Assessment of Life Cycle Modeling Systems as Prediction Tools for a Possible Attenuation of Recombinant Ebola Viruses.

Ebola virus (EBOV) causes hemorrhagic fever in humans with high case fatality rates. In the past, a number of recombinant EBOVs expressing different reporters from additional transcription units or as fusion proteins have been rescued. These viruses are important tools for the study of EBOV, and their uses include high throughput screening approaches, the analysis of intercellular localization of viral proteins and of tissue distribution of viruses, and the study of pathogenesis in vivo. However, they all show, at least in vivo, attenuation compared to wild type virus, and the basis of this attenuation is only poorly understood. Unfortunately, rescue of these viruses is a lengthy and not always successful process, and working with them is restricted to biosafety level (BSL)-4 laboratories, so that the search for non-attenuated reporter-expressing EBOVs remains challenging. However, several life cycle modeling systems have been developed to mimic different aspects of the filovirus life cycle under BSL-1 or -2 conditions, but it remains unclear whether these systems can be used to predict the viability and possible attenuation of recombinant EBOVs. To address this question, we systematically fused N- or C-terminally either a flag-HA tag or a green fluorescent protein (GFP) to different EBOV proteins, and analyzed the impact of these additions with respect to protein function in life cycle modeling systems. Based on these results, selected recombinant EBOVs encoding these tags/proteins were then rescued and characterized for a possible attenuation in vitro, and results compared with data from the life cycle modeling systems. While the results for the small molecular tags showed mostly good concordance, GFP-expressing viruses were more attenuated than expected based on the results from the life cycle modeling system, demonstrating a limitation of these systems and emphasizing the importance of work with infectious virus. Nevertheless, life cycle modeling system remain useful tools to exclude non-viable tagging strategies.

Quantification of Ebola virus replication kinetics in vitro.

Mathematical modelling has successfully been used to provide quantitative descriptions of many viral infections, but for the Ebola virus, which requires biosafety level 4 facilities for experimentation, modelling can play a crucial role. Ebola virus modelling efforts have primarily focused on in vivo virus kinetics, e.g., in animal models, to aid the development of antivirals and vaccines. But, thus far, these studies have not yielded a detailed specification of the infection cycle, which could provide a foundational description of the virus kinetics and thus a deeper understanding of their clinical manifestation. Here, we obtain a diverse experimental data set of the Ebola virus infection in vitro, and then make use of Bayesian inference methods to fully identify parameters in a mathematical model of the infection. Our results provide insights into the distribution of time an infected cell spends in the eclipse phase (the period between infection and the start of virus production), as well as the rate at which infectious virions lose infectivity. We suggest how these results can be used in future models to describe co-infection with defective interfering particles, which are an emerging alternative therapeutic.

The cost of insecurity: from flare-up to control of a major Ebola virus disease hotspot during the outbreak in the Democratic Republic of the Congo, 2019.

The ongoing Ebola outbreak in the eastern Democratic Republic of the Congo is facing unprecedented levels of insecurity and violence. We evaluate the likely impact in terms of added transmissibility and cases of major security incidents in the Butembo coordination hub. We also show that despite this additional burden, an adapted response strategy involving enlarged ring vaccination around clusters of cases and enhanced community engagement managed to bring this main hotspot under control.

An evolutionary insight into emerging Ebolavirus strains isolated in Africa.

On July 19, 2019, the World Health Organization declared the current Ebolavirus (EBOV) outbreak in Congo Democratic Republic (COD) a public health emergency of international concern. To address the potential threat of EBOV evolution outpacing antibody treatment and vaccine efforts, a detailed evolutionary analysis of EBOV strains circulating in different African countries was performed. Genome composition of EBOV strains was studied using multivariate statistical analysis. To investigate the patterns of evolution of EBOV strains, a Bayesian Markov Chain Monte Carlo approach was used. Two different genetic lineages, with a distinct genome composition gave rise to the recent EBOV outbreaks in central and western Africa. Strains isolated in COD in 2018 fall into two different genetic clusters, according to their geographical location of isolation. Different amino acid substitutions among strains from these two clusters have been found, particularly in NP, GP, and L proteins. Significant differences in codon and amino acid usage among clusters were found. Strains isolated in COD in 2018 belong to two distinct genetic clusters, with distinct codon and amino acid usage. Geographical diversity plays an important role in shaping the molecular evolution of EBOV populations.

The ability of single genes vs full genomes to resolve time and space in outbreak analysis.

BACKGROUND: Inexpensive pathogen genome sequencing has had a transformative effect on the field of phylodynamics, where ever increasing volumes of data have promised real-time insight into outbreaks of infectious disease. As well as the sheer volume of pathogen isolates being sequenced, the sequencing of whole pathogen genomes, rather than select loci, has allowed phylogenetic analyses to be carried out at finer time scales, often approaching serial intervals for infections caused by rapidly evolving RNA viruses. Despite its utility, whole genome sequencing of pathogens has not been adopted universally and targeted sequencing of loci is common in some pathogen-specific fields. RESULTS: In this study we highlighted the utility of sequencing whole genomes of pathogens by re-analysing a well-characterised collection of Ebola virus sequences in the form of complete viral genomes (≈19 kb long) or the rapidly evolving glycoprotein (GP, ≈2 kb long) gene. We have quantified changes in phylogenetic, temporal, and spatial inference resolution as a result of this reduction in data and compared these to theoretical expectations. CONCLUSIONS: We propose a simple intuitive metric for quantifying temporal resolution, i.e. the time scale over which sequence data might be informative of various processes as a quick back-of-the-envelope calculation of statistical power available to molecular clock analyses.

Assessment of a health facility based active case finding system for Ebola virus disease in Mbandaka, Democratic Republic of the Congo, June-July 2018.

BACKGROUND: The ninth outbreak of Ebola Virus Disease (EVD) in the Democratic Republic of the Congo occurred in Équateur Province from 8 May-24 July 2018. A system of health facility (HF)-based active case finding (ACF) was implemented in Mbandaka, a regional capital with four confirmed EVD cases, following completion of contact tracing. The goal of this HF-based ACF system was to look for undetected EVD cases among patients that visited HFs beginning one week prior to the system's implementation. METHODS: From 23 June - 24 July 2018, ACF teams visited HFs in Mbandaka and reviewed all medical records as far back as 17 June for any consultations meeting the suspected EVD case definition. The teams then assessed whether to validate these as suspected EVD cases based on factors such as recovery, epidemiological links, and their clinical judgement. ACF teams also assessed HFs' awareness of EVD symptoms and the process for alerting suspected cases. We calculated descriptive statistics regarding the characteristics of reviewed consultations, alert cases, and visited HFs. We also used univariate and multivariate random effects logistic regression models to evaluate the impact of repeated ACF visits to the same HF on the staff's awareness of EVD. RESULTS: ACF teams reviewed 37,746 consultations, of which 690 met the definition of a suspected case of EVD. Two were validated as suspected EVD cases and transferred to the Ebola Treatment Unit for testing; both tested negative. Repeated ACF visits to the same HF were significantly associated with improved EVD awareness (p < 0.001) in univariate and multivariate analyses. CONCLUSION: HF-based ACF during EVD outbreaks may improve EVD awareness and reveal many individuals meeting the suspected case definition. However, many who meet this definition may not have EVD, depending on the population size covered by ACF and amount of ongoing EVD transmission. Given the burdensome procedure of testing suspected EVD cases, future HF-based ACF systems would benefit from improved clarity on which patients require further testing.

Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment.

BACKGROUND: The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. METHODS: We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. FINDINGS: The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Équateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. INTERPRETATION: The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

Phylodynamic assessment of intervention strategies for the West African Ebola virus outbreak.

Genetic analyses have provided important insights into Ebola virus spread during the recent West African outbreak, but their implications for specific intervention scenarios remain unclear. Here, we address this issue using a collection of phylodynamic approaches. We show that long-distance dispersal events were not crucial for epidemic expansion and that preventing viral lineage movement to any given administrative area would, in most cases, have had little impact. However, major urban areas were critical in attracting and disseminating the virus: preventing viral lineage movement to all three capitals simultaneously would have contained epidemic size to one-third. We also show that announcements of border closures were followed by a significant but transient effect on international virus dispersal. By quantifying the hypothetical impact of different intervention strategies, as well as the impact of barriers on dispersal frequency, our study illustrates how phylodynamic analyses can help to address specific epidemiological and outbreak control questions.

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.

Beyond Ebola treatment units: severe infection temporary treatment units as an essential element of Ebola case management during an outbreak.

In the course of the Ebola outbreak in West Africa that was witnessed since early 2014, the response mechanisms showed deficits in terms of timeliness, volume and adequacy. The authors were deployed in the Ebola campaign in the West African country Liberia, where by September 2014 the changing epidemiological pattern made reconsiderations of guidelines and adopted procedures necessary. A temporary facility set up as a conventional Ebola Treatment Unit in the Liberian capital Monrovia was re-dedicated into a Severe Infections Temporary Treatment Unit. This facility allowed for stratification based on the nosocomial risk of exposure to Ebola virus for a growing subgroup of admitted patients that in the end would turn out as Ebola negative cases. At the same time, adequate diagnostic measures and treatment for the non-Ebola conditions of these patients could be provided without compromising work safety of the employed staff. The key elements of the new unit comprised a Suspect Cases Area similar to that of conventional Ebola treatment units for newly arriving patients, an Unlikely Cases Area for patients with a first negative Ebola PCR result, and a Confirmed Negative Cases Area for patients in whom Ebola could be ruled out. The authors, comprising representatives of the Liberian Ministry of Health and Social Welfare, as well as infectious disease specialists from the German Ebola Task Force are presenting key features of the adapted concept, and are highlighting its relevance in raising acceptance for outbreak counter-measures within the population at stake.

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead.

The 2014 Ebola outbreak, the largest recorded, took us largely unprepared, with no available vaccine or specific treatment. In this context, the World Health Organization declared that the humanitarian use of experimental therapies against Ebola Virus (EBOV) is ethical. In particular, an experimental treatment consisting of a cocktail of three monoclonal antibodies (mAbs) produced in tobacco plants and specifically directed to the EBOV glycoprotein (GP) was tested in humans, apparently with good results. Several mAbs with high affinity to the GP have been described. This review discusses our current knowledge on this topic. Particular emphasis is devoted to those mAbs that have been assayed in animal models or humans as possible therapies against Ebola. Engineering aspects and challenges for the production of anti-Ebola mAbs are also briefly discussed; current platforms for the design and production of full-length mAbs are cumbersome and costly.

Epidemiology and Management of the 2013-16 West African Ebola Outbreak.

The 2013-16 West African Ebola outbreak is the largest, most geographically dispersed, and deadliest on record, with 28,616 suspected cases and 11,310 deaths recorded to date in Guinea, Liberia, and Sierra Leone. We provide a review of the epidemiology and management of the 2013-16 Ebola outbreak in West Africa aimed at stimulating reflection on lessons learned that may improve the response to the next international health crisis caused by a pathogen that emerges in a region of the world with a severely limited health care infrastructure. Surveillance efforts employing rapid and effective point-of-care diagnostics designed for environments that lack advanced laboratory infrastructure will greatly aid in early detection and containment efforts during future outbreaks. Introduction of effective therapeutics and vaccines against Ebola into the public health system and the biodefense armamentarium is of the highest priority if future outbreaks are to be adequately managed and contained in a timely manner.

Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.

Confirming Ebola virus disease (EVD), a deadly infectious disease, requires real-time RT-PCR, which takes up to a few hours to yield results. Therefore, a rapid diagnostic assay is imperative for EVD diagnosis. A rapid nucleic acid test based on recombinase polymerase amplification (EBOV-RPA) was developed to specifically detect the 2014 outbreak strains. The EBOV-RPA assay was evaluated by testing samples from suspected EVD patients in parallel with RT-PCR. An EBOV-RPA, which could be completed in 20 min, was successfully developed. Of 271 patients who tested positive for Ebola virus by RT-PCR, 264 (sensitivity: 97%, 95% CI: 95.5-99.3%) were positive by EBOV-RPA; 101 of 104 patients (specificity: 97%, 95% CI: 93.9-100%) who tested negative by RT-PCR were also negative by EBOV-RPA. The sensitivity values for samples with a Ct value of <34, which accounted for 95.59% of the samples, was 100%. Discordant samples positive by RT-PCR but negative by EBOV-RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%, consistent with those of RT-PCR. The EBOV-RPA assay showed 97% sensitivity and 97% specificity for all EVD samples tested, making it a rapid and sensitive test for EVD diagnosis.

Potential Impact of Sexual Transmission on Ebola Virus Epidemiology: Sierra Leone as a Case Study.

BACKGROUND: Sexual transmission of Ebola virus disease (EVD) 6 months after onset of symptoms has been recently documented, and Ebola virus RNA has been detected in semen of survivors up to 9 months after onset of symptoms. As countries affected by the 2013-2015 epidemic in West Africa, by far the largest to date, are declared free of Ebola virus disease (EVD), it remains unclear what threat is posed by rare sexual transmission events that could arise from survivors. METHODOLOGY/PRINCIPAL FINDINGS: We devised a compartmental mathematical model that includes sexual transmission from convalescent survivors: a SEICR (susceptible-exposed-infectious-convalescent-recovered) transmission model. We fitted the model to weekly incidence of EVD cases from the 2014-2015 epidemic in Sierra Leone. Sensitivity analyses and Monte Carlo simulations showed that a 0.1% per sex act transmission probability and a 3-month convalescent period (the two key unknown parameters of sexual transmission) create very few additional cases, but would extend the epidemic by 83 days [95% CI: 68-98 days] (p < 0.0001) on average. Strikingly, a 6-month convalescent period extended the average epidemic by 540 days (95% CI: 508-572 days), doubling the current length, despite an insignificant rise in the number of new cases generated. CONCLUSIONS/SIGNIFICANCE: Our results show that reductions in the per sex act transmission probability via abstinence and condom use should reduce the number of sporadic sexual transmission events, but will not significantly reduce the epidemic size and may only minimally shorten the length of time the public health community must maintain response preparedness. While the number of infectious survivors is expected to greatly decline over the coming months, our results show that transmission events may still be expected for quite some time as each event results in a new potential cluster of non-sexual transmission. Precise measurement of the convalescent period is thus important for planning ongoing surveillance efforts.

Blood-Borne Pathogens: A Canadian Blood Services Centre for Innovation Symposium.

Testing donations for pathogens and deferring selected blood donors have reduced the risk of transmission of known pathogens by transfusion to extremely low levels in most developed countries. Protecting the blood supply from emerging infectious threats remains a serious concern in the transfusion medicine community. Transfusion services can employ indirect measures such as surveillance, hemovigilance, and donor questioning (defense), protein-, or nucleic acid based direct testing (detection), or pathogen inactivation of blood products (destruction) as strategies to mitigate the risk of transmission-transmitted infection. In the North American context, emerging threats currently include dengue, chikungunya, and hepatitis E viruses, and Babesia protozoan parasites. The 2003 SARS and 2014 Ebola outbreaks illustrate the potential of epidemics unlikely to be transmitted by blood transfusion but disruptive to blood systems. Donor-free blood products such as ex vivo generated red blood cells offer a theoretical way to avoid transmission-transmitted infection risk, although biological, engineering, and manufacturing challenges must be overcome before this approach becomes practical. Similarly, next generation sequencing of all nucleic acid in a blood sample is currently possible but impractical for generalized screening. Pathogen inactivation systems are in use in different jurisdictions around the world, and are starting to gain regulatory approval in North America. Cost concerns make it likely that pathogen inactivation will be contemplated by blood operators through the lens of health economics and risk-based decision making, rather than in zero-risk paradigms previously embraced for transfusable products. Defense of the blood supply from infectious disease risk will continue to require innovative combinations of surveillance, detection, and pathogen avoidance or inactivation.

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics.

The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC's ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC's performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings.