ebola outbreak in West Africa The European Centre for Disease Prevention and Control also has information on the Ebola outbreak in West Africa

International audience; Lassa fever is a hemorrhagic fever endemic to West Africa and caused by Lassa virus, an Old World arenavirus. It may be fatal, but most patients recover from acute disease and some experience asymptomatic infection. The immune mechanisms associated with these different outcomes have not yet been fully elucidated, but considerable progress has recently been made, through the use of in vitro human models and nonhuman primates, the only relevant animal model that mimics the pathophysiology and immune responses induced in patients. We discuss here the roles of the various components of the innate and adaptive immune systems in Lassa virus infection and in the control of viral replication and pathogenesis.

The events leading to death in severe cases of Lassa fever (LF) are unknown. Fatality seems to be linked to high viremia and immunosuppression, and cellular immunity, rather than neutralizing antibodies, appears to be essential for survival. We previously compared Lassa virus (LV) with its genetically close but nonpathogenic homolog Mopeia virus (MV), which was used to model nonfatal LF. We showed that strong and early activation of antigen-presenting cells (APC) may play a crucial role in controlling infection. Here we developed an in vitro model of dendritic-cell (DC)-T-cell coculture in order to characterize human T-cell responses induced by MV- or LV-infected DCs. Our results show very different responses to infection with LV and MV. MV strongly and durably stimulated CD8 + and CD4 + T cells, showing early and high activation, a strong proliferative response, and acquisition of effector and memory phenotypes. Furthermore, robust and functional CD4 + and CD8 + cytotoxic T lymphocytes (CTL) were generated. LV, however, induced only weak memory responses. Thus, this study allows an improved understanding of the pathogenesis and immune mechanisms involved in the control of human LV.

On August 8 2014, the World Health Organization (WHO) declared the outbreak of Ebola Virus Disease(EVD) evolving in West Africa since December 2013, a Public Health Emergency of International Concern (PHEIC). It is expected that the outbreak of Ebolavirus Disease (EVD) in West Africa will lead to increased testing of individuals in Europe for EVD. The severity of the situation in West Africa warranted a critical appraisal of the laboratory preparedness and response for EVD, with a focus on information needs for laboratories involved in diagnostics of rare viral diseases associated with the European Network for the Diagnostics of "Imported" Viral Diseases", ENIVD. Essential knowledge and knowledge gaps for an adequate laboratory response focusing on virus properties, infection kinetics, tests specifics and field performances were identified. An inventory of the laboratory capacity for EVD diagnostics among ENIVD laboratories was made. (C) 2014 Elsevier B.V. All rights reserved.

International audience; Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), which primarily targets human dendritic cells (DC) and macrophages (MP). Massive numbers of viral particles are released with no effect on the viability, activation or maturation of these cells. LV does not inhibit the activation of cells induced by sCD40L or LPS. We report here the consequences of exogenous activation of LV-infected human DC and MP for viral replication. The activation of cells with lipopolysaccharide or exogenous poly(I-C) and the transfection of cells with poly(I-C) strongly inhibited LV replication, at least partly by inducing type I interferon (IFN) synthesis. In contrast, cell stimulation with sCD40L did not induce type I IFN responses or inhibit LV release. Recombinant type I IFNs strongly inhibited LV replication in both cell types, whereas IFNgamma and IFNlambda did not. The modest type I IFN production observed in LV-infected MP, but not in DC, was involved in controlling LV replication in MP. These results provide an explanation for the slower replication of LV in MP than in DC, and suggest that type I IFNs are crucial in the control of LV.

Lassa virus (LV) and Mopeia virus (MV) are closely related members of the Arenavirus genus, sharing 75% amino acid sequence identity. However, LV causes hemorrhagic fever in humans and nonhuman primates, whereas MV cannot induce disease. We have previously shown that antigen-presenting cells (APC)—macrophages (MP) and dendritic cells (DC)—sustain high replication rates of LV but are not activated, suggesting that they play a role in the immunosuppression observed in severe cases of Lassa fever. Here, we infected human APC with MV and analyzed the cellular responses induced. MV infection was productive in MP and even more so in DC. Apoptosis was not induced in either cell type. Moreover, unlike DC, MP were early and strongly activated in response to MV, as shown by the increased surface expression of CD86, CD80, CD54, CD40, and HLA-abc and by the production of mRNA encoding alpha interferon (IFN-α), IFN-β, tumor necrosis factor alpha and interleukin-6. In addition, MV-infected MP produced less of the virus than DC, which was related to the fact that these cells secreted IFN-α. Thus, the strong activation of MP is probably a major event in the control of MV infection and may be involved in the induction of an adaptive immune response in infected hosts. These results may explain the difference in pathogenicity between LV and MV.

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), an old-world Arenavirus. Little is known about the immune responses that occur during the disease, but protection seems to be linked to the induction of cellular responses specific for viral glycoproteins. Conversely, severe Lassa fever may be associated with immunosuppression. We studied the infection of human dendritic cells (DC) and macrophages (MP) by LV. Both these cell types are susceptible to LV infection. Viral nucleoprotein was detected in DC and MP, and high and moderate viral titers were obtained with culture supernatants of DC and MP, respectively. LV did not induce apoptosis in DC and MP. These cells were not activated by LV infection. No change was observed in the expression of surface molecules involved in activation, costimulation, adhesion, and Ag presentation following LV infection, or in the functional properties of DC. Inflammatory cytokine production was not detected at the mRNA or protein level after LV infection of DC and MP. Thus, MP, and particularly DC, are crucial targets for LV and are probably involved in the early replication of LV from the initial site of infection. The lack of activation and maturation of cells following infection may be associated with the immunosuppression observed in severe LV infection.