Influenza A viruses (IAVs) are common infections of certain avian reservoir species, and they periodically transfer to mammalian hosts. These cross-species jumps are usually associated with sporadic outbreaks, and on rare occasions lead to the establishment of a lineage in the new host species. The immune pressure exerted by the new host on the emergent virus forces it to evolve and adopt strategies to evade immunity in order to survive in nature. Understanding the biological mechanisms that allow successful inter-species transmission and adaptation to mammals is crucial to develop the theoretical tools required to predict and/or control emergence of new viruses in humans and animals. H3N8 equine influenza virus (EIV) represents an interesting model to study the dynamic of within-host variation of an avian-origin IAV. Indeed, this virus has emerged from birds in 1963 and has circulated in horse populations for more than fifty years despite the availability of vaccines. Evidence of evolution of EIV virulence factor non-structural protein 1 (NS1) also exists. NS1 is the main viral antagonist of the host interferon (IFN) response, and it relies on different strategies for overcoming these responses, which varies depending on the viral strain. While some NS1 proteins effectively block the induction of IFN and IFN stimulated genes (ISGs), others block general gene expression at a post-transcriptional level, and therefore reduce the synthesis of IFN and ISGs indirectly. Importantly, little is known about the contribution of these NS1 functions to EIV infection phenotype and adaptation to horses. In this work, we characterised NS1 proteins spanning the entire EIV lineage and showed that NS1s from different time periods after EIV emergence counteract the IFN response using different and mutually exclusive mechanisms. While EIVs circulating in the early 1960s blocked general gene expression by a NS1-mediated blockade of the cleavage and polyadenylation specificity factor 30 (CPSF30), NS1s from contemporary EIVs specifically inhibit the induction of ISGs by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially during EIV evolution, E186K substitution and C-terminal truncation. These changes in NS1 allowed contemporary EIVs to replicate in the presence of high levels of IFN. The results shown here with EIV indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

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Cardiovascular disease is one of the leading causes of death in the Western world. One of the most common causes is the rupture of unstable atherosclerotic plaques, which can lead to thrombus formation, occlusion of the artery and myocardial infarction. Therefore there is a need for treatments that stabilise vulnerable plaques. Gene therapy has the potential to provide a novel treatment for this. To maximise therapeutic gene expression and minimize any potential adverse effects due to unwanted transgene expression in non-target tissues, a gene delivery vector specifically targeted to areas of atherosclerotic vasculature is required. The vector is also required to efficiently infect cells to produce relatively long term transgene expression, be stable in blood, non-toxic, non-immunogenic and producible at high titres. Viral vectors, particularly those based on adenovirus (Ad) and adeno-associated virus (AAV) have many of the desired features, but transduce vascular cells relatively inefficiently and in a non-selective manner. Methods of altering their tropism have been established and could be utilised to develop vectors with a high degree of selectivity for atherosclerotic plaques. Detargeting of vectors can be achieved by mutating regions of the virus capsid that are thought to bind the native cellular receptors and retargeting to novel cell types is achieved by inserting peptide ligands into the virus capsid. The aim of this study was to develop atherosclerotic plaque targeted vectors and to characterise Ad and AAV vector platforms in this regard. Two approaches were taken. In the first approach three previously identified plaque targeting peptides were tested for their ability to target viral vectors to atherosclerotic plaques. The second approach involved performing phage display in a mouse model of plaque rupture to identify novel peptides that specifically target unstable plaques. Further work was carried out to characterise and develop methods for using an AAV2 based peptide library as a novel tool for biopanning. This work has provided further characterisation of Ad and AAV platform vectors that may be utilised in the development of vectors with a highly selective tropism.

Innate immunity is a branch of the immune system that is responsible for controlling the early events of pathogen infection. One of the key components of the innate immune systems arsenal are the interferon (IFN) cytokines. IFNs are small signalling proteins that are released by cells in response to invading pathogens, and viruses in particular. They are named for their ability to interfere with viral replication. The result of IFN signalling is the up-regulation of a diverse collection of genes termed interferon-stimulated genes (ISGs). These genes act in synchrony to limit the replication of viruses. The protein products of ISGs are involved in a multitude of cellular pathways that limit replication and additionally intercept viral proteins and nucleic acid directly. Some of these ISGs are mediators of an important cell-death response, apoptosis. Apoptosis is a vital component of innate immune signalling and controls viral replication by sacrificing the infected cell to limit further infection of neighbouring cells. The function of specific ISGs in mediating this response is poorly understood.

The aphid transmitted Polerovirus Turnip yellows virus (TuYV) was found to be widespread with high incidences in oilseed rape (OSR) across Europe. UK, France, Germany and Poland all having >90% TuYV incidence in some OSR crops. From the 179 whole TuYV genomes sequenced in this study the phylogenetic analyses indicated three distinct genetic groups in the UK, two of which were also detected in Europe. These three genotypes were also distinct from the original sequenced TuYV-FL. These groups are proposed to be distinct species due to their genetic distance based on the most variable gene ORF5 and phylogenetic analyses of ORF1, ORF3, ORF4 and ORF5. Mixed TuYV infection was uncommon and only two plant samples had genetically distinct isolates. Whole genome analysis also provided valuable information on two recombination hotspots located within TuYV genes ORF3 and ORF5. Investigation into the epidemiology of TuYV revealed many weed and crop species as hosts, including sugar beet, which it was previously thought not to infect. TuYV isolates detected infecting weed plants in the UK were successfully transmitted to OSR. Previously undescribed hosts, verbascum, geranium, teasel, spear thistle, dock and previously described hosts in the Brassicaceae, Compositae and Lepidium families were found in the UK. A full-length infectious clone of a UK isolate of TuYV has been produced, this will allow further assessment of TuYV in the future. The infectious clone was able to cause systemic infection of TuYV and was aphid transmissible. The Arabidopsis thaliana gene knock-out study did not reveal a single eIF gene or gene linked to virus movement or silencing that could provide extreme broad-spectrum resistance. The gene eIF(iso)4G.1 was able to give a broad-spectrum quantitative resistance, and the potential of eIF3D.2 as well as sucrose symporters SUC1 and SUC2 as candidates for extreme TuYV resistance were discovered. This understanding of the epidemiology and diversity of TuYV is being used to develop strategies for control.

This work concerns infection of avian erythrocytes by influenza and two other viruses. The first section investigates the production of viral polypeptides and infectious progeny virions from avian erythrocytes infected with fowl plague virus, Newcastle disease virus and Semliki Forest virus. In the second section a closer examination is made of viral polypeptide synthesis in avian erythrocytes following infection with avian and/or human influenza A strains. The third section investigates the distribution of newly synthesized influenza viral polypeptides between the erythrocyte nucleus and cytoplasm. The fourth and fifth sections are concerned with viral replication in erythrocytes at different stages of differentiation.

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Hepatitis C virus (HCV) is emerging as one of the leading causes of morbidity and mortality in individuals infected with HIV and has overtaken AIDS-defining illnesses as a cause of death in HIV patient populations who have access to highly active antiretroviral therapy. For many years, the clonal analysis was the reference method for investigating viral diversity. In this thesis, a next generation sequencing (NGS) approach was developed using 454 pyrosequencing and Illumina-based technology. A sequencing pipeline was developed using two different NGS approaches, nested PCR, and metagenomics. The pipeline was used to study the viral populations in the sera of HCV-infected patients from a unique cohort of 160 HIV-positive patients with early HCV infection. These pipelines resulted in an improved understanding of HCV quasispecies dynamics, especially regarding studying response to treatment. Low viral diversity at baseline correlated with sustained virological response (SVR) while high viral diversity at baseline was associated with treatment failure. The emergence of new viral strains following treatment failure was most commonly associated with emerging dominance of pre-existing minority variants rather than re-infection. In the new era of direct-acting antivirals, next generation sequencing technologies are the most promising tool for identifying minority variants present in the HCV quasispecies populations at baseline. In this cohort, several mutations conferring resistance were detected in genotype 1a treatment-naïve patients. Further research into the impact of baseline HCV variants on SVR rates should be carried out in this population. A clearer understanding of the properties of viral quasispecies would enable clinicians to make improved treatment choices for their patients.

Dengue virus (DENV) is one of the most important arbovirus infections which continues to be spread to many parts of the world. The widespread distribution of the vector Aedes sp, DENV genetic evolution, emergence of a new serotype, global warming, environmental changes, population growth and human mobility are some of the factors affecting DENV transmission. From the many studies conducted on DENV, there is still a lack of integrated research that includes several aspects that affect DENV transmission at a local scale. The aims for this study was to conduct an integrated study of DENV tranmission, covering entomology, DENV, and socio-economic and environmental factors using Banyumas Regency, Java Indonesia, as a model area. The uniqueness of demography, socioeconomy and environment of each area emphasizes the importance of this research. For the entomology factors, this study found that traditional larvae indices such as House Index (HI), Breteau Index (BI) and Container Index (CI), which have been applied for many decades in entomology surveys, are not relevant measurements for determing mosquito populations. These findings supported previous findings that larvae indices cannot predict the transmission risk level and is not correlated with DENV incidence. In this study, adult mosquito collections were found to be a better measurement of risk of DENV transmission. A high vertical transmission rate was also confirmed in an endemic area, which is possibly one explanation for DENV persistence in that area. From a knowledge, awareness and practice (KAP) survey, there is no correlation between knowledge, awareness and practice of DENV prevention and control, and there is also no association between KAP of people with the mosquito infestations in the area of study. This finding leads to the need for better strategies such as education campaigns about DENV prevention to ensure not only an increase in knowledge but also this knowledge translates into practices. During collection of serum samples from DENV infected patients a higher number of adult age groups reported DENV cases, indicating an age group shift from children to adults. Most of the samples (89% ) from positive result of IgG/IgM test had a secondary infection by serological test, which likely increases the possibility of developing severe clinical manisfestations. Many publications believe that secondary infection by different serotypes could cause severe DENV infection. Unfortunately, the serotyping and genotyping of the patient samples could not be completed due to time constraints, so the information of circulating serotypes and genotypes could not be obtained. It would be interesting to further analyse the serotypes and then correlate them with the less or more severe clinical manifestations and also capture the spread of disease from pylogenetic trees from the genotyping results. Based on spatio and spatio-temporal models, it can be concluded that socioeconomic factors, particularly the level of education and the employment structure were the most important risk factors of DENV infection. It was also revealed that enviromental factors had only a little influence on DENV infection, in contrast with many previous beliefs that global warming and environmental changes are the main factors of DENV infection. Human mobility was proposed to be the main explanation of this phenomenon since more educated people and people with good job type tend to have higher exposure to DENV infection due to their movement from home to work places or public areas. This also complements the fact that more adults reported DENV infection during the patient sample collection, suggesting that adult age groups possibly have a higher risk of DENV infection due to higher mobility, which means higher exposure to DENV infection. The possibility of having a secondary infection is also higher in adults since there has been more time to have the first infection and then the second infection. In order to complete this integrated study, the influence of temperature on mosquito immunity, in particular the RNA interference (RNAi) response was tested. Based on RNAi activity in 24°C, 28°C and 32°C, RNAi activity was slightly more efficient following the increase of temperature. In addition, the infection of Aag2 cells with SFV showed that the increasing temperature will result in lower virus replication. We can assume that the lower or higher temperature only contributes a minor effect on RNAi machinery in vitro. In conclusion, this integrated epidemiological study finds that current entomology surveys are not relevant, because they are not associated with the risk of transmission. In addition, socioeconomic factors rather than environmental factors are proposed to be the most significant factor for DENV infection. Findings such as age shift, secondary infection, human mobility and a high vertical transmission rate are important information which could help the public health sector in their planning and action on DENV prevention and control strategies.