Poultry health and welfare are important for maintaining sustainable and safe food production. In the UK alone, in excess of 900 million broiler chickens are farmed annually with demand continuously increasing. Avian pathogenic Escherichia coli (APEC) is the causative agent of the extraintestinal syndromic poultry disease colibacillosis, which has a major impact on poultry health and is a considerable economic burden to the global poultry industry. The need for disease control is of paramount importance. Little is known about the epidemiology, population dynamics and infection biology of APEC in UK broiler chickens. This knowledge would contribute to the implementation of control measures. This investigation comprised: one longitudinal field study in commercial flocks aimed at simultaneously identifying potential APEC (pAPEC) in the intestinal tract of healthy birds and extraintestinal E. coli in diseased birds, one study determining the contribution of E. coli to early flock mortalities and a series of in vitro experiments and genetic analyses characterising both extraintestinal and avian faecal E. coli isolated from UK broiler chickens. E. coli were subjected to virulotyping, phylogenetic typing, macro-restriction pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The extraintestinal E. coli isolates from diseased birds represent a diverse genetic population. Furthermore, as broiler chickens age, the prevalence of pAPEC in the gastrointestinal tract decreases. The intestinal tract of day-old chicks contains considerable levels of pAPEC (24.05% of the faecal population sampled), while ~70% of early mortalities relate to extraintestinal E. coli infections, possibly originating from the gut. In vitro, pAPEC did not appear to invade intestinal epithelial cells, suggesting the respiratory route is likely to be favourable for dissemination, although pAPEC are cytotoxic and may disrupt epithelial integrity prior to dissemination. There were no significant differences in the intracellular persistence of APEC and faecal E. coli in cultured avian macrophages and survival in serum; challenges encountered by E. coli during dissemination. Overall, this investigation highlights the diverse spectrum of E. coli associated with extraintestinal disease in commercial broiler production and the need to determine the contribution of host susceptibility to disease manifestation.

Hantaviruses are a group of rodent-borne viruses. Seoul hantavirus is considered to be the only global hantavirus, although reported human cases outside of Asia are rare. Human infection occurs when breathing in aerosols of excreta from infected rodents. Hantaviruses have been listed as a major impacting factor leading to a rise in acute kidney injury (AKI) throughout the Western world. In the UK, historically, there was evidence for human and animal exposure to hantaviruses demonstrated by the detection of specific antibodies and classic renal disease, however it is only during this study that existence of a UK hantavirus in wild rodent populations has been proven. Since 2012, several cases of acute AKI due to hantavirus infection in the UK have been confirmed. Two cases were from Yorkshire and had documented exposure to wild rats. Wild rodents were trapped from the farm belonging to one of the patient’s and a strain of Seoul virus, named Humber virus, was isolated from rats. Subsequent cases of AKI were in people with exposure to specially-bred pet fancy rats. Rats from one private breeding colony were tested and a second highly similar Seoul virus, named Cherwell virus, was described. Evidence for Cherwell virus was demonstrated in a human sample with genetic data of the virus recoverable from a serum sample. It was 100% identical to the pet rat strain, thus confirming SEOV as the causative agent of the patient’s AKI. These findings have implications for public health as Seoul virus is capable of causing moderate-severe human disease. The overarching aim of the thesis was to confirm hantaviruses cause human infection in the UK and raise clinical awareness; this was achieved.

This investigation examines the efficiency and mechanism of neutralization of influenza A/PR/8/34 virus by three haemagglutinin (HA)-specific monoclonal IgGs (H36, H37 and H9) and their Fabs. The efficiency of neutralization by the Fabs was lower than that of their IgGs. This was concentration-dependent: 50% neutralization (N50) required 104- to 375-fold more Fab than IgG, and at N$ > o required 15- to 208- fold more Fab than IgG. Affinities of the IgGs and Fabs were very similar and thus did not contribute to the difference in their neutralization efficiencies. The mechanism of neutralization of influenza virus by the IgGs was complex. At N90, the majority of the loss of infectivity was attributable to inhibition of virus attachment to cells, whilst at N50 inhibition of attachment accounted for only a minority of the infectivity loss, with the majority attributed to inhibition of fusion. In between N50 and N90 both mechanisms operate simultaneously. In contrast, inhibition of attachment by the H36 and H37 Fabs was the dominant mechanism of neutralization throughout the range of concentrations studied. The H9 Fab did not inhibit virus- attachment, but inhibited virus-cell fusion. Therefore, it was concluded that the difference between the IgGs and their Fabs was the ability of the IgGs to display simultaneous neutralization mechanisms. Post-attachment neutralization (PAN) provides compelling evidence of the ability of antibodies to neutralize by mechanisms other than inhibition of attachment. H36, H37 and H9 IgGs and their Fabs gave PAN at 4°C and 37°C. Efficiency of PAN by the IgGs was lower than the standard neutralization of free virus (STAN). The efficiency of PAN and STAN by the Fabs was similar, except for H9 Fab whose PAN activity was significantly greater than its STAN activity. The mechanism of PAN by both IgGs and their Fabs was inhibition of fusion. The HA of influenza A/PR/8/34 virus goes through a series of conformational intermediates in the fusion process. Examination of the pre-fusion intermediate (created by treating at pH 5 and 4°C) using conformation-specific MAbs detected changes in the viral HA, but these were less extensive than in virus treated at pH 5 and 37°C (post-fusion conformation). The pre-fusion intermediate does not cause fusion, but is a fusion-committed state, as it can subsequently undergo fusion at high efficiency at pH 7.5 when the temperature is raised to 37°C. IgGs and Fabs did not inhibit the haemolysis of CRBCs by the pre-fusion intermediate, nor did they inhibit the conformational changes of the HA that trigger the fusion process. It was concluded that these antibodies interfere sterically with events that occur prior to the formation of the pre-fusion intermediate. The point at which neutralization becomes irreversible was investigated using the three IgGs and a monoclonal IgA. Neutralization of virus in solution was shown to be reversible by removing antibody from the virus with ammonium hydroxide (pH 11.5) (AmOH). The reversibility of neutralization was also examined under conditions designed to mimic the early steps in the virus infectious cycle, (1) virus attachment to cells and (2) low pH-induced virus-cell fusion. Neutralization could be reversed by removal of antibody with AmOH, after neutralization of (1) virus attached to cells and (2) after low pH treatment of virus attached to cells. This suggested that these MAbs do not trigger a permanent loss of virus infectivity, but are required to be bound to the virus for the duration of the neutralization process.

a) History: Semliki Forest Virus (SFV) was originally isolated from a pool of 130 female Aedes abnormalis mosquitoes captured in Bundinyama, Uganda in 1942 (Smithburn and Haddow, 1944). b) Classification: SFV is classified as a member of the genus alphavirus, of the family Togaviridae (Matthews, 1982). It was originally described as an arbovirus ("arthropod-borne" virus) (Casals and Brown, 1954) since it infects vertebrates and is transmitted by mosquito vector. However, later studies showed that the biochemical properties of the arboviruses were variable. For example, some members of the famiy Bunyaviridae were originally classified as arboviruses and these have a genome of three (negative sense) RNA segments whereas SFV has positive sense genomic RNA. A large number of the arboviruses were found to be morphologically similar, sharing certain structural and biochemical characteristics. These were grouped together and termed togaviruses (toga - shroud, cloak, envelope) (Andrewes, 1970). Two major serologically unrelated genera of the Togaviridae were recognised and were termed group A and group B. These were later designated the genera alphavirus (e.g. SFV) and flavivirus (e.g. yellow fever virus) (Wildy, 1971. Later, two other genera of the are the rubiviruses cholera virus) and, family Togaviridae were also recognised and these (e.g. rubella virus) and pestiviruses (e.g. hog to date, there are also five unclassified viruses.

Hepatitis C Virus (HCV) infects hepatocytes, a liver cell type with complex polarity. Virus entry is a multi-step process that is dependent on a number of host proteins including CD81, scavenger receptor BI, and the tight junction proteins claudin-1 and occludin. CD81 is an attachment receptor for the virus, however, internalization is dependent upon CD81 association with claudin-1 to form a coreceptor complex. The presentation and diffusion kinetics of CD81 shows variable conformation between diverse cell types. Using live imaging I show that CD81 diffusion is regulated by cellular localisation and polarity. Whilst the addition of growth factors and cytokines can modulate infection these effects are not consistently reflected by changes in CD81 diffusion, suggesting that CD81 diffusion kinetics alone does not define HCV entry. I demonstrate that the diffusion of the claudin-1 co-receptor also plays a role in defining infection. In addition, I demonstrate a complex interplay between the coreceptors and EGFR signalling that may involve the rearrangement of membrane domains to promote virus entry.

The thymus is a heterogeneous mix of hematopoietic and stromal cells that function to generate a functional, self-tolerant T-cell pool. Although many of these populations are well studied, the role of non-epithelial stroma remains unclear. Thymic mesenchyme has been identified as an important regulator of T-cell egress. Studies of lymphotoxin beta-receptor LTβR have revealed its critical role in T-cell egress as well as the development and function of lymph node mesenchyme. We hypothesized that LTβR regulation of thymic mesenchyme is critical forT-cell egress. To test this we generated \(Wnt-1^{cre}Ltbr^{flox}\) mice to delete LTβR on thymic mesenchyme and revealed this to be non-essential forT-cell egress. Moreover, we generated \(Foxn-1^{cre}Ltbr^{flox}\) mice to delete LTβR on thymic epithelial cell (TEC). Despite the critical role of LTβR in medullary TEC development, T-cell egress was normal. However, deleting LTβR on thymic endothelium using \(Flk-1^{cre}Ltbr^{flox}\) mice revealed an essential role of LTβR regulation of endothelium to control T-cell egress. Our analysis also revealed that T-cell entry into the perivascular space during T-cell egress occurs stochastically. Collectively our findings highlight a novel role for LTβR regulation of thymic endothelium as a critical pathway of T-cell egress.

Hepatitis C Virus (HCV) is a clinically important infection that leads to chronic liver disease and Human Immunodeficiency Virus (HIV) co-infected patients have more rapid progression to severe liver disease and show higher rates of HCV vertical transmission. Hepatocytes are a highly differentiated cell type and support low level HCV replication. Most studies of the viral life cycle use de-differentiated hepatoma cell lines, which are highly permissive. The mechanism behind this difference is poorly understood. We show that dimethylsulfoxide (DMSO) differentiated Huh-7 cells have a 100-fold reduction in permissivity to HCV infection. We confirm that these cells are differentiated and upregulate key liver specific markers including miR122. They are metabolically active and have intact innate signaling pathways in response to infection. We observed a 10-fold reduction in the initiation of replication and a 10-fold loss in extra-cellular particle infectivity. In contrast cell-to-cell dissemination rates were comparable and cell-contact dependent infection of differentiated cells can overcome the restrictions seen in cell-free infection. HCV cell-to-cell transmission can also be mediated by other cell types. T cells are the primary cell supporting HIV-1 infection. We have shown that HCV can bind primary and immortalized T cells and trans-infect hepatoma cells. This requires replicating HIV but is independent of co-receptor engagement. HIV-1 infection of CD4+ T cells induces a significant increase in HCV trans-infection by increased viral binding. T cells provide a vehicle for HIV-1 to promote HCV infectivity, transmission and persistence.

A PSD95/Dlg/ZO-1(PDZ)-binding motif (PBM) in the E6 protein of high-risk, cancer-causing human papillomaviruses (HPV) targets a subset of cellular PDZ domain-containing proteins involved in diverse regulatory processes including cell polarity and proliferation, for proteasome-mediated degradation. Interaction with this select group of PDZ domain-containing proteins is negatively regulated by cAMP-dependent protein kinase (PKA) mediated phosphorylation of the E6 PBM. This thesis has sought to address the hypothesis that the PBM of E6 plays an important role within the HPV life cycle. This study has shown that deletion of the E6 PBM from HPV18 genomes affects the morphology and growth of viral episome-containing human keratinocytes and furthermore links E6 PBM function to viral episome replication (maintenance replication and differentiation-dependent amplification). Loss of negative regulation of the E6 PBM by mutation of the PKA recognition motif was associated with increased cell growth and indeed the growth of wildtype HPV18 genome-containing cells responded to changes in PKA signalling. Constitutive E6 PBM function was also associated with invasion of cells suggesting that malignant progression of HPV-infected cells may be linked to changes in PKA signalling. Modulation of the E6 PBM function in the viral genome-containing cells was associated with a change in protein levels of the PDZ domain-containing protein discs large (hDlg) and changes in the non receptor protein phosphastase PTPN13 specific species.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller’s and infantile diarrhoea in developing countries, and results in considerable mortality in under 5 year olds. Disease is mediated through adhesion of ETEC cells to the intestinal brush border and the secretion of the heat-stable and/or heat-labile toxin. Whilst the toxins have been well studied it is still not clear how their expression is regulated. This work has defined binding of CRP, H-NS and σ\(^7\)\(^0\) across the genome of the prototypical ETEC strain H10407. We demonstrate a central role for all three factors in regulating pathogenicity in ETEC H10407. Hence, we show that CRP directly regulates expression of both \(estA2\) and \(estA1\), which encode the heat-stable toxins. Furthermore, CRP indirectly represses expression of the heat-labile toxin. This work also identifies a role for CRP in controlling transcription of a small open reading frame, imbedded within a gene, at the 3’ end of an operon encoding a type I secretion system.

The E6 protein of oncogenic α-human papillomaviruses encodes a conserved C-terminal PDZ binding motif (PBM). Conservation of an overlapping kinase recognition site has shown that phosphorylation negatively regulates the E6:PDZ interaction. Many E6 PDZ targets are associated with signalling complexes that regulate cell proliferation and polarity. This study used primary keratinocyte-based models of HPV16 and 18 life cycles to investigate the expression of PDZ targets and the functional role of PBM phosphorylation. The data indicate that changes in total levels of major E6 PDZ targets are not associated with E6-PBM activity. Interestingly, transcription profiles of E6-PBM targets DLG1, PATJ and PTPN13 are dramatically changed in the presence of HPV16 and 18, whilst others remain unaffected. Further analysis of transcriptional changes of DLG1 revealed upregulation of specific alternatively spliced isoforms, including a novel isoform containing an exon previously thought to be intronic. This investigation revealed that phosphorylation of the PBM is linked to oncoprotein stability, presenting a potential regulatory mechanism of E6 PDZ interactions during the virus life cycle. Together, these data offer interesting new perspectives on interactions between oncogenic HPV types and PDZ domain-containing targets and indicate that deregulation of their function by the virus may occur through multiple mechanisms.