Background - The internal elements of the Influenza-A virus, exhibit high levels of conservation and offer a more consistent target for the immune system amidst the diversity of potential strains. T-cell responses to these proteins have been shown to correlate with protection and deceased symptom severity during infection. Yet the epitopes and T-cell receptor (TCR) repertoires that underpin these important responses have not been analysed in detail at the molecular level. Results - These responses were analysed by the development of an HLA-DR1 restricted epitope mapping platform in chapter 3, followed by its application in finding DR1-restricted epitopes within the three internal proteins in chapter 4. Two of these epitopes were analysed by X-ray crystallography to understand their presentation and complement HLA-binding algorithm data. Identification of epitopes that gave robust and reproducible responses allowed analysis of responding T-cell populations by HLA-multimer staining on flow cytometry and subsequent clonotypic analysis of TCR repertoires in chapter 5. The clonotypic repertoire data was interpreted and then information in response to a single epitope was aligned with structural data in chapter 6 to further understand the molecular interactions that shape these responses. Conclusions - This work generated several novel HLA-DR1 restricted epitopes, crystal structures and TCR repertoire information that both expands existing knowledge of CD4+ T-cell responses, and confirms the potential of the conserved influenza proteins as targets in future vaccination research.

Zoonoses involve interactions between at least three species: the pathogen and two hosts, one of which is human and the other a non-human (vertebrate) animal. More than 60% of human infectious diseases are zoonotic, and many have a wildlife host. Urbanisation and human population growth have increased the demand for food and land resources, which have increased interaction between humans, domestic animals and wildlife and thus the potential for cross-species transmission of infections. Most studies of such systems take place in tropical and developing countries where population change and biodiversity makes the emergence of high profile infections (eg Ebola and SARS) more likely. This study, however, focuses on four well known infections within the UK: bovine tuberculosis, water-borne cryptosporidiosis and giardiasis, and campylobacteriosis. The aim of this study was to investigate, using four infectious diseases of economic and public health importance in the UK as study systems, the role of wildlife in the epidemiology of multihost, zoonotic infections. Bovine tuberculosis (bTB) is an important zoonosis in many parts of the world, but human infection is rare in the UK owing to a policy of 'test and cull' in cattle and pasteurisation of milk. However, there has been an epidemic of bTB in British cattle in recent decades, the control of which is complicated by infection in badgers (Meles meles) and controversy over the control of wildlife infection. I investigated TB in badgers in the Cheshire area, located on the edge of the epidemic in England, in collaboration with various stakeholders. Using a road-kill survey, I found M. bovis in 20 of 94 badgers: the estimated prevalence of 21.3% (95% CI 14.2-30.6) is comparable to the county-level prevalence found at the core of the epidemic. That all isolates were spoligotype SP25, suggests this is an expansion of infection from neighbouring counties. The directionality of any cross-species transmission of bTB between wildlife and livestock cannot be ascertained from this project. However, it showed that using road-killed badgers is a valuable approach to sampling, especially if combined with the engagement of stakeholders. Cryptosporidium spp and Giardia duodenalis are protozoa that can cause diarrhoea in many mammals including humans. Llyn Cowlyd, a major water reservoir in North Wales, had seen annual summer peaks of uncharacterised Cryptosporidium spp cysts, without human disease. My study aimed to determine the source(s) of the contamination and to investigate G. duodenalis in the same system. Water samples were collected from the reservoir and feeder streams, and faecal samples from livestock and wild rodents. In total, 97 rodents were sampled: 35 (35.7% CI 95% 26.9-45.6%) were positive for Cryptosporidium spp. and 11 (11.2% CI 95% 6.4-19%) for Giardia spp. Of cryptosporidia detected, 55% were novel genotypes and only 5% C. parvum (zoonotic). Of 11 livestock samples, only two samples were positive for C. parvum and G. duodenalis. All the rodent Giardia belonged to an apparently novel assemblage while livestock Giardia belonged to non-zoonotic assemblage E. The water samples contained C. ubiquitum, C. parvum, and G. duodenalis assemblages E, A (zoonotic), and the novel rodent assemblage. Campylobacter jejuni and C. coli, are common causes of diarrhoeal disease in humans. Infection is common in a wide range of livestock and wildlife species, usually, however, without disease. The aim of this study was to investigate the potential role of wild birds in the epidemiology of campylobacteriosis on dairy and poultry units already studied in depth as part of a larger research programme. In total, 299 birds were sampled and 14 (4.7% CI 95% 2.8-7.7%) were positive for C. jejuni. Multi-locus sequence typing showed each isolate to be different, and many of these sequence types found in wild birds have not been associated with human disease. Overall, these results show that while at first sight wildlife might be assumed to be potential sources of zoonotic infections, further characterisation of the agents involved often revealed separate cycles of infection in wildlife, livestock and humans.

Since 2003, highly pathogenic avian influenza (HPAI), subtype H5N1, has spread across the Asian, African and European continents at an exceptional rate. To date, H5N1 remains primarily a pandemic within poultry populations with limited onward transmission to humans. Since there have been a limited number of human cases throughout the world, epidemiologic uncertainties exist regarding the extent of contact necessary to result in successful transmission between infected poultry and humans. In this thesis I undertook two large-scale surveys to evaluate poultry movement and the extent of interaction between humans and poultry to better define the risks of sustained transmission of H5N1 in poultry and onward transmission to humans. The thesis begins with a review of current knowledge on the epidemiology of HPAI, specifically subtype H5N1, and current options for its control worldwide and specifically within Cambodia. The first half of the thesis presents the methodology and results from a large-scale cross sectional survey of 3,600 rural subjects from 115 villages in six provinces throughout Cambodia. The results from this survey are used to explore animal ownership and husbandry, poultry mortality experienced and poultry mortality reporting, and the extent and frequency of poultry handling behaviours of subjects and how they differ by age and gender. The second half of the thesis presents results from a second cross-sectional survey of 715 rural villagers, 123 rural, peri-urban and urban market sellers and 139 middlemen from six Provinces and Phnom Penh, which was conducted to evaluate poultry movement and trading practices. The results from this survey are used to construct poultry movement networks using social network analysis techniques, to identify critical points for surveillance and to understand the potential transmission and control of HPAI over this network and to identify a spatial model to predict poultry movements. Finally in the last chapter the key findings are presented and discussed in the context of HPAI transmission in the region.

Background: Insecticide treated nets (ITNs) and indoor residual spraying (IRS) are the preferred techniques for malaria vector control in Africa, where their application has already contributed to significant reductions in the burden of the disease. Even though both methods are commonly used together in the same households, evidence of greater health benefits due to these combinations as opposed to use of either ITNs or IRS alone has been minimal and inconclusive. Objectives and methods: The main aim of this research was therefore to contribute to this essential evidence, by way of experimental hut studies and mathematical simulations. I investigated whether there would be any added protective advantages when any of three selected long lasting insecticidal nets (LLINs) are combined with any of three selected IRS chemicals, as opposed to using any of the treatments alone. Data generated from the experimental but studies was then input into an optimised deterministic mathematical model, simulating a typical malaria endemic village. Results and conclusions: Both the field studies and the simulations showed that any synergies or redundancies resulting from LLIN/IRS combinations are primarily a function of modes of action of active ingredients used in the two interventions. Where LLINs are already present, addition of IRS would be redundant unless the IRS chemical is highly toxic, but where IRS is the pre-existing intervention, these combinations always confer improved protection. Therefore, IRS households should always be supplemented with nets, preferably LLINs, which not only protect house occupants against mosquito bites, but also kill additional mosquitoes. Finally, where resources are limited, priority should be given to providing everybody with LLINs and ensuring that these nets are consistently and appropriately used, rather than trying to implement both LLINs and IRS in the same community at the same time.

Genetic drive systems have the potential to modify entire insect populations in a few years through the super-Mendelian inheritance of a genetic trait. We describe a novel gene drive system called CRISPRh, which acts as a selfish genetic element in the malaria mosquito Anopheles gambiae. CRISPRh is a synthetic allele containing CRISPR/Cas9 endonuclease designed to specifically target and insert a copy of itself into its homologous chromosome in the germline of heterozygous individuals. Using a gene targeting approach, we identified three genes that confer recessive female sterility upon disruption. By inserting CRISPRh at each of these genes, we achieved super-Mendelian inheritance of CRISPRh alleles, ranging from 91 to 99.6% of the progeny of heterozygotes. Population modelling predicts that these high rates of transmission and a recessive knockout phenotype would enable CRISPRh alleles to spread rapidly through a wild mosquito population, causing population suppression through the generation of sterile females. We tested the potential of one CRISPRh allele to invade a caged population of naïve mosquitoes and observed an average increase from 50-76.4% over six generations, demonstrating the first gene drive designed to spread through a natural vector population. The CRISPRh constructs described here generate strong unintended fertility effects and are highly susceptible to target site resistance, making them unsuitable for malaria control. We expect that simple modifications can solve these issues and allow the development of gene drives for the suppression of mosquito populations to levels that do not support malaria transmission.

Glycans represent a class of macromolecules that exhibit vital biological roles in living organisms. They are not only essential for maintaining the normal functionalities of a cell, but are also involved in many pathogenic processes. The influenza A virus binds to glycan receptors that are expressed on the surface of respiratory epithelial cells of human airway and thereby initiates infection. Deciphering the structural features of glycans and comprehending their functional implications are thus crucial to expand our understandings of the disease. To validate the alternative models that are used in the studies of influenza, we generated the glycomic profiles from in vivo and in vitro experimental systems by mass spectrometry. A combination of MALDI-TOF MS, MALDI-TOF-TOF MS/MS, GC-EI-MS and enzymatic digestion experiments were utilised to characterise the structure of glycans. The ferret has been used as an experimental animal to investigate the transmission and replication of influenza viruses. To verify the validity of this model, we carried out glycomic characterisation of ferret respiratory tissues to complement the data that was generated from human airway tissues. The mass spectrometric analysis indicates that the respiratory glycosylation of ferret highly resembles that of human, although distinctive expression of glycans displaying the Sda epitope are detected exclusively in ferret. Nonetheless, in comparison to other lab animals such as mouse and swine, ferret remains a better alternative model for studying the pathogenicity of influenza viruses. In the second project, we generated the glycomic profiles from human and ferret respiratory epithelial cells that were cultured under experimental conditions. Glycosylation patterns between these two in vitro systems are largely comparable, except the presence of the Sda epitope in ferret cells. However, when compared to their corresponding in vivo tissues, diminished structural repertoires especially the high-mass structures were observed.

The emergence of novel influenza A viral lineages to which there is limited or no immunity remains an on-going concern for human and animal public health. At least three of the pandemics reported in human hosts during the 20th and 21st century are believed to have emerged as a result of reassortment between human, avian and swine viruses circulating at the time. The emergence of novel lineages with pandemic potential is frequently believed to occur as a consequence of reassortment. The ability to predict when and how the next novel lineage with pandemic potential will arise remains challenging. Additionally, the dynamics of infection in systems where multiple subtypes are known to circulate remain poorly understood. Gaining insight into these biological processes is of vital importance if we are to understand how much co-infection is occurring, and hence the opportunity for reassortment. This thesis uses data analysis, novel algorithmic methods applied to genomic data and mathematical modelling to develop an understating of the dynamics of influenza A infection within non-human hosts. It seeks to analyse patterns of reported reassortment in influenza A across all hosts, geographic regions and time periods. Chapters 3 and Chapter 4 use data on reported reassortment events to identify additional, previously unreported suspected reassortant viral lineages, and develop a quantitative definition of significant reassortants. These chapters use publicly available sequence data to understand to what extent published data on reassortants reflects the possible extent of reassortment that can be detected with an iterative clustering algorithm. They develop methods that can be used to assign a rank score to suggest whether an individual isolate is a suspected reassortant. Methods presented in these chapters are applied to all publicly available influenza A sequence data from two different databases. The estimation of epidemiological parameters such as R0 and the time of virus introduction into a population (Ts) are of vital importance to infer in the event of an outbreak of a novel influenza virus n a poultry production facility. This thesis assesses the performance of different surveillance strategies to estimate R0 and Ts using simulated surveillance data from an outbreak of H7N9 in commercial poultry. Since the detection of novel H7N9, it has been isolated from numerous live bird markets. Live birds markets are known to harbour diverse subtypes of avian influenza within numerous different susceptible hosts. Co-infection with two different influenza viruses is a prerequisite for reassortment to occur therefore the final results chapter explores the conditions required to generate the multi-strain dynamics of different AI subtypes and the resulting co-infection dynamics that emerge. Taken together, through these topics addressed in this thesis we understand more broadly the possible true extent of reassortant detectable in publicly available sequence data. How surveillance data collection can be optimised to help estimate epidemiological parameters in the event of an outbreak, and the possible mechanisms that generate the multi-strain dynamics within a live bird market.

No description available.

BACKGROUND: A big proportion of populations in sub-Saharan Africa still battle with poverty and intense exposure to infections including chronic worm infections. Studies of cognitive effects of worms have not been consistent regarding which functions are affected and whether deworming is beneficial. However, executive functions could be particularly susceptible to impairment. The home environment is reported to affect child development. However, its unique contribution in Ugandan children exposed to various factors has not been estimated. This PhD research therefore aimed to [I] examine effects of maternal and childhood worm infections and their treatment on executive function and other developmental outcomes in infancy and preschool-age children; and [2] investigate the independent effects of the home environment on child mental performance, taking into account the confounding effects of other socio-demographic exposures. In the same sample, [3] relationships between executive functions were examined in order to understand how they may be disrupted by worms and other adverse factors. METHODS: This research was conducted within the ongoing Entebbe Mother and Baby Study (EMABS) in which 2500 pregnant women were randomised to receiving a single dose of albendazole or placebo and praziquantel or placebo in a 2x2 design. 983 infants born to these women were assessed at age 15 months on executive function, and other abilities to measure the effect of maternal worm infections and their treatment on these functions. The infants were then randomised to quarterly doses of albendazole or placebo from age 15 months and were assessed again at age five years (N=870) on measures of executive function, general ability and motor function to examine effects of childhood worm infections and their treatment on performance on these functions. Relevant health and socio-demographic date were collected and used in the analyses. 200 children were assessed on an extended battery of executive function measures and their scores were used to explore the structure and relationships between executive functions . Home environments of 163 children were examined using an adapted version of the Home Observation for Measurement of the Environment (HOME). RESULTS: Maternal Mansonella perstans and Strongyloides stercoralis infection were negatively associated with infants' performance on the A not B task (a measure of executive function) and language respectively but anthelminthic treatment did not show significant effects overall. In childhood, worms were negatively associated with performance on measures of working memory (Verbal Fluency, Counting Span, and Running Memory), attention (Picture Search), and general ability (Block Design). However, treatment with quarterly albendazole did not have significant effects on performance on measures of cognitive functioning. The home environment contributed to cognitive performance above and beyond the effect of other factors. Low-to-moderate inter-construct correlations were observed among executive functions and three components were extracted from the four domains. Planning was most related to inhibition with working memory also contributing to the best fit model. CONCLUSION: Both infant and childhood developmental outcomes suggest that executive functions may particularly be susceptible to impairment by worm infections but treatment may not reverse the effects. Furthermore, the results show that the home environment affects the child independently of SES, parental education and other global measures. Correlations and factor loadings indicate unity among the executive functions, particularly between planning and a combination of inhibition and working memory,

No description available.