abstract: Ebola hemorrhagic fever (EHF) is a severe and often fatal disease in human and nonhuman primates, caused by the Ebola virus. Approximately 30 years after the first epidemic, there is no vaccine or therapeutic medication approved to counter the Ebola virus. In this dissertation, a geminiviral replicon system was used to produce Ebola immune complex (EIC) in plant leaves and tested it as an Ebola vaccine. The EIC was produced in Nicotiana benthamiana leaves by fusing Ebola virus glycoprotein (GP1) to the C-terminus of heavy chain of 6D8 monoclonal antibody (mAb), which is specific to the 6D8 epitope of GP1, and co-expressing the fusion with the light chain of 6D8 mAb. EIC was purified by ammonium sulfate precipitation and protein A or protein G affinity chromatography. EIC was shown to be immunogenic in mice, but the level of antibody against Ebola virus was not sufficient to protect the mice from lethal the Ebola challenge. Hence, different adjuvants were tested in order to improve the immunogenicity of the EIC. Among several adjuvants that we used, Poly(I:C), which is a synthetic analog of double-stranded ribonucleic acid that can interact with a Toll-like receptor 3, strongly increased the efficacy of our Ebola vaccine. The mice immunized with EIC co-administered with Poly(I:C) produced high levels of neutralizing anti-Ebola IgG, and 80% of the mice were protected from the lethal Ebola virus challenge. Moreover, the EIC induced a predominant T-helper type 1 (Th1) response, whereas Poly(I:C) co-delivered with the EIC stimulated a mixed Th1/Th2 response. This result suggests that the protection against lethal Ebola challenge requires both Th1 and Th2 responses. In conclusion, this study demonstrated that the plant-produced EIC co-delivered with Poly(I:C) induced strong and protective immune responses to the Ebola virus in mice. These results support plant-produced EIC as a good vaccine candidate against the Ebola virus. It should be pursued further in primate studies, and eventually in clinical trials. / Dissertation/Thesis / Ph.D. Plant Biology 2010

Ebola virus (EBOV) causes severe disease in humans characterized by high case fatality rates and significant immune dysfunction. A hallmark of EBOV infection is the formation of viral inclusions in the cytoplasm of infected cells. These 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 gene expression and genome replication, little is known about their role in the host response to infection. The cellular stress response is an antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Related to this response is the post-transcriptional regulation of RNA mediated by stability elements called AU-rich elements (AREs) and their associated binding proteins (ARE-BPs), many of which are found in SGs. Because these processes have antiviral implications, many viruses have evolved strategies to interfere with SG formation, or appropriate ARE-BPs to benefit viral replication. However, it is unknown if EBOV interacts with these cellular systems. Here, we show that SG proteins were sequestered within EBOV inclusions where they formed distinct granules that colocalized with viral RNA. The inclusion-bound aggregates were not canonical SGs, and did not lead to translational arrest in infected cells. EBOV did not induce cytoplasmic SGs at any time post infection, but was unable to overcome SG formation induced by additional stressors. Despite the sequestration of SG proteins, canonical SGs did not form within inclusions. At high levels of expression, viral protein 35 (VP35), the viral polymerase co-factor that also mediates various immune evasion functions, disrupted SGs formation independently of eIF2α phosphorylation. Finally, we found that the cellular ARE-BP tristetraprolin (TTP) specifically targeted the 3’untranslated region (UTR) of the viral nucleoprotein (NP) mRNA and promoted its degradation. Interestingly, TTP was not found within viral inclusions, leading us to speculate that inclusions might serve to prevent viral RNA from encountering TTP. These results indicate that EBOV interacts with the cellular stress response and associated RNA regulatory proteins in ways that promote viral replication.

The aim of this thesis is to analyse EU response towards the Ebola outbreak that developed in spring and summer 2014 in Western Africa. The emphasis is put on the EU external action towards the Ebola crisis in the frame-work of the EU development policy and humanitarian aid provided by the EU while identifying the financial framework, institutional structure, key players and the role of the EU Members. It was a common endeavour of whole range of global actors, national governments and many regional or local stakeholders that led to elimination of the threat represented by the Ebola Virus Disease. The European Union traditionally empha-sized multilateral approach that is in compliance with the EU middle power capacities. The de-velopment policy financial mechanisms within the EU have two resources, the EC and the Mem-ber States which cumulatively contributed twice as much as the European Commission to re-solving the crisis.

The thesis is focused on the epidemic of Ebola between years 2014 and 2015 and response of the Czech Republic. The main objective of the thesis is to assess the strategy of the Czech Republic in order to eliminate import of Ebola. The thesis is using study of documents and its effort is a complex overview of the given matters. Used documents are especially information and reports of international and Czech organizations, regulations, expert articles, electronic inquiries and media reports. There was defined 5 research questions. First question is focused on the demographic and epidemiological characteristics of the epidemic in West Africa. Second question was analysing steps, which were performed within the system of preparedness for highly contagious infections in the Czech Republic. I have divided the whole strategy of the Czech Republic into 10 points. They consist of the enforcing of International Health Regulation (2005), Decision no. 1082/2013/EU of the EP and of the Council, National action plan for the case of event, which is subject to International Health Regulation and two directives, implementation of special measures on international airports, conclusion of cooperation agreements, transmission of information within the Czech Republic, simulation actions and humanitarian aid. Third question was analysing the respondence of arrival cards at the Václav Havel Airport in Prague. Respondence was approx. 40 %. (Jágrová, 2016a) Fourth question is assessing the efficiency of implemented measures using the recorded cases of suspicion. Although there was not any case of infection in the Czech Republic, it may be concluded, that implemented measures are efficient. Fifth question is focused on the assessment of implemented measures and suggestions, how the response of the Czech Republic may be improved. Preparedness of the Czech Republic was assessed as very good by experts. Also it may be concluded from results, that preparedness is sufficient. Suggested measures, how the response of the Czech Republic may be improved, are related to the system of rapid alert of the public, creation of team for humanitarian aid, maintaining of sufficient capacities and realization of simulation actions.

abstract: In this dissertation the potential impact of some social, cultural and economic factors on Ebola Virus Disease (EVD) dynamics and control are studied. In Chapter two, the inability to detect and isolate a large fraction of EVD-infected individuals before symptoms onset is addressed. A mathematical model, calibrated with data from the 2014 West African outbreak, is used to show the dynamics of EVD control under various quarantine and isolation effectiveness regimes. It is shown that in order to make a difference it must reach a high proportion of the infected population. The effect of EVD-dead bodies has been incorporated in the quarantine effectiveness. In Chapter four, the potential impact of differential risk is assessed. A two-patch model without explicitly incorporate quarantine is used to assess the impact of mobility on communities at risk of EVD. It is shown that the overall EVD burden may lessen when mobility in this artificial high-low risk society is allowed. The cost that individuals in the low-risk patch must pay, as measured by secondary cases is highlighted. In Chapter five a model explicitly incorporating patch-specific quarantine levels is used to show that quarantine a large enough proportion of the population under effective isolation leads to a measurable reduction of secondary cases in the presence of mobility. It is shown that sharing limited resources can improve the effectiveness of EVD effective control in the two-patch high-low risk system. Identifying the conditions under which the low-risk community would be willing to accept the increases in EVD risk, needed to reduce the total number of secondary cases in a community composed of two patches with highly differentiated risks has not been addressed. In summary, this dissertation looks at EVD dynamics within an idealized highly polarized world where resources are primarily in the hands of a low-risk community – a community of lower density, higher levels of education and reasonable health services – that shares a “border” with a high-risk community that lacks minimal resources to survive an EVD outbreak. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2018

RNA viruses have the fastest evolutionary rates amongst protein-coding organisms on the planet. Ease of sequencing, advanced techniques of analysis and global health and economic concerns have all contributed to the recognition of RNA viruses as a robust research platform. Phylogenetic methods have been at the forefront of analytical techniques used to understand the dynamics of RNA viruses - during natural circulation in populations and in individual hosts, within epidemics, across species barriers and over billions of years that viruses have been around. Most of the work presented in this thesis employs phylogenetic incongruity arising from reassortment and recombination to gain insights into the genomes and populations of RNA viruses. Chapter 2 explores the selection regimes Ebola virus has experienced following a year of circulation in humans inWest Africa, as well as its recent history. Chapter 3 investigates the extent of recombination in MERS-CoV, a novel human pathogen with an obscure epidemiology, which is suggestive of frequent co-infection of some hosts. Chapter 4, on the other hand, documents a pattern of non-intuitive linkage between some segments of the human-endemic influenza B virus genome and explores its potential to speciate. Chapter 5 builds upon chapter 4 and attempts to describe small-scale reassortment between two segments of influenza B virus and the overall migration patterns of influenza B virus in Scotland. Chapter 6 exploits the independence of segments of influenza D virus, a recently described cattle pathogen, and coalescent theory to disentangle the origins of this virus. This thesis exemplifies the success of modern sequencing methods, which, together with the use of sophisticated analytical techniques, have uncovered a wealth of information hidden away in molecular sequences of RNA viruses. The work presented herein demonstrates how reticulate evolution can be exploited as a reliable, and sometimes indispensable, marker to improve inference of evolutionary forces in RNA viruses.

One of the primary objectives of infectious disease research is uncovering the direct link that exists between viral population dynamics and molecular evolution. For RNA viruses in particular, evolution occurs at such a rapid pace that epidemiological processes become ingrained into gene sequences. Conceptually, this link is easy to make: as RNA viruses spread throughout a population, they evolve with each new host infection. However, developing a quantitative understanding of this connection is difficult. Thus, the emerging discipline of phylodynamics is centered on reconciling epidemiology and phylogenetics using genetic analysis. Here, we present two research studies that draw on phylodynamic principles in order to characterize the progression and evolution of the Ebola virus and the human immunodefficiency virus (HIV). In the first study, the interplay between selection and epistasis in the Ebola virus genome is elucidated through the ancestral reconstruction of a critical region in the Ebola virus glycoprotein. Hence, we provide a novel mechanistic account of the structural changes that led up to the 2014 Ebola virus outbreak. The second study applies an approximate Bayesian computation (ABC) approach to the inference of epidemiological parameters. First, we demonstrate the accuracy of this approach with simulated data. Then, we infer the dynamics of the Swiss HIV-1 epidemic, illustrating the applicability of this statistical method to the public health sector. Altogether, this thesis unravels some of the complex dynamics that shape epidemic progression, and provides potential avenues for facilitating viral surveillance efforts.

Between 2014 and 2016, West Africa was struck by the largest ever Ebola epidemic. In Sierra Leone, the outbreak occurred only about a decade after the end of an eleven-year long civil war, which left the country with little capacity to contain the virus. While many have investigated the crisis that the Ebola outbreak caused West African countries, few have turned their attention directly towards the response to it. Following that line of thought, this case study explores how the Ebola response carried out by local, national and international actors played into conflict dynamics in the aftermath of the Sierra Le-onean civil war. By applying the theoretical perspectives of ‘the fortified aid compound’ and ‘dependent agency’, I argue that the response embodied a militarised approach and that it was insensitive towards local customs, which showed in shifting acts of compliance and resistance by beneficiaries. Lastly, by applying the theory of ‘protracted social con-flict’, I argue that conflict dynamics from the civil war were amplified by the Ebola re-sponse to some extent. The study concludes that future responses to epidemics, particu-larly in conflict affected settings, should consider potential negative effects connected to response structures and measures to a greater extent.

Ebola virus (EBOV) infection causes a severe disease in humans and leads to widespread liver necrosis, a dysregulated cytokine response, and coagulopathy. However, little is known about the specific liver response to EBOV infection in humans. Here we present the utilization of an induced pluripotent stem cell (iPSC)-derived hepatocyte platform to define the host response to EBOV infection. We demonstrate that iPSC-derived hepatocytes are a suitable platform for investigating innate immune responses to viral infections. We compared the host response to EBOV infection in iPSC-derived hepatocytes, immortalized hepatocytes, and primary human hepatocytes and identified minimal transcriptomic changes 1 day post infection (dpi). Between 2-3 dpi, EBOV infection led to a significant upregulation of interferon-beta (IFN-β) and select interferon-stimulated genes (ISGs) in iPSC-derived hepatocytes. In addition, the acute phase response and coagulation cascade was downregulated in these hepatocytes, mimicking known liver dysfunction in EBOV disease. Using fluorescent in situ hybridization (RNA-FISH), we showed at single cell resolution that EBOV-infected iPSC-derived hepatocytes express IFN-β, indicating that infected cells mount an antiviral response to EBOV infection. This platform can be utilized to investigate therapeutic targets in human hepatocytes that may attenuate EBOV infection in patients. In addition, we present in this dissertation the development of a minigenome system for the filovirus Lloviu virus (LLOV). LLOV is closely related to EBOV and is known to circulate in bats throughout Europe. The complete sequence of LLOV has yet to be resolved, and therefore investigation of LLOV biology is limited. As part of this work, we established a functional LLOV minigenome system based on sequence complementation of other filoviruses. We demonstrate that the LLOV replication and transcription strategy is generally more similar to ebolaviruses than marburgviruses. We show that a single nucleotide at the 3ꞌ end of the LLOV genome determines specificity of the LLOV polymerase complex. This minigenome system can now be used to elucidate replication and transcription mechanisms employed by this novel filovirus. / 2023-02-19T00:00:00Z

<div>Public health policies increasingly rely on complex models that need to approximate epidemics realistically and be consistent with the available data. Choosing appropriate simplifying assumptions is one of the critical challenges in disease modeling. In this thesis, we focus on some of these assumptions to show how they impact model outcomes. </div><div>In this thesis, an ODE model with a gamma-distributed infectious period is studied and compared with an exponentially distributed infectious period. We show that, for childhood diseases, isolating infected children is a possible mechanism causing oscillatory behavior in incidence. This is shown analytically by identifying a Hopf bifurcation with the isolation period as the bifurcation parameter. The threshold value for isolation to generate sustained oscillations from the model with gamma-distributed isolation period is much more realistic than the exponentially distributed model.</div><div><br></div><div>The consequences of not modeling the spectrum of clinical symptoms of the 2014 Ebola outbreak in Liberia include overestimating the basic reproduction number and effectiveness of control measures. The outcome of this model is compared with those of models with typical symptoms, excluding moderate ones. Our model captures the dynamics of the recent outbreak of Ebola in Liberia better, and the basic reproduction number is more consistent with the WHO response team's estimate. Additionally, the model with only typical symptoms overestimates the basic reproduction number and effectiveness of control measures and exaggerates changes in peak size attributable to interventions' timing.</div><div><br></div>