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Chikungunya virus (CHIKV) and Zika virus (ZIKV) share a common feature, in that the genomes of both viruses consists of a single strand of positive sense RNA, which folds into complex secondary structures. These structures are highly conserved and play essential roles in virus replication. The Tuplin group previously characterised RNA structures within the 5' region of the CHIKV genome, demonstrating that such RNA structures function in a structure dependent manner. We hypothesised that these functional RNA structures could be specifically targeted using antisense oligonucleotides, binding in a sequence specific manner and disrupting key RNA interactions required for efficient virus replication. A panel of locked nucleic acid (LNA) oligonucleotides was shown to bind specific RNA structures in the context of a folded CHIKV RNA molecule. Using a range of methods, LNA oligonucleotides were shown to inhibit virus replication, indicating that RNA elements are accessible within the CHIKV RNA replication complex. Furthermore, LNA oligonucleotides were able to inhibit a CHIKV sub-genomic replicon system, indicating that the oligonucleotides specifically inhibit genome replication. RNA structural elements within the 5' UTR and adjacent capsid coding region of ZIKV were modelled using a combination of SHAPE chemistry, thermodynamic predictions and phylogenetic analysis. ZIKV RNA structure models were generated using both full-length genomic RNA and truncated RNA molecules at both 37 °C and 28 °C, reflecting both mammalian and insect body temperatures. The RNA structural models were used as a basis for reverse genetic analysis of ZIKV RNA structures and a novel pseudoknot interaction was shown to be essential for efficient ZIKV replication. RNA structures within the ZIKV genome were specifically targeted using antisense oligonucleotides, causing impairment of ZIKV replication in both infectious ZIKV and subgenomic replicon systems. Together, these results provide a basis for the development specific antiviral agents targeting CHIKV and ZIKV as no specific agents currently exist.

The Totiviridae is a family of unsegmented, icosahedral, small dsRNA viruses in the realm of Riboviria, which has been historically characterised by host diversity, morphology and host impact on differences in strategies for transmission. Human hosts include parasites like Leishmania, the cause of leishmaniasis a widespread and sometimes fatal disease, Trichomonas, the cause of trichomoniasis, the most common non-viral sexually transmitted infection, and Giardia, which causes giardiasis - an acute or chronic gastrointestinal disease. Eimeria causes serious diseases of domestic animals, particularly chickens, cattle and rabbits. Hosts from which they have been isolated included plant parasitic oomycetes, many yeasts and fungi, red macroalgae (seaweeds), diatoms (single celled algae), woodlice (terrestrial crustaceans), many insects such as flies, mosquitoes, ants and wasps and shrimp (marine crustaceans). However, also fish, freshwater snails that are intermediate hosts to parasites, and plants like papaya, notoginseng, maize, and wild petunias. The totiviruses increase the virulence of the parasites in Leishmania and Trichomonas (hypervirulence) and sometimes decreases fungal virulence (hypovirulence) such as in oats. Totivirus is myocarditis and myonecrosis in salmon, smelt and shrimp, and is asymptomatic in golden shiners. It is not infectious and vertically transmitted in Leishmania, Trichomonas, and other fungi and plants, while in Giardia it is transmitted horizontally by fish, shrimps, papaya. Totiviruses evolve so fast that there is currently no systematic method to search for them. The commercial antibody J2, specific for dsRNA, has been evaluated as such a tool to detect totivirus. While the sensitivity of the antibody was promising, the lack of specificity for dsRNA rendered it useless. To enable a systematic survey, conserved regions in the RNA-dependent RNA polymerase gene of individual virus species and lineages were identified and primers developed for Leishmaniavirus1, Leishmaniavirus2, Leishmania aethiopica virus, Giardiavirus, Eimeriavirus, and Trichomonasvirus. Outside of Leishmaniaviruses, PCR results were limited by the absence of available virus-positive host samples, and the reasons for failures to detect virus in test samples is discussed. The following viruses new to science were discovered: Leishmania infantum virus, Leishmania major virus, Leishmania panamensis virus, Leishmania hertigi virus, Leishmania mexicana virus, Leishmania amazonensis virus, Leishmania venezuelensis virus, Leishmania chagasi virus, Leishmania donovani virus, Leishmania gerbilli virus, and Leishmania tarentolae virus. Outside the current taxonomic grouping of parasites, Totiviruses new to science were discovered in the genus Endotrypanum, in the species Herpetomonas megaseliae, and in the species Blastocrithidia culicis of the Trypanosomatidae. In addition, the first totivirus was discovered in Bodo caudatus (Bodonida: Kinetoplastida), widely expanding the range of vertically transmitted totiviruses and the probable time when these viruses entered their host lineage. Sandflies as most common vectors of Leishmania parasites were investigated with next generation whole genome sequencing for arthropod derived Totiviridae to resolve where the infection came from to Leishmania. Using alignments of all available sequences, a new conserved motif of the RNA-dependent RNA polymerase of dsRNA viruses was discovered. Based on these alignments, a new phylogeny of the totiviruses was reconstructed and generic and whole-family delineations discussed.

Multidrug-resistant bacteria strains possessing extended-spectrum β-lactamases (ESBLs) has become an increasing problem worldwide. Bacteria resistant to antibiotics are mostly enteric, they can contaminate the environment and, through ingestion, enter new hosts to cause infections. Therefore, emphasis was put on isolating ESBL-producing bacteria from treated wastewater. The overall aim of this thesis was to isolate, identify and characterise ESBL-producing bacteria from wastewater followed by isolation and characterisation of bacteriophages specific for these bacteria. Bacterial isolates were recovered after growth on selective media and multiplex PCR was used to amplify SHV, TEM, CTX-M and OXA genes. Biochemical test and whole genome sequencing were applied to identify and characterise the isolated strains. The cell-free supernatants were then used to isolate bacteriophages. A high titer lysate was then used to test the specificity of bacteriophages for different bacterial strains. The nature of phage genetic material was established and morphology of isolated phages was determined by the transmission electron microscopy imaging. Two cold-tolerant bacteria isolated harbouring CTX-M gene were studied by 16S rRNA gene sequence analysis and housekeeping gene sequences analysis which revealed that these isolates showed no close similarity to any known member of the Enterobacteriaceae but are related to the members of Rahnella, Rouxiella and Ewingella genera. The phenotypic characteristics of the two isolates were, however, discrete from these 3 genera. Furthermore, two different bacteriophages infected the two newly identified cold tolerant bacterial strains. These bacteriophages were found to be novel viruses, most likely belonging to the Siphoviridae family, based on their characteristics, morphology, and genome size. In conclusion, all the analysis showed that they two isolates belong to a novel genus in the Enterobacteriaceae family. Given the menacing impact of disease outbreaks caused by contaminated water resources, work presented here identifies novel bacteria with harmful capabilities and potentially offers new tools for environmentally safer treatment to overcome this threat.

Bacteriophages are viruses which infect bacteria specifically. Over the past decades, phage λ has been extensively studied, especially its interaction with the Escherichia coli LamB (EcLamB) protein receptor. Nonetheless, despite the enormous numbers and near-ubiquity of environmental phages, aside from phage λ, there is a paucity of information on other phages which target EcLamB as a receptor. In this study, to answer the question whether there are other EcLamB-targeting phages in the natural environment, a simple and convenient method was developed and used for isolating environmental phages which target a particular surface structure of a particular bacterium, in this case, the EcLamB outer membrane protein. From the enrichments with the engineered bacterial hosts, a collection of EcLamB-targeting phages (ΦZZ phages) were easily isolated. Intriguingly, unlike phage λ, an obligate EcLamB-dependent phage in the Siphoviridae family, the newly isolated ΦZZ phages alternatively recognised EcLamB or E. coli OmpC (EcOmpC) as a receptor when infecting E. coli. Furthermore, ΦZZ phages were suggested to represent new species in the Tequatrovirus genus in the Myoviridae family, based on phage morphology and genomic sequences. Most phages are thought to have a narrow host range due to their exquisite specificity in receptor-recognition. With the ability to optionally recognise two receptors, ΦZZ phages were considered relatively promiscuous. Via the heterologous expression of EcLamB on the bacterial cell surface, the host range of ΦZZ phages was further extended to three different enterobacterial genera. Besides, an interesting selection of evolved phage mutants with broader host range were isolated, and the key mutations involved in their evolution to adapt to new hosts were investigated by genomic analysis. Finally, and importantly, two ΦZZ phages were found to be putative generalised transducers, which could be exploited as tools for DNA manipulations.

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.

Cucumber mosaic virus (CMV) infects over a thousand plant species including many crops. CMV is mainly transmitted between plants by aphids, insects with probing mouthparts that introduce virus particles directly into host cells. The 2b viral suppressor of RNA silencing (VSR) encoded by CMV is a potent counterdefence and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. The 2b VSR not only influences infection, but also host interactions with one of the main insect vectors of CMV, the generalist aphid Myzus persicae. The 2b protein disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). In Arabidopsis, complete inhibition of AGO1 activity is counterproductive to CMV since this triggers antibiosis against aphids and stimulates resistance mechanisms by AGO2. The CMV 1a protein (a replicase component) is able to moderate antibiosis induction by the 2b VSR. This ensures that aphid vectors are deterred from feeding but not poisoned when they feed on CMV-infected Arabidopsis plants. I found that the CMV 1a protein is able to directly inhibit the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in processing-bodies, the 1a protein decreases the proportion of 2b protein molecules available for binding AGO1. This ameliorates 2b-induced viral symptoms and moderates the induction of aphid resistance. However, the 1a-2b protein interaction does not inhibit the VSR activity of the 2b protein. The interaction between the CMV 1a and 2b proteins represents a novel viral regulatory system for VSRs. The finding also provides a mechanism that may explain how CMV, and possibly other viruses, modulate symptom induction and manipulate host-vector interactions.

Bacteriophages (phages) subvert their bacterial hosts through the use of phage-encoded proteins called host takeover factors (HTF). HTF target a number of biological processes and their purpose is to either shut off or modulate host biosynthetic machinery. Despite being the most abundant entities on earth, phages and their HTF remain mostly uncharacterised. The Bacillus subtilis infecting phage, SPO1, encodes 26 genes postulated to be HTF of mostly unknown function. These genes are collectively referred to as the host takeover module (HTM). The first objective of the work described in this thesis was to complete the systematic screen of the SPO1 HTM for HTF that attenuate B. subtilis growth. To this end, the effect of expressing the HTF genes in their respective operons was analysed. The HTF with the most pronounced effect were purified and B. subtilis binding partners found. One such HTF was Gp53 which interacts with the AAA+ ATPase ClpC. The major objective of the work described in this thesis was to characterise in detail the SPO1 HTF Gp53. Using bacterial-two hybrid assays, the binding site of Gp53 on ClpC was mapped and mutational analysis identified residues in both proteins required for the interaction. Gp53 was found to stimulate the ATPase activity of ClpC and compete with the native adaptor, MecA, for binding to ClpC. In vitro degradation assays with ClpC and its protease subunit, ClpP, revealed that Gp53 competition alters normal degradation of native substrates. It also appears that, during infection, Gp53 may alter the degradation of SPO1 proteins Gp39 and Gp40. Thus, the results of this study reveal a novel HTF that modulates the activity of the B. subtilis ClpCP protease in an adaptor-like manner and enables efficient SPO1 phage progeny development.

In nature, bacteria are frequently exposed to predation by bacteriophages and protozoa. These two microbial groups were described as the main causes of bacterial mortality. Yet, bacteria were found to co-exist and thrive in the presence of both predators. This study was established to investigate whether the presence of Acanthamoeba can protect bacteria from killing by bacteriophages. Gentamicin protection assay was used to determine the ability of P. aeruginosa WB1 and E. coli 4s to intracellularly survive within Acanthamoeba castellanii cells. Later, bacteria retrieved form gentamicin protection assay were subjected to phage infection. Phages were applied in a form of single and combination treatments. Results have indicated that P. aeruginosa WB1 and E. coli 4s were not able to intracellularly survive within the amoebal cells and were digested by 99%. And, single-phages were effective in a single-enemy system (phage-bacteria system) but were not effective in multiple-enemies system (in Acanthamoeba presence), while, phage combinations showed higher efficiency in eradicating bacterial numbers in both systems. In addition, this study has investigated the ability of phages to internalise and survive within Acanthamoeba cells. Two experiments were performed, first, the ability of phages to survive inside Acanthamoeba cysts was assessed. Non-nutrient agars were seeded with bacteria infected with phages, then, Acanthamoeba trophozoites were introduced and incubated at room temperature for two weeks to induce encystation. Later, cysts were exposed to acid (2% (v/v) HCL) treatment, UV radiation and extreme heat to assess Acanthamoeba ability to protect phages from hostile conditions. Second, phages (without bacterial hosts) were co-cultured with trophozoites and exposed to UV radiation and extreme heat. Results have indicated that phages were able to intracellularly survive within Acanthamoeba cells. Phages were found more resistant to adverse conditions once trapped within Acanthamoeba cysts, as well as, in the presences of amoebal trophozoites.

Lentiviral vectors are being successfully used as therapeutic agents in a series of clinical applications of gene therapy, genome editing and cancer immunotherapy. 3rd generation HIV-1 derived lentiviral vectors are produced from 4 independent plasmids. Here, I focus on the transfer vector that contains the therapeutic gene and the cis-acting elements that drive its expression including the packaging signal (psi). Lentiviral vector particles carry two copies of transfer vector RNA that become linked via a process known as dimerisation. I focused on improving the infectivity of vectors by targeting their dimerisation and packaging properties based on the hypothesis that WT HIV-1 regulates genome encapsidation tightly by recognising dimeric RNA. The genomic RNA (gRNA) leader region is thought to act as a switch between the monomeric conformation that is associated with translation and the dimeric conformation linked with packaging. I therefore attempted to identify the structures that are important for packaging and optimise those at the expense of structures that are used by the virus for translational control. To do so, I created mutants in the 5’UTR that target regions that play important roles in the process of dimerisation including the Dimerisation Stem Loop (DSL), the U5-AUG duplex formed by sequences located at the beginning of the U5 region and nucleotides surrounding the start of the gag gene, and the polyA stem loop, a region suppressed in the 5’LTR, but known to regulate polyadenylation in the 3’LTR. The introduction of these mutations aimed to create vectors whose RNA is more likely to adopt the dimeric conformation and therefore be packaged. To evaluate this, I developed a novel competitive RT-qPCR assay to measure the relative packaging efficiencies (RPE) of transfer vectors in a competitive co-transfection environment. Biochemical characterisation showed an overall negative effect of the introduced mutations on viral infectivity. Northern Blots confirmed that the propensity of mutated vector RNA to dimerise has increased in the mutants as hypothesised. Here, I report the effect of the dimerisation-stabilising mutations on infectious and physical titres of lentiviral vectors, as well as on their packaging efficiency measured with our novel competitive qPCR assay. Our data suggest that enhancing dimerisation does not automatically lead to better packaging of vector RNA. Despite the improvement of vector RNA dimerisation efficiency and in some cases RPE, the effect of introduced mutations on the ability of the designed transfer vectors to successfully transduce 293T cells was negative, reflecting the multifunctionality of the HIV-1 leader 4 regions and the significance of RNA flexibility. Finally, I explored by Selective 2′-Hydroxyl Acylation Analysed by Primer Extension (SHAPE) the structure of psi in our vector RNAs, in particular studying the influence of regions adjacent to psi on dimerisation and packaging. SHAPE identifies the RNA backbone flexibility, which is an indication of whether nucleotides are base-paired or not. Our single nucleotide level structural analysis revealed that the presence of gag sequences stabilise the psi element of the dimeric RNA, suggesting their role in supporting a stable RNA conformation that can be packaged and offering a potential explanation for their requirement in the transfer vector plasmid for maintenance of infectious titres. These findings will give us better insights into the biology of lentiviral vectors and enable us to design more efficient vectors for a variety of clinical applications.

The orthobunyavirus genus within the Bunyavirales order contains many pathogens of humans and livestock. Their genome consists of three segments of negative-sense, single-stranded RNA and these are encapsidated with polymers of nucleocapsid protein (NP) which binds RNA in a sequence-independent manner to form ribonucleoproteins (RNPs). Formation of RNPs protects the viral genome from the host immune system and has roles in transcription, viral RNA replication and the correct packaging of segments into new virions. Thus, the RNP is an attractive target for the development of antivirals, which is of particular importance as no specific therapies exist for any of the human pathogens of the genus. Within the field there are currently two different hypotheses on the overall structure of the RNP filament, and establishing a definitive model has been hampered by the extreme flexibility and heterogeneity of the filaments. This project aimed to accurately describe the architecture of the orthobunyavirus RNP and produce a model which illuminates its gross structural features, and mechanisms of assembly and RNA binding. Infectious Bunyamwera virus (BUNV) was propagated and purified by ultracentrifugation, allowing the extraction and purification of RNPs from virions, which were then visualized by negative stain electron microscopy (EM). An ensemble of microscopy methods encompassing negative stain EM, single particle cryo-EM and cryo-electron tomography was employed to overcome the extreme heterogeneity of the filaments, and to produce several models of the RNP that permitted the fitting of NP crystal structures and clearly exhibited a helical architecture. This structural information will aid in the development of small molecules which inhibit formation of the orthobunyavirus RNP and which could be investigated further for their therapeutic potential.

A diverse array of viruses capable of infecting both humans and animals constitute the family Paramyxoviridae (Amarasinghe et al. 2019; Fields, Knipe, and Howley 2013). Concerns regarding the interspecies transmission potential paramyxoviruses harboured by wildlife reservoirs has motivated subsequent concerted viral surveillance efforts (Thibault et al. 2017; Drexler et al. 2012). Our understanding of many of these newly identified paramyxoviruses is primarily limited to genome information. However, paramyxovirus genomic variation (Holmes 2010; Kitchen, Shackelton, and Holmes 2011) often impedes our ability to predict the pathogenic potential based upon study of primary sequence alone. Therefore, investigations into paramyxovirus structure and function are vital to elucidating the potential biomedical and economic significance of emerging paramyxoviruses (Zeltina, Bowden, and Lee 2016). The six-bladed beta-propeller receptor binding protein (RBP), responsible for host-cell attachment and initiation of the merger of the viral and host-cell membranes (Fields, Knipe, and Howley 2013; Jardetzky and Lamb 2014), is therefore a central determinant of tissue and species tropism (Thibault et al. 2017; Zeltina, Bowden, and Lee 2016). Characterisation of the RBP, through structural and functional approaches, may enhance our ability to rationalise host range and interspecies transmission potential. Within this thesis, the RBPs of a number of recently classified paramyxoviruses are studied: the pararubulavirus, Sosuga virus (SosV); the narmoviruses, Mossman virus (MosV) and Nariva virus (NarV); the jeilongviruses, J virus (JPV) and Beilong virus (BeiV) (Amarasinghe et al. 2019). In my investigations, I found that despite presentation of several stringently conserved residues associated with sialic acid activity, SosV-RBP lacks hemadsorption and neuraminidase functionality characteristic of closely related viruses, such as mumps virus (Kubota et al. 2016). Absence of activity is rationalised by discovery of structural rearrangements to key sialic acid interacting motifs, such as the triarginyl motif (Crennell et al. 2000), and physicochemical incompatibilities at the expected receptor binding site. Similarly, the jeilongviruses do not display hemadsorption or neuraminidase activity despite conservation of canonical sialic acid interacting motifs. Structural study of the jeilongviral RBPs, has enabled the characterisation of a C-terminal extension that forms a domain that occludes the putative sialic acid site. This provides evidence that jeilongviral RBPs employ an auto-inhibitory function analogous to the one utilised by the RBP of Newcastle disease virus Ulster strain (Yuan et al. 2012). Contrasting pararubulavirus and jeilongvirus RBPs, narmovirus RBPs display a beta-propeller architecture that bears no motifs associated with known modes of paramyxovirus-receptor interactions. However, sequence conservation mapping reveals an extended footprint of elevated amino acid sequence identity on the surface of the protein. Efforts utilising site-directed mutagenesis are being implemented to explore the importance of this region in dictating cell interactions. Through crystallographic and biochemical analysis, this thesis provides a structure-based rationale for understanding the pathobiological independence of the newly defined (Amarasinghe et al. 2019) Pararubula-, Narmo-, and Jeilongviruses from other genera within the Paramyxoviridae, where the RBPs of these viruses are likely incapable of utilising currently known modes of paramyxoviral host-cell recognition. Finally, this work provides a platform to identify the receptors utilised by these unique groups of pathogens.

Because a virus is an obligate cellular parasite, the host is a key part of its environment. Viruses may expand their ecological niche by switching host. Successful host switching can be influenced by ecological and evolutionary factors, genetic constraints and fitness within new hosts. An outcome of host switching is reduced fitness exhibited by viruses, a phenomenon observed in the evolution of viral disease emergence and resistance. To understand the genetic basis of this cost, investigations are required at the genotypic and phenotypic level. A host switching paradigm was developed using the model bacteriophage φX174 which was propagated with its laboratory bacterial host Escherichia coli C and with the novel host Salmonella enterica serovar Typhimurium, LT2 strain IJ750 or Escherichia coli K-12 mutant strain JWO196-2 designated as E. coli K-12gmhB-mut. A chemostat was used to achieve steady-state conditions for propagation of φX174 and bacterial cells. Two experiments were performed using this approach. In the first, φX174 was cultured on E. coli K-12gmhB-mut for 3 days (~206 generations). In the second experiment, φX174 was cultured on E. coli C and S. Typhimurium for four consecutive periods of 10 days (~720 generations), alternating between the two hosts. For the second chemostat experiment, the fitness and attachment rates of each viral population were measured using qPCR in liquid culture in order to identify and characterise fitness costs associated with host-switching. Deep sequencing of chemostat samples was also carried out to identify allelic changes occurring before and after host switches. Viral samples were chosen to capture substitutions associated with each host across the experiment (which might explain observed changes in fitness) and time series were picked to identify the dynamics of adaptation on a new host. Bacterial host strains were not sampled in this study. The phenotype measures indicated the pleiotropic costs of host switching, that is a reduction in phage fitness was observed when this was tested on the host used prior to switching, and this may be explained by changes in the attachment rate. The genotype data revealed sets of changes that could be identified as signatures of adaptation to each host, although control data indicate that these may arise during DNA preparation, implicating synthesis of replicative form DNA in the host as a source of selective constraint. Some host-specific alleles and some shared alleles were identified and their fitness effects were examined in isolation after reconstruction of these alleles in the ancestor via targeted mutagenesis. The fitness effects observed for reconstructed mutants were in the direction expected although they do not fully account for the observed costs of host switching. By analysing different phenotypes and genotypes produced during evolution, a detailed view of φX174’s adaptation to different hosts was obtained. The results support the idea that costs associated with pathogen-host adaptation may be host-specific, associated with specific mutations, acquired early and persist. Examining these is relevant for understanding emerging infectious diseases.

Gene transfer is a technology central to the development of gene therapy and expression of proteins and biological products necessary for designing and producing drug compounds or creating new research methodologies. Consequently, the field has experienced a large bottleneck in economic and time costs when viral vectors are involved. Over the past decade, recombinant mammalian viral vectors have been exploited for these purposes; however, the limitations in their native biology and production methods warrant new vector systems to be investigated and developed. In this thesis, we reinvestigate the humble bacteriophage, a prokaryotic virus, as a potential tool to circumvent the cost limitation of eukaryotic viruses. By combining their genome with that of Adeno-associated virus (AAV), a well-characterised mammalian virus, and developing a novel method of expressing these hybird vectors, we were able to overcome many limitations that these viruses have as separate entities. The proposed vector, termed Phagemid Adeno-associated Virion (PAAV), is as efficacious as traditional vectors, while economically costing a fraction of what is demanded by current practice in the field. The PAAV, was constructed by inserting a recombinant AAV genome into a phagemid expression vector that carries no phage structural genes. The particles are packaged using a custom-designed mammalian-targeted helper virus, resulting in vectors that can be easily produced at a minimum of 2-fold higher yield than the current gold-standard phage vector. We demonstrated through transmission electron microscopy and transducing unit assays that PAAV vectors generated by our method is less than half the size of traditional full-length phage vectors, and through vesicular staining we are able to determine that the PAAV is internalised at almost 2-fold higher than the efficiencies observed in the gold-standard phage vector. We further assessed qualitative and quantitative gene expression efficacies by the PAAV bearing GFP or Luciferase transgenes in various tumour cells, which show a dramatic increase in gene expression by up to over 10-fold of the gold-standard. To demonstrate that the PAAV and its derived vectors can be used as an alternative to DNA transfection, a method central to mammalian virus production, we designed and validated two proof-of-concept methods that are able to produce rAAV using PAAV vectors. Phages are harmless viruses with a safety profile founded by their historic use as antibiotic agents. The PAAV vector system utilizes the economic advantages of phage vectors and combines them with the efficacy of mammalian viral transgenes, offering an efficacious alternative vector that is able to transduce mammalian tumour cells. Furthermore, the PAAV system has the potential to replace conventional transfection, thereby addressing a significant bottleneck in translational research in the field. Taken together, the PAAV offers a novel and advantageous alternative platform to conventional viruses for use in therapeutic and industrial applications.

Prions are infectious agents that cause fatal neurodegenerative diseases in the brain. The wide-accepted protein-only hypothesis states that the misfolded form of prion protein (PrP) is the sole constituent of prions, and the self-propagating process of PrP is considered to play a central role in prion pathogenesis. Prions are believed to propagate when a PrP assembly enters a cell and replicates to produce two or more fibrils, leading to an exponential increase in PrP aggregate number with time. However, the molecular basis of this process has not yet been established in detail. This prion-like replication is also suggested to be the mechanism in the development of other notorious neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. In this thesis, I use single-aggregate imaging to study fibril fragmentation and elongation of individual murine PrP aggregates from seeded aggregation in vitro. From fluorescence imaging of individual PrP aggregates on the coverslip surface, elongation and fragmentation of the PrP assemblies have been directly observed. PrP elongation occurs via a structural conversion from a proteinase K (PK)-sensitive to PK-resistant conformer. Fibril fragmentation was found to be length-dependent and resulted in the formation of PK-sensitive fragments. To gain more insights into the mechanism of the spread of PrP, the quantified kinetic profiles allows the determination of the rate constants for these processes through the use of kinetic modelling. This enables the estimation of a simple framework for aggregate propagation through the brain, assuming that doubling of the aggregate number is rate-limiting. In contrast, the same method was applied to measurement for α-Synuclein (αS) aggregation, which has been suggested to be prion-like and is associated with Parkinson's disease. While αS aggregated by the same mechanism, it showed significantly slower elongation and fragmentation rate constants than PrP, leading to much slower replication rate. Furthermore, the measurements in αS aggregation has been extended to the cellular environment, I use super-resolution imaging to study the amplification of endogenous αS aggregation in cells and the transcellular spread of αS. Endogenous αS showed a clear amplification in number of aggregates with time after seed transduction, and the newly-formed αS aggregates are likely to spread through cell-to-cell transmission. The proteasome was demonstrated to possess a novel disaggregase function for αS fibrils and thus produce more seeds for further replication. It partially explains that αS aggregation in cells was found to replicate at a substantially faster rate than that in vitro. Determining the nature of the oligomers formed during aggregation has been experimentally difficult due to the lack of suitable methods capable of detecting and characterising the low level of oligomers. To address this problem, I have studied the early formation of PrP oligomers formed during aggregation in vitro using various single-molecule methods. The early aggregation of PrP is observed to form a thioflavin T (ThT)-inactive and two ThT-active species of oligomers, which differ in size and temporal evolution. The ThT-active oligomers undergo a structural conversion from a PK-sensitive to PK-resistant conformer, while a fraction of which grow into mature fibrils. These results also enable the establishment of a kinetic framework for elucidating temporal evolution of PrP aggregation and the relationship between oligomers and fibrils. Overall, my research identifies fibril elongation with fragmentation are the key molecular processes leading to PrP and αS aggregate replication, an important concept in prion biology, and provides a simple framework to estimate the rate of prion and prion-like spreading in animals. The results also show that a diverse range of oligomers is formed and co-exist during PrP aggregation which differ both in their structure and properties and provides mechanistic insights into a prion aggregation. The work provides a new quantitative approach to describe the prion-like property in neurodegenerative diseases from a kinetic perspective that can be verified in extending studies in other proteins or in cells.

Phytopathogenic Dickeya species inflict large economic losses on a variety of crops. A lack of effective chemical control methods has generated interest in the use of bacteriophages (phages) as a novel tool for biocontrol. In the last decade, six phages have been isolated in Belgium and Poland using Dickeya solani as the host. Previous work in this laboratory has isolated ninety phages capable of infecting D. solani. The majority have been morphologically classified as members of the Ackermannviridae family. In agreement with findings in Salmonella and Klebsiella species, the capsule of D. solani is a likely receptor of Ackermannviridae family phages. Analysis of D. solani strains carrying reporter fusions suggested that the capsule genes are expressed in response to nutritional stress, however disruption of the capsular polysaccharide cluster did not significantly impact virulence. Experiments assessing capsular polysaccharide as a putative receptor for Ackermannviridae family phages in nosocomial pathogen Serratia produced inconclusive results. Phageresistance due to random transposon mutagenesis identified genes encoding transcription factors and regulators, but none directly linked to capsular polysaccharide production. Thirteen phages were capable of infecting a wider host range of Dickeya species. Morphological and genomic analysis showed that six were Podoviridae family members, whilst the other seven were Myoviridae family members. These are part of the recently defined 'hairy Myoviridae', characterised by a distinct morphology. Another member of this grouping was isolated during this study, but is more closely related to phages of Erwinia amylovora. A subset of the Ackermannviridae family phages were shown to be capable of facilitating transduction. This makes them unsuitable for use in the environment due to the risk of deleterious horizontal gene transfer. This is also true for the Myoviridae family members, but not for one of the Podoviridae family members. This phage could therefore be a promising candidate for therapeutic use.

Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.

With the emerging global threat of antimicrobial resistance, diagnosis and treatment of infectious diseases has become more difficult than ever. We need to consider new paradigms in therapy and innovative alternatives that can allow us to rapidly detect bacteria at the point-of-care and efficiently eliminate pathogens without promoting the dissemination of virulent factors. This thesis aims to develop a new alternative for the development of new applications for therapy and diagnostics of bacteria. Here we use synthetic phage-inducible chromosomal islands (PICIs) to combine the two concepts of diagnostics and therapy to "seek & destroy" specific bacteria. These novel biosensors open a series of applications for the detection and elimination of the pathogen by using the pathogen itself to modify its genome specifically to produce a reporter (seek) and/or a killing switch (destroy). Here we demonstrate that PICIs can achieve high transfer and sensitivity compared to phages, while improving a methodology to study the effects on the viability and detection of pathogens. We engineered PICIs to carry a series of synthetic gene circuits in order to enable the detection of cells and potential sorting of pathogens, while avoiding the spread of antimicrobial genes onto the target cell. Further modifications prove that PICIs can be used as a Trojan-horse to deliver lethal payloads into target cells. Here, we exploited the PICI components by which they highjack the phage machinery to promote their spread. Taken together, this reinforces the recent interests for developing alternative ways to diagnose and treat bacterial infections. This thesis opens a range of applications in using other types of PICIs recently characterised as Synthetic Biology tools for portable diagnostics and prophylactic methods.

Background: In December 2010, a new genotype of respiratory syncytial virus (RSV) with a 72-nucleotide duplication within the attachment (G) gene was identified in Ontario, Canada, and named ON1. Using the ON1 as a unique tag, this study aimed to understand; (1) how new RSV variants are introduced, spread and persist in communities, (2) the genomic signatures that define the emergent RSV variants and whether such substitutions may be associated with potential fitness advantages, and (3) the patterns of RSV spread across geographically defined regions (local and global). Methods: Partial G gene (n=483) and whole genome (n=184) sequence datasets collected between 2010 and 2016 were analyzed using genetic diversity, phylogenetics and statistical methods to understand the molecular epidemiology of RSV in Kilifi County, Coastal Kenya. Further, Kenyan (partial G gene; n=2526) and global (full G gene, n=2238; whole genome, n=1194) sequence datasets collected between 1977 to 2016 were analysed in a Bayesian framework for the inference of the phylogeographic history of local and global RSV spread, respectively. Results: Following initial detection of the genotype ON1 in Kilifi in 2012, there was rapid replacement of the previously circulating RSV group A genotype GA2 by ON1 in subsequent epidemics. While this suggests elevated fitness of ON1 viruses, there was no clear evidence of altered pathogenicity of ON1 relative to GA2 in Kilifi. Signature amino acid substitutions were identified between surface proteins (G, F), polymerase (L) and matrix M2-1 proteins of Kilifi ON1 and GA2 viruses, suggesting co-evolution amongst antigenic and non-antigenic genes of RSV variants. Genetic and phylogenetic analyses reaffirmed previous conclusions that each RSV epidemic is characterized by the frequent introduction of multiple variants, few of which persist across epidemics. Finally, the phylogeographic analyses predicted the northern hemisphere to be the major source population of RSV into the tropics and the southern hemisphere and virus spread between locations in close proximity to be important for virus persistence within a country. Conclusions: Tracking the ON1 tag offered important insights into RSV evolution and transmission. The use of whole genome sequencing and surveying all the variation throughout the genome will be crucial for greater understanding, and potentially improved control, of this important pathogen. However, there is a need for a more targeted approach to RSV surveillance and sequencing that will help build a better picture of RSV spread at different scales.

Vaccinia virus (VACV) morphogenesis is controlled by a temporal cascade enacted by intermediate and late promoter sequences, in conjunction with virally encoded transcription factors. VACV morphogenesis culminates with the formation of two structurally and functionally distinct virion morphotypes from a single mature virus (MV) progenitor; the intracellular mature virus (IMV) and extracellular enveloped virus (EEV). IMV are typified by their single membrane and the presence of the A26 protein, which is expressed at 10 hours post infection. EEVs are characterized by their double membrane and suite of virally encoded proteins, the most crucial of which is the F13 protein expressed at 4 hpi. Previous investigations have implicated the temporal expression of A26 protein as a regulatory switch, negatively regulating the formation of EEV during late times of infection. However, reverse genetic approaches have refuted this claim. Despite this, these studies do not consider the temporal organisation of key IMV and EEV proteins, in addition to their potential to regulate one another. The aim of this study was to investigate the concept of altering the temporal regulation of both A26 and F13 to generate novel phenotypes, exploring the mechanism underpinning IMV vs EEV balance. Recombinant viruses were generated with F13L and A26L alleles expressed under the control of either F13L or A26L promoter sequences. When A26L was introduced under the intermediate F13L promoter sequence a significant plaque size reduction was observed. In addition, a further deduction was observed when F13 expression was delayed under the late A26 promoter. Temporally advanced A26 expression significantly altered its rate of association with the key MV membrane protein A27, which is required for EEV formation. When A26 was expressed under the intermediate F13L promoter, incorporation of A26 on virions was significantly enhanced when compared to A26 expressed under its native promoter sequence. This was correlated with a small reduction in EEV formation. The data presented in this thesis reveals the A26 protein as a potential negative regulator of EEV formation. The temporal segregation of A26 from the EEV morphogenesis proteins; F13 and A27 underpins the transition from EEV to IMV production. In addition, this thesis introduces the concept of altering temporal regulation to explore the constraints of the poxviral genome and its ability to acquire variation. These findings will assist in the refinement of sequencing algorithms used to characterise novel pathogen populations.

Viruses harbouring double-stranded RNA (dsRNA) genomes are highly diverse and their host range spans from bacteria to fungi, plants and mammals. dsRNA viruses share a number of characteristics including the possession of icosahedrally symmetric capsids which shelter their genomes. As many other icosahedral capsids, those of dsRNA viruses have been extensively characterised by cryogenic electron microscopy (cryo-EM). In contrast, in situ structures of genomes and genome-associated proteins such as RNA-dependent RNA polymerases (RdRPs) are scarce due to their characteristic asymmetry and high degree of heterogeneity. This thesis sets to expand the arsenal of image processing algorithms aimed at overcoming such hurdles and to resolve the architectures of capsid interiors in quiescent and mRNA-producing i.e. transcribing φ6 nucleocapsids (QNCs and TNCs), as well as rotavirus QNCs. The φ6 genome is organised in spooled fashion, bearing strong resemblance to the canonical arrangement of viral dsDNA genomes. In φ6 QNCs 12,000 base pairs accounting for over 90% of the entire genome are modelled. φ6 TNCs depict the concerted rearrangements required to activate transcription, namely the outer capsid disassembly, inner capsid expansion and RdRP reorganisation. Rotavirus QNCs paint an entirely different picture as the RdRP-dsRNA interplay displays such extraordinary flexibility that thousands of unique capsid interior arrangements are made possible. Taken together, these results further the understanding of virus capsid interiors and highlight important disparities among dsRNA viruses.

Ebola virus (EBOV) was first identified in 1976 and, since then, small outbreaks have been occurring in central and West African countries. The largest EBOV outbreak until now, taking place from year 2013 to 2016, left more than 28.000 cases and 11.000 deaths. EBOV is a highly contagious and virulent pathogen with no FDA-licensed therapeutic available yet. Huge efforts are currently being made for the development of effective antiviral therapeutics and the investigation of viral evolution dynamics in correlation with its virulence. Viruses are obliged parasites of the host cell in order to accomplish every stage of their biological life cycle. For viral RNA synthesis, occurring in the cell cytoplasm, Ebola virus must interact with host proteins, which at the same time can be exploited as potential antiviral targets. In this study, coimmunoprecipitation and high-throughput label-free proteomics are used to elucidate novel protein associations between EBOV VP35, L and host factors. Biological importance of the host proteins DYNLL1 and CALM on Ebola virus life cycle is assessed by using small molecule inhibitors in an EBOV minigenome system in cellular culture, resulting in a significant decrease of viral replication when either of the cellular factors is antagonised. The study of VP35 is taken further and the phenotypic changes on the protein functionality during the early beginning of the 2013-2016 West African outbreak are characterised, demonstrating slight differences on the viral protein performance that could help understand better Ebola virus virulence. Overall, this thesis provides a better understanding of the interactions that Ebola virus establishes with its host, the implications that single mutations can have on viral proteins functionality and the EBOV pathogenesis.

RNA viruses present a large group of viruses that contains many important human pathogens. Chikungunya virus is an Alphavirus transmitted by tiger mosquitoes, causing a febrile disease that often leads to very disabling, sometimes chronic, joint and muscular pain that can last for several weeks up to months. The Picornaviridae family including enterovirus A71, coxsackievirus B3, poliovirus, enterovirus D68 and rhinoviruses cause various different clinical symptoms and diseases like hand-foot-and-mouth disease, poliomyelitis, or the common cold. For none of these viruses direct-acting antivirals are on the market yet, stressing the need to design novel compounds that could target these viruses and that may enter into (pre-)clinical development soon. The replication cycle of RNA viruses requires specific viral proteins that replicate the viral genome and fulfil other crucial functions within the host-cell but are not packed into new viral particles. These non-structural proteins present excellent targets to inhibit the viral replication and were therefore investigated using computer-aided techniques in order to find novel antiviral compounds. Pharmacophore screening and docking were used to select molecules from large chemical libraries that were then tested in cell-based antiviral assays for their activities. Then the compounds were synthesised and improved using classic medicinal- chemistry modifications. For chikungunya several different compounds with low micromolar activity could be identified. For the picornaviruses several inhibitors were reported, but the exact mode-of-action on their molecular target (2C protein) was unknown. Possible sites and interactions were explored using site identification tools, docking and molecular dynamics simulations. In collaboration with virologists and structural biologists this lead to the clarification of the mode-of-action of fluoxetine, which exhibits a stereoselective activity on 2C. In addition, a series of novel inhibitors with broad-spectrum activity against the described picornaviruses was developed.

The latent dsDNA viruses of the genus Phaeovirus (family Phycodnaviridae, clade Nucleo-cytoplasmic Large DNA Viruses; NCLDVs) employ genome integration in their brown algae hosts (class Phaeophyceae). The only phaeoviruses described in detail infected the order Ectocarpales, though Phaeovirus major capsid protein (MCP) occurs in 4 kelp (order Laminariales) species. Phaeoviruses are a major knowledge gap because brown algae are ecologically and economically important and have independently evolved complex multicellularity. This study aimed to investigate kelp Phaeovirus morphology, evolution, host range, distribution, host impacts, and genomics. Microscopy of Laminaria digitata gametophytes revealed particles and cell morphology typical of Phaeovirus infections. This putative Laminaria digitata virus 1 (LdV-1) infection, unlike the Ectocarpales phaeoviruses, often occurred in vegetative cells. L. digitata Phaeovirus symptoms were ~3 times more common in 18 versus 15 oC culture, but overall were uncommon and highly variable. No impact on gametophyte reproduction was observed. Broad-scale MCP PCRs and subsequent phylogeny identified 4 novel kelp phaeoviruses, placing the phaeoviruses of Ecklonia maxima, Ecklonia radiata, and Undaria pinnatifida in subgroup A, a Macrocystis pyrifera Phaeovirus in subgroup C, and a Saccharina japonica Phaeovirus in the novel subgroup D. Kelp phaeoviruses may follow the Ectocarpales Phaeovirus evolutionary trend of genome reduction (in subgroups B, C, and D versus A). Combined with all available data, 26 % of kelp were Phaeovirus MCP-positive. Genomic data from LdV-1 and 3 available kelp genomes contained Phaeovirus orthologs from the following putative, integrated phaeoviruses: LdV-1, Ecklonia radicosa virus (ErcV), Saccharina japonica virus (SjV), and Undaria pinnatifida virus (UpV). Subsequent phylogeny of 9 Phaeovirus core genes showed similar subgroups as before and non-core orthologs had implications for Phaeovirus evolution. For kelp phaeoviruses, this study has revealed a partial infection cycle, preliminary observations of viral symptoms, a broader distribution and host range, and evolutionary insights for both viruses and hosts.

The emergence of infectious diseases is increasing globally, whilst biodiversity is being lost at an unprecedented rate. The amphibian chytrid fungus (Batrachochytrium dendrobatidis) has driven the extinction of more species than any other known pathogen. This loss was particularly severe among Neotropical amphibians, with several infamous extinctions from Costa Rica. Although chytrid has been attributed to all enigmatic population declines in Costa Rica, its presence has only been investigated from a few populations. Alongside chytrid, another pathogen, ranavirus (Ranavirus spp.), is considered a significant threat to amphibian populations. Very little is known about ranaviruses outside of temperate regions, but its recent confirmation from Costa Rica has provided an opportunity to investigate both pathogens amongst declining amphibian populations. The aim of this thesis was to improve the knowledgebase of chytrid and ranaviruses in the tropics, using Costa Rica as a model system, investigate cryptic diversity among local amphibian hosts and to optimise and determine suitable diagnostics that could be used to aid ranavirus research globally. The Área de Conservación Guanacaste (ACG), northern Costa Rica was chosen as my main study location for the investigation of hosts and pathogens. The ACG is comprised of high habitat diversity that is representative of multiple tropical habitats. Samples from the ACG were used to compare four ranavirus assays to determine appropriate diagnostics. Museum specimens collected between 1976 and 1989 were used to examine how long ranaviruses have been present in northern and central Costa Rica and whether they were present during outbreaks of chytrid. The presence of ranavirus was also examined amongst highly threatened amphibian populations in tropical regions of Australia. I identified six candidate species of amphibian, indicating current inventories of Neotropical amphibians to be incomplete. Among the populations of these candidate species, ranavirus and chytrid are present. Chytrid was commonly found in wet forest habitats, whereas ranavirus was detected among all habitat types. However, ranavirus prevalence increased with temperature. Both pathogens were non-randomly distributed among host taxonomic families and ranavirus was detected in Costa Rican reptile species, representing the first detection of this pathogen from this group in Central America. Comparisons of ranavirus diagnostics indicated some assays to exhibit poor sensitivity and generate false positives. Using this information and a highly sensitive assay I detected ranavirus from populations sampled from prior to and during amphibian declines. Infected individuals included extinct and severely declined species. I also detected this pathogen from the order Gymnophiona, which, to my knowledge, has not been found infected with ranavirus previously. Additionally, I detected ranavirus from highly threatened Australian amphibian populations. Future research priorities should include the description of candidate species identified here using morphometrics, bioacoustics and nuclear DNA. If confirmed as true species, their conservation status should be assessed. The results of pathogen surveys among Costa Rican populations will allow researchers to identify areas and taxa more likely to be infected within the tropics. My findings from comparing ranavirus assays will facilitate the generation of highly accurate results, hence improve estimates of distribution and impacts of ranavirus. My thesis has improved the knowledge of both hosts and ranaviruses in Costa Rica, but also in tropical systems more generally, and will help guide future researchers with the effective detection of ranaviruses from these under-studied systems.

Enteric pathogens form a large part of infectious diseases which contribute to a bulk of the healthcare costs. Enteric infections are usually contracted via the faecal-oral route or through contact with contaminated surfaces. Treatment by antibiotics is becoming increasingly ineffective due to the growing number of antibiotic resistant strains. Anti-microbial resistance poses a serious threat to the future of healthcare worldwide and necessitates the search for alternate forms of therapy. Bacteriophages (phages), are viruses which specifically infect and lyse bacteria. To introduce phages as a viable form of therapy, route of administration needs to be considered carefully. Model phages with broad host ranges are ideal for therapy however oral delivery to the lower gastro-intestinal (GI) poses several challenges. The acidic stomach environment can be detrimental to phages, rendering them inactive during passage. To overcome this challenge and improve the stability of phage during encapsulation and storage, this PhD research has been conducted. pH responsive polymers, Eudragit and alginate were used to develop composite microparticles which protected phage from acidic pH (pH 1-3). A novel method of acidifying oil was developed for crosslinking droplets in vitro to avoid the use of harsh solvent systems that can cause phage inactivation. Platform microfluidic technology was employed for phage encapsulation for the first time. Monodispersed droplets and particles were produced, offering fine-tuning of droplet diameter to tailor the release and pH protection of encapsulated phage. Process scale-up was attempted using membrane emulsification (ME) to produce larger volumes of encapsulated phage. In vitro and in-situ models investigated the efficacy of encapsulated phage-bacterial killing. Industrial scale method of spray drying, and electrospinning were also used to demonstrate the versatility of the formulation. Tableting dry powder phage, showed an effective method for producing solid dosage forms for therapy. Additionally, electrospun phage fibres also showed the potential use of pH responsive formulations in addressing wound infections. Improvement in encapsulated phage storage stability was observed with the addition of trehalose in the formulation. This research underpins the need for testing phage encapsulation for site-specific delivery and offers insight into the potential use of commercially available technologies.

Introduction: RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids. Methods: This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer's MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation. Results: The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B. Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV "tries" to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.

The recent emergence of SARS and MERS and the discovery of novel coronaviruses in animals and birds suggest that the Coronavirus family encompasses more members that have not yet been identified. This study describes the design and validation of a novel pan-Coronavirus PCR and its application in virus discovery in samples obtained from 813 European rodents and shrews encompassing seven different species. Novel alphacoronaviruses were detected in the species Rattus norvegicus, Microtus agrestis, Sorex araneus and Myodes glareolus. These new viruses, together with the recently described Lucheng Rn coronavirus found in China, form a distinct rodent/shrew-specific clade within the coronavirus phylogeny and genomic analysis suggests a very ancient origin rather than the global spread of these rodent and shrew viruses. These data greatly extend the knowledge of wildlife reservoirs of alphacoronaviruses and provide important insight into their origins. In addition, further virus discovery in two vole samples, using a next-generation sequencing metagenomics approach, revealed the presence of novel viruses belonging to the poxvirus, astrovirus, rotavirus, gammaherpesvirus and influenza virus families. These studies provide important insight into the true extent of virus diversity in wildlife animals.

Plant viruses are usually linked to plant or crop infection and disease, with many examples of viral infection causing social, environmental and economic damage in plants and crops described worldwide. Therefore, the choice of any particular management strategy which helps to avoid or reduce virus infection and disease in the field, needs to be specifically studied and evaluated according to the etiology of each pathogen, transmission and impact. Field samples were collected from carrot crops during the period 2014-2016 across different regions and countries and were tested using real-time Polymerase Chain Reaction (RT-qPCR). Results revealed high levels of viral infection and showed the elevated presence of previously undescribed viruses such as Carrot torrado virus1 (CaTV1) and Carrot closterovirus 1 (CtCV1). Higher levels of infection found in year-round cultivation fields also indicated that this type of practice, widely used in the UK, might be detrimental and act in favour of virus infection. Further studies identified Cow Parsley and Hogweed as possible virus reservoirs of the tested viruses and identified the aphid species Myzus persicae, as the vector responsible of CaTV1 transmission. Early detection of a pathogen from a field sample relies on the development and availability of a specific, sensitive and rapid diagnostic method. A RT-qPCR diagnostic assay has been developed and validated for the detection of CaTV1 according to the requirements established in the European Plant Protection Organization bulletin. Next Generation Sequencing (NGS) was used to study viral communities in carrots and a new viral sequence from the genus Carlavirus was identified in samples from the UK. By comparison with specific targeted diagnostics, this technique was shown to be a valuable approach for field surveillance and rapid detection of all the viruses present in a sample. The use of more modern and sophisticated techniques such as NGS, has led to the discovery of high numbers of new viruses with unknown impact. As with CaTV1, many viruses have been found to not cause any acute disease in the infected plants. However, in this study some asymptomatic viruses, recently identified using NGS, were found to be more abundant in herbicide resistant than in wild-type sensitive black grass plants. Although no relationship between the presence of these viruses in the plants and the development of herbicide resistance could be established, the effects of asymptomatic infections are discussed.

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.

Influenza A virus (IAV) replication and transcription occur in the host cell nucleus; a feature which means both the viral genome (vRNA) and mRNA must be exported from the nucleus to the cytoplasm. The mechanism by which vRNA nuclear export is achieved has been well characterised, but how viral mRNAs are exported is poorly understood. The cellular NXF1-dependent mRNA export pathway has been shown to be involved in the export of some viral mRNAs, but how they are recruited to this pathway is unknown. Prior work from our laboratory showed that segment 7 mRNA was inefficiently exported to the cytoplasm in a sub-viral ‘minireplicon’ system, providing the first indication that there were viral requirements for IAV mRNA nuclear export. Further addition of individual viral polypeptides was tested and the effect on segment 7 mRNA export was analysed by fluorescent in situ hybridization (FISH) and confocal microscopy. This identified the NS1 protein as the viral factor required for efficient segment 7 nuclear export. Mutational studies on NS1 were carried out to unveil the mechanistic role of this protein in viral mRNA nuclear export, by plasmid transfection as well as in the context of recombinant viruses. These approaches indicated that both functional domains of NS1 were necessary to preserve the mRNA export function. Furthermore, these mutant proteins were used to examine the association between NS1 and the NXF1-dependent pathway in the context of mRNA nuclear export. Protein-protein and protein-RNA binding assays indicated that interactions between NXF1 and NS1, and NXF1 and segment 7 mRNA were necessary, but not sufficient to promote segment 7 viral mRNA export. Lastly, the role of NS1 protein in the nuclear export of viral mRNAs from other genome segments was studied. The intracellular localisation of most viral mRNAs was not affected by the absence of NS1 or the presence of an export-incompetent NS1 mutant protein. However, segment 4 mRNA exhibited a similar phenotype to segment 7 mRNA in showing a dependence on NS1 for efficient nuclear export. Overall, the results presented in this dissertation suggest that NS1 acts as an adaptor protein between the viral RNA synthesis machinery and cellular export pathway. This provides deeper insights for the characterization of a recently identified function of the IAV NS1 protein, of being required for the efficient nuclear export of mRNA from “late” kinetic class viral genes.

Vaccinia virus (VACV) is a large double-stranded DNA virus with a cytoplasmic site of replication. It has a complex life cycle that produces two distinct infectious virion forms, Intracellular Mature Virions (IMVs) and Extracellular Enveloped Virions (EEVs). The host cell microtubule trafficking machinery is hijacked by the virus at three distinct positions of the viral life cycle. After virus entry, the virus cores are transported to pre-nuclear sites where they form viral factories that ultimately produce fully functional and infectious IMVs. A small proportion of IMVs are further transported to sites of wrapping, where they are enveloped by a host-derived double membrane to form Intracellular Enveloped Virions (IEVs). The IEVs are then transported to the cell periphery to facilitate efficient viral spread. The viral proteins A36, F12 and E2 together with the kinesin-1 microtubule motor protein are thought to be involved in IEV egress from the site of wrapping to the cell periphery, although the exact mechanism of movement is unclear. Until recently, A36 was the only known protein to interact with the kinesin-1 motor through kinesin light chain (KLC), but F12 has also been shown to interact with KLC through E2. The precise mechanism of how the IEV interacts with and activates the kinesin-1 motor protein is unclear, and this study explores the interactions of IEV proteins with KLCs in detail, mapping interactions between KLC and A36 or F12/E2. A36, F12 and E2 also show no sequence or predicted structural homology to any other known proteins, and structural studies were performed in an attempt solve their 3D structure. The CRISPR-Cas9 targeted genome editing tool was also utilised to knockout different KLC isoforms in multiple cell lines to assess their contribution to IEV egress as well as cellular trafficking. These studies will provide insight into the mechanisms behind the spatial and temporal control of kinesin motor activity in the cell.

Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.

Plant viruses can cause many plant diseases, which result in substantial damage to crop production. To overcome viral infections, plants evolved RNA silencing which can recognise viral RNAs during their replications and slice them into small RNA (sRNA) using antiviral nucleases called DICER or Dicer-like (DCL). The resulting virus-derived small interfering RNA (vsiRNA, 21-24 nucleotides) then guides effector nucleases, namely ARGONAUTE (AGO), to cleave viral RNAs in the cytoplasm in a nucleotide-specific manner. However, the activity of vsiRNA is not restricted to the control of viral RNA accumulation. Virus-derived sRNAs can regulate host gene expression if host mRNAs share sequence complementarity with vsiRNAs. Interestingly, vsiRNAs are also able to target and methylate homologous DNA sequences in the nucleus indicating that vsiRNAs have potential to regulate endogenous genes at transcriptional level by modifying the epigenetic status of gene promoter sequences. This mechanism is referred to as transcriptional gene silencing (TGS). Thus, RNA silencing opens up new strategies to stably and heritably alter gene expression in plants. However, the mechanisms and efficacy of plant virus-induced TGS are largely unknown. The aim of my PhD was to investigate the molecular and environmental factors that are involved in virus-induced epigenetic modifications in the infected plants and in their progeny. First, I examined the required sequence complementary between sRNAs and their nuclear target sequence. I demonstrated for the first time that nuclear-imported vsiRNAs can induce RNA-directed DNA methylation (RdDM) and subsequently heritable virus-induced transcriptional gene silencing (ViTGS) even when they do not share 100% nucleotide sequence complementarity with the target DNA. This finding reveals a more dynamic interaction between viral RNAs and the host epigenome than previously thought. Secondly, I explored how environmental stimuli such as light and temperature can affect the efficacy of ViTGS. I found that ViTGS is greatly inhibited at high temperature. Using RNA-seq, I established that inefficient ViTGS at high temperature is due to the limited production of secondary sRNAs that may limit the initiation, amplification and spreading of virus-induced DNA methylation to neighbouring cells and down generations. Lastly, I studied the link between the viral suppressors of RNA silencing (VSRs): viral proteins that can interfere with plant RNA silencing and ViTGS. I established that VSRs of certain viruses can impair TGS in infected tissues, suggesting that viruses may alter the epigenome and consequently plant gene expression in the infected plants and their progeny. Collectively, my work reveals how viruses can re-program the epigenome of infected plants, and deepens our knowledge of how we can harness pathogens to modify the epigenome for plant breeding.

Chikungunya virus (CHIKV) causes an acute fever with debilitating joint pain. Spread by the Aedes species of mosquito, recent increases in global temperature, and mutations in the viral glycoproteins have facilitated outbreaks worldwide with huge economic burden. Despite the recent resurgence of CHIKV, there are currently no vaccines or antiviral agents available. CHIKV, an alphavirus, possesses a positive sense, single stranded RNA genome that encodes four non-structural proteins (nsPs). The CHIKV nsP3 possesses an N-terminal macro domain, a domain found in the proteins of all species, and are defined by their ability to bind ADP-ribose. It is unclear what role the nsP3 macro domain contributes to CHIKV replication. Initially, a panel of cell lines was validated in terms of their physiological relevance and ability to support the replication of the CHIKV replicon and infectious virus. The phenotypes of a panel of mutants in the ADP-ribose binding pocket of the nsP3 macro domain were assessed in the context of a sub-genomic replicon and infectious virus in a range of relevant cell lines. Comparison of this data to the known biochemical properties of the nsP3 macro domain from the literature, indicated that ADP-ribose binding was crucial to CHIKV replication. In addition, this data suggested a role for the nsP3 macro domain in antagonising cellular innate immune pathways. ADP-ribose signalling has been implicated in the activation of the NFкB pathway therefore potential for the nsP3 macro domain to interfere with this cell signalling pathway was investigated. It was demonstrated that CHIKV did not activate the pathway and that expression of nsP3 actively was inhibitory. Furthermore, macro domain mutants with reduced ADP-ribose binding were unable to inhibit the pathway. It is therefore proposed that the CHIKV nsP3 macro domain is a virulence factor, able to suppress the host NFкB pathway to facilitate viral replication.

Paleovirology is the study of viruses over evolutionary timescales. Contemporary paleovirological analyses often rely on sequence data, derived from organism genome assemblies. These sequences are the germline inherited remnants of past viral infection, in the form of endogenous viral elements and the host immune genes that are evolving to combat viruses. Their study has found that viruses have exerted profound influences on host evolution, and highlighted the conflicts between viruses and host immunity. As genome sequencing technology cheapens, the accumulation of genome data increases, furthering the potential for paleovirological insights. However, data on ERVs, EVEs and antiviral gene evolution, are often not captured by automated annotation pipelines. As such, there is scope for investigations and tools that investigate the burgeoning bulk of genome data for virus and and antiviral gene sequence data in the search of paleovirological insight.

Louping ill virus (LIV; Flavivirus, Flaviviridae) is a poorly characterised animal pathogen of significant economic concern within the UK. Transmitted by ticks, LIV predominantly causes disease in ruminants and grouse, resulting in substantial losses. LIV is closely related to another Flavivirus, tick-borne encephalitis virus (TBEV), a significant human pathogen. Conversely, human cases of LIV are rarely reported and it is not considered a human pathogen. The phylogenetic relationship between LIV and TBEV is unclear and the molecular mechanisms that underpin host restriction in these viruses are poorly understood. The work presented in this thesis describes the characterisation of LIV using both phylogenetic and molecular approaches. Phylogenetic analysis, utilising a dataset of over 22 LIV genomes sampled over 84 years, indicates that LIV has spread throughout the UK predominantly by localised transmission events. However, several long-distance dispersal events are evident, indicating that LIV spread has likely been exacerbated by human processes such as livestock movement. Despite the LIV dataset spanning almost a century, a molecular clock signal could not be reliably estimated. This raises doubts about the clock rates which have been estimated for other closely related tick-borne flaviviruses. Previously it has been shown that the TBEV non-structural (NS) proteins do not act as type-I interferon (IFN) antagonists, quite unlike other Flavivirus NS proteins. Therefore, to facilitate comparison between LIV and TBEV, the possible antagonistic actions of the LIV NS proteins was investigated. Several LIV NS proteins were identified which function as antagonists throughout the IFN induction cascade. Additionally, the structure of the LIV subgenomic flavivirus RNA (sfRNA) was modelled and compared to TBEV, and it was found that both exert antagonistic effects within the IFN induction pathway. Finally, herein is described the development of the first LIV reverse genetics system based on circular polymerase extension reaction (CPER). This powerful tool can be used in the future to produce chimeric viruses which would allow further investigation into the factors governing host restriction and virulence in tick-borne flaviviruses. In summary, the results described within this thesis provides insight into the evolution of LIV and elucidates the immune evasion strategies employed by LIV during infection. Additionally, the outputs of this thesis provide important tools to further investigate the mechanisms governing host restriction and virulence within the tick-borne flavivirus sub-complex.

Plant viruses post a significant risk to both global food security, and industrial agriculture, however very little is known regarding their molecular mechanisms. Despite intensive study since the discovery of a multitude of plant virtual movement proteins, it remains unknown how they transverse the plasmodesmata, and thus move between cells. The CMV virus is widespread, infecting over a thousand plant species, and yet the means by which the movement protein CMV 3a associates to cellular membranes, targets itself and viral genomes to plasmodesmata have not been described. This study initially attempted to purify the CMV 3a protein from bacterial expression for structural and biophysical studies to examine viral protein and host membrane interactions. The study also began mapping the CMV 3a protein surface to investigate protein localisation and membrane attachment in planta, identifying structural features, including two potentially amphipathic helices which bear further investigation for potential roles in membrane association. Finally, this thesis examined the potential for the lipid modification S-acylation (Palmitoylation) as a membrane anchor, across a range of viral movement proteins. Describing this modification of viral movement proteins for the first time, S-acylation was demonstrated to not only be widespread, but potentially play different roles across a range of plant virus movement systems. This information is vital for the advancement of the field's understanding of the cell to cell movement of plant viruses, and the potential development of control strategies; and hence the safeguarding of global food security.

Norovirus has been identified as a common cause of gastroenteritis worldwide, and food as a transmission vehicle has been well documented. Standardised detection methods exist for the detection of norovirus from fresh produce and molluscan bivalves, whilst detection methods for a wider range of food matrices that may be implicated in transmission of norovirus do not currently exist. The detection of norovirus in foods suspected to be implicated in transmission is paramount for appropriate outbreak investigation. The contamination of foods other than shellfish and fresh produce often occurs via food handlers. The proportion of norovirus that is typically transferred from food handlers to food also remains unknown. Understanding this is necessary in order to estimate the risk of infection and the burden of gastroenteritis caused by norovirus that is attributable to food contaminated by food handlers. These questions were addressed by the development of a combined capture, concentration and quantitative detection protocol with the aim to enhanced norovirus recovery from a range of food types. A food surface wash and norovirus capture method that was sensitive, reduced processing time, and increased throughput capacity was applied to a range of ready to eat foods. An automated nucleic acid extraction method which further reduced processing time and increased throughput was validated. Finally the validated method demonstrated that two real time RT-PCR assays currently used for the detection of norovirus in shellfish and fresh produce or in faecal samples were comparable overall, and hence either could be used in combination with the norovirus capture, concentration and extraction protocol described in this thesis. The protocol was applied to a range of food matrices and resulted in < 1% to 55% recovery of norovirus GI and < 1% to 25% recovery of norovirus GII. The optimised protocol was then used to quantify virus transfer between food handlers hands and to food, in simulation experiments where food handlers’ gloved hands were artificially contaminated prior to preparation of a sandwich. This enabled norovirus transfer to food items and to other food handlers to be measured at each stage. Quantitative data demonstrated that 5.9 ± (SD ± 0.1) log10 cDNA copies/μl of norovirus GII inoculum, resulted in a percentage recovery of between 3.0% and 0.02% from Food Handlers and 7.8 ± (SD ± 0.1) log10 cDNA copies/μl of norovirus GI inoculum resulted in a percentage recovery between 9.6% and 0.004% from Food Handlers. The average percentage recovered from sandwich pieces over six replicates was 0.2% for norovirus GII and 1.2% for norovirus GI. The method and protocols developed could be rolled out to official control laboratories and aid foodborne outbreak investigation by allowing testing of food categories that currently are not investigated. Furthermore, this work demonstrated the extent of norovirus transfer from hands to food ingredients and the environment and could be used in risk assessment models. Further work applying these protocols to quantify the transfer from contaminated hands using a range of viral loads will be useful in determining risk more accurately, and to monitor and investigate food premises by introducing this as an additional food and hand hygiene marker.

Human noroviruses (HuNoVs) are distributed globally, affect all age groups and place a significant burden upon health services. The diversity of this RNA virus is thought to play a significant role in the persistence of HuNoVs as the main cause of non-bacterial gastroenteritis globally. Molecular diagnostics have been critical for understanding the epidemiology of outbreaks and sporadic cases, and to design and implement effective intervention strategies and disease control measures. Immunocompromised individuals are widely considered to be a reservoir for epidemic variants of HuNoV and whilst there are studies investigating the emergence of novel strains in an immunocompetent general population, reports at the the individual level are scarce. Three separate methodologies were developed to characterise HuNoV persistence in acute convalescent and chronic infection. First, a standardised quantification method to accurately quantify the most prevalent HuNoV genogroup. Second, a PGM-MB capture method to select HuNoV prior to massively parallel sequencing (MPS). Third, an assay to measure host specific coproantibody responses to three epidemic variants from different epochs. Quantification of longitudinal samples from individuals with acute or chronic HuNoV infection showed the virus distribution was homogenous in stool and an RNA external standard, in contrast to DNA, did not underestimate virus titre. HuNoV PGM-MB capture meant near complete viral genomes could be recovered at variable mean coverage. A bioinformatics pipeline demonstrated over the course of chronic infection allele frequencies were much more variable. In acute infection, minor alleles were present at a much lower frequency, but potential immune escape mutants were present. Immune escape mutants existed as minority variants or conserved mutations in the consensus sequence, and were in the presence of HuNoV specific-coproantibody, which were mapped to the protein surface. In HuNoV chronic infection, immune pressure is variable or non-existent, and therefore epidemic variants could emerge over long periods of infection by random chance. However, under immune pressure exerted by coproantibodies, escape variants may be seen. In three individuals, acute HuNoV symptomatic infection occurred despite the presence of specific secretory Ab responses to the VLP classed as the closest phylogenetic relative. The closest relative (Sydney 2012), differed at two amino acids, one of which has been previously described (A340T) as belonging to an epitope, and another which can be classed as having a potential role in immune escape (A323T). A single individual with acute HuNoV infection established a more prominent response to an earlier strain of HuNoV, rather than two contemporary strains, which proposes a role for Original Antigenic Sin (OAS) or Antigenic Seniority in the secretory Ab immunity. Finally, the use of MPS in outbreak tracking was assessed and compared to the currently used amplicon and Sanger based method. Overall both methods showed significant correlation. However, MPS provided greater depth and the ability to identify variants among samples within an outbreak that represented consensus changes in one or more samples from the same outbreak. This meant that the MPS data would have been able to link all the samples into a single outbreak or transmission network, where the current Sanger sequencing may not have been able to link them all.

Vaccinia virus (VacV) is a large dsDNA virus belonging to the Poxviridae family. Due to its size, the VacV replication cycle occurs entirely within the cytoplasm of infected cells. This exposes the virus to the many intracellular DNA sensors found in human cells. Since its discovery in 2013, cyclic GMP-AMP Synthase (cGAS) has been shown to be the predominant cytoplasmic DNA sensor in many cell types, including keratinocytes. Once activated, cGAS generates the second messenger cGAMP to trigger IFNβ production, through the adaptor protein STING. VacV can limit IFN production by inhibiting this pathway at multiple points, but little is known about whether VacV is able to inhibit DNA sensing directly. To investigate this, intracellular DNA sensing within keratinocytes was analysed during VacV infection. Results showed that within 4 hours of infection VacV was able to induce the loss of cGAS to undetectable levels, effectively blocking the cell IFNβ response. Additional DNA sensors that cooperate with cGAS such as IFI16 remained unaffected, showing that cGAS is essential for the IFN response to VacV. Inhibition of host degradation pathways did not prevent cGAS loss during VacV infection, suggesting cGAS is directly targeted for enzymatic cleavage. This demonstrates a potential new mechanism VacV employs to limit DNA sensing through cGAS, although the viral proteins responsible remain elusive. Identification of these proteins may provide new treatments for certain autoimmune disease where overactivation of cGAS contributes to disease phenotype.

Vaccinia virus (VACV), a large double-stranded DNA virus, is a close relative of smallpox virus. Two distinct infectious forms of virions are produced by VACV: Single-membrane mature virions (MVs) and double-membrane enveloped virions (EVs). VACV is thought to exploit cell motility as a means to enhance the spread of infection. A single viral protein, F11, contributes to this by mediating the inhibition of RhoA signalling in order to facilitate cell retraction. However, F11 is not sufficient for VACV-induced cell motility, indicating that additional viral factor(s) must be involved. We showed that vaccinia virus growth factor (VGF), a homologue of EGF, promotes infected cell motility to facilitate the spread of infection. We found that VGF secreted from early infected cells is cleaved by the metalloprotease ADAM10 whereupon it acts largely in a paracrine fashion to direct cell motility. Real-time tracking of cells infected in the presence of EGFR/MEK/FAK/ADAM10 inhibitors, or with VGF and F11 deleted viruses, revealed defects in radial velocity and directional migration efficiency during plaque formation, leading to impaired cell-to-cell spread of infection. Intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. These results demonstrate how poxviruses hijack EGFR-induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo. In addition, we developed the open-source software VirusMapper that combines super-resolution microscopy and single-particle averaging to map the localisation of proteins within viral particles. We found that the viral entry fusion complex (EFC) localises to the tips of virions. Furthermore, we found that infected cells produce VACV-triggered extracellular vesicles (VEVs) that contain EV membrane proteins but are devoid of virions. VEVs are phosphatidylserine-positive, suggesting that they may use apoptotic mimicry to dampen the immune response of neighbouring cells, thereby enhancing virus spread.

Recent years have witnessed virus disease outbreaks that have caused considerable morbidity and mortality. As 80% of human viruses are zoonotic, outbreaks can be prevented by active surveillance of wildlife for pathogens with a potential for cross-species transmission. Rodents serve as important reservoirs for many zoonoses and epizooses. In this thesis, a cDNA library of 976 rodents, shrews, moles and birds was constructed. Molecular methods using inosine-containing degenerate primers were developed for the screening of novel viruses of the families, Arenaviridae and Filoviridae. Filovirus RNA was absent in all specimen screened. Lymphocytic choriomeningitis mammarenavirus (LCMV) RNA was detected in 4 Mus musculus (19%). Further screening of specimens obtained from a zoo outbreak resulted in the detection of LCMV RNA in a total of 20 M musculus, 2 Geoffroy’s marmoset, 1 black and white colobus and 1 Black-crested gibbon from two separate sampling sample sets. Viral RNA was detected in non-human primates (NHP) not previously reported. Rodents screened at the zoo after the outbreak were negative for LCMV RNA. Phylogenetic analyses of full-length glycoprotein precursor (GPC) of 4 NHP-derived and 4 rodent-derived LCMV revealed a clustering of the former with other lineage-I LCMV GPC sequences. Molecular characterisation of the novel LCMV strains uncovered a consistency in amino acid substitutions across primate-derived and rodent-derived LCMV strains at position 211 of GPC which may be a possible determinant for cross-species transmission. Analysis of the alpha-dystroglycan receptor of rodents and NHPs revealed residues that might influence GPC binding affinity and therefore drive virus evolution. Pseudoviruses constructed from GPC of rodent-derived LCMV displayed higher infectivities in hepatocellular carcinoma cells than their NHP-derived LCMV counterpart which is indicative of the existence of another unidentified host receptor. Together, the results establish a framework for further investigation of the molecular basis for cross-species transmission of LCMV between rodent and primate hosts.

Background Shiga toxin producing Escherichia coli (STEC) O157 causes severe gastrointestinal disease and haemolytic uremic syndrome, and has a major impact on public health worldwide with regular outbreaks and sporadic infection. Phage typing, i.e. the susceptibility of STEC O157 strains to a bank of 16 bacteriophages, has been used in the UK to differentiate STEC O157 for the past 25 years and the phage type (PT) can be an epidemiological marker of strains associated with severe disease or associated with cases that occur from foreign travel. However, little is known about the molecular interactions between the typing phages (TP) and STEC O157. The aims of this thesis were to use whole genome sequencing to elucidate the genetic basis for phage typing of STEC O157 and through this understand genetic differences between strains relevant to disease severity and epidemiology. Results Sequencing the STEC O157 TPs revealed that they were clustered into 4 groups based on sequence similarity that corresponded with their infectivity. Long read sequencing revealed microevolutionary events occuring in STEC O157 genomes over a short time period (approximately 1 year), evidenced by the loss and gain of prophage regions and plasmids. An IncHI2 plasmid was found responsible for a change in Phage Type (PT) from PT8 to PT54 during two related outbreaks at the same restaurant. These changes resulted in a strain (PT54) that was fitter under certain growth conditions and associated with a much larger outbreak (140 as opposed to 4 cases). TraDIS (Transposon directed Insertion site sequencing) was used to identify 114 genes associated with phage sensitivity and 44 genes involved in phage resistance, emphasising the complex nature of identifying specific genetic markers of phage susceptibility or resistance. Further work is required to prove their phage-related functions but several are likely to encode novel phage receptors. Deletion of a Stx2a prophage from a PT21/28 strain led to a strain that typed as PT32, supporting the concept that the highly pathogenic PT21/28 lineage I strains emerged from Stx2c+ PT32 strains in the last two decades by acquisition of Stx2a-encoding prophages. Conclusions This body of work has highlighted the complexity of bacteriophage interaction and investigated the genetic basis for susceptibility and resistance in E. coli. The grouping of the TPs showed that resistance or susceptibility to all members of a typing group was likely to be caused by one mechanism. IncHI2 was identified as one of the markers for the PT54 phenotype. The Stx2a prophage region was associated with the switch from PT32 to PT21/28, although PT32 strains containing both Stx2a and Stx2c-encoding prophages have been isolated and can provide insights into phage variation underpinning the susceptibility to the relevant typing phages. The TraDIS results indicated that susceptibility or resistance was governed by multiple genetic factors and not controlled by a single gene. The significance of LPS for initial protection from phage adsorption was evident and a number of novel genes controlling phage susceptibility and resistance identified including the Sap operon and stringent starvation protein A respectively. While SNP-based typing provides an excellent indication of the evolution and relatedness of strains, phage typing can provide real insights into short term evolution of the bacteria as PTs can be altered by mobile elements such as prophages and plasmids. This study has shown that, although complex, genetic determinants for PT can be mined from the genome and allow us to understand the evolution of this zoonotic pathogen between host species and during outbreaks.