Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

[Recent advances in the development of vaccines against hemorrhagic fevers caused by arenaviruses]. / Les fièvres hémorragiques causées par les arénavirus : de récentes avancées vaccinales.

Arenaviruses are a global threat, causing thousands of deaths each year in several countries around the world. Despite strong efforts in the development of vaccine candidates, vaccines against Lassa fever or Bolivian and Venezuelan hemorrhagic fevers are yet to be licensed for a use in humans. In this synthesis, we present the arenaviruses causing fatal diseases in humans and the main vaccine candidates that have been developed over the past decades with an emphasis on the measles-Lassa vaccine, the first Lassa vaccine ever tested in humans, and on the MOPEVAC platform that can potentially be used as a pan-arenavirus vaccine platform.

Title: Les fièvres hémorragiques causées par les arénavirus : de récentes avancées vaccinales. Abstract: Le développement de vaccins contre les arénavirus est un enjeu global. En effet, plusieurs milliers de personnes meurent chaque année de la fièvre de Lassa en Afrique occidentale et les virus Machupo, Guanarito ou Chapare continuent de ré-émerger en Amérique du Sud. Pourtant, il n'existe à ce jour aucun vaccin validé pour une utilisation dans l'espèce humaine pour lutter contre ces arénavirus. Dans cette synthèse, nous présentons les différents arénavirus causant des maladies mortelles chez l'espèce humaine et les principaux candidats vaccins développés au cours des dernières décennies contre ces virus. Nous décrivons plus particulièrement le vaccin rougeole-Lassa, premier vaccin contre la fièvre de Lassa à avoir été testé dans l'espèce humaine, et la plateforme MOPEVAC qui permet de générer avec succès des vaccins mono- ou multivalents contre potentiellement tous les arénavirus pathogènes connus.

Hemostasis defects underlying the hemorrhagic syndrome caused by mammarenaviruses in a cynomolgus macaque model.

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.

Immunogenicity, safety, and tolerability of a recombinant measles-vectored Lassa fever vaccine: a randomised, placebo-controlled, first-in-human trial.

BACKGROUND: Lassa fever is a substantial health burden in west Africa. We evaluated the safety, tolerability, and immunogenicity of a recombinant, live-attenuated, measles-vectored Lassa fever vaccine candidate (MV-LASV). METHODS: This first-in-human phase 1 trial-consisting of an open-label dose-escalation stage and an observer-blinded, randomised, placebo-controlled treatment stage-was conducted at a single site at the University of Antwerp, Antwerp, Belgium, and involved healthy adults aged 18-55 years. Participants in the dose-escalation stage were sequentially assigned to a low-dose group (two intramuscular doses of MV-LASV at 2 × 104 times the median tissue culture infectious dose) or a high-dose group (two doses at 1 × 105 times the median tissue culture infectious dose). Participants in the double-blinded treatment stage were randomly assigned in a 2:2:1 ratio to receive low dose, high dose, or placebo. The primary endpoint was the rate of solicited and unsolicited adverse events up to study day 56 and was assessed in all participants who received at least one dose of investigational product. The trial is registered with ClinicalTrials.gov, NCT04055454, and the European Union Drug Regulating Authorities Clinical Trials Database, 2018-003647-40, and is complete. FINDINGS: Between Sept 26, 2019, and Jan 20, 2020, 60 participants were enrolled and assigned to receive placebo (n=12) or MV-LASV (n=48). All 60 participants received at least one study treatment. Most adverse events occurred during the treatment phase, and frequencies of total solicited or unsolicited adverse events were similar between treatment groups, with 96% of participants in the low-dose group, 100% of those in the high-dose group, and 92% of those in the placebo group having any solicited adverse event (p=0·6751) and 76% of those in the low-dose group, 70% of those in the high-dose group, and 100% of those in the placebo group having any unsolicited adverse event (p=0·1047). The only significant difference related to local solicited adverse events, with higher frequencies observed in groups receiving MV-LASV (24 [96%] of 25 participants in the low-dose group; all 23 [100%] participants in the high-dose group) than in the placebo group (6 [50%] of 12 participants; p=0·0001, Fisher-Freeman-Halton test). Adverse events were mostly of mild or moderate severity, and no serious adverse events were observed. MV-LASV also induced substantial concentrations of LASV-specific IgG (geometric mean titre 62·9 EU/ml in the low-dose group and 145·9 EU/ml in the high-dose group on day 42). INTERPRETATION: MV-LASV showed an acceptable safety and tolerability profile, and immunogenicity seemed to be unaffected by pre-existing immunity against the vector. MV-LASV is therefore a promising candidate for further development. FUNDING: Coalition for Epidemic Preparedness Innovations.

Rapid protection induced by a single-shot Lassa vaccine in male cynomolgus monkeys.

Lassa fever hits West African countries annually in the absence of licensed vaccine to limit the burden of this viral hemorrhagic fever. We previously developed MeV-NP, a single-shot vaccine protecting cynomolgus monkeys against divergent strains one month or more than a year before Lassa virus infection. Given the limited dissemination area during outbreaks and the risk of nosocomial transmission, a vaccine inducing rapid protection could be useful to protect exposed people during outbreaks in the absence of preventive vaccination. Here, we test whether the time to protection can be reduced after immunization by challenging measles virus pre-immune male cynomolgus monkeys sixteen or eight days after a single shot of MeV-NP. None of the immunized monkeys develop disease and they rapidly control viral replication. Animals immunized eight days before the challenge are the best controllers, producing a strong CD8 T-cell response against the viral glycoprotein. A group of animals was also vaccinated one hour after the challenge, but was not protected and succumbed to the disease as the control animals. This study demonstrates that MeV-NP can induce a rapid protective immune response against Lassa fever in the presence of MeV pre-existing immunity but can likely not be used as therapeutic vaccine.

A MOPEVAC multivalent vaccine induces sterile protection against New World arenaviruses in non-human primates.

Pathogenic New World arenaviruses (NWAs) cause haemorrhagic fevers and can have high mortality rates, as shown in outbreaks in South America. Neutralizing antibodies (Abs) are critical for protection from NWAs. Having shown that the MOPEVAC vaccine, based on a hyperattenuated arenavirus, induces neutralizing Abs against Lassa fever, we hypothesized that expression of NWA glycoproteins in this platform might protect against NWAs. Cynomolgus monkeys immunized with MOPEVACMAC, targeting Machupo virus, prevented the lethality of this virus and induced partially NWA cross-reactive neutralizing Abs. We then developed the pentavalent MOPEVACNEW vaccine, expressing glycoproteins from all pathogenic South American NWAs. Immunization of cynomolgus monkeys with MOPEVACNEW induced neutralizing Abs against five NWAs, strong innate followed by adaptive immune responses as detected by transcriptomics and provided sterile protection against Machupo virus and the genetically distant Guanarito virus. MOPEVACNEW may thus be efficient to protect against existing and potentially emerging NWAs.

Pathogenesis of recent Lassa virus isolates from lineages II and VII in cynomolgus monkeys.

The area of Lassa virus (LASV) circulation is expanding, with the emergence of highly pathogenic new LASV lineages. Benin recently became an endemic country for LASV and has seen the emergence of a new LASV lineage (VII). The first two outbreaks in 2014 and 2016 showed a relatively high mortality rate compared to other outbreaks. We infected cynomolgus monkeys with two strains belonging to lineage II and lineage VII that were isolated from deceased patients during the 2016 outbreak in Benin. The lineage VII strain (L7) caused uniform mortality. Death was associated with uncontrolled viral replication, unbalanced inflammatory responses characterized by increased concentrations of pro- and anti-inflammatory mediators, and the absence of efficient immune responses, resembling the pathogenesis associated with the prototypic Josiah strain in monkeys. The lineage II strain (L2) showed apparently lower virulence than its counterpart, with a prolonged time to death and a lower mortality rate. Prolonged survival was associated with better control of viral replication, a moderate inflammatory response, and efficient T-cell responses. Transcriptomic analyses also highlighted important differences in the immune responses associated with the outcome. Both strains caused strong inflammation in several organs. Notably, meningitis and encephalitis were observed in the cerebral cortex and cerebellum in all monkeys, independently of the outcome. Due to their apparently high pathogenicity, emerging strains from lineage VII should be considered in preclinical vaccine testing. Lineage II would also be beneficial in pathogenesis studies to study the entire spectrum of Lassa fever severity.

A single-shot Lassa vaccine induces long-term immunity and protects cynomolgus monkeys against heterologous strains.

A safe and protective Lassa virus vaccine is crucially needed in Western Africa to stem the recurrent outbreaks of Lassa virus infections in Nigeria and the emergence of Lassa virus in previously unaffected countries, such as Benin and Togo. Major challenges in developing a Lassa virus vaccine include the high diversity of circulating strains and their reemergence from 1 year to another. To address each of these challenges, we immunized cynomolgus monkeys with a measles virus vector expressing the Lassa virus glycoprotein and nucleoprotein of the prototypic Lassa virus strain Josiah (MeV-NP). To evaluate vaccine efficacy against heterologous strains of Lassa virus, we challenged the monkeys a month later with heterologous strains from lineage II or lineage VII, finding that the vaccine was protective against these strains. A second cohort of monkeys was challenged 1 year later with the homologous Josiah strain, finding that a single dose of MeV-NP was sufficient to protect all vaccinated monkeys. These studies demonstrate that MeV-NP can generate both long-lasting immune responses and responses that are able to protect against diverse strains of Lassa virus.

Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques.

Lassa virus (LASV) is endemic in West Africa and induces a viral hemorrhagic fever (VHF) with up to 30% lethality among clinical cases. The mechanisms involved in control of Lassa fever or, in contrast, the ensuing catastrophic illness and death are poorly understood. We used the cynomolgus monkey model to reproduce the human disease with asymptomatic to mild or fatal disease. After initial replication at the inoculation site, LASV reached the secondary lymphoid organs. LASV did not spread further in nonfatal disease and was rapidly controlled by balanced innate and T-cell responses. Systemic viral dissemination occurred during severe disease. Massive replication, a cytokine/chemokine storm, defective T-cell responses, and multiorgan failure were observed. Clinical, biological, immunological, and transcriptomic parameters resembled those observed during septic-shock syndrome, suggesting that similar pathogenesis is induced during Lassa fever. The outcome appears to be determined early, as differentially expressed genes in PBMCs were associated with fatal and non-fatal Lassa fever outcome very early after infection. These results provide a full characterization and important insights into Lassa fever pathogenesis and could help to develop early diagnostic tools.

Rational design of universal immunotherapy for TfR1-tropic arenaviruses.

Certain arenaviruses that circulate in rodent populations can cause life-threatening hemorrhagic fevers when they infect humans. Due to their efficient transmission, arenaviruses pose a severe risk for outbreaks and might be exploited as biological weapons. Effective countermeasures against these viruses are highly desired. Ideally, a single remedy would be effective against many or even all the pathogenic viruses in this family. However, despite the fact that all pathogenic arenaviruses from South America utilize transferrin receptor 1 (TfR1) as a cellular receptor, their viral glycoproteins are highly diversified, impeding efforts to isolate cross-neutralizing antibodies. Here we address this problem using a rational design approach to target TfR1-tropic arenaviruses with high potency and breadth. The pan-reactive molecule is highly effective against all arenaviruses that were tested, offering a universal therapeutic approach. Our design scheme avoids the shortcomings of previous immunoadhesins and can be used to combat other zoonotic pathogens.

Vaccines inducing immunity to Lassa virus glycoprotein and nucleoprotein protect macaques after a single shot.

Lassa fever is a major threat in Western Africa. The large number of people living at risk for this disease calls for the development of a vaccine against Lassa virus (LASV). We generated live-attenuated LASV vaccines based on measles virus and Mopeia virus platforms and expressing different LASV antigens, with the aim to develop a vaccine able to protect after a single shot. We compared the efficacy of these vaccines against LASV in cynomolgus monkeys. The vaccines were well tolerated and protected the animals from LASV infection and disease after a single immunization but with varying efficacy. Analysis of the immune responses showed that complete protection was associated with robust secondary T cell and antibody responses against LASV. Transcriptomic and proteomic analyses showed an early activation of innate immunity and T cell priming after immunization with the most effective vaccines, with changes detectable as early as 2 days after immunization. The most efficacious vaccine candidate, a measles vector simultaneously expressing LASV glycoprotein and nucleoprotein, has been selected for further clinical evaluation.

Fatal Case of Lassa Fever, Bangolo District, Côte d'Ivoire, 2015.

Lassa fever has not been reported in Côte d'Ivoire. We performed a retrospective analysis of human serum samples collected in Côte d'Ivoire in the dry seasons (January-April) during 2015-2018. We identified a fatal human case of Lassa fever in the Bangolo District of western Côte d'Ivoire during 2015.

Trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells.

Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.

Immune parameters and outcomes during Ebola virus disease.

BACKGROUND: The West African Ebola virus epidemic from 2014-2016 highlighted the lack of knowledge about the pathogenicity of the virus and the factors responsible for outcome. A performant and rapid diagnosis is of crucial importance, as is overcoming the difficulty of providing high-quality patient management during such an extensive outbreak. Here, we propose to study the role of the immune mediators during Ebola virus disease and to define some molecules of importance in the outcome. METHODS: Plasma from Guinean patients sampled during the outbreak were analyzed using RT-qPCR, magnetic bead assay, ELISA, and high-quality statistical analyses. We also performed a transcriptomic analysis in leukocytes samples. Therefore, we deeply characterized the immune responses involved in Ebola virus disease. RESULTS: We evaluated the immune patterns depending on the outcome of the disease. Survivors presented an efficient and well-balanced immune response, whereas fatalities were characterized by an intense inflammatory response, overexpression of multiple cytokines, and a "chemokine storm." The plasma concentration of most of the parameters tested increased until death. Statistical analyses also allowed us to define a panel of markers highly predictive of outcome. CONCLUSION: The immune response observed in fatalities was highly similar to that characterizing septic shock syndrome. Our results suggest that immune responses can play a major pathogenic role during severe Ebola virus infection and argue in favor of therapeutic approaches that act on both viral replication and the induction of shock syndrome. FUNDING: French Ministry of Foreign Affairs, the Agence Française de Développement, and the Institut Pasteur.

A 40-Month Follow-Up of Ebola Virus Disease Survivors in Guinea (PostEbogui) Reveals Long-Term Detection of Ebola Viral Ribonucleic Acid in Semen and Breast Milk.

BACKGROUND: With the increasing frequency and impact of Ebola virus disease (EVD) outbreaks illustrated by recent epidemics, a good understanding of the extent of viral persistance or ribonucleic acid (RNA) detection in body fluids from survivors is urgently needed. METHODS: Ebola viral RNA shedding was studied with molecular assays in semen (n = 1368), urine (n = 1875), cervicovaginal fluid (n = 549), saliva (n = 900), breast milk (n = 168), and feces (n = 558) from EVD survivors in Guinea (PostEbogui cohort, n = 802) at a regular base period until 40 months after inclusion. RESULTS: Twenty-seven of 277 (9.8%) male survivors tested positive for Ebola RNA in at least 1 semen sample. The probability of remaining positive for Ebola RNA in semen was estimated at 93.02% and 60.12% after 3 and 6 months. Viral RNA in semen was more frequent in patients with eye pain (P = .036), joint pain (P = .047), and higher antibody levels to Ebola virus antigens (nucleoprotein [P = .001], glycoprotein [P = .05], and viral protein-40 [P = .05]). Ebola RNA was only rarely detected in the following body fluids from EVD survivors: saliva (1 of 454), urine (2 of 593), breast milk (2 of 168), cervicovaginal secretions (0 of 273), and feces (0 of 330). Ribonucleic acid was detected in breast milk 1 month after delivery but 500 days after discharge of Ebola treatment unit (ETU) in 1 woman who became pregnant 7 months after discharge from the ETU. CONCLUSIONS: The frequency and potential long-term presence of viral RNA in semen confirmed that systematic prevention measures in male survivors are required. Our observation in breast milk suggests that our knowledge on viral reservoir in immune-privileged sites and its impact are still incomplete.

E3 Ligase ITCH Interacts with the Z Matrix Protein of Lassa and Mopeia Viruses and Is Required for the Release of Infectious Particles.

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release.

Lassa virus activates myeloid dendritic cells but suppresses their ability to stimulate T cells.

Lassa virus (LASV) is responsible for a viral hemorrhagic fever in humans and the death of 3,000 to 5,000 people every year. The immune response to LASV is poorly understood, but type I interferon (IFN-I) and T-cell responses appear to be critical for the host. We studied the response of myeloid dendritic cells (mDC) to LASV, as mDCs are involved in both IFN-I production and T-cell activation. We compared the response of primary human mDCs to LASV and Mopeia virus (MOPV), which is similar to LASV, but non-pathogenic. We showed that mDCs produced substantial amounts of IFN-I in response to both LASV and MOPV. However, only MOPV-infected mDCs were able to activate T cells. More surprisingly, coculture with T cells completely inhibited the activation of LASV-infected mDCs. These differences between LASV and MOPV were mostly due to the LASV nucleoprotein, which has major immunosuppressive properties, but the glycoprotein was also involved. Overall, these results suggest that mDCs may be important for the global response to LASV and play a role in the outcome of Lassa fever.

A Vaccine Platform against Arenaviruses Based on a Recombinant Hyperattenuated Mopeia Virus Expressing Heterologous Glycoproteins.

Several Old World and New World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antivirals, or specific treatments are available, except for Junín virus. However, protection of nonhuman primates against Lassa fever virus (LASV) is possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified by using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in NP of MOPV, we found that this activity was essential for multiplication in antigen-presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPVExoN6b) that is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to those of MOPV, and (iii) still promotes a strong type I interferon (IFN) response. MOPVExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV or Lujo, Machupo, Guanarito, Chapare, or Sabia virus in order to broaden specific antigenicity while preserving the hyperattenuated characteristics of the parental strain. Our MOPV-based vaccine candidate for LASV, MOPEVACLASV, was used in a one-shot immunization assay in nonhuman primates and fully protected them from a lethal challenge with LASV. Thus, our hyperattenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.IMPORTANCE Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus and early care for treatment and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyperattenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected nonhuman primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic, and efficacious in nonhuman primates.

Modeling Ebolavirus Budding with Virus Like Particles.

About 15 years ago, several groups initially described the release of virus like particles (VLPs) upon expression of Ebola virus VP40 in mammalian cells. Further development of the protocol later allowed for the dissection of the Ebola virus budding mechanism and for the identification of critical VP40 residues involved in this process. VLPs are now produced routinely in several laboratories as a tool to study virus entry or egress and have even been proposed as vaccine candidates against Ebola virus disease. Here we described protocols for the production and the analysis of Ebola virus VLP release.

Clinical, virological, and biological parameters associated with outcomes of Ebola virus infection in Macenta, Guinea.

BACKGROUND. The pathogenesis of Ebola virus (EBOV) disease (EVD) is poorly characterized. The establishment of well-equipped diagnostic laboratories close to Ebola treatment centers (ETCs) has made it possible to obtain relevant virological and biological data during the course of EVD and to assess their association with the clinical course and different outcomes of the disease. METHODS. We were responsible for diagnosing EBOV infection in patients admitted to two ETCs in forested areas of Guinea. The pattern of clinical signs was recorded, and an etiological diagnosis was established by RT-PCR for EBOV infection or a rapid test for malaria and typhoid fever. Biochemical analyses were also performed. RESULTS. We handled samples from 168 patients between November 29, 2014, and January 31, 2015; 97 patients were found to be infected with EBOV, with Plasmodium falciparum coinfection in 18%. Overall mortality for EVD cases was 58%, rising to 86% if P. falciparum was also present. Viral load was higher in fatal cases of EVD than in survivors, and fatal cases were associated with higher aspartate aminotransferase (AST) and alanine aminotransferase (ALT), C-reactive protein (CRP), and IL-6 levels. Furthermore, regardless of outcome, EVD was characterized by higher creatine kinase (CPK), amylase, and creatinine levels than in febrile patients without EVD, with higher blood urea nitrogen (BUN) levels in fatal cases of EVD only. CONCLUSION. These findings suggest that a high viral load at admission is a marker of poor EVD prognosis. In addition, high AST, ALT, CRP, and IL-6 levels are associated with a fatal outcome of EVD. Damage to the liver and other tissues, with massive rhabdomyolysis and, probably, acute pancreatitis, is associated with EVD and correlated with disease severity. Finally, biochemical analyses provide substantial added value at ETCs, making it possible to improve supportive rehydration and symptomatic care for patients. FUNDING. The French Ministry of Foreign Affairs, the Agence Française de Développement, and Institut Pasteur.