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The structure of the herpesviruses particle is characterised by an icosahedral capsid surrounded by a proteinaceous tegument layer and is enclosed by a lipid envelope. The understanding of the structure of the capsid, primarily through the use of cyro-electron microscopy, is greater to than of the tegument, due to the typically amorphous nature of the tegument. The interaction between the capsid and tegument has been well studied, unveiling interactions limited to the capsid vertices involving two minor capsid proteins, pUL17 and pUL25, and the large tegument protein pUL36. In herpes simplex virus – 1 (HSV-1), pUL17 and pUL25 form the capsid vertex-specific component (CVSC), a heterodimeric structure which resides in top of triplexes between peripentonal hexons. pUL36 has been suggested to connect the CVSC to the penton and to the rest of the tegument proteins. Recent studies on the gammaherpesvirus Karposi’s sarcoma-associated herpesvirus (KSHV) and the alphaherpesvirus pseudorabies virus (PrV) have questioned both the protein content of the CVSC and the organization of pUL17 and pUL25. As well as the composition of the CVSC, these studies have provided further insight as to the location of tegument assembly to the capsid, a subject that remains a highly contested. In order to clarify the content of the CVSC, virus mutants with deletions of the large inner tegument protein pUL36, and a second inner tegument protein, pUL37, were analysed using cryo-electron microscopy and icosahedral reconstructions. The examination and comparisons of these mutants with wild-type HSV-1 revealed that the CVSC is not only formed by pUL17 and pUL25 as originally reported, but also pUL36, as suggested in the most recent studies. In addition, comparisons of the capsid structure of the pUL36 deletion mutant with a mutant with a full deletion of pUL34, a protein implicated in nuclear egress as part of the nuclear envelopment complex (NEC) and therefore cannot exit the nucleus, suggest that at least part of pUL36 is present on nuclear capsids. Further analyses of the mutant virus with pUL36 deleted using immunofluorescence and Western blotting, along with the capsid reconstruction of a pUL36 deletion mutant which retains only the N-terminal 361 codons suggest that the C-terminal end of pUL36 is present in the nucleus. The work presented here offers additional evidence to clarify the roles of pUL17, pUL25 and pUL36 in tegument assembly. In particular, structural analysis has implied that the contributions of pUL36 to the nuclear capsid stabilizes the CVSC structure from a structural stand-point, emphasizing it’s importance as a multifunctioning protein which acts as a bridge between the capsid and tegument.

The spore forming bacterium, Bacillus anthracis is the aetiological agent of anthrax. The 2001 US anthrax letter attacks and the 2009‐2010 outbreak of injectional anthrax in the UK highlighted the importance of early detection and confirmation of this agent, both for patient outcome and forensic investigations. A reliable and consistent method was used in this study to safely simulate blood cultures with B. anthracis and used to determine the time to positive detection. This was performed with different strains and with varying concentrations of inoculum. An inverse linear relationship was observed with all strains and used to estimate the bacterial blood concentration of anthraxpatients based on data gathered from the literature and front‐line laboratories in the UK. The study explored a method to potentially reduce the turnaround times for the confirmation of B. anthracis at the national reference laboratory. Serum separator tubes were used to concentrate the bacteria from simulated blood cultures. A simple wash step was performed prior to performing confirmatory phenotypic tests and inactivation for rapid molecular detection. A comparison of test results with and without serum separator tube processing was made for B. anthracis and bacterial isolates referred during the outbreak of injectional anthrax. Simulated mixed blood cultures of B. anthracis and possible common contaminants were also tested. Compared to routine methods, confirmatory phenotypic test results were achieved 24 hours sooner using the method. The simple wash step and inactivation was sufficient to provide nucleic acid for molecular confirmatory assays and genotyping. A new ‘sample to answer’ platform, the Biofire Filmarray® was also trialled and correctly identified B. anthracis directly from simulated blood culture and provided results within one hour. Aspects relating to potential biosafety concerns for processing B. anthracis blood cultures were explored. The data generated suggests the aerosol risk is low for B. anthracis. Viability of material on microscopy slides was examined and the data supports the recommended use of alcohol fixation for slide preparation. There has been no previous evidence reported for sporulation occurring in blood culture bottles and the study findings suggest this is possible five days post positive detection. Interactive e‐learning modules have been produced to disseminate the study outcome. The e‐learning is intended for front‐line laboratories to raise awareness for the safe handling and laboratory identification of B. anthracis.

Neisseria gonorrhoeae is a public health concern due to increasing numbers of cases of gonorrhoea and the ability of the organism to develop antimicrobial resistance. N. gonorrhoeae can harbour β-lactamase plasmids that encode TEM-1 penicillinase, but do not produce extended spectrum β-lactamases (ESBLs). ESBLs are active against the last remaining option for gonorrhoea monotherapy, ceftriaxone. The aim of this research was to establish what resistance mechanisms in N. gonorrhoeae could abolish the effectiveness of ceftriaxone for the treatment of gonorrhoea. Investigations into the genetic diversity of blaTEM alleles in gonococcal isolates from 2012 detected a high proportion of blaTEM-135 alleles (27%). Only a single specific mutation near the β-lactamase active site could result in TEM-135 penicillinase evolving into an ESBL. Electroporation was established for the transformation of native gonococcal resistance plasmids into N. gonorrhoeae, and was then used in attempts to transfer the enteric plasmid pEK204 (harbouring blaCTX-M-3 and blaTEM-1) into gonococcal strains. Electroporation and natural transformation were additionally used to transform gonococci with blaCTX-M-3 and blaTEM-10 genes. Five transformants were detected using blaTEM-10 and these all showed increased minimum inhibitory concentrations of ceftriaxone. The lack of success in uptake of pEK204 and blaCTX-M-3 was probably due to large plasmid size and lack of recombination site, respectively. Nevertheless, gonococcal β-lactamase plasmids were successfully transferred into clinical strains of the multidrug-resistant clone N. gonorrhoeae ST1407, suggesting that this could also happen in natural mixed gonococcal infections. In summary, it is encouraging that no further blaTEM alleles were detected and that N. gonorrhoeae was not able to express a CTX-M-type ESBL. However, the expression of TEM-10 ESBL is concerning and this work is the first report of ESBL activity in gonococci, albeit in vitro. It is essential to continue antimicrobial susceptibility surveillance and to develop molecular surveillance to detect rapidly the emergence of an ESBL in N. gonorrhoeae.

Background: Chlamydia trachomatis is the commonest curable sexually transmitted infection in the UK. The prevalence is shared equally by men and women. A National Chlamydia Screening Programme (NCSP) has been introduced in England, supported by advances in testing technologies which enable non-invasive sampling methods to be used in non-healthcare settings. The NCSP tests nearly twice as many women as men and is more likely to test men in non-healthcare settings. Men are seen as an important, but difficult to reach group. Little is known about where men prefer to access testing and whether or not nontraditional settings, such as football clubs, are acceptable. Methods: 1) A national stratified random probability sample survey of men aged between 18 and 35 years resident in Great Britain, exploring attitudes to self-collected testing for Chlamydia, acceptability of venues to collect testing kits, health seeking and sexual risk behaviours. 2) Qualitative interviews with men who play amateur football. It explores the acceptability of three different, club-based, testing pathways; Health-care professional promoted; Peer-led promoted; and poster-led promoted. Results: Men are well engaged with existing health services and find selfcollected testing kits for Chlamydia highly acceptable. Healthcare settings are the most acceptable venues to access testing although sports settings are acceptable to a minority. Attitudes to testing in football clubs are influenced by factors relating to men’s characteristics, promoter characteristics and the impact of testing on time and effort involved. Conclusions: Whilst non-healthcare settings can be used to reach some men for Chlamydia testing, existing services are already well accessed and offer considerable opportunities to test more men. More should be done to ensure men are able to access testing within the context of daily living, without significantly impacting on the time needed to pursue their main interests.

The purpose of this multi-partnered project was the production of a fully integrated POC system, combining automated nucleic acid extraction in a centrifugally operated microfluidic disk (the LabDisk), with loop mediated isothermal amplification (LAMP) and optical detection, capable of detecting the sexually transmitted pathogens Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis in clinical urine and swab samples. LAMP is a novel nucleic acid amplification method, designed to amplify target nucleic acid in a highly specific and rapid manner, under isothermal conditions. The work detailed in this thesis presents the development of a rapid total nucleic acid extraction process, based on the capture of target nucleic acid by magnetic silica beads, optimised for use on the LabDisk platform. The extraction process was capable of the purification of target nucleic acid from a clinical sample within 5 minutes, and was robust when challenged with a range of inhibitory compounds potentially encountered in samples for STI testing. The system was capable of tolerating N. gonorrhoeae (1 x 105 CFU/ml) urine suspensions containing samples containing 50% total blood volume, 1x108 E. coli cells per ml, and 10mg/ml of BSA, without any effects on the downstream amplification time of the N. gonorrhoeae specific LAMP assay. A freeze dried lysis buffer pellet was developed, that was able to increase the sample volume, thereby decreasing the time to detection, whilst minimising the stored fluid volume on the LabDisk. LAMP assays were designed for the detection N. gonorrhoeae and M. genitalium, and the limits of detection and specificity of the assays were evaluated. The N. gonorrhoeae ORF1 assay was able to detect a minimum of 20 copies of the N. gonorrhoeae genome per reaction, whilst the M. genitalium pdhD assay was capable of detecting 16 genome copies. The tolerance of the ORF1 LAMP assay to urea, and blood, was found to be 1.8M, and 20% reaction volume, respectively. The increased tolerance of the LAMP assay to these inhibitors in comparison to PCR demonstrates the suitability of LAMP when processing urine samples for STI’s. To our knowledge this is the first application of LAMP technology for the detection of these organisms, and the first attempt at commercialising a fully integrated molecular diagnostics system based on LAMP.

Background: Despite the acknowledged importance of economic assessments for public health interventions at the human-animal-ecosystem interface, there are currently limited economic methodologies for doing so. In this thesis studies were undertaken to ascertain the economic impact of interventions to control trypanosomiasis and taeniasis/cysticercosis in south-east Uganda and northern Lao PDR respectively. Also, in Uganda studies were done to find out if demand of draft cattle would be an important economic driver for spreading trypanosomiasis due to inter-district trade. Method: In Uganda, a one year recall cross-sectional baseline survey and an 18 month longitudinal survey of 660 households was conducted; to determine the benefits and changes due to restricted application of deltamethrin insecticide to only the legs, belly and ears of cattle. During the 18 month study, the households participating in the study were divided into six regimes depending on the type of intervention done in their cattle and these were; diminazine injection only, deworming only, no treatment and those had 25%, 50% and 75% of the total village cattle sprayed. Thus, the first three regimes were those households that had their cattle not sprayed with insecticide at all as opposed to the last three. Additionally, cattle trade data was collected for network and value chain analysis in all markets in Tororo and Namutamba districts from 199 cattle traders. In northern Lao PDR, stochastic modelling was done to determine the burden of neurocysticercosis associated epilepsy and soil transmitted helminthes. A cross-sectional study was carried out in 49 households, focusing on the prevalence of cysticercosis and soil transmitted helminths before and after a twelve month intervention to control a hyperendemic focus of Taenia solium. The village data was then extrapolated to the wider northern Lao PDR population. Results: The Uganda study indicated that the restricted application of deltamethrin in cattle induced change of USD 31 per head of adult bovine per year; this was the change in income that directly occurred due to restricted spraying of cattle with deltamethrin. During the intervention period, the annual difference in income between those households that had their cattle sprayed using restricted application protocol and those that did not was USD 123; and this was significant (t= 7.18, p= < 0.001). Analysis of variance using households that had their cattle receive no treatment as control showed that restricted application of deltamethrin significantly increased household income compared to diminazine aceturate injection and deworming of cattle only. The incremental benefit cost ratio of spraying 0% to 25% of the cattle was found to be the highest (16:1) compared to spraying 25% to 50% (3:1) and 50% to 75% (1:1) of the cattle. Cattle trade network and value chain analysis revealed that the key cattle markets from which trypanosomiasis is likely to spread into Tororo District are Molo, Namutumba and Soroti. Also, it was found that the risk of spread of human African trypanosomiasis from south-east to north-west Uganda is high due to the increased demand for male cattle for draft work. In northern Lao PDR, 5,094 (95% CI: 25.6-28,940) DALYs were estimated to be imposed annually due to Taenia solium associated epilepsy, with 446.4 (95% CI: 2.2- 2,536) DALY imposed per 100,000 person-years. Due to the high benefits to pig production, the net monetary cost per DALY averted for simultaneously controlling T. solium, soil transmitted helminthes and classical swine fever was only USD 14, which fell to USD 11 if the separable cost method were applied. If the intervention did not target pigs, then the cost per DALY averted was USD 44; well below the current standard for ’very cost effective ‘of the 1 year’s per capita GDP. Conclusion: This study provided empirical evidence for evaluating the impact of quantifying the benefits of controlling zoonotic diseases in the livestock sector (Uganda case study) and in both livestock and human health populations (Lao PDR case study); this economic assessment approach can be used for planning future integrated health interventions. The results of this study support the policy of preventing the spread of infection by spraying at least 25% of the cattle using RAP, as well as injecting all cattle in key livestock markets in south east Uganda with diminazine aceturate to prevent HAT. In northern Lao PDR, simultaneous control of T. solium, soil transmitted helminths and classical swine fever is the most cost-effective approach. There are still difficulties in incorporating human and animal parameters into a single analytical framework; consequently there is a need to adapt the approaches undertaken in this study to the analysis of other zoonotic diseases in different settings to improve on their robustness.

Medical cartography can provide valuable insights into the epidemiology and ecology of infectious diseases, providing a quantitative representation of the distribution of these pathogens. Such methods therefore provide a key step in informing public health policy decisions ranging from prioritising sites for further investigation to identifying targets for interventions. By increasing the resolution at which risk is defined, policymakers are provided with an increasingly informed approach for considering next steps as well as evaluating past progress. In spite of their benefits however, global maps of infectious disease are lacking in both quality and comprehensiveness. This thesis sets out to investigate the next steps for medical cartography and details the use of species distribution models in evaluating global distributions of a variety of zoonotic diseases of public health importance. Chapter 2 defines a methodology by which global targets for infectious disease mapping can be quantitatively assessed by comparing the global burden of each disease with the demand from national policymakers, non-governmental organisations and academic communities for global assessments of disease distribution. Chapter 3 introduces the use of boosted regression trees for mapping the distribution of a group of vector-borne diseases identified as being a high priority target, the leishmaniases. Chapter 4 adapts these approaches to consider Ebola virus disease. This technique shows that the West African outbreak was ecologically consistent with past infections and suggests a much wider area of risk than previously considered. Chapter 5 investigates Marburg virus disease and considers the variety of different factors relating to all aspects of the transmission cycle that must be considered in these analyses. Chapters 6 and 7 complete the mapping of the suite of viral haemorrhagic fevers by assessing the distribution of Crimean-Congo haemorrhagic fever and Lassa fever. Finally, Chapter 8 considers the risk that these viral haemorrhagic fevers present to the wider African continent, quantifying potential risk of spillover infections, local outbreaks and more widespread infection. This thesis addresses important information gaps in global knowledge of a number of pathogens of public health importance. In doing so, this work provides a template for considering the global distribution of a number of other zoonotic diseases.