Ebola Virus Disease (EVD)

Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein–protein interactions on a functional level.

Up to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014–2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host’s innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.

Vesicular stomatitis virus (VSV) expressing the Ebola virus (EBOV) glycoprotein (GP) in place of the VSV glycoprotein G (VSV/EBOV-GP) is a promising EBOV vaccine candidate which has already entered clinical phase 3 studies. Although this chimeric virus was tolerated overall by volunteers, it still caused viremia and adverse effects such as fever and arthritis, suggesting that it might not be sufficiently attenuated. In this study, the VSV/EBOV-GP vector was further modified in order to achieve attenuation while maintaining immunogenicity. All recombinant VSV constructs were propagated on VSV G protein expressing helper cells and used to immunize guinea pigs via the intramuscular route. The humoral immune response was analysed by EBOV-GP-specific fluorescence-linked immunosorbent assay, plaque reduction neutralization test and in vitro virus-spreading inhibition test that employed recombinant VSV/EBOV-GP expressing either green fluorescent protein or secreted Nano luciferase. Most modified vector constructs induced lower levels of protective antibodies than the parental VSV/EBOV-GP or a recombinant modified vaccinia virus Ankara vector encoding full-length EBOV-GP. However, the VSV/EBOV-GP(F88A) mutant was at least as immunogenic as the parental vaccine virus although it was highly propagation-restricted. This finding suggests that VSV-vectored vaccines need not be propagation-competent to induce a robust humoral immune response. However, VSV/EBOV-GP(F88A) rapidly reverted to a fully propagation-competent virus indicating that a single-point mutation is not sufficient to maintain the propagation-restricted phenotype.

Viruses of the genus Ebolavirus are the causative agents of Ebola virus disease (EVD), of which there have been only 25 recorded outbreaks since the discovery of Zaire and Sudan ebolaviruses in the late 1970s. Until the west African outbreak commencing in late 2013, EVD was confined to an area of central Africa stretching from the coast of Gabon through the Congo river basin and eastward to the Great Lakes. Nevertheless, population serological studies since 1976, most of which were carried out in the first two decades after that date, have suggested a wider distribution and more frequent occurrence across tropical Africa. We review this body of work, discussing the various methods employed over the years and the degree to which they can currently be regarded as reliable. We conclude that there is adequate evidence for a wider geographical range of exposure to Ebolavirus or related filoviruses and discuss three possibilities that could account for this: (a) EVD outbreaks have been misidentified as other diseases in the past; (b) unidentified, and clinically milder, species of the genus Ebolavirus circulate over a wider range than the most pathogenic species; and (c) EVD may be subclinical with a frequency high enough that smaller outbreaks may be unidentified. We conclude that the second option is the most likely and therefore predict the future discovery of other, less virulent, members of the genus Ebolavirus.

On 23 March 2014, the World Health Organization issued its first communiqué on a new outbreak of Ebola virus disease (EVD), which began in December 2013 in Guinée Forestière (Forested Guinea), the eastern sector of the Republic of Guinea. Located on the Atlantic coast of West Africa, Guinea is the first country in this geographical region in which an outbreak of EVD has occurred, leaving aside the single case reported in Ivory Coast in 1994. Cases have now also been confirmed across Guinea as well as in the neighbouring Republic of Liberia. The appearance of cases in the Guinean capital, Conakry, and the transit of another case through the Liberian capital, Monrovia, presents the first large urban setting for EVD transmission. By 20 April 2014, 242 suspected cases had resulted in a total of 147 deaths in Guinea and Liberia. The causative agent has now been identified as an outlier strain of Zaire Ebola virus. The full geographical extent and degree of severity of the outbreak, its zoonotic origins and its possible spread to other continents are sure to be subjects of intensive discussion over the next months.

Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40–RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.

Ebola virus causes rapidly progressive haemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DCs), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T-cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse DCs. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86 and major histocompatibility complex class II. Further, it suppresses the induction of cytokines such as interleukin (IL)-6, IL-12, tumour necrosis factor α and alpha/beta interferon (IFN-α/β). Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4+ T cells. Addition of type I IFN to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolishes its activity, whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference in DC function with resultant deficiency in T-cell function, which may contribute to the profound virulence of Ebola virus infection.

Adenoviral vectors can be used to generate potent humoral and cellular immune responses to transgene products. Use of adenoviral vectors based on non-human isolates may allow for their utilization in populations harbouring neutralizing antibodies to common human serotypes. A vector chimera was constructed using simian adenovirus 22 (a serotype belonging to the species Human adenovirus E) and simian adenovirus 21 (a serotype belonging to the species Human adenovirus B) expressing the Ebola (Zaire) virus glycoprotein (Ad C5/C1-ZGP). This chimeric adenovirus vector was used as a model to test its efficacy as a genetic vaccine and comparisons were made to a vector based on the commonly used human adenovirus C serotype 5 (Adhu5-ZGP). Ebola glycoprotein-specific T- and B-cell responses were measured in B10BR mice vaccinated with either Adhu5-ZGP or Ad C5/C1-ZGP vectors. Both vectors resulted in Ebola glycoprotein-specific gamma interferon-expressing T cells, although the Ad C5/C1-ZGP vector appeared to induce lower frequencies with kinetics slower than those elicited by the Adhu5-ZGP vector. The total immunoglobulin G response to Ebola glycoprotein was similar in sera from mice vaccinated with either vector. Two rhesus macaques vaccinated with the Ad C5/C1-ZGP vector were found to mount T-cell and antibody responses to the Ebola glycoprotein. It was found that a single administration of the chimeric Ad C5/C1-ZGP vector protected mice against a lethal challenge with a mouse-adapted strain of the Ebola (Zaire) virus.