Logan Banadyga

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.

Assessing the contribution of interferon antagonism to the virulence of West African Ebola viruses.

The current Ebola virus (EBOV) outbreak in West Africa is unprecedented in terms of both its size and duration, and there has been speculation and concern regarding the potential for EBOV to increase in virulence as a result of its prolonged circulation in humans. Here we investigate the relative potency of the interferon (IFN) inhibitors encoded by EBOVs from West Africa, since an important EBOV virulence factor is inhibition of the antiviral IFN response. Based on this work we show that, in terms of IFN antagonism, the West African viruses display no discernible differences from the prototype Mayinga isolate, which corroborates epidemiological data suggesting these viruses show no increased virulence compared with those from previous outbreaks. This finding has important implications for public health decisions, since it does not provide experimental support for theoretical claims that EBOV might gain increased virulence due to the extensive human-to-human transmission in the on-going outbreak.

An Improved Reverse Genetics System to Overcome Cell-Type–Dependent Ebola Virus Genome Plasticity

Reverse genetics systems represent a key technique for studying replication and pathogenesis of viruses, including Ebola virus (EBOV). During the rescue of recombinant EBOV from Vero cells, a high frequency of mutations was observed throughout the genomes of rescued viruses, including at the RNA editing site of the glycoprotein gene. The influence that such genomic instability could have on downstream uses of rescued virus may be detrimental, and we therefore sought to improve the rescue system. Here we report an improved EBOV rescue system with higher efficiency and genome stability, using a modified full-length EBOV clone in Huh7 cells. Moreover, by evaluating a variety of cells lines, we revealed that EBOV genome instability is cell-type dependent, a fact that has significant implications for the preparation of standard virus stocks. Thus, our improved rescue system will have an impact on both basic and translational research in the filovirus field.

A hamster model for Marburg virus infection accurately recapitulates Marburg hemorrhagic fever

Marburg virus (MARV), a close relative of Ebola virus, is the causative agent of a severe human disease known as Marburg hemorrhagic fever (MHF). No licensed vaccine or therapeutic exists to treat MHF, and MARV is therefore classified as a Tier 1 select agent and a category A bioterrorism agent. In order to develop countermeasures against this severe disease, animal models that accurately recapitulate human disease are required. Here we describe the development of a novel, uniformly lethal Syrian golden hamster model of MHF using a hamster-adapted MARV variant Angola. Remarkably, this model displayed almost all of the clinical features of MHF seen in humans and non-human primates, including coagulation abnormalities, hemorrhagic manifestations, petechial rash, and a severely dysregulated immune response. This MHF hamster model represents a powerful tool for further dissecting MARV pathogenesis and accelerating the development of effective medical countermeasures against human MHF.

Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging

Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.

Small Animal Models for Studying Filovirus Pathogenesis

Filovirus small animal disease models have so far been developed in laboratory mice, guinea pigs, and hamsters. Since immunocompetent rodents do not exhibit overt signs of disease following infection with wild-type filoviruses isolated from humans, rodent models have been established using adapted viruses produced through sequential passage in rodents. Rodent-adapted viruses target the same cells/tissues as the wild-type viruses, making rodents invaluable basic research tools for studying filovirus pathogenesis. Moreover, comparative analyses using wild-type and rodent-adapted viruses have provided beneficial insights into the molecular mechanisms of pathogenicity and acquisition of species-specific virulence. Additionally, wild-type filovirus infections in immunodeficient rodents have provided a better understanding of the host factors required for resistance to filovirus infection and of the immune response against the infection. This chapter provides comprehensive information on the filovirus rodent models and rodent-adapted filoviruses. Specifically, we summarize the clinical and pathological features of filovirus infections in all rodent models described to date, including the recently developed humanized and collaborative cross (CC) resource recombinant inbred (RI) intercrossed (CC-RIX) mouse models. We also cover the molecular determinants responsible for adaptation and virulence acquisition in a number of rodent-adapted filoviruses. This chapter clearly defines the characteristic and advantages/disadvantages of rodent models, helping to evaluate the practical use of rodent models in future filovirus studies.

Closer than ever to an Ebola virus vaccine

Forty years ago, in 1976, hemorrhagic fever outbreaks in former Zaire (now the Democratic Republic of the Congo) and Sudan devastated the local communities [1]. In Zaire, an international response team consisting of scientists and medical doctors from the USA, Europe, and Africa identified a filamentous virus similar to Marburg virus (MARV) as the causative agent. The pathogen, which infected 318 people and took the lives of 280 around a Belgian Catholic Mission Hospital in Yambuku, was named Ebola virus (EBOV) after the nearby Ebola river [1]. Since then, EBOV outbreaks have occurred sporadically in Central Africa but have never affected more than a few hundred people, due in large part to geographical isolation and rapid quarantines. The situation changed in December 2013, when the largest documented EBOV epidemic began in the small village of Meliandou, Guinea [2]. A 2-year-old boy developed a hemorrhagic fever, and the disease spread quickly to other people in nearby villages. It took 3 months until the infectious agent of the disease was identified – EBOV. For the first time, EBOV was found outside of Central Africa, and it spread quickly from Guinea to the neighboring countries Sierra Leone and Liberia, reaching rural communities as well as the heavily populated capital cities. On 8 August 2014, the World Health Organization (WHO) declared the West African EBOV epidemic a global health emergency [3], and the status was not lifted until 29 March 2016, almost 2 years later. The epidemic had devastated Guinea, Liberia, and Sierra Leone, causing almost 30,000 human infections with over 11,000 fatalities [4]. After 40 years of research, were there still no countermeasures? We do have candidates for vaccines and therapeutics, but their development and licensure has been rather complicated. Unlike Malaria or HIV, EBOV does not have a reputation for causing millions of cases every year, and the interest from pharmaceutical companies, as well as research funding agencies, in developing and licensing a vaccine or treatment was therefore minimal. Moreover, the virus circulates in Africa where the majority of people have no means to afford medication and vaccines. After the terrorist attacks on 11 September 2001, as well as the subsequent anthrax attacks that began shortly after, the threat that EBOV and other pathogens posed as agents of bioterrorism spurred government agencies to dramatically increase research funding. Research into countermeasure development was expanded, and within several years the first successful vaccine and treatment studies against EBOV infection in animal models were published [5]. Before the West African EBOV epidemic, only two vaccines were tested in human phase I clinical trials with limited success [5]. However, after the global health emergency was declared by the WHO in summer 2014, several experimental vaccine approaches and treatment options, which had previously been successfully tested in nonhuman primates (NHPs), were accelerated into human clinical trials [6]. Among these were the vesicular stomatitis virus (VSV)-based vaccine, VSV-EBOV (also known as rVSV-ZEBOV), and a chimpanzee adenovirus type 3 (cAd3)-based vaccine, cAd3-EBO. The cAd3-EBO vaccine was developed in the last 5 years at the Vaccine Research Center at the National Institutes of Health (NIH) in the USA, building off of several advances made with the human adenovirus 5 platform [7]. The VSVEBOV was developed in the early 2000s at the Public Health Agency of Canada (PHAC) and tested in collaborative projects in Canada and the USA [8]. In 2010, NewLink Genetics acquired the license to produce and market the VSV-EBOV vaccine, and in early 2014, PHAC had about one thousand doses available for phase I clinical trials. Merck acquired the license to produce and market the VSV-EBOV vaccine in late 2014, and with this support, phase I human clinical trials were started in October 2014. At the same time, GlaxoSmithKline and the NIH launched the first phase I clinical trials for the cAd3-EBO vaccine. In the following year, both vaccines were shown to be immunogenic with limited adverse effects [9–11], prompting phase II and III clinical trials in West Africa. While no phase III vaccine efficacy data are currently available for the cAd3-EBO vaccine, the VSV-EBOV vaccine showed promising efficacy in Guinea using a ring-vaccination approach [12]. Just this past December, the second report of the study was published demonstrating that the VSV-EBOV vaccine is indeed highly efficacious and may have contributed to controlling the later stage of the outbreak in Guinea [13]. Remarkably, after the minimum time to immunity (10 days) had been reached, the study documented no new EBOV cases among those who received the vaccine immediately, compared to 16 new infections in the delayed vaccination (control) group. Nevertheless, important questions about the durability of

Quantification of RNA Content in Reconstituted Ebola Virus Nucleocapsids by Immunoprecipitation

Immunoprecipitations are commonly used to isolate proteins or protein complexes and assess protein-protein interactions; however, they can also be used to assess protein-RNA complexes. Here we describe an adapted RNA immunoprecipitation technique that permits the quantification of RNA content in Ebola virus nucleocapsids that have been reconstituted in vitro by transient transfection.