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Summary During inflammatory diseases, cancer, and infection, the cGAS/STING pathway is known to recognize foreign or self-DNA in the cytosol and activate an innate immune response. Here, we report that negative-strand RNA paramyxoviruses, Nipah virus (NiV), and measles virus (MeV), can also trigger the cGAS/STING axis. Although mice deficient for MyD88, TRIF, and MAVS still moderately control NiV infection when compared with wild-type mice, additional STING deficiency resulted in 100% lethality, suggesting synergistic roles of these pathways in host protection. Moreover, deletion of cGAS or STING resulted in decreased type I interferon production with enhanced paramyxoviral infection in both human and murine cells. Finally, the phosphorylation and ubiquitination of STING, observed during viral infections, confirmed the activation of cGAS/STING pathway by NiV and MeV. Our data suggest that cGAS/STING activation is critical in controlling paramyxovirus infection and possibly represents attractive targets to develop countermeasures against severe disease induced by these pathogens., Graphical abstract, Highlights • RNA sensors are insufficient for the effective control of paramyxovirus infection • STING adaptor protein is involved in controlling Nipah virus infection in mice • cGAS/STING axis is primordial for optimal production of IFN-I against NiV and MeV • STING protein can be activated during infections by RNA viruses, Immune system; Molecular biology; Virology

Crimean-Congo hemorrhagic fever virus (CCHFV) is a virus that causes severe liver dysfunctions and hemorrhagic fever, with high mortality rate. Here, we show that CCHFV infection caused a massive lipidation of LC3 in hepatocytes. This lipidation was not dependent on ATG5, ATG7 or BECN1, and no signs for recruitment of the alternative ATG12-ATG3 pathway for lipidation was found. Both virus replication and protein synthesis were required for the lipidation of LC3. Despite an augmented transcription of SQSTM1, the amount of proteins did not show a massive and sustained increase in infected cells, indicating that degradation of SQSTM1 by macroautophagy/autophagy was still occurring. The genetic alteration of autophagy did not influence the production of CCHFV particles demonstrating that autophagy was not required for CCHFV replication. Thus, the results indicate that CCHFV multiplication imposes an overtly elevated level of LC3 mobilization that involves a possibly novel type of non-canonical lipidation. Abbreviations: BECN1: Beclin 1; CCHF: Crimean-Congo hemorrhagic fever; CCHFV: Crimean-Congo hemorrhagic fever virus; CHX: cycloheximide; ER: endoplasmic reticulum; GFP: green fluorescent protein; GP: glycoproteins; MAP1LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; n.i.: non-infected; NP: nucleoprotein; p.i.: post-infection; SQSTM1: sequestosome 1.

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.

Clinical follow-up of Ebola virus disease (EVD) survivors revealed a persistence of clinical symptoms and higher risk of mortality. Long-term analyses of the immune and inflammatory profiles of EVD survivors are currently lacking. Here, we evaluate immune profile status and gene expression profiles in 35 Guinean EVD survivors (EBOV_S) from the last West African outbreak, a median of 23 months (IQR [18-25]) after discharge from the Ebola treatment center. We show a persistent increase of several biomarkers of inflammation, immune activation and gut tissue damage in EBOV_S compared to healthy donors living in the same area. These results are confirmed by phenotypic characterization of immune cell subsets revealing increases in activation marker expression and the frequencies of CD8+ T cells, exhausted B cells, non-classical NK cells and circulating dendritic cells. All survivors have EBOV-specific IgG antibodies and robust and polyfunctional EBOV-specific memory T-cell responses. Deep sequencing studies of the genes expressed in blood revealed a significant enrichment in genes associated with antiviral responses in EBOV_S. This study assess the long-term persistence of immunological dysfunction in survivors and identify a set of biological and genetic markers that could be used to define a signature of “chronic Ebola virus disease (CEVD)”.

Lors de l’epidemie de maladie a virus Ebola qui a frappe l’Afrique de l’Ouest en 2014, l’OMS a souligne le besoin de tests de diagnostic rapide (TDR), simples d’emploi, aisement deployables et facilement utilisables sur le terrain. Le TDR (Lateral Flow Assay) Ebola eZYSCREEN® a ete developpe dans ce contexte d’urgence avec des anticorps monoclonaux diriges contre la glycoproteine d’enveloppe du virus. Deux versions ont ete industrialisees, l’une pour des echantillons de sang total et l’autre pour des echantillons de serum et de plasma. Ces deux versions ont une limite de detection analytique de 105 pfu/ml, la stabilite est au minimum de 393 jours a 30 °C et 120 jours a 45 °C. Une etude de validation, independante, non retrospective, a ete realisee en situation reelle, pendant la flambee epidemique a Conakry et au centre de traitement Ebola de Coyah sur 144 patients. Les sensibilites et specificites des TDR sont respectivement de 65,3 et 98,9 % sur sang total et de 74,5 et 100 % sur serum. Les performances observees avec la version sang total sont a prendre avec precaution en raison de contraintes logistiques, dues au contexte particulier de cette crise Ebola, qui ont complique la realisation de cette etude, avec notamment un delai moyen de trois jours entre le prelevement et la realisation des tests, ce qui est en dehors des specifications. Neanmoins, ce test de terrain, simple d’emploi, facilement deployable, qui ne necessite pas, contrairement aux tests de laboratoire, d’appareillage sophistique ni meme d’electricite, peut contribuer a la chaine diagnostique de la maladie a virus Ebola, en tenant compte de ses avantages, stabilite et specificite elevees, mais aussi de sa limite de sensibilite par rapport aux techniques moleculaires de laboratoire, qui restent la reference pour le diagnostic de la maladie a virus Ebola. Le TDR Ebola eZYSCREEN® a obtenu le marquage CE-IVD.

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

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak. ispartof: Nature vol:524 issue:7563 pages:97-101 ispartof: location:England status: published

Background. A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. Methods. The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcriptionpolymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. Results. The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged 74 years (90%) and low in patients aged 1019 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD 1 . In particular, very little is known about human immune responses to Ebola virus (EBOV) 2,3 . Here, we have for the first time evaluated the physiology of the human T cell immune response in EVD patients at the time of admission at the Ebola Treatment Center (ETC) in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we have identified an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by high percentage of CD4 and CD8 T cells expressing the inhibitory molecules cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death-1 (PD-1), which was correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation despite comparable overall T cell activation. Concommittant with virus clearance, survivors mounted a robust EBOV-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.