Allison Groseth

Mutation rate and genotype variation of Ebola virus from Mali case sequences

Evolution in the Ebola virus outbreak Has rapid mutation produced alarming new virus characteristics in the 2013–2015 Ebola virus outbreak in West Africa? Hoenen et al. sequenced isolates obtained 9 months into the epidemic from cases in Mali. The nucleotide substitution rate was consistent with rates estimated from past Central African outbreaks. In contrast, analysis of sequence data from early in the outbreak indicated rapid mutation. This more recent finding offers confidence that diagnostic methods, vaccines, and other treatment interventions will remain effective. Nevertheless, vigilance must be maintained: A few mutations can radically change the biological properties of other RNA viruses. Science, this issue p. 117 During the current outbreak in West Africa, Ebola virus has not mutated faster than historically observed. The occurrence of Ebola virus (EBOV) in West Africa during 2013–2015 is unprecedented. Early reports suggested that in this outbreak EBOV is mutating twice as fast as previously observed, which indicates the potential for changes in transmissibility and virulence and could render current molecular diagnostics and countermeasures ineffective. We have determined additional full-length sequences from two clusters of imported EBOV infections into Mali, and we show that the nucleotide substitution rate (1.3 × 10–3 substitutions per site per year) is consistent with rates observed in Central African outbreaks. In addition, overall variation among all genotypes observed remains low. Thus, our data indicate that EBOV is not undergoing rapid evolution in humans during the current outbreak. This finding has important implications for outbreak response and public health decisions and should alleviate several previously raised concerns.

Infection of Naïve Target Cells with Virus-Like Particles: Implications for the Function of Ebola Virus VP24

ABSTRACT Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription, an indispensable step in natural infection. Here we describe a ZEBOV iVLP system which overcomes this limitation and show that VP24 is essential for the formation of a functional ribonucleoprotein complex.

Both matrix proteins of Ebola virus contribute to the regulation of viral genome replication and transcription.

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans and non-human primates. While the role of the EBOV major matrix protein VP40 in morphogenesis is well understood, nothing is known about its contributions to the regulation of viral genome replication and/or transcription. Similarly, while it was reported that the minor matrix protein VP24 impairs viral genome replication, it remains unclear whether it also regulates transcription, since all common experimental systems measure the combined products of replication and transcription. We have developed systems that allow the independent monitoring of viral transcription and replication, based on qRT-PCR and a replication-deficient minigenome. Using these systems we show that VP24 regulates not only viral genome replication, but also transcription. Further, we show for the first time that VP40 is also involved in regulating these processes. These functions are conserved among EBOV species and, in the case of VP40, independent of its budding or RNA-binding functions.

Oligomerization of Ebola Virus VP40 Is Essential for Particle Morphogenesis and Regulation of Viral Transcription

ABSTRACT The morphogenesis and budding of virus particles represent an important stage in the life cycle of viruses. For Ebola virus, this process is driven by its major matrix protein, VP40. Like the matrix proteins of many other nonsegmented, negative-strand RNA viruses, VP40 has been demonstrated to oligomerize and to occur in at least two distinct oligomeric states: hexamers and octamers, which are composed of antiparallel dimers. While it has been shown that VP40 oligomers are essential for the viral life cycle, their function is completely unknown. Here we have identified two amino acids essential for oligomerization of VP40, the mutation of which blocked virus-like particle production. Consistent with this observation, oligomerization-deficient VP40 also showed impaired intracellular transport to budding sites and reduced binding to cellular membranes. However, other biological functions, such as the interaction of VP40 with the nucleoprotein, NP, remained undisturbed. Furthermore, both wild-type VP40 and oligomerization-deficient VP40 were found to negatively regulate viral genome replication, a novel function of VP40, which we have recently reported. Interestingly, while wild-type VP40 was also able to negatively regulate viral genome transcription, oligomerization-deficient VP40 was no longer able to fulfill this function, indicating that regulation of viral replication and transcription by VP40 are mechanistically distinct processes. These data indicate that VP40 oligomerization not only is a prerequisite for intracellular transport of VP40 and efficient membrane binding, and as a consequence virion morphogenesis, but also plays a critical role in the regulation of viral transcription by VP40.

Inclusion bodies are a site of ebolavirus replication.

ABSTRACT Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

A Novel Life Cycle Modeling System for Ebola Virus Shows a Genome Length-Dependent Role of VP24 in Virus Infectivity

ABSTRACT Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. IMPORTANCE Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories.

Current ebola vaccines

Introduction: Ebolaviruses cause severe viral hemorrhagic fever in humans and non-human primates (NHPs), with case fatality rates of up to 90%. Currently, neither a specific treatment nor a vaccine licensed for use in humans is available. However, a number of vaccine candidates have been developed in the last decade that are highly protective in NHPs, the gold standard animal model for ebola hemorrhagic fever. Areas covered: This review analyzes a number of scenarios for the use of ebolavirus vaccines, discusses the requirements for ebolavirus vaccines in these scenarios and describes current ebolavirus vaccines. Among these vaccines are recombinant adenoviruses, recombinant vesicular stomatitis viruses (VSVs), recombinant human parainfluenza viruses and virus-like particles. Interestingly, one of these vaccine platforms, based on recombinant VSVs, has also demonstrated post-exposure protection in NHPs. Expert opinion: The most pressing remaining challenge is now to move these vaccine candidates forward into human trials and toward licensure. In order to achieve this, it will be necessary to establish the mechanisms and correlates of protection for these vaccines, and to continue to demonstrate their safety, particularly in potentially immunocompromised populations. However, already now there is sufficient evidence that, from a scientific perspective, a vaccine protective against ebolaviruses is possible.

MINIGENOMES, TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLES AND BEYOND: REVERSE GENETICS SYSTEMS FOR FILOVIRUSES AND OTHER NEGATIVE STRANDED HEMORRHAGIC FEVER VIRUSES

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research.

RNA Polymerase I-Driven Minigenome System for Ebola Viruses

ABSTRACT In general, Ebola viruses are well known for their ability to cause severe hemorrhagic fever in both human and nonhuman primates. However, despite substantial sequence homology to other members of the family Filoviridae, Reston ebolavirus displays reduced pathogenicity for nonhuman primates and has never been demonstrated to cause clinical disease in humans, despite its ability to cause infection. In order to develop a tool to explore potential roles for transcription and replication in the reduced pathogenicity of Reston ebolavirus, we developed an RNA polymerase I (Pol I)-driven minigenome system. Here we demonstrate successful Reston ebolavirus minigenome rescue, including encapsidation, transcription, and replication, as well as the packaging of minigenome transcripts into progeny particles. The Pol I-driven Reston ebolavirus minigenome system provides a higher signal intensity with less background (higher signal-to-noise ratio) than a comparable T7-driven Reston ebolavirus minigenome system which was developed simultaneously. Successful Reston ebolavirus minigenome rescue was also achieved by the use of helper plasmids derived from the closely related Zaire ebolavirus or the more distantly related Lake Victoria marburgvirus. The use of heterologous helper plasmids in the Reston ebolavirus minigenome system yielded levels of reporter expression which far exceeded the level produced by the homologous helper plasmids. This comparison between minigenomes and helper plasmids from different filovirus species and genera indicates that inherent differences in the transcription and/or replication capacities of the ribonucleoprotein complexes of pathogenic and apathogenic filoviruses may exist, as these observations were confirmed in a Lake Victoria marburgvirus minigenome system.

In Vitro and In Vivo Characterization of Recombinant Ebola Viruses Expressing Enhanced Green Fluorescent Protein

To facilitate an understanding of the molecular aspects of the pathogenesis of Zaire ebolavirus (ZEBOV) infection, we generated 2 different recombinant viruses expressing enhanced green fluorescent protein (eGFP) from additional transcription units inserted at different positions in the virus genome. These viruses showed in vitro phenotypes similar to that of wild-type ZEBOV (wt-ZEBOV) and were stable over multiple passages. Infection with one of the viruses expressing eGFP produced only mild disease in rhesus macaques, demonstrating a marked attenuation in this animal model. However, in mice lacking signal transducer and activator of transcription 1, both viruses expressing eGFP caused lethal cases of disease that were moderately attenuated, compared with that caused by wt-ZEBOV. In mice, viral replication could be easily tracked by the detection of eGFP-positive cells in tissues, by use of flow cytometry. These findings demonstrate that the incorporation of a foreign gene will attenuate ZEBOV in vivo but that these viruses still have potential for in vitro and in vivo research applications.