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The rail industry needs to address a number of important operational challenges in the foreseeable future. First of all, the safety of rail transport needs to be maintained at an absolute maximum matching the achievements of the European airline industry of zero fatalities. Secondly, promote sustainable growth to support increasing demand for both passenger and freight rail transport. Thirdly, support the implementation of measurable innovations and improvements that help increase capacity of current infrastructure through enhanced availability. Finally, maximise the environmentally benign character of railway transport through exploitation of novel technologies such as hydrogen trains and advanced electrification employing renewable energy sources. This project, primarily focused on the UK Rail infrastructure, investigated the benefits arising from a holistic approach in the application of Remote Condition Monitoring (RCM) as a critical means for the accurate, efficient, reliable and cost-effective evaluation of key railway infrastructure assets and rolling stock. This work involved the use of several techniques and innovative methodologies based primarily on Acoustic Emission (AE) and vibration analysis in order to address the evaluation requirements for different components of interest. The results obtained have been very promising and present rail infrastructure managers and rolling stock operators with new opportunities for improved and more reliable operations. This work has led to the instrumentation of multiple sites across the UK rail network enabling measurements to be carried out on various assets under actual operational conditions. At Cropredy an integrated high-frequency vibro-acoustic RCM system has successfully been installed on the Chiltern railway line on the way from London to Birmingham. This customised system has been fully operational since 2015 measuring more than 200 passenger and freight trains every day moving at speeds up to 100 miles per hour (MPH). Prior to the installation of the system at Cropredy a Certificate (PA05/06524) of Acceptance was issued by Network Rail which after being renewed recently is now valid until September 2021. The system is due for an upgrade in the following stage of development, employing wireless sensors and advanced energy harvesting devices which are being developed under a collaborative Engineering and Physical Sciences Research Council (EPSRC) project between Exeter and Birmingham Universities, Network Rail, Swiss Approval UK and Quatrro. The widespread implementation of the techniques and methodologies researched will give rise to significant potential impact with respect to the effectiveness of maintenance strategies, particularly in terms of cost efficiency, improved availability of railway assets and better planning of available resources. As modern rail transport moves towards 24-hour railway, the inspection, maintenance and track renewal and upgrade regime will need to be re-thought at a fundamental level. Effective RCM will be a key factor in realistically enabling true round the clock operations. The results presented in this thesis have been part of a six-year research effort with a clear focus on addressing the true industrial need. The findings of this work have led to a re-think within Network Rail regarding the new possibilities arising from the effective use of RCM in designing and implementing more efficient and cost-effective railway operations whilst helping reduce the cost. The use of autonomous sensing systems in the future will change the inspection and maintenance strategies currently used shifting towards a truly prognostic operational strategy.

The thesis identifies goals of proportionate dispute resolution (PDR). The operation of the decision-making and dispute resolution processes relating to the provision of remedial help for children with special educational needs (SEN) is examined to assess attainment of these goals. A factual basis is established for the analysis by describing the relevant legislative provisions and what is known about their operation from existing empirical research. The exercise of discretion is examined. A theoretical framework is devised to enable consideration of the balance of trade-offs; collective goals and individual interests; and adequacy of redress. The SEN decision-making and appeals processes are analysed with reference to this framework. Parties to SEN disputes are parents and Local Education Authorities (LEAs). Attainment of PDR goals by the formal SEN dispute resolution mechanisms is assessed and the mechanisms compared. In 2002, obligations were imposed upon LEAs to provide informal disagreement resolution services in the form of conciliation and mediation. The strengths and weaknesses of these dispute resolution models are considered with reference to theoretical and empirical works. The effect of their introduction is then assessed with reference to the framework and attainment of PDR goals. None of the formal or informal dispute resolution mechanisms assure attainment of all of the PDR goals. Neither does the operation of the system as a whole. Analysis of the children's services complaints model using the framework reveals that this model assures attainment of all PDR goals and affords adequate redress. The model appears to resolve problems identified in the SEN dispute resolution process, and to be a promising candidate both for reform of that process and for a unified system of education and children's services complaints. The role of children in the process and possibilities for one-door access and a single system are considered.

Supernovae, the explosive deaths of certain classes of stars, are among the most broadly significant phenomena in astrophysics, contributing to our understanding of an enormous range of topics including the origin of chemical elements as well as the basic composition and ultimate fate of the Universe writ large. Despite this, many open questions remain concerning both the detailed physics that underlies the explosions themselves and the stellar systems that can give rise to them. In this dissertation I present a series of four observational studies that help to answer some of these questions, using statistical samples to constrain the origins of thermonuclear supernovae and probe the progenitor distribution of core-collapse supernovae, and performing in-depth case study analyses to better understand the unusual events ASASSN-14ms and ASASSN-18tb. I utilize a variety of data sources, but this work relies most heavily on images obtained by the All-Sky Automated Survey for Supernovae (ASAS-SN) and the Transiting Exoplanet Survey Satellite (TESS) and on spectra obtained by the Large Binocular Telescope (LBT) and the South African Large Telescope (SALT).

Background: Adenoviruses are an endemic cause of human infection with 104 types across 7 species currently identified. Clinical syndromes vary but infection in transplantation can be severe. Deficiencies in current classification methods are apparent as the number of novel types continue to grow. Whole genome sequencing is employed in investigation of pathogenicity, drug resistance, transmission dynamics and nosocomial outbreak investigation but is rarely used in human Adenoviruses. Bioinformatic processing of sequencing data is particularly challenging as no commercial solutions or best practice guidelines currently exist. This project aimed to develop a method for direct enrichment of Adenovirus DNA from clinical samples for whole genome sequencing alongside generation of a validated open access pipeline for data analysis to update current knowledge of Adenovirus epidemiology. Method: A custom Agilent SureSelectXT RNA oligonucleotide Target Enrichment system was designed for adenovirus enrichment direct from faecal, blood and respiratory tract secretions for subsequent illumina sequencing. Minor modifications were made to the SureSelectXT low input protocol to improve suitability for non-human applications. A bespoke three stage Unix coded bioinformatics pipeline was constructed and validated through direct comparison of open-source tool performance. Illumina sequencing data from multiple Adenovirus types was used to determine suitability of quality trimming parameters, genome assembly and variant calling tool for inclusion. Genomic similarity of isolates was assessed at a whole genome and classical typing gene level phylogenetically and through pairwise distance models to assess current adenovirus epidemiology. Results: Adenovirus whole genomes could be enriched directly from various clinical sample matrices with a success rate of 94%. Sequence data quality trimming using Trimmomatic did not impact whole genome sequence generation regardless of parameters used. No significant difference was observed in genome assembly using BWA-MEM and Bowtie2 however variant calling tool performance was more inconsistent with LoFreq reporting the most variants. Starting concentration of Adenovirus DNA heavily influenced enrichment success where Adenovirus species or type did not. Species C Adenovirus most commonly found at 49% closely followed by B and A. Genetic differences between Adenoviruses of the same type were not always apparent from classical typing gene assessment but required whole genome analysis. Higher sequence divergence between Adenovirus types than previously observed was found during assessment of the hexon, penton and fiber genes Conclusions: This modified custom SureSelectXT protocol is a robust method for generating Adenovirus whole genome sequences from clinical samples and suitable bioinformatic pipelines can be generated using only open-source tools. Adenovirus epidemiology in the North West has remained largely unchanged however phylogenetic analysis has highlighted deficiencies in current typing methods and confirms the need for whole genome analysis in outbreak investigation.

Pressure ulcers are one of the most common secondary complications for people with spinal cord injury, and add $10 billion annually to healthcare costs in the US. They are the most frequently seen preventable hospital acquired condition. Recent pressure ulcer research has added examination of anatomical risk factors, mainly fat and muscle characteristics, to the many previously identified risk factors. Translation of the new anatomical-based risk assessment theories is contingent on development of clinical techniques for measurement and better understanding of relationships with known factors, which has slowed the integration of this research into clinical settings. This study was designed to help bridge this gap between lab and clinic, by examining how anatomical features affect both tissue deformation and interface pressure. Six participants – two control and four with spinal cord injury – underwent MRI imaging while seated on a variety of seat cushions and in an unloaded condition, as well as pressure mapping. Three dimensional models of the tissue were created from the images. Significant anatomical differences were observed between the two groups. People with SCI lack muscle under the ischial tuberosity when sitting. The results suggest that tissue thickness was the anatomical feature most indicative of pressure ulcer risk. Greater unloaded thickness was associated with lower interface pressure and less change in tissue volume under seated loads, signifying a decrease in pressure ulcer risk. Higher deformation asymmetry – an imbalance between the change in tissue volume from one side of the buttocks to the other – also suggested increased pressure ulcer risk. Deformation asymmetry is particularly important because it can be partially corrected by adjusting sitting posture. These important characteristics should be used to direct further efforts to implement a more personalized risk model based upon the anatomy of the tissues at risk of breaking down.

Very little is known about the origins, evolution and resistance dynamics of human mastadenovirus (HAdV) infection. Despite an association with significant morbidity and mortality amongst the immunocompromised population, current treatment strategies have a weak evidence base. Existing diagnostic tools are inadequate for influencing management decisions or infection control strategies. Furthermore, a recent surge in HAdV cases, amongst a haematopoietic transplant population, including five deaths, led this study to use whole genome sequencing (WGS) to investigate. To gain a complete transmission picture for the first epidemiology chapter, outbreak and non-outbreak HAdV sequences (54 sequences from 37 patients) were compared with GenBank sequences and a database of previously sequenced HAdVs. An improved bait set for WGS was used. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships paired with epidemiological data from routine Infection, Prevention and Control (IPC) activity. During this time period two formal outbreaks had been identified by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single nucleotide polymorphisms. One of the formal IPC outbreaks was confirmed, one was not. Of the five HAdV-associated deaths, three were unlinked, the remaining two considered the source of transmission events. Existing antivirals used to treat HAdV infections target the DNA polymerase of the virus. Immunosuppressed patients, particularly children, are susceptible to overwhelming HAdV infection. The subsequent chapter of this thesis interrogates variants identified within the DNA polymerase. Linking sequence data with patient data and published mutations, the association between antiviral therapy response/failure and mutations is described. None of the previously reported confirmed or putative resistance mutations were identified in this patient cohort. The clinical consequences of unmitigated HAdV transmission events are high. Focusing on two high risk wards using WGS, this study identified six transmission events that, over prolonged periods, would have gone unnoticed using traditional polymerase chain reaction and epidemiology. Mixed infection is frequent (10% of patients), providing a sentinel source of recombination and superinfection. Prolonged HAdV viraemia is associated with poor outcomes; two of the ten patients failing to respond to therapy in this study did not survive. A few HAdV-C5 and for the first time HAdV-A31 mutations warrant further investigation to confirm or refute their association with antiviral resistance. Resistance is likely to be multifactorial but existing diagnostic methods fail to provide the reassurance that toxic antivirals should be withdrawn or continued. Immunosuppressed patients harbouring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of this study, HAdV WGS is being incorporated routinely into a clinical algorithm to monitor antiviral resistance, prevent transmission and influence IPC policy in real-time.

Background: Human cytomegalovirus (HCMV) is the most prevalent cause of congenital infections, non-hereditary sensorineural hearing loss and other serious neurological sequelae in childhood. Predicting the consequences of an in utero infection is hindered by the lack of a deep understanding of the pathogenesis of congenital HCMV infections. Viral and host factors may influence the outcome, and viral factors may include variability in genes encoding of the essential envelope glycoproteins (gB, gM, gN, gH, gL, gO and gUL128-gUL131A). Objectives: The aims of the current study were to (1) develop a deep-sequencing-based method to genotype HCMV glycoproteins in single and mixed genotype infections and (2) assess the genetic polymorphism of these glycoproteins in children with confirmed congenital and unconfirmed congenital or postnatal infections, immunocompromised patients and immunocompetent individuals to investigate whether specific genomic variants may be correlated with different forms of HCMV disease. Methodology: A multiplex polymerase chain reaction (PCR) coupled with MinION nanopore sequencing and a novel bioinformatics pipeline was used. Results: The ability of the protocol to detect single and mixed genotypes was evaluated using five HCMV laboratory strains and mock mixed samples that were prepared from these strains. This method accurately detected all the expected single genotypes in the five laboratory strains and low-frequency minor genotypes down to the abundance of 1-10% in the mock mixed infection samples with a sequence accuracy of > 99%. The genomic variants of the glycoprotein genes were investigated in 109 clinical isolates from the 3 clinical groups. To verify the sequencing results, a subset of the samples was also subjected to the restriction fragment length polymorphism and direct sequencing methods. The developed protocol detected HCMV with a limit of detection of 102-103 IU/ml with an accuracy of > 99%. The sequencing protocol detected some or all of the glycoprotein genes in 109 samples and differentiated the glycoprotein gene sequences at the subtype level. gB.1, gM.2, gN.4 (4d), gH.2, gL.4 and gO.1 (1a) were the most dominant single genotypes in the group of children with congenital/postnatal infections, and a significant correlation was only found for gM.2 and gH.2. While gB.1, gM.3, gN.4, gH.1 and gH.2, gL.4 and gO.2 were prevalent genotypes in the immunocompromised group, gB.1, gM.1, gN.1 and gN.4, gH.1, gL.4 and gL.3 and gO.1 were found in the immunocompetent group. Significant associations were found only for gM.3 and gO.2 in the immunocompromised group and only for gO.1 in the immunocompetent group. The sequences of the UL128, UL130 and UL131A genes demonstrated low levels of amino acid variability among a subset of 39 clinical isolates (0.6-3%, 0.5-2.4% and 0.8-1.6%, respectively). Variabilities were phylogenetically grouped. High rates of mixed genotypes were detected in all studied groups. Conclusion and future directions: Using a novel multiplex PCR and deep sequencing, it was found that high rates of mixed HCMV infections frequently occurred in children with congenital/postnatal infections and the other studied groups. The HCMV strains causing such infections were extremely diverse. The study results suggest that the incidence of mixed infections has been underestimated in previous studies. Understanding the role of mixed genotypes in influencing replication within the host could aid the development of a virologic prognostic marker of the consequences of congenital infections.

Hepatitis C virus (HCV) is a major worldwide health issue, infecting approximately 71.1 million people worldwide, with 143 000 thought to be chronically infected in England. The vast majority of these (92.1%) infections are found in injecting drug users. A major breakthrough in screening has been achieved in the past few years with the introduction of increased screening for HCV, specifically targeting the injecting drug user population. Dried blood spots were shown to be a suitable sample type to use in these circumstances, overcoming previous issues caused by poor venous access and limited availability of sufficiently trained phlebotomists and increasing testing in this population. Despite the improvements brought about through the use of this sample type several issues still remain including: - A large population of injecting drug users are still unaware of their hepatitis C infection. - Testing from dried blood spots uses commercial CE marked HCV serological assays, but these assays are not CE marked for use with dried blood spots. - Optimisation of commercial assays has increased the sensitivity achievable from dried blood spots to a high level (97-100%) this does not match that available from serum samples. Only an assay specifically designed for dried blood spot samples will be able to increase the sensitivity past what is currently achievable. - Reliable identification of acute HCV infection is not currently possible. The HCV Core, NS3, NS4, NS5a and NS5b proteins were produced using flashBAC™ ultra baculovirus expression system. Low protein yield in NS4, NS5a and NS5b protein production was overcome with the use of a baculovirus transfer vector incorporating an anti-apoptotic vankyrin expression cassette. Proteins were purified using immobilized metal affinity chromatography. Solubility issues in the NS4, NS5a and NS5b proteins were overcome using urea or guanidine based solubilisation. Purified proteins were optimised in individual ELISAs, and their performance assessed for the detection of anti-HCV in both serum and dried blood spot samples. Results from the individual ELISAs were combined to give a multi analyte array type profile. Performance for detection of anti-HCV in serum samples was high, with a sensitivity of 91.6% while maintaining a specificity of 100%. Performance with DBS samples was higher, giving a sensitivity and specificity of 100%. This matched the performance of the commercial Roche Elecsys® anti-HCV assay and improved on the performance of the commercial Ortho anti-HCV assay for screening of anti-HCV from DBS samples, while removing the need for anti-HCV confirmation testing in the majority (93.5%) of samples.

Polyomavirus-associated nephropathy (PyVAN) occurs in recipients of kidney transplantation. The development of PyVAN can cause a decrease or complete loss of the function of the kidney. Two of the human polyomaviruses BKPyV, JCPyV are known to be involved in the pathogenesis of PyVAN, and a further virus HPyV9 has been found in renal transplant recipients. A multiplex quantitative polymerase chain reaction (qPCR) for BKPyV, JCPyV, and HPyV9 DNA, together with an internal control molecule, was used to investigate polyomavirus infection in the late transplant period. Results for BKPyV, JCPyV were reported in IU/mL. The dynamic range, linearity and reproducibility of the assay was good (intra- and inter-assay variation for each viral target of < 1% or less, with a wide range of target concentrations). In a cohort of 466 renal transplant recipients investigated late after transplant, blood samples from 27 were positive for BKPyV with a viral load of between log 2.97 IU/ml to log 8.48 IU/mL; 15 were positive for JCPyV with generally lower viral loads (log 3.55 and 6.56 IU/mL); 4 were HPyV9 positive but with only very low viral load (log 0.42 to log 1.25 copies/mL). The age range, sex, and ethnicity of positive patients were similar to those of the population from which the samples were drawn. BKPyV was detected most frequently (in 27 of the 466 patients; 5.8%), the BKPyV positive samples tended to be collected earlier after transplantation, with a median of 2.33 years (maximal follow up post-transplant was 34 years). Fewer patients were JCPyV positive even though the JCPyV positive blood samples tended to be even earlier after transplant (median collection time of 0.38 years). The low rate of detection and low viral load detected in HPyV9 positive samples does not support the role of HPyV9 in disease causation both early and late after transplant. The VP1 proteins of BKPyV, JCPyV and HPyV9 were produced in insect cells using a baculovirus protein expression system using pOET8.VE2 transfer vector with flashBAC™ expression system and purified using nickel chelate purification of Histidine tagged proteins. Preliminary analysis of the protein was made in ELISA, but additional optimisation of expression and purification of the antigens will be required for use in the serological investigation of polyomavirus infection.

The purpose of the present study was to explore whether the phonological encoding difficulties that have been demonstrated in children who stutter persist in adults whose stuttering persists. This hypothesis was investigated by comparing the phonological working memory of adults who stutter (AWS) and adults who do not stutter (AWNS) using non-word repetition and phoneme elision tasks. Twenty-four adults (age range = 17;9 to 46;11 mean age = 28;2): 12 AWS and 12 AWNS matched on gender and age participated in this study. A total of 48 non-words consisting of an equal number (N = 12 per syllable length category) of two-syllable, three-syllable, four-syllable and seven-syllable non-words were selected for use in the non-word repetition and phoneme elision tasks. In the non-word repetition task, results showed a significant interaction between fluency group and syllable length for the 7-syllable length category only, indicating that AWS require a significantly higher mean number of attempts than AWNS. Results of the phoneme elision task revealed a significant main effect for syllable length with both groups demonstrating a significant reduction in accuracy as the non-words increased in length, but there was no significant interaction between fluency group and syllable class length. Potential implications of these findings are presented along with recommendations for future research.

Background: Intravenous drug use remains the single biggest risk factor for the acquisition of Hepatitis C. The majority of centres in the UK will not treat those who are actively injecting drugs or have only very recently stopped. The main barrier to treatment is a perception that the patient will simply become reinfected. There is no firm data to support this and it is this gap in knowledge that this project will address. Aims and Objectives: Firstly, to determine the reinfection rate in a cohort of drug users (current and past) with Hepatitis C who have been previously treated and cured. Secondly, to determine long term outcomes in those who injected drugs at the time of treatment and compare this to past injecting drug users. Methods: This is a prospective study looking at reinfection rates based on a cohort of intravenous drug users that have been previously treated and achieved a sustained virological response (SVR). The HCV database at North Manchester Hospital of all patients started on treatment since 2004 was accessed. Those who were injecting drugs in the 6 months prior to treatment including those who continued to inject during treatment were identified. These are the 'current' drug using group. The other group of patients that stopped injecting drugs at least 6 months prior to commencing treatment were also identified. These are the 'past' drug using group. The sample size was 60. The patients were followed up for at least 2 years to collect data on outcomes including reinfection rates using annual HCV RNA (with subsequent genotyping if positive) and liver disease progression using standard liver function blood tests and a Fibroscan (a non-invasive method to assess liver fibrosis and cirrhosis). Quality of life data was also collected at each visit. Results: The total duration of follow up post SVR in this study was 248.6 years. There were two cases of reinfection. The rate of reinfection in all participants was 0.80 cases per 100 person years (95% confidence interval [CI], 0.10 to 2.88). The mean MCS scores were found to significantly decline (p=0.001) in those who were non cirrhotic, not reinfected and not consuming alcohol in excess. Further research is needed in this area.

HCMV is a common viral pathogen that infects most of the world's population by early adulthood. It is typically asymptomatic in immunologically healthy individuals but causes severe disease in immunocompromised patients and congenitally infected infants. HCMV glycoproteins are highly polymorphic, and various types of associations have been suggested between glycoprotein types and the pathogenicity of the virus. Several studies on viruses other than HCMV have related the glycosylation of the viral glycoproteins to virulence. This project aimed to determine whether there is a robust relationship between the individual glycoprotein sequence and its glycosylation, how this influences the growth characteristic of the virus and whether this is related to its pathogenicity. Glycosylation patterns of 89 clinical specimens of different infection categories and specimen types were correlated with genetic sequence alterations of the virus glycoproteins (gB, gH, gL, gM, gN, gO), followed by determining whether mutation results in specific changes in glycosylation. The aim was approached using a cell culture model and a quantitative lectin-based assay (ELLA). A significantly increased glycosylation level for the following genotypes: mixed gH, gN4a, gO4, mixed gL was detected. Whereas a decreased pattern was found to be associated with gH1, gH2, gN3a, gO1a and gL2 genotypes (P < 0.05). Glycoproteins of strains isolated from respiratory specimens were significantly highly glycosylated compared to the blood and urine samples, and from blood specimens compared to the urine samples (P < 0.05). Furthermore, strains from congenitally infected infants and urine samples had a significantly higher growth rate than others tested. No direct association between the virus growth and its virulence was found. These findings demonstrate that glycosylation of glycoproteins in HCMV is affected by the glycoprotein polymorphisms and signifies a potentially important mechanism for avoidance of antibody-mediated neutralization, which, in turn, facilitates HCMV pathogenicity. This phenomenon requires further study and may have application for the selection of novel targets for diagnosis, vaccine development and other preventive measures to combat diseases caused by this virus.

Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV.

Human Cytomegalovirus (HCMV) is the most common cause of viral congenital infection in the world. Around 5-10% of HCMV infected children are symptomatic at birth, and 50-90% of these develop severe manifestations with a 30% mortality rate. Among the asymptomatic children at birth, 10-15% develop late sequelae. The major cell entry glycoproteins of HCMV form three complexes: gC-I containing gB; gC-II containing gM & gN; and gC-III containing gH, gL, and gO (or UL128-131). These entry glycoproteins are polymorphic, producing different glycoprotein genotypes. The polymorphic nature of the glycoproteins as well as their ability to elicit neutralizing antibodies made them of interest in correlating them with the severity and outcome of the disease. This study aimed to develop a robust system to identify clusters of glycoprotein genotypes and to correlate them with disease manifestation. PCR assays of high sensitivity were used to identify all six glycoproteins. The PCR products were digested using restriction enzymes (RFLP) to identify the glycoprotein genotypes. Available laboratory strains (AD169, Towne, Davis, Toledo, and Merlin) as well as 112 clinical samples were amplified and genotyped using the assay, and their glycoprotein genotype profile was determined. A reliable sensitive assay was successfully developed to identify all glycoprotein genotypes including a novel gM assay using PCR/RFLP. The clinical samples were grouped according to disease manifestation (Group 1: congenital/postnatal patients (subgroup 1A: confirmed congenital patients & subgroup 1B: patients with either congenital or postnatal infection), Group 2: immunocompetent patients, Group 3: immunocompromised patients (subgroup 3A: immunocompromised patients with primary infection, subgroup 3B: immunocompromised patients with recurrent infection & subgroup 3C: immunocompromised patients with unconfirmed primary or recurrent infection)). Genotype gB1 was found predominantly prevalent in congenital/postnatal and immunocompromised patients, while gB3 was the most common genotype in immunocompetent patients. This result along with the phylogenetic analysis performed in this study suggest a relationship between gB genotypes and the immune response of the patients, where gB3 may be positively selected by host immune pressure. The novel gM assay genotyped the highly conserved gene (UL100) into three distinct genotypes; gM3 genotype associated with the congenital/postnatal group; which may provide an insight into understanding viral attachment and spread into the host cell. In congenital/postnatal infection, gH1 (72.7%) and gL4 (65.1%) were the most prevalent genotypes (gH1= 32/44, gL4= 28/43; P=0.000). In immunocompetent patients, mixed gH and mixed gL genotypes significantly correlated with the group, and in the immunocompromised group gH2 and mixed gL genotype were the most common genotypes (51.1% and 46.9% respectively). Glycoproteins gO, gH and gL are components of gC-III complex and gO1 was found to be the most prevalent gO genotype in all infection types (Group 1= 32.1%, Group 2= 85.7%, Group 3= 18.8%; P < 0.05). Also, in congenital/postnatal infection gN and gO were found to significantly link with each other and this is expected since both glycoproteins are highly polymorphic and are located on adjacent gene loci in HCMV genome (gN1+gO1a (P=0.000), gN3a+gO4 (P= 0.000)). The specific gN-gO linkages found here could be potential indicators for congenital/postnatal infection. In congenital/postnatal infection group, gH had significant linkages to gN and gO (gH1+gN1 (P=0.023, gH1+gO1a (P=0.013)) suggesting that interlinked selection of glycoprotein genotypes in the gC-II and gC-III complexes is involved in the development of congenital infection. High viral loads were found trending with immunocompromised patients, while low viral loads were significantly associated with mixed infected patients. This study has shown significant associations between a number of glycoproteins and congenital infection. Previously ignored glycoproteins gM and gL have been shown to be potentially of significant interest in this study and a larger study to confirm this is needed. In most cases the pattern of glycoprotein genotypes in congenital infection is more similar to that of immunocompromised than immunocompetent patients and it is possible that immune pressure is selecting for or against particular glycoprotein genotypes. The relationship between mixed infection and sample type may offer opportunities for development of prognostic biomarkers for congenital disease and further work is warranted.

To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

The development of a safe and effective HIV vaccine remains challenging due to its high antigenic variability. Poxviruses are large, stable, and have a track record of use as human vaccine candidates. Recombinant fowlpox virus 9 (rFP9), a highly attenuated host range-restricted poxvirus strain, has been safely administered to humans with no ill effects, and is known to be immunogenic. This thesis describes the construction of complex rFP9 encoding various sequences of SIVmac239. The SIVmac239/macaque model is widely used for HIV vaccine development. The ultimate aim of this work was to combine the advantages of FP9 with those of live attenuated SIV to produce a safe yet hopefully effective model HIV vaccine candidate. Transfer plasmids for five different insertion sites within the FP9 genome were designed and constructed. Homologous recombination (HR) of adjacent FP9 sequences was employed to facilitate the integration of SIVmac239 sequences into the FP9 genome. Positive rFP9 were identified by blue colouration in presence of X-gal using a transient colour selection (TCS) technique, and the final markerless pure recombinants were confirmed by PCR. Expression of the target SIV proteins in the presence of T7 polymerase has been demonstrated by immunocytochemical (ICC) staining and Western blotting (WB) assays. Expression was also quantified by enzyme-linked immunosorbent assay (ELISA) in various cell lines at multiple time points. Five different unique rFP9 have been constructed through this project. All SIVmac239 open reading frames (ORFs) save nef have been integrated into the FP9 genome, and protein expression demonstrated where possible. Moreover, a single rFP9 vector expressing the defective SIVmac239 genome driven by T7 RNA polymerase has been successfully constructed and validated using a green fluorescent protein marker.rFP9 showed appropriate transgene expression in both avian and mammalian cells, although at different levels. The expression efficiency of rFP9 was finally compared to another attenuated poxvirus vector, modified vaccinia Ankara (MVA). Comparing the protein expression levels between rFP9 and rMVA was quite difficult because different poxvirus promoters (early/late in rFP9; intermediate in rMVA) were used to direct the transcription of the T7 RNA gene. Given this limitation, although generally higher levels of expression were seen with rFP9, this cannot be attributed to the FP9 with any certainty.

Introduction: Epidemiological studies have indicated that 5-38% of influenza like illnesses (ILI) develop into severe disease due to, among others, factors such as; underlying chronic diseases, age, pregnancy, and viral mutations. There are suggestions that dual or multiple virus infections may affect disease severity. This study investigated the association between co-infection between influenza A viruses and other respiratory viruses and disease severity. Methodology: Datum for samples from North West England tested between January 2007 and June 2012 was analysed for patterns of co-infection between influenza A viruses and ten respiratory viruses. Risk of hospitalization to a general ward ICU or death in single versus mixed infections was assessed using multiple logistic regression models. Results: One or more viruses were identified in 37.8% (11,715/30,975) of samples, of which 10.4% (1,214) were mixed infections and 89.6% (10,501) were single infections. Among patients with influenza A(H1N1)pdm09, co-infections occurred in 4.7% (137⁄2,879) vs. 6.5% (59⁄902) in those with seasonal influenza A virus infection. In general, patients with mixed respiratory virus infections had a higher risk of admission to a general ward (OR: 1.43, 95% CI: 1.2 – 1.7, p = <0.0001) than those with a single infection. Co-infection between seasonal influenza A viruses and influenza B virus was associated with a significant increase in the risk of admission to ICU/ death (OR: 22.0, 95% CI: 2.21 – 219.8 p = 0.008). RSV/seasonal influenza A viruses co-infection also associated with increased risk but this was not statistically significant. For the pandemic influenza A(H1N1)pdm09 virus, RSV and AdV co-infection increased risk of hospitalization to a general ward, whereas Flu B increased risk of admission to ICU/ death, but none of these were statistically significant. Considering only single infections, RSV and hPIV1-3 increased risk of admission to a general ward (OR: 1.49, 95% CI: 1.28 – 1.73, p = <0.0001 and OR: 1.34, 95% CI: 1.003 – 1.8, p = 0.05) and admission to ICU/ death (OR: 1.5, 95% CI: 1.20 – 2.0, p = <0.0001 and OR: 1.60, 95% CI: 1.02 – 2.40, p = 0.04). Conclusion: Co-infection is a significant predictor of disease outcome; there is insufficient public health data on this subject as not all samples sent for investigation of respiratory virus infection are tested for all respiratory viruses. Integration of testing for respiratory viruses’ co-infections into routine clinical practice and R&D on integrated drugs and vaccines for influenza A&B, RSV, and AdV, and development of multi-target diagnostic tests is encouraged.

Aims: To explore the clinical significance of EBV infection in adult renal transplant recipients when detected in the late post-transplant period. Methods: (1) A prospective observational study recruiting 499 stable adult kidney transplant recipients with serial blood sampling for EBV DNAemia and assessment of clinical outcomes and associated factors. (2) A retrospective analysis of PTLD incidence, timing and outcomes in relation to EBV infection. Results: EBV DNAemia in stable kidney transplant recipients is common, found in 46% of recruited individuals screened over 1 year, with persistent DNAemia seen in 10%. DNAemia prevalence increased significantly with time from transplant (p<0.0001) from 16% within 1 year of transplant to 66% in those transplanted for 20-24 years. High baseline DNA levels predicted persistence of DNAemia. Time adjusted analyses showed significant association of DNAemia with EBV seronegative status and previous PTLD and low DNAemia rates with Mycophenolate Mofetil (MMF) use and lymphopenia. The mechanism did not appear to be directly linked to MMF induced B cell depletion. Chronic high viral load detection was significantly associated with time from transplant, EBV seronegative status at transplant, ciclosporin use and plasma detection of DNA. No significant differences in overall patient survival at 3 years, clinical symptoms or clinical findings such as anaemia, thrombocytopenia or rate of decline in renal function were seen between stable transplant recipients with and without EBV DNAemia. PTLD incidence also increases with time from transplant and was greatest during the 10th-14th post-transplant years. Disease was EBV positive in 68% cases. No statistically significant differences in overall patient survival, or overall disease complete response rates were seen in relation to recipient EBV serostatus or EBV status of PTLD histology. Conclusions: EBV DNAemia prevalence increases with time from transplant but was not associated with worse patient or graft survival or specific symptoms. PTLD incidence including EBV negative disease also increases with time from transplant but response rates and survival were not influenced by EBV serostatus or histological status.

Development of a broadly cross-protective capsular group B meningococcal (MenB) vaccine has been hampered by poor capsular immunogenicity and often diverse and poorly cross-protective subcapsular antigens. The MenB MC58 strain genome has facilitated the discovery of novel, relatively conserved vaccine candidates. The four-component MenB (4CMenB) investigational vaccine contains factor H-binding protein (fHbp; variant 1), neisserial heparin-binding antigen (NHBA), Neisserial adhesin A (NadA) and PorA P1.4-containing outer membrane vesicles. The latter are known to elicit protection against homologous strains. Clinical trials have demonstrated protective responses in infants and adults against isolates expressing homologous PorA or fHbp (subvariant 1.P1), or heterologous NadA (variant 2). Cross-protective responses have also been demonstrated in adults and, to a lesser extent, infants, against isolates expressing heterologous fHbp variant 1 subvariants. The contribution of NHBA is still poorly understood. MenB currently accounts for 87% of invasive meningococcal disease in England and Wales. The proportion of disease due to the ST-269 clonal complex (cc269) peaked at 45.6% in 2006 and is currently approximately 24.2%. The aims of this study were (i) to genotypically assess potential 4CMenB coverage against recent English and Welsh invasive disease isolates and, specifically, cc269 isolates from England and Wales and other countries, (ii) to compare phenotypic expression levels of the 4CMenB antigens (excluding PorA) among typical cc269 isolates, and (iii) to assess 4CMenB responses against typical cc269 isolates among healthy adults administered 4CMenB.Full length alleles for fHbp variant 1, NHBA and NadA variants 1, 2 and 3 were present in 64.6%, >99% and 7.1%, respectively, of English and Welsh invasive disease isolates from 2007/8. Between 67.5% and >99% (adults) or 25.7% and 43.5% (infants) of the isolates were predicted to be covered by 4CMenB. cc269 comprised two antigenically distinct lineages (clusters) centred around ST-269 and ST-275, respectively. These accounted for 57% and 40% of cc269 in 2007/8. Both clusters effectively lacked nadA and PorA P1.4. The predominant fHbp;NHBA profiles represented by the respective clusters were 1.P15;P0021 and (1.P13 or 2.P19);P0017. Between 77.4% and 100% (adults) or 2.2% and 27.1% (infants) of cc269 isolates from 2007/8 were predicted to be covered by 4CMenB. Estimates for infants were conservative due to e.g. the exclusion of NHBA. Serum bactericidal antibody (SBA) analyses targeting typical fHbp variant 1-expressing cc269 strains, indicated high levels of coverage among adults administered 4CMenB. Notable differences among genotypically matched isolates e.g. in terms of SBA geometric mean titres, were not reflected in the relative fHbp and NHBA expression levels. Such differences may lead to conflicting estimates of coverage in infant populations. Whilst these are investigated further it seems prudent to use typical isolates giving mid-range responses when assessing SBA, and therefore protection, among infants. Potential 4CMenB coverage of cc269 and the broader meningococcal population in England and Wales was high among adults and encouraging among infants when compared to that of existing MenB vaccines.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen with ability to establish a persistent infection in the host for many years. Diseases are more serious in congenitally infected infants and immunocompromised individuals such as AIDS patients and transplant recipients. It rarely causes disease among immunocompetent individuals. The envelope glycoproteins of HCMV (gB, gH, gN, gM, gL and gO) are essential for viral infectivity as they are involved in attachment and penetration of the host cell, cell to cell viral transmission and fusion of infected cells. Also, they are known to be important targets for humoral and cell mediated immune responses against the virus. The hypothesis states that HCMV genotypes are constantly mutating and evolving, then is controlled in a setting of immunocompetence, but in immunocompromised patients, congenitally infected patients or possibly within the CNS, the virus is able to evade the immune system and genotypes may evolve which are more pathogenic and/or neuroinvasive. In current study, a total of 669 CSF samples were collected from Malawian children with neurological illness that have been tested for the presence of HCMV. A comparison study population was also studied which contained 26 HCMV isolates obtained from the Manchester Royal Infirmary Clinical Virology Laboratory. The isolates were originally collected from congenitally CMV infected infants or immunocompromised children and adults showing symptoms of HCMV disease. Positive HCMV samples were screened for glycoproteins (B, H, N, L and O) and then glycotyped by restriction fragment length polymorphisms and sequencing analysis. HCMV DNA was found to be present in 46 (6.7%) out of 669 CSF samples. In 19 of these HCMV positive samples, glycoprotein genotypes (B, H, N, L and O) were identified. The predominant glycoprotein genotypes in the CSF population were as follows, gB type 3(63.2%), gH type 1(73.2%), gN type 4 (50%), gL type 4 (55.8%) and gO type 1 (60%), whilst in the comparison study population (control group) the most common glycoprotein genotypes were; gB type 3 (52.3%), gH type 1 (52%), gN type 4 (60%), gL type 4 (56%) and gO type 1(48%). No statistically significant difference was found between glycoprotein genotypes among the CSF samples and comparison study population. However, when linkage between glycoprotein types was studied and analysed differences between the two populations emerged. The particular glycoprotein linkage (gH1, gN1 and gO1) occured six times in the CSF study population with severe and fatal outcome as it occured four times with fatal outcome, and in two other patients, one was HIV positive and one with sequelae.Novel linkages between gO and gN genotypes were also found (gN3b/gO3), (gN4a/gO4), (gN4b/gO2b), (gN4b/gO3), (gN4d/gO1c), (gN1/gO1c), and (gN4c/gO4). The (gN1/gO1c) combination was occurred three times, but only in CSF study population and was associated with fatal outcomes, two patients died and one was HIV positive patients.RFLP assay was found to have limited efficacy in identification of HCMV genotypes, as discrepancies in genotype results between RFLP and sequencing were found in all glycoprotein genotypes. Thus, sequencing analysis was more accurate and recommended for identification of the HCMV genotypes.In conclusion, investigation of different glycoprotein genotypes in CSF samples has shown no significant correlation between single glycoprotein and disease outcome. Recombination between HCMV strains may lead to progression of disease. However, studying a large group of CSF samples positive for HCMV from immunocompromised patients may clarify the correlation between glycoprotein types and neurological outcome, and lead to improved strategies for treatment and prevention of HCMV associated disease.

Encephalitis is a matter for serious public health concern because of the high morbidity and mortality associated with many cases. Epidemiological studies have shown that viral encephalitis (VE) is more common than the sum of encephalitis caused by all other pathogens. However, more than 95% of cases have no known cause. Thus, there is a significant need to develop a sensitive method for the diagnosis of these unknown cases. Previous sequence independent amplification (SIA) assays have proved successful in detecting new viruses in many biological samples but not in CSF samples. This may be due to the relatively low sensitivity of most available methods as CSF usually contains lower concentrations of pathogen than most other samples. A known problem with these types of assays is the annealing of the random primers to human DNA which facilitates preferential amplification of background human DNA. Thus, large scale sequencing is usually required to detect a virus, which in turn reduces the detection sensitivity to more than 1000 viral copies/µl, a CSF concentration that is rarely seen in cases of VE.This project was designed to develop a highly sensitive SIA assay for novel nucleic acid identification that could be used in testing CSF samples obtained from patients with neurological diseases of unknown cause. The study started with evaluation of two existing SIA assays commonly used for virus discovery; whole genome amplification (WGA) and random PCR (r-PCR). Sequential modification and adaptation of these methods was carried out to increase their sensitivity. Ultimately, a novel primer (Sa primer) that showed no binding to most human DNA sequences in GenBank was designed and synthesised. Its 3' end was tagged with 6 and 7 random nucleotides generating 2 r-primers; Sa-6 and Sa-7. The sensitivity of the r-primers was checked in a novel assay developed during this project and named Sa-SIA using known concentrations of HCMV and HSV-1. CSF samples from Malawian children were then tested using the developed assay. Results showed that adaptation of the existing WGA and r-PCR assays allowed detection of up to 1300 viral copies/µl. When the novel primers developed in this project were used in a random PCR assay (Sa-r-PCR), it was found that using Sa-6 primer 130, 13, and 1.3 HCMV copies/µl could be detected with 100, 60, and 50% efficiency respectively. When using Sa-7 primer, the same concentrations of virus were detected with 100, 42, and 28.6% efficiency. DNase-1 treatment of the samples pre-extraction resulted in an improvement in viral detection sensitivity in samples with a high background of host DNA. Starting with template concentrations of 11000, 110, 11, and 1.1 HSV-1 copies/µl, viral detection efficiency was increased from 33.3, 10, 0, and 0% to 92, 55.6, 16.7, and 0% respectively when pre-extraction DNase-1 treatment preceded Sa-r-PCR using Sa-6 primer. The final developed assay (Sa-SIA) consisted of centrifugation, DNase-1 treatment, DNA extraction, Sa-r-PCR using Sa-6 and Sa primers, gel electrophoresis, band excision, cloning, small scale sequencing (sequencing of ≤ 20 positive clones from one constructed DNA library), and bioinformatics. It had a detection sensitivity of 1.3-11 viral copies/µl. When this assay was applied to stored CSF samples, one 448bp sequence was identified which gave 96% coverage with 81% identity to Torque teno midi virus-1 and 93% coverage with 81% identity to small anellovirus-2. A 236bp sequence from another CSF sample showed 66% coverage with 97% homology to an unclassified sequence previously identified in a viral genomic survey of stool sample in an earlier published study. In conclusion, the standardised method had been shown to detect 1.3 to 11 viral copies/µl of two viruses; HCMV and HSV-1. The detection of these viruses was achieved with only small scale sequencing. Application of this method to CSF samples has shown promising results. However, this method could be followed by more advanced post amplification analyses such as next generation sequencing.

Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.