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Bacteriophages, viruses that obligately infect bacteria, represent the most abundant and diverse biological entities on the planet, with key and complex ecological roles in all environments where they have been studied. Agricultural wastes and manures (i.e., cattle slurry) are economically important fertilisers that are applied to land. Despite the widespread use of slurries, there is a paucity of knowledge regarding the microbial composition within them. The first part of this thesis sought to optimise viral metagenomics, for the study of viral communities in nature. As the study of uncultivated viral genomes is underpinned by known complete viral genomes, I assessed the current extent of viral sequencing to provide the most complete reference database possible in an updated and reproducible fashion. This led to the INPHARED database, now published in PHAGE and available on GitHub; a community resource that provides genomes and annotation files to aid in viral genomic and metagenomic analyses. Furthermore, I investigated biases in the current collection of phage genomes and demonstrated that clear biases towards phages of a small subset of clinically relevant bacteria. Subsequently, I sought to benchmark sequencing technologies and assembly approaches for the recovery of viral genomes from viral metagenomes. This work, in part published in Microbiome and under review in Microbial Genomics, demonstrated that choice of sequencing technology and assembly algorithm will have significant impacts on downstream analyses and estimates of viral diversity. Overall, these analyses demonstrated that a combination of long and short read sequencing approaches performed best at recovering viral genomes in a mixed community. The second part of this thesis applied the understandings described above to the dairy farm environment. I utilised long- and short-read sequencing to characterise the viral community of agricultural slurry in a longitudinal study, as well as modelled slurry tanks that contained agricultural antimicrobials, and the dairy cattle gut across life stages. Analysis of the cattle slurry virome, now published in Microbiome, revealed a diverse and novel community that was stable over time, despite constant influx and efflux of material. Notably, there was widespread phage carriage of a virulence determinant- VapE-that is associated with bovine mastitis-causing pathogens such as Streptococcus spp. Subsequent experiments with mock slurry tanks revealed the slurry virome may be influenced by the presence of footwash, although the reasons for this remain unclear. Analysis of the dairy cow virome uncovered 1,338 predicted complete phage genomes, the most of a single virome study to date. The phages within the dairy cow gut were largely novel, and their community composition changed over key life stages. The results within this thesis have advanced the methodology of viral metagenomics approaches in general, and show that viruses likely play important ecological roles within agricultural environments, including augmenting the virulence of relevant veterinary pathogens.

Acute febrile illnesses (AFI) are a significant cause of morbidity in Uganda and sub-Saharan Africa (SSA). The epidemiology and burden of disease due to viral AFI are unknown in Uganda despite an active surveillance system at the Uganda Virus Research Institute (UVRI). This work describes the epidemiology of arboviruses and other AFI from data and samples stored (from 2016 to 2019) at the UVRI, providing a candid picture of the spatiotemporal distribution and seasonality of arboviruses in Uganda. Furthermore, for the arbovirus-negative specimens stored at the UVRI, a virus discovery approach was applied using the widely affordable conventional Polymerase Chain Reaction (PCR) method and later augmented by high throughput sequencing (HTS). We identified the age group 5-14 (years) most at risk of arboviral infections. Generally, arboviruses were more prevalent during the first rainy season from March to May, with yellow fever virus (YFV) as the most prevalent arbovirus. A significant spatiotemporal cluster was observed in the central region, where most arbovirus activities occurred. Among AFI, conventional PCR detected enteroviruses (Coxsackie A4, B1 viruses and human echovirus E4), measles virus (MeV) genotype B3 and West Nile virus (WNV) lineage two. The MeV was phylogenetically analysed and determined to be different from previously circulating B3 strains in Uganda but identical to strains in neighbouring Kenya and Rwanda. Furthermore, the WNV, although similar to the ancestral 1937 strain, had acquired a motif (NYSTQ) in the envelope protein similar to that found in lineage one WNV strains. In addition, HTS discovered viruses undetected by PCR, such as Hepatitis C virus, Pegiviruses, and sequences belonging to Orbiviruses. The ever-decreasing costs of sequencing will support the application of PCR augmented by regular HTS in the surveillance of AFI and, thus, capture emerging and re-emerging viruses.

Morbilliviruses are responsible for some of the most devastating outbreaks of disease in animals, accompanied by high morbidity and mortality rates. Canine distemper virus (CDV) and peste des petits ruminants virus (PPRV) are highly promiscuous morbilliviruses which have widely expanded their host range in recent decades. However, the extent of infection in atypical hosts and the genetic impact upon endangered, vulnerable species remain poorly understood, particularly in East Africa. The development of a highly accurate serological method capable of differentiating between circulating morbilliviruses is required. Further, the need for longitudinal and clinical monitoring of animals is essential to understand the infection dynamics occurring in susceptible and emerging hosts. Finally, there is a lack of research on the impact which decades of morbillivirus outbreaks have had on the critically endangered African wild dogs of Tanzania, which is paramount to understanding how this species can be further protected and preserved. This thesis comprises two studies. The first study aims to confirm the presence of PPRV and CDV in cattle, sheep, and goats from ten households across the Serengeti District in Northern Tanzania. This work involves a longitudinal serological study underpinning active infection of animals, using a pseudotyped-virus neutralization assay (PVNA) to detect highly specific antibodies to PPRV and to CDV. This study also monitored clinical signs to investigate the disease manifestations of infections in livestock hosts, using a logistic regression model to test for associations between infection and signs of disease in each species. The second study aims to clarify whether genetic change has occurred in the wild dog population of the Serengeti over recent decades, by investigating neutral (microsatellite) and adaptive (MHC DRB1) genetic markers from published data (Marsden et al, 2012), and recently developed data following a fatal CDV outbreak in 2017. It also aims to determine whether there is evidence of natural selection on adaptive markers, and evidence of genetic variation attributable to factors beyond demographic change. Results from these studies demonstrate that cattle in a mixed livestock setting become naturally infected with PPRV with no associated clinical disease. Data also show for the first time that cattle, sheep, and goats become naturally infected with CDV with no associated clinical disease. CDV seropositivity was detected predominantly in cattle. Infections occurred throughout the study with no patterns associated between household and infection, indicating widespread circulation of both viruses beyond the household level. The source of infection remains to be established, although livestock trade and sporadic outbreaks in domestic dogs are likely sources of PPRV and CDV infection, respectively. This work provides strong evidence that PPRV sub-clinically infects cattle in multi-host livestock communities, and that CDV sub-clinically infects livestock, predominantly cattle. This thesis provides the first analysis of genetic changes in the Serengeti population of African wild dogs. This work utilized the pseudotype-based virus neutralization assay to detect CDV-specific neutralizing antibodies in sera of wild dogs which survived the 2017 CDV outbreak, while RT-qPCR was used to detect CDV in tissue samples from deceased wild dogs. Results showed an overwhelming proportion of PCR positives and lack of protective immune response in deceased and surviving animals, respectively. Allele frequency data showed fluctuations in DRB diversity over time coinciding with CDV outbreaks. Heterozygosity varied for both neutral and immune markers over time but showed no excess or evidence of population bottleneck. Bayesian analysis of microsatellites found some genetic structuring over time (K = 2). The test for selection indicated balancing selection during at least two time points at the DRB, in keeping with CDV outbreaks. This study provides strong evidence of population structuring and the potential link between adaptive markers and disease outbreaks in wild dogs.

Viruses are abundant, diverse, and ubiquitous across soil habitats. By regulating microbial dynamics through host cell lysis, and augmenting host metabolism through the expression of auxiliary metabolic genes, bacteriophages (phages) are integral members of the marine food web. Critically, this has highlighted the knowledge gap in our understanding of viruses in soil ecosystems. The advent and accessibility of next-generation sequencing has uncoupled the investigation of soil viruses from their lab cultivation with hosts. This approach was subsequently taken here to address the following questions: 1) What is most prevalent infection strategy of soil viruses, and what drives the shifts between virulence and lysogeny? 2) What are the ecological drivers of spatial variation in soil viral diversity? 3) How do soil viruses influence soil biogeochemistry? In Chapter 2, a multi-'omic approach was taken to investigate the influence of crop rotation on the composition and spatiotemporal activity of soil viral communities. This was the first time that ssRNA phages were described at the root surface, which in turn expanded the number of Leviviricetes genomes by more than five times. Chapter 2 also provides seminal evidence of crop rotation-driven impacts on microbial communities extending to viruses, such that the novel principal of "viral priming" was presented. In Chapter 3, a publicly available metagenomic data set was leveraged to describe virushost interactions throughout the soil depth profile. This uncovered evidence of viruses augmenting the remineralisation of soil carbon across the markedly underexplored niches of subsurface soil. Furthermore, by investigating viral micro diversity across the soil depth profile for the first time, subsurface soil was found to host distinctive antagonistic co-evolution between viruses and their hosts. In summary, this thesis extends our understanding of the ecological roles of microbial viruses across soil microbiomes. The further characterisation of these roles across spatial, temporal, and trophic scales could inform global actions to mitigate climate change, promote bioremediation, and support sustainable agriculture. Most importantly, this work implores that future soil microbiome studies integrate viruses and recognise their interactions with microbes, higher plants, and even humans.

Infectious agents and especially viruses have had a tremendous impact on all stages of the evolution of the living matter. Being only composed of a few biomolecules they are often seen as a relic of processes from the early emergence of life on earth, but also they have contributed throughout evolution in all kingdoms of life. Throughout human history, infectious diseases have played significant role in the route of history, both regarding total death toll as well as policy changes. However, the highly dynamic nature of viral evolution in combination with overlapping mechanisms between the virus and the host poses a significant challenge for antiviral drug design based on traditional methods. This thesis examines the structural and dynamical properties of nucleic acid sequences from viruses as potentially broad-spectrum and robust antiviral target. The introductory chapter briefly introduces nucleic acid structures and previous work from the Hannon group on nucleic acid targeting and reviews viral processes throughout the different classifications. It also introduces theoretical and computational methods that could shine light on the generally elusive dynamical landscape of nucleic acid structures. After methodology is described in chapter a 2, fundamental theoretical understanding of dynamic molecular processes is discussed in chapter 3. Here, structure and dynamics of coordination compounds are discussed, in an effort to examine those away from crystal structure limitations. Employing Density Functional Theory (DFT), different spin states of complex molecules are discussed with the potential implications to metastable states within a chemical species. In chapter 4, work from the earlier chapters is applied in the understanding of structure and dynamics of HIV-1's TAR RNA, the most well-characterised RNA structure in literature. Having validated the Molecular Dynamics approach against known properties of TAR RNA, comparing metastable states identified through a Markov state model of the system with published NMR data, its potential interactions with supramolecular cylinders are examined resulting in a proposed dominant binding mode in agreement with previous experimental results. Chapter 5 introduces a pipeline for in silico optimisation of molecules targeting RNA given only the sequence of a novel virus (in this case SARS-CoV2). From sequence, secondary and tertiary structure are proposed and using molecular dynamics the conformational landscape of some of its metastable states can be sampled, exposing potentially key regions that could be targeted and change the RNA's behaviour and thus potentially disturbing the viral replication cycle. The predictions of the pipeline have been experimentally verified in collaboration with the Grzechnik group and in cellulo effects are also being observed and reported. In Chapter 6, DNA structures are investigated this time, specifically G quadruplexes (G4s). After a very brief exploration of the interaction of previously characterized mono-nuclear complexes with human G4s (MYC and H-Telo) at the MD level of theory, the chapter focuses on the newly identified (2018) unique structure of HIV-1 LTR G4. This G4 structure features a stem on top of a guanine quartet potentially creating a high affinity target aiming to change the dynamics of the structure. The previously established methodology in mapping molecular interactions between ligands and nucleic acids is also applied here and a global minimum can be suggested although further simulations and coordination with experiments is needed for conclusive mapping of the interaction. Chapters 4 and 5 have been published and are presented here verbatim, as well as parts of Chapter 3 whereas Chapter 6 has not been published.

Human papillomavirus type 16 (HPV16) is present in >90% of HPV-driven head and neck squamous cell carcinomas (HNSCC) whereas HPV18, the second most common high-risk HPV type, is rarely detected. To understand HPV16 pathogenesis in the head and neck, we developed isogenic HPV16 and HPV18 episome-containing primary human tonsil keratinocytes models, which accurately recapitulate the molecular phenotype of the tonsils, the primary site of HPV-driven HNSCC. Differentiation in organotypic raft culture showed evidence of productive infection including viral DNA amplification and expression of viral late proteins. Long-read Nanopore RNA-sequencing was used to characterise and compare HPV16 and HPV18 transcriptome organisation in undifferentiated cells. These studies demonstrated higher activity of the major viral early promoter in HPV16 cells, contributing to higher expression of HPV16 E7 oncogenes. Further, HPV18 cells showed increased levels of transcripts encoding E8^E2, a repressor of early viral transcription, suggesting that HPV18 elicits greater E8^E2-mediated regulatory control of the early promoter than HPV16. Finally, short-read Illumina sequencing was performed to investigate virus-mediated transcriptional reprogramming of host transcriptomes. Here, HPV16 was shown to establish a more immunosuppressive environment than HPV18 by strongly downregulating activities of central transcriptional immune mediators including NF-kB, IFN-α, and IFN-γ, impairing expression of critical antiviral effector genes like CD40, CCL2, and IL1B. This could provide an essential survival advantage for HPV16-infected cells in the tonsils.

The last decade has seen the emergence of multi-drug resistant pathogens as a lead- ing cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including hon strange alterations, constitutively tic growth, and control over phage repli- cation. However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to these problems, this thesis aims to provide generally applicable tools to enable rapid and scal- able engineering of phage-based therapies using cell-free systems. To this end, a novel rapid cell-free phage engineering pipeline, the SpyPhage system, is developed. The system uses a "scaffold" bacteriophage which is engineered to incorporate a SpyTagmoiety on its capsid head to enable rapid post-synthetic modification of their surfaces with SpyCatcher fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and a novel de novo genome assembly protocol, the SpyTag protein is targeted as a capsid fusion to K1F,a phage targeting the pathogenic strain E. coli K1. A cell-free system is used to achieve the first known demonstration of (1)cell-free assembly and(2) a post-translational decoration of the phage surface with therapeutic fusion proteins. This cell-free phage production pipeline proposed enables the generation of multiple phenotypi- cally distinct phage with a single underlying "scaffold" genotype. These phages could be- come the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations. The increasing incidence of bacterial infections caused by antibiotic-resistant pathogens worldwide also underlines the need to develop novel diagnostic tools enabling the early initiation of targeted antimicrobial therapy. One promising possibility is to unite the high specificity and sensitivity of phage- based applications with the speed and sensitivity provided by microfluidic devices. As a prerequisite for developing such systems, a simple and efficient method is presented for the directional immobilization of phages on the surface of Polydimethylsiloxane (PDMS) chips, a material commonly used for building such devices. This work utilises the covalent interaction between two proteins: SpyTag, expressed by the phage, and BslA-Spycatcher fusion protein, purified and immobilized on PDMS surfaces. The use of SpyTagged phage enables the faciledirectional immobilisation of phage on to PDMS-based microfluidicde- vices. This technique serves to illustrate a generally applicable solution to develop the next generation of phage-based bio-sensors.

Regional adaptation and end-use potential for dry common beans (Phaseolus vulgaris L.) provides an opportunity for sustainable innovation in the UK agrifood system to support the 'Great Food Transformation' required for addressing global problems in nutrition and environmental degradation caused by food production. In this work, molecular genetic methods were applied for both responsive and strategic disease resistance breeding objectives of the Warwick dry bean breeding programme. This includes confirming predictions by J Taylor for genetically distinct sources of recessive halo blight resistance in two P. vulgaris accessions from Africa using an allelism test in one example and genotyping-by-sequencing to map resistance in the second example. In response to a novel virus outbreak in trial fields, this research applied high throughput sequencing for viral diagnostics and detected the pathogenic potyvirus Bean yellow mosaic virus (BYMV) using total RNA sequencing and metagenomic analyses. A 1.5 Mb mapping interval was defined for a major effect recessive resistance locus to BYMV in the Warwick pre-breeding material on P. vulgaris chromosome 3. Furthermore, whole genome resequencing of parental lines revealed polymorphisms in candidate genes for both halo blight resistance (HB4.2 locus defined by Tock et al., 2017) and the novel BYMV resistance mapped here, as well as providing insights into the relatedness of one parent lines to an English heirloom variety, Ireland Creek Annie. Additionally, through the lens of consumer behaviour research, consumer attitudes towards pulses and interest in adding homegrown white haricot beans to the UK diet, particularly as a dry ingredient, was investigated. The methods applied to provide this foundational evidence include a systematic literature review examining perceptions and attitudes to pulses, and an online consumer behaviour experiment that investigated effects of packaging country-of-origin and cooking-time messages had on consumer perceptions of dry white haricot beans.

Viruses that circulate in wildlife can have devastating health and economic consequences when they enter human and livestock populations. Efforts to mitigate the burden of these zoonotic pathogens currently tend to focus on the spillover hosts; however, since this approach does not address the reservoir of disease, it prolongs the risk of re-emergence as viruses continue to circulate unimpeded in wildlife. Vaccination of wildlife reservoirs has the potential to prevent spillover, but is hampered by the logistical challenge of delivering vaccine to and achieving sufficient coverage in large, reclusive animal populations. Whilst orally available vaccines held inside edible baits have seen success in combating disease in some wildlife reservoirs, unique dietary requirements or behaviours can preclude the use of this strategy. The obligate blood-feeding common vampire bat Desmodus rotundus, reservoir host of rabies virus and primary source of rabies cases throughout Latin America, is one species in which edible baits are unsuitable, and other management strategies have thus far failed. Virally vectored transmissible vaccines which utilise the replicative capabilities of live viral vectors to spread autonomously between hosts, offer a potential solution. However, progress towards real world use of such vaccines is hampered by the selection of vaccine vectors which will prove both safe and efficacious. A betaherpesvirus recently identified in vampire bats (Desmodus rotundus betaherpesvirus; DrBHV) presents a promising candidate vector. In this thesis, I aimed to identify the key characteristics of DrBHV, and evaluate its biological and epidemiological suitability to vector a transmissible vaccine targeting rabies virus. The results presented here are based on field-collected samples, subjected to a combination of PCR, deep sequencing, and computer modelling. In Chapter 2, I aimed to assess the prevalence and host specificity of DrBHV, and the similarity of its genome composition to other betaherpesviruses currently considered for use as vaccine vectors. I used PCR to amplify a conserved region of the herpesvirus genome in saliva samples from Peruvian bats, with 96.9% of vampire bats testing positive, regardless of demographic group. Sanger sequencing of these regions and those from other positive bats revealed specificity of DrBHV to the Phyllostomidae family. Thus, DrBHV is able to spread efficiently within vampire bats, with only rare infection of closely related and cohabitating bat species. Metagenomic sequencing was able to construct a full genome consensus sequence for DrBHV similar in length and composition to betaherpesviruses in other species. This sequencing also showed the presence of multiple strain infections, suggesting that DrBHV may have the capacity to superinfect individuals, evading the host immune response. I aimed to further explore superinfection and DrBHV diversity in Chapter 3, by the amplification and deep sequencing of the highly variable gene encoding glycoprotein B. I identified eleven strains of DrBHV which varied in prevalence and geographic distribution across Peru. The phylogeographic structure of these strains was predictable from both host genetics and landscape topology, informing long-term DrBHV-vectored vaccine deployment strategies. Multi-strain infections were observed in 79% of infected bats and resampling of marked individuals showed strain acquisitions by already infected individuals, implying that pre-existing immunity and strain competition are unlikely to inhibit vaccine spread. Finally, in Chapter 4, I used the strain-specific prevalence data to fit models of DrBHV transmission. I identified the most likely model to include lifelong, persistent infection with cycles of latency and re-activation, a mechanism which would allow vaccinated individuals to boost their own immunity, and continually transmit vaccines to other bats throughout their lifetime. Simulations of vaccine spread show that a DrBHV-vectored vaccine can reach a population equilibrium coverage of >80% after a single introduction of vaccine, resulting in a 95% decrease in the size of rabies outbreaks. Furthermore, ongoing vaccine transmission is able to maintain these levels of vaccine coverage long-term, even in the presence of realistic levels of reversion, negating the need for recurrent and costly vaccination campaigns. In summary, the work presented in this thesis supports DrBHV as a candidate to vector a transmissible vaccine targeting a major source of rabies in Latin America and shows how accessible genomic data can enlighten vector selection and deployment strategies for transmissible vaccines. This work constitutes a fundamental step towards what would be the first deployment of a transmissible vaccine to prevent spillover of a zoonotic virus, thus allowing the management of disease to shift from reactive damage control to proactive prevention.

Emerging viral zoonoses are a significant global health concern, brought into sharp focus in recent times by the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent global pandemic. Other emerging and re-emerging zoonotic viruses also continue to present significant threats to public health, including Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus that can cause severe disease and high case fatality rates in people, but with a complex ecology that remains poorly understood in many areas of the world, particularly in Africa. Serological tools are vital for understanding emerging zoonotic viruses both at a population- and individual-level. Population-level surveillance of animal and human populations can shed light on patterns of viral circulation and aid in identification of risk factors associated with infection, while individual-level serological investigations help to characterise the immune response to infection, providing insights into the nature of protective immunity, possible vaccine targets and improving methods of disease detection. The initial focus of this thesis was the development of a diagnostic enzyme-linked immunosorbent assay (ELISA) for detection of CCHFV, and its subsequent use to explore the epidemiology of the virus in livestock and people in northern Tanzania. However, the start of the coronavirus disease 19 (COVID-19) pandemic part way through this research resulted in an additional set of research questions relating to SARS-CoV-2 diagnostic assay performance and epidemiology during the first wave of the pandemic in Glasgow, UK. This thesis therefore explores diagnostics assay development and epidemiology of CCHFV and SARS-CoV-2. Firstly, an indirect ELISA for detection of anti-CCHFV antibodies in livestock was developed and optimised. It showed good potential for use as an in-house assay for detection of CCHFV exposure in animals. Secondly, the performance of two indirect ELISA assays against the S1 subunit of the SARS-CoV-2 spike protein (S1) and the receptor binding domain (RBD) was investigated, establishing a cut-off value for interpretation of these assays, determining sensitivity and specificity, and exploring measures of assay precision. Both assays showed good ability to distinguish between positive or negative serum samples for anti-SARS-CoV-2 IgG antibodies. These ELISAs were then used to investigate levels of SARS-CoV-2 exposure in a patient population in Glasgow during the first wave of the COVID-19 pandemic, demonstrating that overall seroprevalence remained low throughout this period. Additionally, ELISA responses were compared against levels of neutralising antibodies (NAbs), measured using HIV(SARS-CoV-2) pseudotype virus neutralisation assays (PVNAs), demonstrating heterogeneity in IgG and NAb responses, and highlighting an association between disease severity and higher levels of IgG and neutralising antibodies. The epidemiology of CCHFV in northern Tanzania was explored through analysis of a large cross-sectional study of livestock and people in linked households, using a commercially produced species-independent ELISA. This study demonstrated for the first time that CCHFV is circulating in northern Tanzania. High levels of exposure were found in cattle (n = 1530/3098, 49.4%), goats (n = 823/2475, 33.3%), and sheep (n = 582/2124, 27.4%) across the region, and an overall seroprevalence of 15.1% (n = 53/351) was observed in people, despite an absence of confirmed clinical disease in the country (Temur et al., 2021). Substantial heterogeneities were observed in levels of exposure between study sites for both livestock and people, indicating that local context is important for determining exposure to CCHFV. However, patterns of village-level exposure varied between people and livestock, possibly suggesting different drivers of exposure. Risk factors associated with CCHFV seropositivity were also investigated in livestock, and demonstrated that increasing age, and extensive, pastoral agro-ecological settings were associated with higher levels of exposure. Additionally, a novel association was identified between pig keeping and higher exposure in cattle. The work presented in this thesis demonstrates the application of serological methods for investigation of two important emerging and re-emerging zoonotic viruses, SARS-CoV-2 and CCHFV. This research adds substantially to our knowledge of CCHFV epidemiology in northern Tanzania, demonstrating high levels of exposure to the virus in livestock populations and highlighting its potential as a public health concern in the country, while the work on SARS-CoV-2 provided important information on population-level immunity and the nature of the immune response during the early phases of the global pandemic.

Zika virus infection arose as a public health issue during late 2015 and 2016 on the American continent. ZIKV epidemic was especially interesting since the previous report of patients infected by the virus only showed mild symptoms including fever, rash, and joint pain, however, the appearance of neurological symptoms including Guillain Barre syndrome in adults and microcephaly on new-borns and other clinical features that were grouped into Zika congenital syndrome. This change on the outcome of the infections resulted in many questions, what changed on the virus that favoured neuroinvasion? are these changes linked to mutations that suffered the virus? The envelope protein of the virus was the best candidate to try to explain the infection of neural cells and the later development of the reported syndromes since this is the first protein that interacts with the hosts' cells and changes in the amino acid sequence can lead to new interactions between the virus and the host. In this study, experiments were designed to try to answer these questions, including identifying mutations on the envelope protein that had a direct impact on the cellular tropism. As a way to dissect the entry process and the interaction between the cells and the mutations on the glycoprotein, a pseudotype model was used. Different strategies were used to try to produce pseudotypes that incorporate the Zika protein including adjusting the amounts of backbone and heterologous glycoprotein, use different cell lines, and supplementing other viral proteins that could enhance particle production. Other approaches were tested, including the generation of plasmids that contain the viral genome, viral production and harvest and the amplification of viral fragments to produce infectious viruses from PCR products. Altogether these experiments showed that the flaviviruses pose a challenge to the development of pseudo particles and other methodologies due to its replication cycle and the singularities of this viral family.

Clinical isolates of influenza A virus (IAV) typically form a pleomorphic population of virions that present as a continuum of morphologies broadly classified as filaments, bacilli, and spheres. Laboratory strains of IAV, however present mainly as spherical and bacilliform particles, suggesting a role for filaments in vivo. How these filaments form is not fully understood, but it has previously been shown that mutations in the viral matrix protein (M1) can be determinants of filament formation. In this work we show that filament formation also depends on multiple other genetic factors. To this end, we compared two IAV strains A/equine/Ohio/03 (O/2003) and A/equine/South Africa/4/03 (SA/2003) and found that SA/2003 could form filaments while O/2003 could not, despite no differences in their M1 sequences. To map the genetic basis of this difference, we generated reassortant viruses between O/2003 and SA/2003 and identified segments 1 (encoding polymerase basic protein 2, PB2), 4 (haemagglutinin, HA) and 6 (neuraminidase, NA) as determinants of morphology. We established that single mutations in segments 4 and 6, which alter the HA and NA proteins, alter virion morphology. To our surprise, we also identified three synonymous mutations in segment 1 of the virus that were determinants of filament formation despite not altering any known protein. We then extended this work to unravel the associated mechanisms of this change and found despite some differences in the activity of NA, contribution of HA to filament production, and differences in segment 1 RNA structure, there was no clear underlying mechanism. Given, that we were unable to identify the mechanisms associated with the change in morphology, we further extended this work to identify the factors involved in morphogenesis. To characterize IAV filament morphogenesis we employed cryogenic electron tomography (Cryo-ET) of vitrified equine fibroblasts (E. Derm). Although we were unable to identify any additional factors associated with IAV budding, we were able to generate a robust pipeline for studying filament formation. These results show that M1 is not the only determinant of IAV morphology, and that the ability to form filaments, a poorly studied but natural characteristic of IAV infection, is in fact modulated by multiple proteins and RNA determinants.

Since the late 1960s UV irradiation of Herpesviruses has been used to study various aspects of cell biology, including the cellular immune response to virus infection. Previous studies have demonstrated that UV irradiation of herpesvirus virions strongly stimulates the type I and II IFN response to viral infection. However, the spatial and temporal kinetics of recruitment of immune signalling actuated in response to UV irradiated virus infection have remained poorly understood, due to limitations of viral DNA (vDNA) detection. Utilizing 5-Ethynyl-2' deoxycytidine (EdC) labelling of HSV-1 DNA in combination with click chemistry, we were able to directly visualize vDNA and characterize the recruitment of host immune factors to infecting wild type (WT) or UV irradiated viral genomes. UV irradiation ofHSV-1 virions induced the premature cytoplasmic release of vDNA in a microtubule-dependent manner. An increase in vDNA thymine dimer formation was observed that restricted genome decompacting once released from the capsid. HSV-1 genomes in the cytoplasm exhibited an enhanced recruitment of cGAS and STING following UV irradiation compared to non-UV irradiated cytoplasmic genomes. Depletion of cGAS led to the loss of STING recruitment, demonstrating a sequential recruitment of cellular PRRs. ChIP analysis further supported this finding as cGAS interaction with HSV-1 genomes was enhanced following UV irradiation, whilst STING demonstrated no interaction at all. Conversely, UV irradiation reduced frequency of recruitment of key PML-NB proteins to vDNA in the nucleus. ChIP analysis demonstrated the interaction between UV irradiated vDNA and PML to be significantly reduced compared to WT HSV-1 genomes. The click labelling was further extended to human Cytomegalovirus (hCMV) to investigate if these immune defences are conserved among Herpesviruses or HSV-1 specific. Collectively, our analysis shows multiple host factors to alter their respective recruitment and/or vDNA binding properties in response to infection following UV irradiation.

Respiratory viruses are the cause of significant disease burden and coinfections with more than one virus constitute between 10-30% of viral respiratory infections. Interactions among respiratory viruses are recognised for their importance in influencing viral dynamics, however direct virus-virus interactions are poorly understood. Influenza A virus (IAV) and respiratory syncytial virus (RSV) are important respiratory pathogens that share epidemiological characteristics, including timing of seasonal peaks of infections, and biological characteristics, including cellular tropism within the respiratory tract. To characterise interactions between IAV and RSV during coinfection, we developed an in vitro model in A549 cells, a cell line derived from the human lung. Analysis of viral growth kinetics and viral dynamics by live cell imaging showed that, while IAV replication appears unaffected by coinfection with RSV, RSV replication is significantly decreased in coinfection. Imaging of coinfected cells stained for IAV and RSV nucleoproteins and glycoproteins show that they localise to the same regions of the plasma membrane, suggesting there may be opportunity for viral interactions during viral assembly. To further explore this hypothesis, virus particles budding from coinfected cells were examined using super-resolution confocal microscopy. Filamentous structures extended from coinfected cells, that incorporated glycoproteins from both viruses in distinct patches along the filament. The ultra-structure of these filaments, determined by cryo-electron tomography, revealed the formation of chimeric viral particles (CVPs) that contained genomes and structural features from both IAV and RSV. Additionally, coinfection by IAV and RSV generated pseudotyped RSV filaments that incorporate IAV glycoproteins. Functional assays using a sialidase showed that CVPs can facilitate entry of IAV into cells that were stripped of IAV entry receptors, demonstrating CVPs possess expanded receptor tropism. To determine the likelihood for CVP formation in the airway epithelium, we coinfected primary differentiated human bronchial epithelial cell (hBEC) cultures at air-liquid interface. We observed that IAV and RSV infect ciliated epithelial cells and identified foci of coinfection. IAV and RSV proteins both localised at the apical surface of coinfected cells, providing opportunity for interactions to occur during viral assembly. Additionally, IAV and RSV replication kinetics and cytopathic effect in hBEC cultures reflected trends observed in the in vitro cell model, suggesting that viral interactions may be conserved between simplified and representative airway models. Overall, this project characterises interactions between IAV and RSV during coinfection and we show that coinfection by IAV and RSV results in formation of a novel class of viral particles. By expanding viral tropism, formation of CVPs may alter viral dissemination within the respiratory tract, potentially impacting disease outcomes for a coinfected individual. Further, by defining a previously unknown source of viral interaction with implications on viral structure, we contribute more widely to the understanding of the properties of IAV and RSV, and their infection biology as a whole.

The total dependence of a virus on its host for its survival leads to a fundamental entanglement with its host's cellular machinery. This drives a coevolutionary relationship that leaves an imprint of the host in viral genomes. The aim of this thesis was to develop machine learning approaches to identify and exploit these host predictive signals. We present methods that use these signals both to build classifiers that can assign putative information to virus genomes and to locate the discriminative features on viral proteins thereby identifying regions that are important in the host relationship. The first step aimed to identify discriminative features that capture the different aspects of the virus host relationship. We generated a range of feature sets from alternative representations of the viral genomes that each aimed to exploit the different levels of biological information present. We used a supervised machine learning approach to compare a range of feature sets for their ability to predict host taxonomic information. Next, we opened these "black box" classifiers and to extract the discriminative information learnt by the model to identify regions of a viral protein that are associated with their host relationship. We used the 'local' nature of some of the predictive feature sets to transform an amino acid sequence into host signals. Finally, we developed a multi-view generative mixture model, MVC, to tease apart the complex signals that are embedded in viral genomes via different evolutionary processes. This Bayesian approach uses the clustering of the data defined by labels of interest to guide the features associated with those labels into the "relevant view". The MVC model is able to identify features associated with weak effect in the data.

The staphylococcal type VII secretion system (T7SS) is responsible for the export of several virulence factors. Some T7SS effectors are associated with long-term persistence of Staphylococcus aureus in murine models and modulation of epithelial and dendritic cell death, although the molecular mechanisms underlying T7SS-mediated modulation of cellular processes remain unclear. The aim of this study was to investigate the role of staphylococcal T7SS during macrophage infection. Isogenic mutants lacking either single or multiple secretion substrates of the T7SS (EsxC, EsxB, EsaE and EsxD) or EssC, an ATPase driving protein export were studied in comparative in vitro assays using THP-1 macrophages and in vivo mouse models. Time-lapse microscopy over the course of 24 hrs revealed that intracellular S. aureus caused the formation of pore-induced intracellular traps (PIT)-like structures, indicative of lytic cell death, in macrophages. Wild type (WT) S. aureus infections resulted in an early peak in cell death and inflammatory cytokine release which was absent or markedly shifted with T7SS mutant infections. WT induced early cell death was attributed to the T7SS promotion of the necroptosis pathway. Importantly T7SS mutants were not defective at causing necroptosis, but promotion of this pathway appeared to occur at later timepoints, suggesting other S. aureus virulence factors can also cause the onset of necroptosis in macrophages. Interestingly, in low-dose skin infections, T7SS mutants survived better than the WT and demonstrated enhanced dissemination to distal sites. However, WT was more successful in high-dose infections which indicates dose dependent changes in the effect of the T7SS. Finally, using interdisciplinary techniques a microfluidic air-way-blood-vessel-on-a-chip was designed, fabricated and evaluated for the investigation of S. aureus-host interactions under shear stress. Overall, this project provided insight into specific roles of the T7SS in macrophages and questions whether that the T7SS is always beneficial for S. aureus during infection.

Viruses have evolved to utilise host cell factors that promote viral replication and inactivate host cell factors that inhibit viral replication. For adenovirus, early region proteins modulate the functional activity of pro-viral and anti-viral host cell proteins in order to promote viral replication. In this regard, adenovirus early region proteins, E1B-55K, E4orf3 and E4orf6 engage with the cellular Ubiquitin-Proteasome System, both independently and cooperatively to inactivate anti-viral host cell proteins. The principal aims of this thesis were to: investigate further how adenovirus E1B-55K, E4orf3 and E4orf6 proteins function in isolation to modulate host cell factors through engagement with the Ubiquitin-Proteasome System; and consider the role of the cellular chromatin remodelling complex, FACT in adenovirus infection. In order to study their functions in isolation we have developed, and characterized, clonal U2OS cell lines that express human Ad5 and Ad12 early region proteins in a tetracycline-responsive manner. Using site-directed mutagenesis coupled with pharmacological inhibition of the proteasome and Cullin Ring Ligases we determined that Ad5 E4orf6 is subject to ubiquitylation on K95 and K171 and undergoes proteasome-dependent degradation in a Cullin Ring Ligase-dependent manner. Using quantitative mass spectrometry we also established that Ad12 E4orf6, but not Ad5 E4orf6, engages with Cullin Ring Ligases to promote the proteasome-mediated degradation of core histone proteins. In this regard, both adenovirus 5 and adenovirus 12 were shown to promote the loss of core histones during infection but the mechanisms underlying this process require further investigation. Using a combination of immunoflouresence, GFP pull-down and RNA interference allied with Western blot analyses work presented here also established that FACT complex components, SPT16 and SSRP1 associated with the RPA complex at viral replication centres during infection and that ablation of FACT gene expression affected adenovirus early region gene product expression during infection. In consideration of previous published work and the data presented here, this study indicates that adenovirus utilises the Ubiquitin Proteasome System at multiple levels to regulate both host cell protein and adenovirus early region protein function during infection and suggests that cellular histones limit adenovirus replication. Moreover, our data suggests that the histone chaperone protein complex, FACT has pro-viral activities during adenovirus infection. Taken together, our studies have identified potential new functions for adenovirus early region proteins and have developed useful resources for studying viral protein-host protein interactions.

Respiratory syncytial viruses of humans (hRSV) and animals (bovine, bRSV) are the leading cause of lower respiratory tract infections - bronchiolitis and pneumonia - in their respective hosts. They pose serious health risks to young calves, children under 5, the elderly and immunocompromised. RSV is an enveloped, single-stranded negative-sense RNA virus taxonomically classified within the Orthopneumovirus genus in the Pneumoviridae family. The only available treatment option for hRSV is monoclonal antibody based immunoprophylaxis. There is no approved vaccine for hRSV, while bRSV vaccines are of varying effectiveness. RSV can suppress the host immune response and prevents the generation of long-term immunity. RSV encodes two accessory proteins (NS1 and NS2) which are well established to block interferon signalling. Previous studies have also shown that the viral SH protein plays a role in pathogenesis by inhibiting the NF-κB pathway, an important regulator of the host innate immune response. However, the exact mechanisms employed are less well characterised. We aimed to understand the role of the RSV SH protein and found that expression reduced NF-κB signalling at the level of both IκBα degradation and subunit p65 nuclear translocation, when wild type virusinfected cells were compared to cells infected with viruses lacking SH expression. However, the differences were minimal, especially when NF-κB transactivation was assessed in cells ectopically expressing SH in the absence of all other viral proteins. Interestingly however, during these experiments we identified separate RSV-mediated antagonism of the NF-κB pathway, with a mechanism entirely distinct from the NS1, NS2 and SH proteins. In both hRSV and bRSV infected cells we demonstrated that the p65 subunit of NF-κB is sequestered to perinuclear viral inclusion bodies (IBs). These were formed in the cytoplasm of RSV infected cells and were separately confirmed as the sites of viral RNA replication and synonymous with viral inclusion bodies. We also found that captured p65 is unable to translocate to the nucleus following TNF-α stimulation, highlighting the antagonistic nature of this event.

Zika virus (ZIKV, Flaviviridae), like other emerging arboviruses, poses a considerable threat to human health. It is estimated that approximately half of the world's population is at risk from contracting a mosquito-borne arboviral infection, and this was exemplified during the 2015/16 ZIKV outbreak in the Americas. ZIKV infection is thought to be largely asymptomatic, although ZIKV disease has previously been characterised by mild symptoms such as a maculopapular rash, conjunctivitis, and fever. However, recent outbreaks have been associated with an increased incidence of Guillain-Barré syndrome, and a pattern of neurological and developmental symptoms in neonates which is now termed congenital Zika syndrome. Despite intense efforts, no therapeutic or vaccine has been developed. As such, it is vital that further fundamental research is conducted to discover novel host-virus interactions in both vector and host systems, which may allow development of targeted interventions. Here, multiple approaches were used to generate basic tools for ZIKV research, and siRNA screens and data from mass-spectrometry based proteomics were utilised to uncover important host interactors of ZIKV. A study investigating the Aedes aegypti immune response was conducted, and the classical RNAi effector Argonaute 2 (Ago2) was not found to be antiviral, whereas PIWI 4 was. Data from a previous proteomics experiment suggested that glucose-regulated protein 78 kDa (GRP78) may interact with ZIKV E. In this study, co-immunoprecipitation and immunofluorescence was used to verify that GRP78 interacts with ZIKV E in both mammalian and Aedes aegypti cell culture. GRP78 is a key modulator of the unfolded protein response (UPR), and while small-molecule inhibitors (EGCG and HNK) of the GRP78-mediated UPR did not inhibit ZIKV infection, EGCG was able to inhibit ZIKV entry independent of GRP78, likely through direct binding of the virion. Further study of GRP78 revealed that while it is not important for entry, replication, or egress of ZIKV, it did aid viral translation. Depletion of GRP78 with siRNA resulted in a loss of coordination of viral replication factories and relieved a virus-specific inhibition of host translation. Furthermore, STRING analysis of GRP78 host-interactors followed by a targeted siRNA screen revealed that DnaJC1 is also a pro-viral factor. DnaJC1 has previously been shown to coordinate GRP78 localisation to ribosome exit tunnels, and so may contribute to ZIKV infection through GRP78, though this was not assessed in this study. Additionally, by using a circular polymerase extension reaction (CPER) system, a reverse genetics ZIKV was generated. This CPER ZIKV represents a genetically stable source of virus which can be easily modified and can support future research. Collectively, the data herein informs on important ZIKV interactions in both arthropod vector and mammalian systems, and highlights tools and techniques that can be used to conduct future fundamental ZIKV research.

Hepatitis B virus (HBV) infects liver cells resulting in hepatitis B infection. In approximately 90% of childhood infections and 10% of adult cases the virus establishes a chronic, lifelong infection. In 2015, it was estimated that approximately 3.5% of the global population were living with chronic HBV infection. Long-term complications of chronic HBV infection such as cirrhosis and hepatocellular carcinoma cause a significant disease burden. The World Health Organisation has set a goal to eliminate the infection by 2030. However, current treatment for HBV does not cure the infection, and most people require the treatment for life. Therefore, a better understanding of the virus lifecycle and viral intra-host evolution is crucial in the development of antiviral drugs. The error-prone nature of the HBV polymerase and combined with high replication rate result in genetic diversity manifesting as a quasispecies in a chronically HBV- infected host. However, the impact of genetic diversity in HBV infections is still poorly understood; while antigenic differences and drug resistant forms of HBV have been assigned to specific viral polymorphisms, the consequence of the majority of genetic variants have not been interrogated. In this study, genetic diversity of HBV infections was investigated using a nanopore sequencing platform to determine longitudinal HBV evolution within hosts. The data extends previous knowledge that greater viral diversity is observed in HBV e-antigen (HBeAg)-negative infection and the rate of evolution in the nonoverlapping region is much higher than that of the overlapping region. The HBV surface proteins (HBsAg) comprise the Large (L), Middle (M) and Small (S). The HBsAg gene is completely overlapped by the polymerase gene in a -1 frameshift. Mutations occurring in one open reading frame (ORF) can also impact on the other; therefore, it is challenging to investigate the phenotype of HBsAg mutations without altering phenotypic behaviour of the polymerase. Moreover, HBV research requires Biosafety Level (BSL)-3 containment, which hinders research progress. To address these limitations, retroviral pseudotyped viruses (PVs) were utilised in this study to investigate HBV entry and the effect of viral polymorphisms. Retroviruses pseudotyped with the HBV glycoproteins, possess the structural proteins of retrovirus and HBV surface glycoproteins and only require BSL-2 laboratory facilities, which offers an alternative to overcome the drawbacks of handling HBV in vitro. This study developed novel pseudotyping techniques to investigate the HBV entry pathway. A sodium taurocholate co-transporting polypeptide (NTCP) overexpressing hepatocellular carcinoma cell line was developed to determine the impact of receptor expression on HBV entry. A new cell line (293T.gtD4.S) constitutively expressing the S protein of a genotype D isolate has also been developed. Using an in-house engineered lentiviral vector pNL4.3.Nluc.R-E- and a plasmid expressing L co- transfecting 293T.gtD4.S cells, the infectivity of HBV PVs was quantified and monitored. The results revealed the importance of L/S ratio in HBV infectivity.

Previous studies have shown that addition of recombinant prion into a cell free prion replication assay - PMCA inhibits the formation of PrPSc. Previously naturally existing versions of ovine prion protein were tested: rARQ, rARR and rVRQ within this assay. Of these, rVRQ was the most potent inhibitor of amplification of different scrapie isolates (IC50 value 120 nM) and bovine BSE (IC50 - 171 nM). The main aim of this study was to produce additional molecular clones for expression of recombinant ovine prion protein where codon 136 had been mutated to code for different amino acids. These rPrPs were tested in dose response experiments in order to investigate whether the change at 136 position in ovine PrP could impact on the ability to inhibit or stop the prion protein misfolding compared to previously tested rVRQ. In order to produce rPrP mutants at codon 136, site-directed mutagenesis was used. All rPrPs were purified by metal affinity chromatography taking advantage of the metal binding properties of PrP molecule. All mutated rPrPs were added to protein misfolding cyclic amplification (PMCA) at different concentration and compared to rVRQ. After amplification, samples were digested with Proteinase K (100 µg/ml) and quantified on immunoblots. The best inhibitors were tested with different ovine scrapie (ARQ/VRQ, VRQ/VRQ, AHQ/VRQ), bovine BSE and ovine BSE isolates (ARQ/ARQ). The results showed that three of the recombinant prion proteins: rRRQ, rKRQ and rPRQ (with arginine, lysine and proline at 136 position, respectively) were found to inhibit the PrPSc misfolding significantly better than naturally occurred rVRQ. The structure of rPrP variants and amino acid substitution at 136 position were analysed and different length peptides containing the valine, arginine, lysine and proline at 136 position were designed. None of these peptides analysed in PMCA gave similar levels of inhibition to the equivalent full length recombinant prion protein response. Moreover, structural analysis showed that introduction of longer amino acids at position 136 did not alter the whole scaffold of prion protein. In addition, the longer side chains for arginine136 and lysine136 or pyrrolidine loop in proline could result in more interatomic bonding in comparison to valine136 and therefore could act to stabilize the whole PrP molecule. Furthermore, the presence of the longer side chains of arginine136 and lysine136 would not predict further structure changes because of the 'structural' pocket present on the opposite site of position 136 in ovine PrP. The Rov9 cell line could be persistently infected with processed (heated and sonicated) scrapie brain homogenate SSBP1 (VRQ/VRQ) and SSBP1 derived, NaPTA precipitated PrPSc. In both cases, PrPres was detected in cell lysates from induced with 1 µg/ml doxycycline and when 500 µg of total protein was digested with 20 µg/ml of PK followed by PrPres concentration by centrifugation. The best inhibitory rPrPs were used in experiments to prevent the infection with SSBP1 isolate or reduce the PrPres in persistently infected Rov9 cells. As a result, addition of 250 nM of rRRQ, rKRQ and rPRQ prevented the infection of Rov9 cells at culture passage 1. The rPrP variants showed more promising results than natural rVRQ. In contrast, no significant reduction of PrPres was observed when persistently infected Rov9 cells were treated with 250 nM of either variants or natural rPrPs for 4 days. Overall, this work demonstrated a novel therapeutic approach for prion diseases using recombinant prion proteins. The recombinant protein treatment was effective not only in scrapie model but also among other TSEs and therefore these rPrPs or analogous strategy could be applied as potential human TSE therapeutic.

Respiratory syncytial virus (RSV) is an enveloped virus with a negative sense, single-stranded RNA genome. RSV is responsible for upper respiratory tract infections such as rhinitis and croup mainly in babies and young children, but it can also infect the elderly and the immunocompromised. If RSV infection progresses into the lower respiratory tract then more severe diseases such as bronchiolitis and pneumonia can occur, which can lead to death. There is no licensed RSV vaccine and the only licensed antivirals are monoclonal antibodies, such as palivizumab, which are for prophylactic treatment only. During the RSV lifecycle, intra-cytoplasmic compartments named inclusion bodies (IBs) form. It is thought that RSV carries out transcription and replication within IBs. IBs contain viral proteins, viral RNA and some cellular host proteins. They have also been described to form through phase-phase separations and exist as membraneless structures. Here we employed a multi-modal correlative imaging approach to explore RSV cytoplasmic compartments during RSV infection, aiming to better understand RSV replication. Our use of RSV genome-specific fluorescent probes discovered a new RSV-induced cytoplasmic organelle: genome-rich bodies, GRBs. GRBs appear to be distinct from IBs, containing much of the viral genome within the cell as evidenced through confocal laser scanning microscopy. Using soft X-ray cryotomography we found that GRBs are granular, membranous cytoplasmic structures. Cryo-focussed ion beam milling followed by electron cryotomography revealed that GRBs are enclosed by an outer lipid bilayer and contain many internal membrane bound compartments, some of which have multiple membrane layers. Within the internal compartments are ring-like and tubular structures which we hypothesise are RSV nucleocapsids - viral genomes and anti-genomes coated by the RSV nucleoprotein. Morphologically identical structures were also seen within RSV filaments budding out of infected cells. The application of multiple imaging modalities has enabled the discovery of GRBs on different resolution scales, generating a detailed description of GRB structure and morphology. Further investigation to explore mechanistic aspects of these structures may inform the development of future RSV interventions.

Equine sarcoids are tumours of dermal fibroblasts affecting all equid species including horses/ponies, donkeys, and mules. Their UK prevalence is estimated at ~5% (National Equine Health Survey) and The Donkey Sanctuary UK has identified that sarcoids are a significant problem in their animals. Bovine papillomavirus types 1 and 2 (BPV-1/2) are widely accepted to be causative agents of these tumours, with other factors also required for tumour development. There is a contradiction in the current evidence regarding viral/tumour transmission; there are reports suggesting spread between equines, but infective virus has not been isolated from sarcoids. This PhD project used a range of interdisciplinary methods to investigate both epidemiological and molecular aspects of sarcoids. Methods The sanctuary population demographics were described, and a text mining approach (WordStat) was used to identify animals with several categories and subcategories of disease. The number of animals in each category/subcategory per 100 animal years at risk (AYAR) was calculated to assess the prevalence of sarcoids, compared to other diseases. Univariable and multivariable binary logistic regression modelling (Stata) were performed to identify risk factors for sarcoid development. Details on "friendships" between animals were included and, to validate these, an observational behavioural study was carried out by collecting nearest neighbour data. DNA extracted from sarcoid lesions and Stomoxys calcitrans flies from the sanctuary was used in PCR reactions to amplify the E5 (Early 5) gene or LCR (Long Control Region) section of the viral genome to confirm whether BPV-1/2 DNA was present, and these results were compared to lesions' histopathology diagnoses. Amplified LCRs were sequenced to determine the BPV type and subtype present and phylogenetic trees were created using FastTree (Geneious). Antibody to the major viral capsid protein was used to capture viral particles in donkey sarcoid and fly samples. DNA attached to the capsid protein was then amplified by PCR (immune capture-PCR assay (IC-PCR)) to determine whether the samples contained potentially infective viral particles. RNA was extracted from normal skin and sarcoid samples from horses and donkeys, and from established cell lines (normal equine fibroblasts, BPV-1-infected equine fibroblasts and sarcoid fibroblasts). microRNA (miRNA) and messenger RNA (mRNA) sequencing were performed by the University of Liverpool and reads were mapped to the horse (EquCab3.0) and BPV-1/2 genomes. The host mRNA and miRNA profiles of samples were normalised and analysed by Principal Components Analysis. The viral copy number was calculated for each sample and the BPV expression profile determined for BPV positive samples using normalised viral read counts, to determine any correlations between viral and host variables. Evidence for the existence of papillomaviral miRNAs was explored.

In the Americas, arbovirus transmission is concentrated within urban settings in tropical zones, where high human population densities and environmental conditions enhance the survival and reproduction of Aedes aegypti. Since its re-emergence in South America in the 70's, dengue virus has been expanding and increasing in urban settings where it is now endemic. Additionally, the recent arrival of new arboviruses into the region, such as chikungunya (2013) and Zika virus (2015), have triggered major epidemics leading significant public health and economic impacts. These pathogens are linked in sharing a common mosquito vector in Ae. aegypti. Given the absence of effective licenced vaccines, vector control is thus the primary strategy for reducing the transmission of all of these pathogens. Effective vector control and public health preparedness require detailed understanding of vector ecology and human exposure risk within foci of transmission. Both vector populations and viral dynamics are highly dependent on environmental conditions, but the nature of environmental impacts likely depends on local ecological context. Ecuador bears an important burden of arboviral transmission in South America. Most transmission is concentrated in coastal cities where dengue is endemic and rising, and major outbreaks of chikungunya and Zika have recently occurred. However, there has been limited investigation of vector ecology in these rapidly expanding urban settings, and its association with seasonal patterns of arboviral transmission. To address this gap, this study aimed to assess the environmental drivers of Ae. aegypti ecology, infection rates and arboviral transmission within two major urban hotspots in Coastal Ecuador. This was accomplished through a series of field studies of vector ecology, laboratory analyses of arboviruses, and modelling investigations designed to identify environmental determinants of human exposure and infection incidence. The first chapter reviews what is known about the most important Aedes-borne viruses and their vectors in South America, with particular focus on Ecuador, vector control, and the global and regional disease burden. The second chapter presents results from a field study carried out in a urban neighbourhood of Quinindé, Ecuador, that evaluated a novel trapping method, the Mosquito Electrocuting Trap (MET), for direct estimation of human exposure to Ae. aegypti bites. The third chapter describes results of a 6-month field study aimed to characterize the environmental determinants of Ae. aegypti abundance and distribution, behaviour and arboviral infection rates within two cantons in Coastal Ecuador at the tail end of the 2016-17 Zika outbreak. The fourth chapter presents an analysis of seasonal and annual variation in dengue, chikungunya and Zika virus within these 2 cantons, and associations with climatic and entomological variables. The fifth chapter discusses the key results of each of the chapters and the implications of the findings towards an effective vector control in Ecuador and beyond the country. It was found that the MET was effective for measuring Ae. aegypti host-seeking behaviour, and generated representative estimates of their biting rate and activity time relative to the standard BG-sentinel trap (BGS). Analysis of Ae. aegypti ecology indicated that its abundance varied significantly between cantons, neighbourhoods within cantons associated with urbanization gradient, temporal timing of collection and past rainfall. Additionally, there was significant variation in Ae. aegypti resting behaviour (resting in or outside houses) between cantons. This demonstrates the existence of heterogeneity in Ae. aegypti population dynamics and behaviour between and within the study sites, which highlights the importance of localized surveillance to guide vector control. Likewise, arboviral incidence of dengue and chikungunya (as reported to the health system) also differed between the two study sites, being dengue 1.5 and chikungunya 2.5 times higher in Portoviejo than in Quinindé during the peaking week. The seasonal pattern of disease incidence varied among the three arboviruses, with a difference of 5 weeks between each of their peak of incidence. Intra-annual incidence was also found to be linked with climatic and entomological variables, with dengue and chikungunya incidence being positively associated with temperature and rainfall, while Zika incidence negatively associated with such climatic variables. Outdoor Aedes collections with Prokopack aspirators and BGS were positively related to dengue incidence, while indoor Prokopack aspirations were negatively associated with this variable. The interannual incidence of dengue differed between years analysed (2013-2018) with 2015 being the year with highest dengue incidence. Such findings highlight the importance of conducting focalized epidemiological surveillance on each site, but also differentiating between arboviruses, rather than assuming they all will follow dengue trends. Findings from this work have provided new entomological and epidemiological information to the study sites and despite the short period of study, fine spatial scale heterogeneity was detected in arbovirus transmission dynamics.

Adenoviruses have evolved to inactivate host cell responses to DNA damage through interaction with host cell proteins and by targeting some of these proteins for ubiquitin-mediated proteolysis. Indeed, previous studies have suggested that early region proteins dysregulate ATR signalling pathways during infection by sequestering the MRE11-RAD50-NBS1 complex and promoting the proteasomal degradation of TopBP1. Moreover, the cellular E1B-55K-interacting protein, E1B-AP5 modulates ATR kinase activity during infection. Work presented here has established that adenovirus targets the cellular replication, and DNA damage response protein, SMARCAL1 for degradation in an E1B-55K/E4orf6 and Cullin Ring ligase-dependent manner. As such, we determined that the phosphorylation of SMARCAL1 residues, S123, S129 and S173 by ATR and CDK kinases promoted SMARCAL1 degradation during infection. We also determined that SMARCAL1 recruitment to viral replication centres during infection was partially dependent upon SMARCAL1 phosphorylation but was mainly dependent upon its ability to interact with the RPA complex. We also determined that E1B-55K interacted directly with SMARCAL1 in adenovirus E1-transformed cells and could modulate cellular DNA replication. Indeed, E1B-55K expression initially enhanced DNA replication fork speed but ultimately, promoted replication fork stalling. We propose that SMARCAL1 is targeted for degradation by adenoviruses to inhibit host cell DNA replication.

High-risk human papillomaviruses (HPV) infect squamous epithelia and cause hyperproliferative lesions that can progress to cancer. During infection, the virus interacts with and regulates the host kinome, which has important implications for viral and cellular protein function. The cyclic AMP (cAMP) dependent protein kinase A (PKA) phosphorylates several viral proteins and altered activity of this pathway may be relevant for oncogenic progression of HPV infections; however, whether the virus modulates the activity of PKA during infection is unclear. Using a bioluminescent resonance energy transfer-based approach, HPV18 replication was consistently associated with increased activity of the PKA pathway, occurring concomitantly with increased levels of cAMP; this was partially dependent on the HPV E5 oncoprotein. Additionally, biochemical analysis showed that the virus interacted with a nuclear regulator of PKA RII signalling, the A kinase-anchoring protein 95 (AKAP95). This interaction was mediated by the PSD95/DLG/ZO-1 (PDZ) binding motif of the E6 oncoprotein, requiring the polarity protein hScrib and PKA. Intriguingly, E6 regulated AKAP95 PKA binding and silencing of the AKAP in a physiological HPV18 replication model showed that it regulated expression of E6 targets Dlg1, hScrib and p53. Thus, E6 targeting of AKAP95 may be relevant for control of nuclear PKA signalling in order to facilitate virus replication.

Ficolins are polymorphic liver-expressed pattern recognition receptors (PRRs) that contribute to the innate surveillance of virus infections, recognising carbohydrates such as N-acetyl glucosamine, which are components of glycoproteins found on the surface of different viruses. While human ficolin-2 has been demonstrated to bind and inhibit entry of hepatitis C virus (HCV) particles, the antiviral activity of ficolins found in other mammalian species is unknown. For the first time, recombinant mouse and non-human primate (NHP) ficolins were cloned, expressed and purified to determine their interaction with HCV and non-primate/Equine hepacivirus (NPHV/EqHV), and the impact on virus entry. HCV and EqHV have been recently reported as closely related within the hepacivirus genera (family Flaviviridae). This relatedness may provide insights on the potential use of EqHV as a model for the study of HCV. Both viruses encode two glycoproteins (E1 and E2), which are found on the surface of the viral envelope. These glycoproteins facilitate the entry into the host and are targets for host immune recognition molecules like ficolins. HCV and EqHV pseudoparticles (pp) were used to determine the neutralising ability of ficolins from different species. An EqHVpp model system for the investigation of entry and neutralisation of EqHV infection was created and validated. In this study, for the first time, the neutralisation of hepacivirus by non-human ficolins was assessed. These findings may provide better insight into the divergent evolution of these genes in mammals. It is possible that ficolins might serve as a future therapeutic anti-viral agent for hepaciviruses.

Cryptococcus neoformans is an opportunistic fungal pathogen, which is widespread in the environment. Due to its global distribution and association with pigeon excreta, human exposure to this pathogen is frequent but initial infection is rapidly cleared or contained in immunocompetent hosts. In immunocompromised hosts, however, C. neoformans can cause severe disease often with dissemination to the central nervous system. Without rapid treatment disseminated infection is invariably lethal. With the rise of transplant and cancer therapy patients, the pool of vulnerable hosts is ever larger. Thus, studies of the interaction between Cryptococcus neoformans and the immune system are paramount for the development of novel treatment options. In this study, different aspects of the interaction between C. neoformans and phagocytic cells, such as macrophages and dendritic cells, were explored. One of the key features of Cryptococcal pathogenesis is its ability to persist and replicate within macrophages, as well as its ability to escape via a novel non-lytic mechanism known as vomocytosis. The impact of vomocytosis on disease progression is not yet fully appreciated, but it has been linked to dissemination of the fungal cell within the host. Vomocytosis is a highly regulated process, influenced by both fungal and host parameters. Macrophage polarisation is one such parameter, whereby alternatively activated macrophages show diminished occurrence of vomocytosis events. In this work, the interplay between two modulators of vomocytosis was explored. It was found that inhibition of the atypical MAP kinase ERK5, a known regulator of vomocytosis, limits alternative activation of macrophages, thus, providing a link between both phenomena. Cryptococcal meningitis occurs mainly in HIV+ patients. The effect of chronic viral exposure on vomocytosis has not been studied before. My work revealed that viral exposure significantly enhances vomocytosis, acting through low levels of type-I interferons. Preliminary work expanded this finding to include bacterial co-infection settings, which can also elicit a type-I interferon response. Lastly, the consequences of prolonged exposure to Cryptococcal cells or components on antigen presentation and antigen-induced T cell proliferation were explored. This setting emulates long-term latent infection within the lungs of immunocompetent hosts, which are frequently exposed to this fungus. The presence of Cryptococcal capsule components negatively impacts antigen-induced T cell proliferation by altering the antigen processing capabilities of antigen presenting cells. Overall, this work highlights the complex interaction between C. neoformans and the innate immune system and underscores the importance of incorporating biological context cues into our experimental systems.

Feline leukaemia virus (FeLV) infects domestic cats and other wild felids worldwide. FeLV exposure has three potential outcomes, namely abortive, regressive and progressive infection. The early interactions between FeLV and the haematologic and lymphatic systems of the host determine the pathogenesis of infection and the immune response appears to have a major influence on disease progression. The aim of this thesis was to investigate current diagnostic tests for FeLV infection as well as the humoral immune response of cats naturally exposed to FeLV and to determine whether an assay could be developed that would predict the outcome of infection. A total of 489 samples from 432 domestic cats, from several cohorts, were investigated, as well as 113 samples from 112 non-domestic exotic feline species. Firstly, the development and optimisation of three study specific methods is described. The first assay to be developed was a novel enzyme-linked immunosorbent assay (ELISA) to detect antibodies recognising FeLV-A and FeLV-B surface unit (SU) glycoproteins in cat plasma. Secondly, a product enhanced reverse transcriptase (PERT) assay to detect reverse transcriptase in culture fluids from in vitro peripheral blood mononuclear cell (PBMC) cultures was optimised. Thirdly, a quantitative polymerase chain reaction (qPCR) assay was developed to simultaneously detect FeLV provirus and the housekeeping gene control feline.

Rabies is an encephalitic disease with a mortality rate that approaches 100% in the absence of treatment. The World Health Organisation recognises rabies as a neglected tropical disease due to its disproportionate impact on poor and vulnerable populations in developing countries. Whilst vaccines exist to help prevent and control rabies, these are not always available or accessible to those at high risk. Rabies currently can only be treated effectively prior to the onset of symptoms and as immunoglobulin treatments are expensive and in short supply, and current vaccines require multiple doses to reach full efficacy, full courses of post-exposure prophylaxis may not be completed, leaving vulnerable individuals at risk. Rabies is a zoonotic viral disease that is predominantly caused by the rabies virus. The rabies virus has a broad host range and is capable of infecting most mammalian species, though its main reservoir, and the source of most human infections, are canids, particularly dogs. Rabies can also be caused by non-rabies lyssaviruses, a group of viruses related to rabies virus that are mainly found in bats. Lyssaviruses have been isolated globally and whilst there are very few reports of rabies encephalitis caused by non-rabies lyssavirus species in humans, the general lack of awareness of lyssaviruses in bats can lead to delays in seeking treatment and increase the risks of developing rabies once exposed. Current vaccines are highly effective against rabies virus, but display less effectiveness against other lyssavirus species, with little to no protection provided against the more diverse lyssaviruses. In this project, a lyssavirus pseudotype panel was generated using a range of lyssavirus species. This pseudotype panel was then used to investigate the neutralisation and tropism profiles of the lyssaviruses in question. In developing the pseudotype panel, it was identified that the choice of glycoprotein expression plasmid was vital for the generation of highly infectious pseudotype particles, and that the pI.18 plasmid was superior to pcDNA3.1. A novel chimp adenovirus-based rabies vaccine was compared to a current human rabies vaccine for its capacity to elicit rabies-neutralising antibodies. A single dose of the novel vaccine was revealed to produce a comparable antibody response to that produced by the human vaccine following a full three-dose schedule. Finally, the lyssavirus pseudotype panel was used to infect several animal cell lines to determine whether the viruses displayed any evidence of host adaptation. Some of the lyssavirus species showed increased infectivity in cells corresponding to their original host species, relative to a lab-adapted rabies virus strain.

This study aims to develop novel diagnostic assays for zika virus (ZIKV) and equine influenza viruses (EIV) by • Generating pseudotyped ZIKV as a surrogate for wild-type virus in the virus neutralisation (VN) test • Producing plant-expressed ZIKV non-structural (NS) 1 protein as a surrogate for wild-type virus in the ELISA • Identifying lineage-specific antibodies against EIV using Next Generation Phage Display (NGPD) to replace antibodies produced by immunizing animals for use in ELISA. Co-circulation and cross-reaction among flaviviruses have become the major obstacle for an accurate diagnosis. Antigenic drift and shift of influenza viruses underline the importance of close monitoring and surveillance for precise diagnosis and vaccine production. This study aimed to develop novel diagnostic assays for emergent/re-emergent viruses, namely, Zika virus (ZIKV) and equine influenza virus (EIV) using various techniques including pseudotyping, plant expression and next-generation phage display (NGPD) to improve their current diagnostic assays. Vesicular stomatitis virus (VSV) backboned pseudotype virus (PV) was generated by co-transfecting producer cells with commercially available VSV plasmids. ZIKV envelope protein (ZIKV E) was used to replace the VSV glycoproteins to generate VSV-backboned ZIKV PV, which showed no infectivity. However, VSV backboned Chikungunya virus (CHIKV) PV used as a control exhibited similar luciferase activity as the VSV PV (~1×107 RLU/ml), indicating successful production of VSV-backboned CHIKV PV. The virus budding site of ZIKV is different from CHIKV and VSV, which was identified as one of the possible reasons for the failure of infectivity of ZIKV PV. Future work would be to mutate the ER-retention signals in the transmembranes of ZIKV prME for its translocation to the plasma membrane and to incorporate furin proteases during pseudotyping for the ZIKV E to achieve a mature and infectious form. Next, a ZIKV non-structural protein 1 (NS1) ELISA was developed using ZIKV NS1 proteins expressed in plants. Unlike envelope proteins, NS1 does not elicits flavivirus cross-reactive neutralisation antibodies but induces a non-neutralising yet virus-specific antibody response. NS1 gene was codon-optimised, synthesised and inserted into a plant expression vector pEAQ-HT to obtain expressed protein. Preliminary results using the resultant ELISA showed around 95% sensitivity and 95% specificity using 20 ZIKV positive human serum samples and 19 Dengue-only positive samples. For future work, a bigger sample size should be tested for the proper validation of this plant produced NS1 ELISA. Finally, antibody peptide sequences that specifically recognise a strain of EIV from clade 2 and not the strain from clade 1 were identified using NGPD and data analysis. It is important to be able to distinguish different clades to keep current equine vaccine effective for the circulating strain. Future work would include the recovery of the peptide sequences using inverse PCRs and testing their specificities against other EIV strains in the two clades. Identified antibody peptides could be used to replace ferret antisera in the haemagglutination test for characterising circulating EIVs.

Zika virus (ZIKV) is a member of the Flavivirus genus of the family Flaviviridae. Members of this genus possess a single-stranded positive-sense RNA genome which are flanked by 5’ and 3’ untranslated regions. Previously, ZIKV infection was thought to cause symptomatic infection in 20% of patients, characterised by a non-purulent rash and a mild fever. However, recent ZIKV outbreaks have seen the emergence of novel neurological sequelae associated with ZIKV infection. These symptoms affected the central and peripheral nervous systems (CNS and PNS, respectively) of neonates and adults (congenital ZIKV syndrome and Guillain-Barré syndrome, respectively). Consequently, prior to commencement of this thesis, the knowledge of ZIKV interactions within the CNS and PNS was limited. Therefore, the primary aim of this thesis was to expand the current knowledgebase of ZIKV infection within cells of the CNS and PNS. The work presented herein assessed available tools to study ZIKV infection in vitro, including testing previously uncharacterised commercial antibodies targeting the ZIKV envelope (ZIKV E) and NS1 proteins. Subsequently, a suitable antibody raised against the ZIKV E was identified and used in downstream analyses. A model system of ZIKV infection of the CNS and PNS using mixed-cell co-cultures derived from mouse spinal cord (CNS) or dorsal root ganglion (PNS) was used to determined cell-type specific susceptibility to ZIKV infection. It was found that cells of the PNS are refractory to ZIKV infection, whereas cells of the CNS are permissive to ZIKV infection. In the CNS, oligodendrocytes and their precursors were the most susceptible cell type to ZIKV infection. Moreover, when infecting CNS co-cultures at a time point which reflects prenatal life through to post-natal life, white matter structures (myelin sheath and axons) are injured. However, neuronal cell bodies remained healthy. Using purified primary neuronal cultures derived from mouse spinal cord, it was determined that neurons were refractory to ZIKV infection in the absence of accompanying glial cells. This suggests axonal damage may not be a result of direct infection of neurons themselves and may be a consequence of oligodendrocyte infection and injury. Transcriptomic analyses of ZIKV-infected CNS co-cultures revealed that ZIKV infection induces the upregulation of genes involved in antiviral responses and inflammatory pathways, including TNF and ROS/NO pathway components; these pathways may be involved in exacerbating injury to white matter structures within ZIKV-infected CNS-co-cultures. In summary, the results described in this thesis show that ZIKV can infect the early post-natal CNS. Furthermore, ZIKV-infection of this murine model demonstrates that infection induces the injury and depletion of myelin and axons. This is likely to be due to a combination of effects such as direct viral infection, cell death of oligodendrocytes, and secreted inflammatory factors. Myelination occurs late in foetal development and carries on into early adulthood. These data, in combination with precedent pathological findings, suggest that ZIKV-infected children born absent of microcephaly may develop other, more subtle, neurological sequelae later in life.

Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis with over 70 million people affected worldwide. Recent advances in direct acting antivirals for HCV treatment have resulted in sustained virological response rates of more than 90%. Thus, the World Health Organisation (WHO) has developed a global strategy for elimination of the virus by 2030. Major barriers to elimination include the lack of a point-of-care (POC) test for immediate linkage to care and the development of a HCV vaccine. This first part of this thesis covers experiments aimed towards the development of a novel POC test based on loop mediated isothermal amplification (LAMP). The second section of the thesis addresses the novel phenomenon of secondary spontaneous clearance (SSC), studied as a natural vaccine model to decipher the immune responses involved in spontaneous resolution following treatment relapse. A double-blind study of HCV LAMP was conducted on RNA samples of all major HCV genotypes and viral loads. The sensitivity and specificity of the assay was 90% and 98%, respectively within less than 25 minutes of incubation which fulfils current WHO recommendations. We developed a user-friendly read-out based on a pregnancy-test like lateral flow device. LAMP has minimal equipment and training requirements with previous field studies making it ideal for a future HCV POC test. The two cases of SSC post-treatment relapse were studied in comparison with four treatment failure (TF) patients. In both SSC cases, low levels of neutralising antibody were detected at the time of diagnosis but on relapse, a robust and sustained response was generated that reduced the infectivity of autologous viral pseudoparticles expressing HCV envelope proteins – a feature not present in any control patients. Furthermore, significant differences in levels of Th-1 cytokines were noted prior to treatment in SSC but not TF. This natural vaccine model (prime on acute infection, boost on relapse) adds weight to the importance of adaptive immune responses in resolving HCV infection.

Rift Valley fever is a mosquito-borne viral disease of ruminants, camels and humans. In Tanzania, outbreaks have occurred at intervals of 10 - 20 years with major epidemics reported in 1977, 1997/98 and 2006/2007. Our ability to prevent future epidemics is limited by poor understanding of how the virus circulates between major epidemics. This study aimed to investigate the epidemiology of inter-epidemic RVFV infections in northern Tanzania. This study involved (a) collection and characterisation of mosquitoes; (b) RVFV serological analysis of serum samples from cattle (n=3582), sheep (n=2586), goats (n=3303) and human populations (n=565) collected through cross-sectional household surveys; (c) analysis of risk factors for livestock and human seropositivity; (d) molecular detection of RVFV in mosquitoes and diagnostic materials collected during investigation of 190 livestock abortion events. Generalised Linear Mixed-Effects Models (GLMMs) were used to examine predictors of vector mosquito abundance, and risk factors for RVFV exposure in livestock and humans. Maximum Entropy (MaxEnt) algorithm was used to model vector mosquito habitat suitability and spatial distribution. A total of 2224 mosquitoes were collected including Culex spp (n = 1123), Anopheles spp (n=1006), Mansonia spp (n=56), Aedes spp (n=34), and Coquillettidia spp (n=5) with significant variation in abundance with percentage difference in normalised difference vegetation index (NDVI). No RVFV infections were detected in any of the mosquitoes collected. RVFV seroprevalence was higher in cattle 4.4% (95% CI:3.7-5.1), than in sheep 2.6%, (95% CI: 2.0-3.3) and goats 1.4% (95%CI: 1.0-1.8), with seropositivity in young animals providing evidence of recent virus circulation. Seropositivity in livestock increased with age (OR=1.3, CI: 1.2 - 1.4, p<0.001) consistent with endemic circulation and was associated with a history of abortion in goats (OR=2.5, 95%CI: 1.1 - 5.4, P=0.023) and sheep (OR=2.7, 95%CI: 1.1 - 6.3, P=0.025). Human seroprevalence was 8.5% (95% CI: 6.4 - 11.2) and varied between villages and between households within villages. Handling of aborted material (OR=4.3, 95% CI: 1.7-10.8) and consumption of raw milk (OR=4.1, 95%CI: 1.8 - 9.3, P=0.001) were significant risk factors for human seropositivity. RVFV was detected in a cluster of 14 (7.4%) abortion cases including the milk of three aborting dams. This provides strong evidence for continuous RVFV circulation in livestock between major epidemics in Tanzania and that unboiled milk is an important potential source of infection for people.

Effective surveillance of foot-and-mouth disease (FMD) is of the utmost importance in order to understand the disease risks and implement effective control strategies. Epidemiological data obtained for FMD is mostly obtained through recognition and reporting of clinical cases by farmers which has several limitations. For example, under-reporting of disease is common, due to deficiencies in veterinary infrastructure, the effort involved for sample collection, or the repercussions of control measures for farmers. Diagnostic sample types, usually vesicular epithelium and fluid, are invasive and labour intensive to obtain, and can only be collected from acutely infected animals. Therefore, animals with sub-clinical FMD infection (particularly those in vaccinated herds) may not be identified but may still contribute to disease transmission. It is likely, therefore, that the true prevalence of FMD is not accurately known in parts of the world where the disease is endemic. Consequently, the requirement exists for a simple approach for FMD surveillance that does not rely on farmer reporting. Milk is a non-invasive sample type routinely collected from dairy farms and has been utilised for the surveillance of a number of other diseases. Despite numerous publications suggesting the potential of milk as a valuable sample type for foot-and-mouth disease (FMD) surveillance, empirical studies have mainly focused on the risk of transmission via milk, or the detection of FMD virus (FMDV) in milk from individual animals. This thesis aimed to expand on previous studies to determine the utility of milk and its limitations for the surveillance of FMD at the individual and herd level, using data collected from experimental and field studies. A highly sensitive and specific high-throughput RNA extraction and real-time rRT-PCR was optimised and utilised for FMDV RNA genome identification throughout the project. Using this method, it was demonstrated that FMDV RNA genome could be detected up to 28 days post infection using milk samples collected from individual cows. Further analysis using serotyping or lineage-specific rRT-PCR assays and VP1 sequencing of milk samples collected from individual cows in northern Tanzania highlighted the use of milk as a suitable alternative to more invasive sample types such as epithelium. Additionally, storage and shipment condition simulations performed demonstrated good stability of FMDV RNA genome within milk samples. Following these experiments, the potential use of pooled milk for herd–level FMD surveillance was investigated. Two proof-of-concept pilot studies were performed comparing the rRT-PCR results of pooled milk samples collected from both a large-scale dairy farm in Saudi Arabia and milk pooling facilities supplied by smallholder dairy farms in Kenya, with farmer reports of clinical disease. Results supported laboratory limit of detection studies, demonstrating that FMDV could be detected from milk pools of up 10,000 litres, even when there were low numbers of clinical cases. Furthermore, both studies suggested the detection of subclinical infection in milk samples, where disease was not reported. Data from pilot studies performed in this thesis therefore support the use of milk as a simple, non-invasive approach for herd-level FMD surveillance. Further field studies are required to determine the full utility of this method before it may be implemented for targeted/risk-based surveillance alongside existing surveillance systems to facilitate improved knowledge of FMD epidemiology, or for use in FMD contingency plans.

Background: Following a healthcare incident which has created a risk of blood borne virus transmission, a patient notification exercise can be implemented to inform patients that they have been exposed to an unanticipated and unacceptable risk of harm as a result of medical error. Those managing the incident must consider all the facts of the case as well as the negative and positive implications, before reaching a decision on whether a patient notification exercise is required. One of the goals of notification is to detect the presence of blood borne virus infection within the exposed patient population thus providing the opportunity for treatment. The risk of transmission, within this setting, however, is thought to be extremely low. Patient notification exercises are thought to create stress for patients, carry significant financial costs and reflect poorly on the health service. Most notification exercises identify very few new blood borne virus diagnoses and almost never identify incident-related transmissions (Close et al 2013; Mason et al 2008; Conrad et al 2011; Blatchford et al 2000; Roy et al 2005; Henderson et al 2017). Detection of transmissions and the concept of transparency must be balanced against any negative effects created by the exercise. Currently, incident management teams must make their risk assessment and decision regarding notification without the support of formal guidance and with no up-to-date synthesis of evidence from previous dental incidents. One of the aims of this doctoral study was to design and present an evidence-based decision-making algorithm to aid the incident management team's decision-making process regarding patient notification, following a dental infection control breach. Methods: This doctoral work comprises a scoping review study with three components: a stakeholder consultation, a review of the published and grey incident literature and a policy mapping exercise (Davis et al 2009). In the stakeholder consultation, purposive sampling was utilised to seek out and record the responses of those with experience of managing United Kingdom dental incidents. Participants were interviewed using a specifically developed questioning route and semi-structured interview style. The literature review was used to identify articles which either reported or focused on the management of (a) specific dental incident(s) or were considered to be an opinion piece on large-scale medical patient notification exercises. In regards to the grey literature, the chief investigator was directed to unpublished United Kingdom incident reports by key contacts and organisations. One of the chief investigators' educational supervisors, Dr Roy had access to a number of Scottish reports through her role as senior epidemiologist within Health Protection Scotland and had historically been involved in the management of such incidents occurring in Scotland. The chief dental officers of each devolved United Kingdom nation were contacted and every health protection team across England, Wales and Northern Ireland were emailed. The UK Advisory Panel for Healthcare Workers Infected with Blood Borne Viruses (UKAP 2019) was also contacted and a list of the dental incidents for which they had provided advice, was supplied. Four different data extraction forms were used to collect information from reports and articles based on incident type, with the intention of presenting qualitative data as a narrative synthesis and quantitative data in the form of graphs and tables. In the guidance mapping exercise, an initial list of websites to search was formulated based on recommendations from stakeholder consultation participants, the medical college librarian and university public health lecturers. Further sites were included based on the chief investigator's personal knowledge and utilisation of the resource 'Grey Matters' (Canadian Agency for Drugs and Technologies in Health 2015). Guidance documents were included if their use was cited by those managing incidents or they provided guidance on a) how to grade/assess the risk associated with an infected healthcare worker/infection control incident and/or b) when to notify patients following an infected healthcare worker/infection control incident and/or c) when duty of candour/disclosure standards are triggered. Results: One hundred and forty nine dental incidents from six developed countries occurring between 1990 and 2017 were identified. Around half of infection control incidents (48% of 40) went to notification compared to 14% of those involving infected healthcare workers (n=107). Infection control incidents account for an increasing proportion of those managed (3.6% from 1990-1999 but 35.8% from 2010-2017). The stakeholder consultation, literature and guidance revealed that transmission risk was considered to be the most influential decision factor regarding patient notification, however, level of risk was rarely applied to incident management in a structured or consistent way. There was a consensus that, although very important, risk could not be calculated accurately and that at best, only qualitative descriptions such as 'low', 'very low' or 'negligible' could be applied (Mason et al 2008; Millership et al 2007; Unpublished reports 2001-2017). Minimal information could be drawn from historical 'proven transmission' events as they are rare, occurred long ago and/or lacked contextual detail. The limited conclusions that could be drawn suggested that incidents involving syringe reuse, multi-dose vials, extractions and oral surgery settings present a greater risk of patient-to-patient BBV transmission. Multiple sources suggested that if transmissions (patient-to-patient or healthcare worker to patient) have already been identified, or are strongly suspected, then notification and testing is necessary. However, of the four proven, reported dental blood borne virus transmissions since 1990, not one identified any further transmissions via their associated notification exercises. This suggests that any incident-related transmissions which require detection may all reliably be identified prior to the use of a patient notification exercise. This review suggested that there was significant pressure to be candid with patients following an incident, a mantra which has arisen due to historical paternalism and an awareness of the new Duty of Candour legislation which may override any consideration of risk level (CQC 2014; GMC 2015; Scottish Government 2018). Decision makers often contemplated; what the public would want and their reactions to finding out an organisation had not been open with them. Unsurprisingly, notification was deemed essential when the public were already aware of an incident. The professional and statutory Duty of Candour was often weighed against the perceived opportunity costs of conducting notification, the significant expenditure of time, staff and workload as well as the predicted psychological impact on patients and reputational consequences for the dental profession which could result in patients not seeking dental care. Notification appeared to be conducted based on the need to err on the side of caution. Patient safety was the priority and with a challenging risk assessment, no guidance or information from other incident management teams and unclear Duty of Candour guidance, notification and testing was utilised to respect the importance of transparency and mitigate the risks of both not adhering to legislation and leaving patients undiagnosed. Research gaps: Decision makers were not only hampered by an absence of guidance but also by an inability or struggle to ascertain what others have done in the past. This doctoral work discovered that within the United Kingdom there is no central repository for incident data, incident details are rarely published (15%) and there is a lack of sharing of lessons learned amongst public health teams. When reports are made available or their data are presented in a published journal article, no standardised way of reporting exists and there are inconsistencies in both the amount and detail of information presented. There was great variability in the types of journal that featured articles on patient notification. Creation of a central repository of 'blood borne virus transmission risk' incident information and an increase in publication activity with agreement on the most suitable type of journal is needed.

Vaccines against infectious bronchitis virus (IBV) are produced by repeated passage of virulent IBVs in embryonated hens’ eggs. Although used as a standard method by industry to generate a vaccine, the mechanism behind attenuation remains unclear. Moreover, the genetic changes that occur during the process of attenuation by egg passage largely go unreported. RNA viruses such as IBV do not exist as clonal structures but rather a population of genetically related viruses that interact on a functional scale. The evolution of IBV is purportedly the influence of two factors; the generation of genetic diversity and selection of pre-existing genetic variants. The origin of attenuating mutations as a result of serial egg passaging is a subject of debate. A panel of egg-passaged isolates of IBV, four started using a virulent population and four started using the virulent population’s clone have previously been generated in the lab. The starting, intermediate and final attenuated viruses were deep sequenced using an array of high-throughput sequencing (HTS) techniques. The genetic differences occurring at both consensus- and subconsensus level between virulent and attenuated IBVs are reported here as is the order of mutational appearance; both are used to inform as to the evolutionary pathways leading to attenuation. The use of HTS to study the process of attenuation by egg passaging is novel to the field. During this analysis, it was observed that the 3′ untranslated region (UTR) of IBV undergoes a proportionally higher rate of mutation compared to the rest of the genome. The involvement of the region in governing virus pathogenicity, to elude as to a possible mechanism of IBV attenuation, was studied by reverse genetics exchanging a virulent IBV’s 3′ UTR with that of a non-pathogenic IBV equivalent. Interestingly, this virus was shown to be non-pathogenic when introduced to birds.

Genome editing in plants has provided many new breakthroughs due to the development of different methods, such as CRISPR/Cas9, by which this can now be carried out. Beneficial traits should now be more quickly integrated into breeding lines. The Potyvirus TuMV, affects a wide variety of crop species including Brassicas, causing losses in yield and increasing production costs, therefore broad-spectrum resistance to TUMV is needed. Previous work identified a B. rapa variety, RLR22, which had resistance to TuMV due to a single mutation in an isoform of eIF4E, eIF(iso)4E, truncating the protein. This stops the virus utilising the eIF4F translation initiation complex, which consists of three subunits eIF4E, eIF4A, and eIF4G. This represents an ideal target for developing CRISPR/Cas9 knock outs as equivalent resistance has not yet been found in B. oleracea. Further investigation into the genetic makeup of the eIF4F complex enabled the targeting of specific paralogs that might confer resistance. Bioinformatic analysis of the genes involved identified several novel paralogs of eIF(iso)4e, eIF4A, eIF(iso)4g. This allowed for the design of paralog specific PCR primers and guide RNAs. RNAseq analysis also revealed alternative splicing taking place in all the gene families involved. CRISPR modification of B. oleracea proved difficult, resulting in only one heterozygous edited plant. This is potentially due to lethality of the knock-out of the target gene. In B. rapa, novel transformation techniques were employed to overcome the obstacles of recalcitrance to being transformed in this species. Protoplast protocols were optimised for use in Brassica and these increased transformation rates significantly. However, the use of embryogenesis transcription factors to increase regeneration of plants, and the use of Carbon Nanodots to aid in transformation were unsuccessful. CRISPR modification in Brassicas remains difficult, however, these complications stress the obstacles that must be overcome for industry to implement such technologies into their breeding programs.

Using conventional diagnostic methods to test for plant viruses requires knowledge of the pathogens likely to be associated with a host species. This knowledge can either be applied when using a targeted test method (e.g. ELISA or PCR) to identify required tests, or in bioassay, through knowing which viruses will transmit into which assay host. In the last decade High Throughput Sequencing (HTS) has revolutionised plant virology diagnostics, however, the knowledge and skills of the diagnostic virologist are needed to interpret the results of sequencing and to investigate the myriad of novel viruses reported using this technique. This thesis presents a body of published work and an accompanying linking document focussing on the development and application of these diagnostic technologies in a plant health/biosecurity setting. The thesis explores the use of diagnostic technologies in virus detection and discovery, but also in supporting research applications such as gathering the data necessary to support plant health risk assessment or carry out epidemiological studies on vector efficiency. The publications include a review of new virus records from the United Kingdom over a 35-year period, discussing the factors driving virus discovery such as changes in trade, research focus, and diagnostic technologies. Two case studies are presented which investigate diseases of unknown aetiology utilising contrasting approaches to infer the causal agent/s of disease, one utilising biological demonstration, the other experimental design and statistical analysis. Two publications discuss the evaluation and validation of diagnostic techniques. The final publication describes an investigation into the relative efficiency of transmission of potato virus Y and potato virus A by a range of aphid species. The accompanying linking document discusses each publication in the context of the current literature, as well as discussing alternative approaches to inferring causation where traditional biological approaches may not be possible.

Turnip mosaic virus (TuMV) and Turnip yellows virus (TuYV) detrimentally affect cultivated plant species of high socioeconomic importance. Both plant viruses cause losses through diminished harvest yields and quality of produce worldwide. They particularly impact species of the Brassica genus. Few viral disease management strategies exist, and most are considered neither viable nor durable for long-term protection of important brassica crops. Genetic sources of resistance against TuMV and TuYV have been identified previously, although remain unexploited. Through the development of these resistance sources, use of various genotyping strategies, linkage mapping approaches and quantitative trait loci (QTL) analyses, genomic regions associated with TuYV and TuMV resistances have been defined here. In Brassica juncea, pairs of significant QTLs for two sources of recessive TuMV resistance have been elucidated. Complementation testing of these resistances indicated one resistance gene is shared by the two plant lines investigated. Three Brassica rapa TuMV resistance mapping populations have also been generated. These sources of TuMV resistance proved effective against isolates of TuMV known to overcome most current major sources of resistance; this germplasm represents valuable and highly sought after broad-spectrum TuMV resistance. In Brassica oleracea, a major and a minor QTL accounting together for 46.3% of quantitative TuYV resistance with QTL intervals of 24.2cM and 6cM, respectively, were identified using just 93 first backcross plants. A robust marker loci-filtering strategy has also been developed here. Based upon successive filtering without the need to employ standard measures of marker quality (such as read depth for sequencingbased genotyping), or imputation and correction of genotypic data, unreliable genotyping by sequencing data was rendered useful where conventional filtering strategies could not achieve this. B. oleracea genome-editing (GE) using the CRISPR/Cas9 system to induce TuMV resistance was also explored; edits were introduced but appeared to be lethal, highlighting potential challenges for future GE.

Epstein-Barr virus (EBV) has been associated with several lymphoid and epithelial malignancies, such as Burkitt's Lymphoma and Nasopharyngeal Carcinoma, due to latent infection properties. The only EBV protein produced in all latency profiles of EBV is Epstein-Barr virus nuclear antigen 1 (EBNA1), a protein that forms a homodimer that is responsible for ensuring the replication and persistence of the EBV genome in cells to ensure a lifelong infection. EBNA1 has been explored as a driver for oncogenesis due to its ability to interact with host proteins and cellular promoters, whilst evading the immune system. The EBNA1 domain called the Glycine-Alanine repeat (GAr) causes translation suppression, resulting in the prevention of MHC class I EBNA1 antigen presentation. The GAr mediated self-regulatory properties of EBNA1 may be oncogenic; the full mechanism of which requires elucidation but appears to utilise a host stress response mechanism. This study focuses on revealing possible therapeutic agents for controlling EBV disease through the inhibition of EBNA1 expression or function. In order to inhibit EBNA1 dimerisation function, eight peptides, attached to cell penetrating peptides, were synthesised and designed to mimic the region of EBNA1 responsible for stabilising homodimers. Peptides were applied to human B cell lines to determine toxicity, specificity to EBNA1 cell survival, and the effect on dimerisation through an optimised dimerisation assay. The peptides did not affect EBNA1 specifically and no effect on cell viability or homodimer level was observed. Confocal microscopy revealed that the peptides were unable to enter the nucleus and instead aggregated, indicating they were unsuitable for use as a therapeutic agent. To investigate the GAr mediated self-inhibition mechanism, epithelial cell lines were transfected with GAr containing or lacking EBNA1 constructs, and cellular protein expression level evaluated. Pathway analysis and the evaluation of protein expression changes in latency I and III cell lines and Eμ-EBNA1 transgenic tumour cells revealed PI3K signalling components, C-MYC, E2F1, and MDM2 as candidate host proteins involved in this mechanism. Drug inhibition of proteins involved in these pathways and the effect on EBNA1 was analysed in human BL cell lines and Eμ-EBNA1 tumour cells in culture. C-MYC, PI3K, and MDM2 inhibition caused a reduction in EBNA1 expression, with PI3Kδ inhibition appearing to cause cell death in EBNA1 dependent cell lines. CAL 101, a PI3K p110δ inhibitor that is FDA approved, was then analysed for potential therapeutic properties in vivo using transplanted Eμ-EBNA1 tumour cells that were tagged with RFP to facilitate analysis. Whilst PI3K inhibition was successful in causing Eμ-EBNA1 tumour cell death in culture, this was not translated in vivo. Two in vivo studies, one that targeted dissemination of tumour cells and the other for treating established tumours, both resulted in progressed but variable tumour infiltration that did not differ significantly from the vehicle treated controls. The link between the proteins MDM2, E2F1, C-MYC and PI3K reveals new therapeutic routes to target EBNA1 function and suggests that MDM2 may be involved in the EBNA1 self-inhibition mechanism. In summary, these findings contribute to understanding the mRNA translational stress response caused by the GAr of EBNA1, which acts to increase proliferation signals in EBNA1 containing tumour cells.

This thesis comprises two projects detailing the synthesis and testing of bifunctional molecules. The projects were aimed at addressing the key biological problems of combating drug resistance and the requirement for novel therapeutic strategies. Chapter one details the first of these projects, which was concerned with elucidating the mechanism(s) of drug resistance in parasitic nematodes by using bifunctional fluorescent chemical probes based on the anthelmintic drug ivermectin. The first section presents the relevant background information for the project including information regarding the mechanism of action of ivermectin and the basic anatomy of nematodes, which were used to guide the design of the probes, BLI and FBI. The second section discusses the synthesis of each of the probes as well as their biological assaying used to assess the utility of the probes for the purposes of studying ivermectin resistance. The FBI probe was then administered to both free-living and parasitic nematode strains, which when assessed with fluorescence microscopy, successfully identified the route of ivermectin uptake. Furthermore, the data generated was consistent with impaired drug uptake as an operative mechanism of ivermectin resistance. The final section comprises experimental data regarding the preparation and characterisation of the discussed compounds as well as biological assay data. Chapter two details a project aimed at developing heterobifunctional PROTAC molecules towards the selective degradation of HIV capsid protein in human cells. The first section presents the relevant background information for the project including information on the chosen protein targets, which was used to guide the design of the PROTAC molecules. These molecules are based on the HIV capsid inhibitor PF74. The second section discusses the synthesis of each of the PROTAC molecules. Additionally, the synthesis of (S)- and (R)-PF74 and an epimerisation study of PF74 are discussed. The third section discusses the biological assaying of the enantiomers of PF74, which successfully identified (S)-PF74 as significantly more active than (R)-PF74. In addition, the testing of each of the synthesised PROTACs is presented, which successfully demonstrated inhibition of HIV replication and answered fundamental questions regarding PROTAC target engagement. The final section comprises experimental data regarding the preparation and characterisation of the discussed compounds as well as biological assay data.