Trypanosoma cruzi and the Leishmania donovani complex are parasitic protozoa that, respectively, cause Chagas disease in the Americas, and visceral leishmaniasis, predominantly in South Asia, East Africa, and Brazil. T. cruzi is divided into the lineages TcI-TcVI. The relationship between infecting lineage(s) and spectrum of clinical presentations remains poorly understood. This project developed lineage-specific serology to identify an individual’s history of lineage infection. A high level of polymorphism in the surface mucin TSSA was identified, and lineage-specific synthetic peptides based on this diversity were applied here in ELISA with chagasic sera from endemic countries. Peptide TSSApep-II/V/VI, based on a sequence common to those lineages, was widely recognised by sera from Southern Cone countries, and also unexpectedly by four samples from Ecuador; TSSApep-V/VI, which differs by a single amino acid from TSSApep-II/V/VI, was also recognised in these regions. A single TSSApep-IV reaction was seen in both Colombia and Venezuela. However, TSSApep-I was rarely and weakly recognised among the serum panel. Among the Brazilian patients, a much higher proportion of TSSApep-II/V/VI responders had ECG abnormailities than non-responders (38% vs. 17%, p<0.0001). Rapid diagnostic tests for L. donovani complex infection based on rK39 antigen have lower sensitivity in East Africa compared to South Asia. The homologous sequences of rK39, and of another proposed diagnostic antigen HASPB, were amplified from a panel of East African L. donovani strains, and compared to published sequences, revealing significant diversity from rK39 and South Asian sequences, and non-canonical combinations of HASPB repeats. Cohorts of Indian and Sudanese VL patients were assayed by ELISA for anti- Leishmania IgG levels. There was an overall 46.8 – 61.7 fold lower response in the Sudanese cohort, as calculated by mean reciprocal log10t50 titres, regardless of antigen source, patient gender or age. An investigation into the association of IgG subclass reactivity with VL clinical status revealed significantly elevated IgG1 levels in patients with active (pre-treatment) VL and those with post-therapy relapse compared to those deemed to be cured. A novel prototype rapid immunochromatographic test to detect IgG1 gave > 80% of relapsed VL patients as IgG1 positive, and 80% of cured patients as IgG1 negative.

Chagas disease remains the most important parasitic infection in Latin America. The aetiological agent, Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), is a complex vector-borne zoonosis transmitted in the faeces of hematophagous triatomine bugs (Hemiptera: Reduviidae: Triatominae), and maintained by mammalian reservoir hosts ranging from the southern United States to Argentinean Patagonia. In the absence of chemotherapy, infection is life-long and can lead to a spectrum of pathological sequelae ranging from subclinical to lethal cardiac and/or gastrointestinal complications in up to 30% of patients. T. cruzi displays remarkable genetic diversity, which has long been suspected to contribute to the considerable variation in clinical symptoms observed between endemic regions. Currently, isolates of T. cruzi can be assigned to a minimum of six stable genetic lineages or discrete typing units (DTUs) (TcI-TcVI), which are broadly associated with disparate ecologies, transmission cycles and geographical distributions. The principal mode of reproduction among T. cruzi strains is the subject of an intense, decades-old debate. Despite the existence of two recent natural hybrid lineages (TcV and TcVI), which resemble meiotic F1 progeny, a pervasive view is that recombination has been restrained at an evolutionary scale and is of little epidemiological relevance to contemporary parasite populations. The aim of this PhD project was to investigate T. cruzi genetic diversity through significant development of phylogenetic markers and their application to the characterization of natural parasite population structures and genetic exchange mechanisms. Multiple, single-copy, chromosomally-independent, nuclear housekeeping genes were assessed initially for their ability to allocate isolates to DTU-level, to facilitate higher resolution intra-lineage analyses and finally for their inclusion alongside additional targets in a standardized T. cruzi multilocus sequence typing (nMLST) scheme. For the immediate future, nuclear MLST, using a panel of four to seven nuclear loci, is a robust, reproducible and highly discriminatory method that has potential to become the new gold standard for T. cruzi DTU assignment. To investigate natural parasite population structures and uncover evidence of genetic exchange, a high resolution mitochondrial MLST (mtMLST) scheme, based on ten gene fragments, was developed and evaluated against current nuclear markers (multilocus microsatellite typing; MLMT) using isolates belonging to the oldest and most widely distributed lineage (TcI). Observations of gross nuclear-mitochondrial phylogenetic incongruence indicate that recombination is ongoing, geographically widespread and continues to influence natural populations, challenging the traditional paradigm of clonality in T. cruzi. Application of this combined nuclear-mitochondrial methodology to intensively sampled, minimally-subdivided TcI populations revealed extensive mitochondrial introgression within a disease focus in North-East Colombia as well as among arboreal transmission cycles in Bolivia. Failure to detect any reciprocal nuclear hybridization among recombinant strains ! 4 may be indicative of alternate, cryptic mating strategies in T. cruzi, which are challenging to reconcile with both in vitro parasexual mechanisms of genetic exchange described, and patterns of Mendelian allele inheritance among natural hybrid DTUs. High resolution genotyping of TcI populations was also undertaken to explore the interaction between parasite genetic heterogeneity and ecological biodiversity, exposing the significant impact human activity has had on T. cruzi evolution. Reduced genetic diversity, accelerated parasite dissemination between densely populated areas and mitochondrial gene flow between domestic and sylvatic populations, suggests humans may have played a crucial role in T. cruzi dispersal across the Bolivian highlands. Parallel reductions in genetic diversity were observed among isolates from the Brazilian Atlantic Forest, attributable to ongoing anthropogenic habitat fragmentation. By comparison domestic TcI isolates (TcIDOM) are divergent from their sylvatic counterparts, but also genetically homogeneous, and likely to have originated in North/Central America before distribution southwards. Molecular dating of Colombian TcIDOM clones confirmed that this clade emerged 23,000 ± 12,000 years, coinciding with the earliest human migration into South America. Lastly, Illumina amplicon deep sequencing markers were developed to explore the interaction between parasite multiclonality and clinical status of chronic Chagas disease. An unprecedented level of intra-host genetic diversity was detected, highlighting putative diversifying selection affecting antigenic surface proteases, which may facilitate survival in the mammalian host. In lieu of comparative genomics of representative T. cruzi field isolates, not yet a reality, as is the case with other more experimentally-tractable trypanosomatids, presented herein are some of the highest resolution genotyping techniques developed in T. cruzi to date, which have the potential to expand our current understanding of parasite genetic diversity and its relevance to clinical outcome of Chagas disease.

Tick-borne pathogens represent a large threat to the UK and International Public Health authorities. Due to recent changes in legislation, an increase in animal & human movements and changing climate, the UK may now be at an increased risk of importing exotic tick species and their associated pathogens. It is vital to assess the susceptibility of UK tick species to these highly fatal tick-borne viral zoonoses. To date, studies investigating the interaction of many pathogens with their vectors have been hindered due to the lack of a suitable tick transmission model at high containment. This thesis investigates the intrinsic ability of Ixodes ricinus, the most widely distributed tick in Europe and the UK, to acquire, replicate and transmit both Hazara virus (HAZV, a hazard group 2 surrogate for CCHFV) and Crimean–Congo haemorrhagic fever virus (a hazard group 4 pathogen) addressing their potential to act as competent vectors. During the last decade CCHFV has emerged in new areas within Europe and the principle tick vector of CCHFV has been detected within the UK. The development of methods for use with ticks and highly pathogenic viruses within ticks was an essential part of this work. Firstly techniques for the handling, extraction and storage of RNA obtained from I. ricinus ticks were optimised and different endogenous controls were assessed for their ability to amplify mRNA transcripts for use as endogenous controls. The use of the immersion technique for use with I. ricinus nymphs was optimised and working procedures and protocols for handling ticks at containment level 2 and 4 were established. Ixodes ricinus nymphs are susceptible to infection with HAZV with 100% becoming infected 13 days post-immersion. HAZV was able to establish itself within the key target organs of the tick midgut and salivary glands, produce infectious virus particles and transmit virus to 38% of mice. This artificial method of inoculation was optimised for use within the CL4 environment and was used to show that I. ricinus nymphs were not susceptible to CCHFV via immersion. In addition to horizontal transmission, I. ricinus ticks also demonstrated vertical transmission of HAZV through to the adult stage. This is the first time I. ricinus ticks have been assessed for their susceptibility to HAZV and CCHFV and their use in establishing the first high containment In vivo tick feeding model in Europe.

For human commensal Neisseria meningitidis (Nm) to initiate the pathology of which it is capable it must be able to cross cellular barriers, and once crossed, survive in the blood. It has been established by several studies that susceptibility to infection by Nm increases markedly following viral infection. A potential causative mechanism for this observed temporal effect may include the upregulation of bacterial receptors on host cells in response to virallyinduced cytokines. The initial part of the study presented aims to clarify the effect of inflammatory cytokines in upregulating epithelial receptors targeted by Nm. Results demonstrated that treatment of epithelial monolayers with IFN-V led to de novo synthesis of major Opa receptor, CEACAM1, which in turn, led to increased invasion of host cells by capsulate Opa-expressing bacteria. This is dependent on the activation of Nuclear factor-kappa B (NFKB) as demonstrated by the abrogation of infiltration in the presence of NFKB inhibitors. Once the bacterium has invaded the epithelial layer, the next step in pathogenesis is traversing the endothelial barrier of the host vasculature. Vitronectin (Vn) has been implicated previously in mediating adhesion to and invasion of human brain microvascular endothelial cells (HBMEC) by Opc-expressing Nm. The aim of the second part of the study was to investigate the nature of the Opc-Vn interaction. The data demonstrated that Opc-expressing Nm preferentially bind directly to the activated form of human Vn (aVn) in which sulphotyrosines YS6 and YS9 are exposed. Accordingly, cell association and invasion was abrogated in the presence of both sulphated Vn peptide spanning residues 43-68 and conformation-dependent antibody 8E6 binding at this site. Monomeric preparations of native Vn were also unable to support endothelial interactions with Nm. A second, lower affinity interaction was also observed in the presence of heparin with data indicating that the same region of Opc is responsible for both interactions.

Dengue viruses (DENVs) are mosquito-borne flaviviruses, which are responsible for a wide range of illnesses, from a mild febrile illness to the more serious dengue haemorrhagic fever and dengue shock syndromes. The DENV NS4B is a highly hydrophobic integral ER membrane protein that has been reported to perturb type I interferon (IFN) signalling and has emerged as a major target for anti-viral strategies. The objective of this study was to determine the mechanism of action of two consecutive nucleotide mutations in the NS4B gene (nt 7020 and 7021) that result in the substitution of threonine 66 with alanine, which allow the virus to replicate persistently in mammalian and mosquito cells. Initially, a transient gene expression system was developed to analyse the effect of the mutations on the localisation of NS4B and its ability to perturb the IFN response and the unfolded protein response. The mutations were not found to significantly affect any of these processes. Novel cell lines were then produced that stably expressed the NS4B gene and its mutant counterpart in an inducible fashion. The cell lines were used to identify cellular proteins (DNAJA2, PFN2, FAF2, BAX and PMSG1) that potentially interact with the NS4B proteins using a high-throughput proteomic approach. Finally, HEK293T cell lines that were either stably persistently infected with a virus containing the NS4B mutations or contained a wild type DENV replicon or a replicon containing the NS4B mutations were established. Genome sequencing demonstrated that the NS4B mutations were more stable in a replicon than in the full viral genome. The effect of the NS4B mutations on host cell processes was investigated by transcriptomic analysis using RNAseq. The transcriptomic analysis revealed that canonical cellular pathways were affected by viral replication including IFN signaling, virus recognition, ubiquitination, apoptosis, and ER stress. Further analysis showed that the NS4B mutations affected the ability of the virus and a DENV replicon to effectively suppress IFN signaling and IFN sensitive gene expression. This study provides further insight into the role of the NS4B protein in the viral-host interaction and provides a mechanism to explain how a persistent DENV infection may occur.

Production of metallo-[beta]-lactamase (MBL) proteins protects Gram-negative bacteria from almost all [beta]-lactam antibiotics. MBLs hydrolyse [beta]-lactams, abolishing their antimicrobial activity. One method of restoring [beta]-lactam efficacy against MBL producing organisms would be co-administration with an MBL inhibitor, but to date no such inhibitor is commercially available. This project sought to express and characterise a range of MBLs, with the aims of both understanding structure-function relationships within this enzyme family and aiding the identification of MBL inhibitors. VIM-1 is an MBL commonly identified in resistant clinical isolates.

No description available.

Primary prevention of Human Papillomavirus (HPV) infection through vaccination of young women before sexual debut is recommended by the World Health Organisation. HPV vaccination programmes are being implemented throughout the world, including the United Kingdom, and have the potential to reduce substantially cervical cancer mortality over the long term. To prevent existing inequalities in cervical cancer mortality from widening equitable provision of HPV immunisation is required. Using a mixed methods approach, the aim of this thesis was to examine inequalities of uptake of HPV vaccination by young women. Quantitative research methods were used to ascertain whether inequalities existed and identify factors associated with inequalities in uptake. A systematic review and metaanalysis indicated differences in HPV vaccine initiation by ethnicity and healthcare coverage, but no strong evidence for differences by socioeconomic variables. In the predominantly school-based HPV vaccination programme in the south west of England, uptake was not shown to vary markedly by social deprivation. However, associations with ethnicity and substantially lower uptake in non-mainstream educational settings were observed. Qualitative studies were undertaken to understand factors affecting lower HPV vaccine uptake. A qualitative systematic review and evidence synthesis indicated that decision making about HPV vaccination of young women in high-income countries is dominated by policy makers, healthcare professionals, and parents. Based on cultural perceptions about sexual activity, parents may decide not to allow their daughters to be vaccinated. A case study showed unresolved tension for responsibility of key aspects of the HPV vaccination programme in the south west of England prevents uptake. Overall, the findings from this thesis can be used to inform the development of interventions to increase uptake and address inequalities. A complex intervention, which addresses procedures for consent, increases access by setting of vaccine delivery, and overcomes cultural and literacy barriers faced by minority ethnic groups is recommended.

The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. Dengue is a global health concern with up to 390 million human infections estimated to occur annually. Despite intensive research, the pathogenesis of dengue is not well understood. The use of high-throughput transcriptomic and small interfering RNA approaches has led to the identification of cellular proteins and pathways that are required for, and modulated by, the DENV lifecycle. In comparison to other high-throughput approaches, high-throughput proteomic analysis has not been applied to the investigation of the DENV -host protein interaction. In this study, for the first time, ~table isotope labelling by ~mino acids in fell culture (SILAC) combined with high throughput mass spectrometry (MS) has been used to examine the host cell response to DENV infection. Initially, SILAC-MS analysis was used to investigate the changes that occurred in the proteome of human alveolar epithelial A549 cells in response to DENV infection. Nuclear and cytoplasmic fractions prepared from mock and DENV -2 infected A549 cells were analysed. 2115 and 3098 proteins in nuclear and cytoplasmic fractions respectively were identified and quantified in both the mock and DENV -2 infected A549 cells. The SILAC-MS results were subjected to bioinformatics analysis and validated for eight selected proteins by Western blot and immunofluorescence analysis. Two of the selected proteins, ELKS/Rab6-interactinglCAST family member 1 (ERC 1) and PRA 1 family protein 2 (PRAF2), significantly decreased during infection and were investigated in more detail to determine their relevance to the DENV lifecycle. Knockdown of ERCI but not PRAF2 inhibited DENV -2 replication whereas overexpression of PRAF2 but not ERCI inhibited DENV-2 infection. Co-immunoprecipitation analysis combined with SILAC-MS revealed that ERCI and PRAF2 interacted with the DENV NS5 and NS 1 respectively and cellular proteins involved in trafficking, suggesting that DENV may modulate intracellular transport processes during its release. Comparative analysis of DENV -2 and DENV -4 infected human hepatocyte Huh-7 cells was then done by SILAC-MS. The SILAC-MS results identified common and cell specific pathways that were modulated by DENV infection and identified cellular proteins that were differentially effected by DENV -2 and DENV -4 infection. Furthermore, the analysis of cell specific pathways that were modulated by DENV infection was done by comparison ofDENV-2 infected A549 and DENV-2 infected Huh- 7 cells. Overall the work described in this thesis demonstrated that SILAC-MS is a powerful approach for the analysis of changes in the host proteome upon DENV infection. A number of novel protein changes were identified that can now be furher examined to increase our understanding of DENV replication and identify targets against which antiviral strategies can be developed.

No description available.