Viruses are the most abundant biological agents in the global marine environment. Through cellular lysis viruses influence many biogeochemical and ecological processes, including energy and nutrient cycling, host distribution and abundance, algal bloom control and genetic transfer. Nano- and picophytoplankton are ubiquitous in the world’s oceans and are responsible for a high proportion of the annual global carbon fixation. However, relatively few viruses have been isolated and described that infect these key primary producers and little is known of their diversity, dynamics or propagation strategies. The aims of this study were to detect, isolate and characterise novel marine viruses that infect these important members of the phytoplankton assemblage. Screening of seawater samples for viruses that infect a broad representation of nano and picophytoplankton species was undertaken here. To enable this, a large culture collection of 106 phytoplankton species was established and used to screen seawater samples for viruses on a weekly basis over a two year period. A total of 12 novel viruses infecting the prasinophyte species’ Ostreococcus tauri and Micromonas pusilla were isolated from seawater sampled in coastal waters of the Western English Channel. Viruses were purified by plaque purification or liquid serial dilution techniques. Characterisation of novel virus isolates included growth kinetics, visualisation using transmission electron microscopy, host range analysis and estimates of viral genome sizes using pulsed field gel electrophoresis. Phylogenetic analysis of these viruses was conducted based on the sequence of the conserved DNA polymerase gene. Genome sequencing of two of the viruses infecting O. tauri was completed and revealed many exciting features, including a suite of genes hitherto unreported, or with rare occurrence, in viruses. Evidence is presented for horizontal gene transfer between viruses isolated in this study and their hosts, as well as between other eukaryotic and bacterial sources. Functional characterisation of the viral genomes sequenced and described in this study will provide clearer insights into viral dynamics and evolutionary history.

The innate immune system is a vital part of the body's defences against viral pathogens. RIG-I and MDA5 belong to the retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) family and function as cytoplasmic PRRs that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. RIG-I and MDA5 contribute to antiviral signalling in different ways depending on the virus involved. Coxsackieviruses are positive sense, single-stranded RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. They cause many serious diseases, including viral myocarditis (which can lead on to dilated cardiomyopathy), aseptic meningitis, and pancreatitis. In order to identify which RLR recognises these viruses and which RNA species triggers RLR activation during Coxsackievirus infection, viral ssRNA and replicative intermediates of Coxsackievirus RNA as well as synthetic dsRNA were used in this study. The results revealed that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Confocal microscopy provided unique evidence between the relationship of viral dsRNA and MDA5 while cytokine assays using HEK-MDA5 cells showed a strong immune response to Coxsackievirus and the dsRNA intermediates. This shows very strong evidence that MDA5 is a key sensor of the dsRNA intermediate of Coxsackieviruses. As RIG-Is role in Coxsackie recognition still needs to be verified Huh7 and Huh7.5.1 cells were used and showed no difference in immune response in the absence of RIG-I to Coxsackievirus infection, as well as the isolated ssRNA, suggesting that the VPg group present on the RNA blocks recognition. Furthermore immunoprecipitation experiments showed that in response to Coxsackievirus stimulation, RLRs homodimerise as well as heterodimerise with LGP2, potentially upregulating their activity as a possible mechanism for viral detection. The data presented here show a much clearer role for RLRs in Coxsackievirus infection, while opening new questions as to MDA5s role in the diseases caused by Coxsackieviruses, as well as the specifics behind dimerisation of the RLRs

The Epstein-Barr virus (EBV)-encoded Latent membrane proteins (LMP) 2A and LMP2B are frequently expressed in a number of EBV-associated malignancies. Although the true function of these proteins remains to be elucidated, LMP2A appears to function as a surrogate B-cell receptor in B-cells, preventing BCR signalling and ensuring viral latency. Much less is known about the function of these proteins in epithelial cells, although LMP2A has been shown to modulate Wnt/\(\beta\)-catenin, MAPK and PI3K/Akt signalling, whilst LMP2A and LMP2B have been shown to promote cell motility and to alter the turnover of certain classes of immune receptors. Viral infection of the host cell triggers the innate immune system to promote and sustain the initiation of an anti-viral state. Viruses have developed immune evasion strategies to counteract the effects of this immune response and to prevent recognition of viral antigens by the adaptive immune response. Here novel functions for LMP2A and LMP2B are described with respect to modulation of innate immunity, findings which have implications for the role of these proteins in EBV-driven oncogenesis. The first wave of the innate immune response is mediated by Toll-like receptors (TLRs), a family of PAMP receptors that transduce signals to activate the type I interferon response. Findings presented here show that LMP2A attenuates signalling from a number of these receptors in response to TLR agonist stimulation. These data were observed only in LMP2A expressing cells while those expressing LMP2B showed no alteration of TLR signalling, indicating that the amino-terminal signalling domain of LMP2A controls these functions. Preliminary findings have also identified a role for LMP2A and LMP2B in the control of vesicular trafficking, with both proteins increasing endosomal-lysosomal trafficking and lysosomal acidification, an effect that important for degradation and turnover of receptors. These data present novel mechanisms by which EBV may evade immune responses and in doing so, contribute to the progression of the EBV-associated epithelial cell malignancy, NPC.

Kaposi’s sarcoma-associated herpes virus (KSHV) encodes genes with immunomodulatory potential, one of which is vIRF-2 that shares homology to cellular interferon regulatory factors. The innate antiviral mechanism mediating the type I interferons is an essential host cell defence mechanism limiting viral replication. The aim of this study was to determine the range and type of cellular gene sets and associated biological pathways whose expression is deregulated by vIRF-2. HEK 293-derived cell clones were engineered to express doxycycline-inducible vIRF-2. Interferon (IFN) responses were induced with recombinant (r) IFN-α and measured by an IFN stimulated response elements (ISRE) luciferase reporter gene assay. The effects of vIRF-2 on cell transcriptome profile in response to rIFN-α were determined by DNA microarray analysis and confirmed by immunoblot assay. vIRF-2 protein inhibited activation of ISRE-luc by over 50% and significantly (p<0.05) down-regulated the expression of 57/78 (73%) of rIFN-α regulated genes. The DAVID and GSEA software packages revealed vIRF-2 down-regulates the RIG-I-like receptor, JAK-STAT and Ubiquitin ligase pathways and many gene sets involved in antiviral response, transcriptional regulation and apoptosis. Immunoblot assays demonstrated reduced levels of RIG-I/DDX58, TBK-1, p-38, STAT1, pSTAT1, IRF-9 and OAS3. The biological significance of the vIRF-2 anti-IFN property was demonstrated by the rescue of encephalomyocarditis virus (EMCV) replication in vIRF-2 expressing cells treated with rIFN-α; EMCV was titred by plaque assay on L929 cells. These data confirm the role of KSHV vIRF-2 in negative regulation of the IFN-α/β innate immune response by a mechanism dependent on negative regulation of RIG-I/DDX58, STAT1, IRF-9 and OAS3.

Many viruses exploit and, in some cases, promote host cell endocytic pathways for infection. These pathways include caveolar and clathrin-mediated endocytosis, as well as macropinocytosis. The entry mechanisms of many viruses are not clear cut, with more than one pathway implicated in some cases. Hepatitis C virus (HCV) is a hepatotropic virus associated with liver disease, fibrosis, cirrhosis and hepatocellular carcinoma. There are four co-receptors or “entry factors” for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI) and the tight junction proteins Claudin 1 (CLDN1) and Occludin (OCLN). Clathrin-dependent endocytosis of HCV has been demonstrated in hepatoma cell lines and has also been shown to be the route of entry for co-receptor CD81; however, other endocytic pathways have not been considered. This thesis investigates a role for caveolae in HCV entry. In addition, it has recently become apparent that the epidermal growth factor receptor (EGFR) is required for viral entry into hepatoma cells and that stimulation with EGF results in increased entry and infection. This thesis investigates the role of EGFR in the endocytosis and trafficking of HCV receptors/entry factors, with a particular focus on CD81, using live-cell and super-resolution imaging techniques.

Enterohaemorrhagic Escherichia coli (EHEC) pathogens represent major difficulties to health as they cause infections and in the gastrointestinal tracts of animals. Previous studies revealed that the locus of enterocyte effacement (LEE) pathogenicity island is the major player in triggering the attaching and effacing lesions that induce death of host cells. The first step in its expression is activation of the LEE1 promoter that controls expression of the LEE encoded regulator (Ler) that activates LEE expression. The global regulator of LEE activator (GrlA) transcription is considered to be especially important in this process. Although GrlA binds to the LEE1 promoter and activates transcription initiation in vitro, the fold of activation was only ~ 1.5 fold. The aim of this work was to discover the factors that trigger GrlA activity. Thus, in chapter 3, it is shown how bacterial attachment to host cells triggers GrlA activity to an extent not seen during planktonic growth, with up to ~20 fold activation. Data also show that the level of free unbound GrlA defines the activity of the LEE promoter by GrlA, and the previously characterised GrlR anti-GrlA protein merely serves to buffer the level of GrlA.

A capsule composed of mainly α-glucan forms the most outermost layer of cell envelope of Mycobacterium tuberculosis, the causing agent of tuberculosis. Capsule participates in modulating the immune system responses. In GlgE pathway, one of the three pathways involved in α-glucan synthesis, trehalose in converted to glucan through the activity of four enzymes, trehalose synthase, maltokinase Pep2, maltosyltransferase GlgE and branching enzyme GlgB. It has shown that M. tuberculosis TreS and Pep2 form an octameric complex together. During this study we solved the structure of the complex at 3.6 Å in M. smegmatis. The structure reveals two pairs of Pep2 monomers bind to the opposite sides of the diamond shaped TreS tetramer in a 4 + 4 complex. However, studying the stoichiometry of the complex using other techniques such as: ITC, AUC and SAXS indicates a 4 + 2 complex formation in the solution. Moreover, our data using size exclusion chromatography would suggest that this complex formation is pH dependent and favors complex formation at more acidic pHs. This study is a step forward towards better understanding of the capsule synthesis in M. tuberculosis and highlights the role of complex formation between enzymes as an effective strategy to control the metabolic pathways in mycobacteria.

Plasmids RK2 are broad host range plasmids and they carry genes for antibioticresistance. Their central control operon, encoding two global regulators KorA and KorB, is anatural example of a negatively self-regulated operon. The aim of the work described herewas to use mathematical models to better understand the function and adaptation of KorA and orB regulation, in the context of available experimental data. The deterministic models combined with statistical inference allowed for analyses of he regulation mechanism and hypothesis generation about the system dynamics. Furthermore, the extended model was applied to data from different plasmid hosts, in order to indentify processes that might cause a difference in KorB abundance between the hosts. The stochastic multi-scale models were constructed and used in order to understand evolutionary reasons for such a negative and cooperative autoregulation. The system was tested for optimizations in mRNA production, response time, fluctuation and robustness. Moreover, the effect of cooperative regulation on the response time of the switch between vegetative and conjugative transfer, was also tested. In conclusion, the studies have deepened our understanding of the model plasmid RK2, of negative and cooperative regulation more generally, and generated hypotheses suitable for experimental validations.

Arboviruses (Arthropod-borne viruses) cause acute diseases that are increasingly affecting both human and animal health. Currently, there is a critical lack of understanding about the nature of arbovirus-host interactions in the lymph nodes (LNs), the place where the adaptive immune response is initiated and shaped. In this study, we used bluetongue virus (BTV) and its natural sheep host, to characterise the early events of an arbovirus infection with particular focus on the LNs. Our findings reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. This study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts the stromal network of marginal reticular cells and follicular dendritic cells composing the scaffolding of the follicular area. These cells contribute to antigen presentation and affinity maturation of B-cells for the production of antibodies. Consequently, we observed a loss of germinal centre structure, which hinders B-cell proliferation. This process results in a delayed production of high affinity and virus neutralizing antibodies that is directly related to the virulence of the BTV strain used and the severity of disease. Moreover the humoral immune response to a different antigen is also hampered in BTV-infected animals. Our data show that an arbovirus can evade the host antiviral responses by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.

Human herpesvirus (HHV) –6A and HHV-6B are unique among herpesviruses in their ability to integrate into the telomeres of human chromosomes and be inherited in a Mendelian fashion. Based on current data, inherited chromosomally integrated HHV-6 (iciHHV-6) is present in 0.5-2% of the UK population. There is increasing evidence to suggest that iciHHV-6 is not a dead-end for the virus, and that HHV-6 can be excised from the genomes of iciHHV-6-positive individuals. It is hypothesised that this excision occurs from the formation of T-loops between the end of the telomere and the viral telomere like repeats (TLRs). T-loops form naturally in a cell as part of the complex that protects the end of the chromosomes; however, under certain circumstances these can be excised leading to a loss of telomeric repeats from the chromosome and the formation of circular, extra-chromosomal telomeric sequence. There is increasing evidence that excision can lead to reactivation of the virus and potentially cause disease. Our current understanding of the phenotypic associations of iciHHV-6 is based on case-reports and case-control studies of individual diseases. The work presented in this thesis set out to answer a number of questions regarding the phenotypic consequences, and genome dynamics of iciHHV-6. First, the association between both exogenously acquired HHV-6 and iciHHV-6 and classical Hodgkin lymphoma (cHL) was examined in a case-control study. Whilst exogenously acquired HHV-6 was significantly associated with cHL the virus was present at low levels in DNA extracted from cHL tumours. Virus at such low level was concluded as not having a direct role in the pathogenesis of cHL. A case control study of iciHHV-6 and cHL revealed no difference in the prevalence of iciHHV-6 amongst cases and controls, but identified a single iciHHV-6-positive individual who appeared to have four integrated viral genomes. Secondly, iciHHV-6 was examined in the Generation Scotland: Scottish Family Health Study (GS:SFHS) cohort, in a hypothesis generating study. It was revealed the iciHHV-6 was present at a prevalence of 2.7%. Further analysis showed a statistically significant difference in the prevalence of iciHHV-6 between individuals born in Scotland (2.8%) and England (1.8%). Analysis of disease phenotypes revealed potential associations between iciHHV-6 and breast cancer in an unrelated subset of the GS:SFHS cohort. Also confirmed was the recent 3 report of an association between iciHHV-6 and angina pectoris. Analysis of other variables revealed iciHHV-6-positive females had a lower average Mill Hill vocabulary test score than iciHHV-6-negative females; and that iciHHV-6-positivity was associated with participation in fewer years of education. Thirdly, the iciHHV-6 genome in an LCL generated from an iciHHV-6A-positive individual was shown to be dynamic. The gross structure of the HHV-6 genome is a unique (U) region flanked by direct repeats (DR) (DR.U.DR).Analysis using droplet digital PCR (ddPCR), on DNA extracted at various time points of the LCL culture revealed that viral sub-genomic regions were lost in a proportion of cells, which coincided with a reduction in population doublings of the culture. At the point of reduction of viral copy number, an excess of HHV-6 DRs was noted suggesting that only a portion of the viral genome had been lost in these cells. Gradually the viral copy number returned to approximately one copy per cell. It is likely this was caused by an outgrowth of cells where excision had not occurred. This in vitro model demonstrated that whilst excision of iciHHV-6 genomes is possible, it may be accompanied by a reduction in cell viability. Loss of HHV-6 genomic regions was also examined in 59 iciHHV-6-positive individuals and was infrequent. Only six showed some degree of DR loss. Further to this, inheritance of single direct repeats was observed in six individuals and two families in the GS:SFHS cohort. A novel ddPCR and mathematical model was developed to predict the iciHHV-6 genome configuration in samples where atypical U:DR ratios were observed. Through this, we hypothesise that an iciHHV-6-positive individual who had 4 U regions per cell and 5 DR regions per cell, had an integrated HHV-6A genome concatemer with the configuration (DR.U)4.DR, and hypothesised that this arose from integration of a replication intermediate. Finally, phylogenetic analysis of five regions of 26 iciHHV-6 genomes and their counterparts in exogenously acquired HHV-6 genomes was performed. There was a higher degree of divergence between iciHHV-6A and exogenous HHV-6, along with evidence of a common viral ancestor in four iciHHV-6-positive individuals. This divergence was not observed in iciHHV-6B were very little variation was observed between iciHHV-6B and exogenously acquired HHV-6B. These results shed light on the complex relationship between iciHHV-6 and the human host.