The 3' Untranslated Regions of Ebola Virus mRNAs Contain AU-Rich Elements Involved in Posttranscriptional Stabilization and Decay.

The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.

An introduction to the Marburg virus vaccine consortium, MARVAC.

The emergence of Marburg virus (MARV) in Guinea and Ghana triggered the assembly of the MARV vaccine "MARVAC" consortium representing leaders in the field of vaccine research and development aiming to facilitate a rapid response to this infectious disease threat. Here, we discuss current progress, challenges, and future directions for MARV vaccines.

Avatar Mice Underscore the Role of the T Cell-Dendritic Cell Crosstalk in Ebola Virus Disease and Reveal Mechanisms of Protection in Survivors.

Ebola virus disease (EVD) is a complex infectious disease characterized by high inflammation, multiorgan failure, the dysregulation of innate and adaptive immune responses, and coagulation abnormalities. Evidence accumulated over the last 2 decades indicates that, during fatal EVD, the infection of antigen-presenting cells (APC) and the dysregulation of T cell immunity preclude a successful transition between innate and adaptive immunity, which constitutes a key disease checkpoint. In order to better understand the contribution of the APC-T cell crosstalk to EVD pathophysiology, we have developed avatar mice transplanted with human, donor-specific APCs and T cells. Here, we show that the transplantation of T cells and APCs from Ebola virus (EBOV)-naive individuals into avatar mice results in severe disease and death and that this phenotype is dependent on T cell receptor (TCR)-major histocompatibility complex (MCH) recognition. Conversely, avatar mice were rescued from death induced by EBOV infection after the transplantation of both T cells and plasma from EVD survivors. These results strongly suggest that protection from EBOV reinfection requires both cellular and humoral immune memory responses. IMPORTANCE The crosstalk between dendritic cells and T cells marks the transition between innate and adaptive immune responses, and it constitutes an important checkpoint in EVD. In this study, we present a mouse avatar model in which T cell and dendritic cell interactions from a specific donor can be studied during EVD. Our findings indicate that T cell receptor-major histocompatibility complex-mediated T cell-dendritic cell interactions are associated with disease severity, which mimics the main features of severe EVD in these mice. Resistance to an EBOV challenge in the model was achieved via the transplantation of both survivor T cells and plasma.

Expression of the Ebola Virus VP24 Protein Compromises the Integrity of the Nuclear Envelope and Induces a Laminopathy-Like Cellular Phenotype.

Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication.

Metagenomic Snapshots of Viral Components in Guinean Bats.

To prevent the emergence of zoonotic infectious diseases and reduce their epidemic potential, we need to understand their origins in nature. Bats in the order Chiroptera are widely distributed worldwide and are natural reservoirs of prominent zoonotic viruses, including Nipah virus, Marburg virus, and possibly SARS-CoV-2. In this study, we applied unbiased metagenomic and metatranscriptomic approaches to decipher the virosphere of frugivorous and insectivorous bat species captured in Guéckédou, Guinea, the epicenter of the West African Ebola virus disease epidemic in 2013-2016. Our study provides a snapshot of the viral diversity present in these bat species, with several novel viruses reported for the first time in bats, as well as some bat viruses closely related to known human or animal pathogens. In addition, analysis of Mops condylurus genomic DNA samples revealed the presence of an Ebola virus nucleoprotein (NP)-derived pseudogene inserted in its genome. These findings provide insight into the evolutionary traits of several virus families in bats and add evidence that nonretroviral integrated RNA viruses (NIRVs) derived from filoviruses may be common in bat genomes.

Chikungunya Outbreak in the Republic of the Congo, 2019-Epidemiological, Virological and Entomological Findings of a South-North Multidisciplinary Taskforce Investigation.

The Republic of Congo (RoC) declared a chikungunya (CHIK) outbreak on 9 February 2019. We conducted a ONE-Human-Animal HEALTH epidemiological, virological and entomological investigation. Methods: We collected national surveillance and epidemiological data. CHIK diagnosis was based on RT-PCR and CHIKV-specific antibodies. Full CHIKV genome sequences were obtained by Sanger and MinION approaches and Bayesian tree phylogenetic analysis was performed. Mosquito larvae and 215 adult mosquitoes were collected in different villages of Kouilou and Pointe-Noire districts and estimates of Aedes (Ae.) mosquitos' CHIKV-infectious bites obtained. We found two new CHIKV sequences of the East/Central/South African (ECSA) lineage, clustering with the recent enzootic sub-clade 2, showing the A226V mutation. The RoC 2019 CHIKV strain has two novel mutations, E2-T126M and E2-H351N. Phylogenetic suggests a common origin from 2016 Angola strain, from which it diverged around 1989 (95% HPD 1985-1994). The infectious bite pattern was similar for 2017, 2018 and early 2019. One Ae. albopictus pool was RT-PCR positive. The 2019 RoC CHIKV strain seems to be recently introduced or be endemic in sylvatic cycle. Distinct from the contemporary Indian CHIKV isolates and in contrast to the original Central-African strains (transmitted by Ae. aegypti), it carries the A226V mutation, indicating an independent adaptive mutation in response to vector replacement (Ae. albopictus vs Ae. aegypti).

Ebola Virus Disease Survivors Show More Efficient Antibody Immunity than Vaccinees Despite Similar Levels of Circulating Immunoglobulins.

The last seven years have seen the greatest surge of Ebola virus disease (EVD) cases in equatorial Africa, including the 2013-2016 epidemic in West Africa and the recent epidemics in the Democratic Republic of Congo (DRC). The vaccine clinical trials that took place in West Africa and the DRC, as well as follow-up studies in collaboration with EVD survivor communities, have for the first time allowed researchers to compare immune memory induced by natural infection and vaccination. These comparisons may be relevant to evaluate the putative effectiveness of vaccines and candidate medical countermeasures such as convalescent plasma transfer. In this study, we compared the long-term functionality of anti-EBOV glycoprotein (GP) antibodies from EVD survivors with that from volunteers who received the recombinant vesicular stomatitis virus vectored vaccine (rVSV-ZEBOV) during the Phase I clinical trial in Hamburg. Our study highlights important differences between EBOV vaccination and natural infection and provides a framework for comparison with other vaccine candidates.

Severe Human Lassa Fever Is Characterized by Nonspecific T-Cell Activation and Lymphocyte Homing to Inflamed Tissues.

Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T-cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here, we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF, we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific and antigenically unrelated T cells. However, severe and fatal LF cases were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of nonspecific T cells with homing capacity to inflamed tissues are associated with severe LF.IMPORTANCE Lassa fever may cause severe disease in humans, in particular in areas of endemicity like Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here, we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus and poor Lassa virus-specific effector T-cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflamed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T-cell targeting as a potential postexposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models, such as nonhuman primates.

Comparative pathogenesis of Ebola virus and Reston virus infection in humanized mice.

Filoviruses of the genus Ebolavirus include 6 species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus, case fatality rates can range between 0% and 90%. In order to understand the molecular basis of these differences, it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Nonhuman primates (NHPs) are the gold-standard models for filovirus pathogenesis, but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHPs. Here we used HLA-A2-transgenic, NOD-scid-IL-2γ receptor-knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While markedly less pathogenic than Ebola virus, Reston virus killed 20% of infected mice, a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition, the case fatality ratios of different Ebolavirus species in humans were recapitulated in the humanized mice. Our findings point to humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses, and suggest that further investigations on Reston virus pathogenesis in humans are warranted.

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

Analysis of the Cellular Stress Response During Ebola Virus Infection by Immunofluorescence.

In this chapter, the use of immunofluorescence analysis as a tool to examine stress granule (SG) formation in Ebola virus (EBOV)-infected cells is described. The following protocol focuses on the process of inducing and analyzing the cellular stress response, including treatment of cells with inducers and inhibitors of the SG formation, and also describes EBOV infection, DNA transfection, and the usage of different cell lines.

Analyzing Apoptosis Induction and Evasion in Ebola Virus-Infected Cells.

In this chapter, we describe a Western blot assay for the successful detection of apoptosis in Ebola virus (EBOV)-infected cells. The protocol includes all steps from cell culture, infection of cells, generation of lysates, and analysis using Western blot to detect caspase cleavage as marker of apoptosis.

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions.

UNLABELLED: A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. IMPORTANCE: Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.