ABSTRACT HCMV is a virus which establishes lifelong persistence in its host. In the face of an immune response, reinfection and reactivation may still occur which may be due in part to sequence heterogeneity amongst different strains in proteins involved in viral infectivity. The objectives of this thesis were to perform a comprehensive sequence analysis of neutralising determinants of HCMV amongst clinical isolates, and to determine the biological consequences of virus strains having variant sequences. The nucleotide sequences for regions containing neutralising determinants within the envelope glycoproteins gB and gH were determined for a large number of clinical HCMV strains from AIDS patients and renal transplant recipients. A comprehensive phylogenetic analysis of sequence data for each region was performed and revealed that clinical HCMV strains segregated into a limited number of subtypes at each site. The segregation was also region dependent, with the suggestion of recombination events not only between the gH and gB loci, but also within the gB locus at two different genetic sites. Analogous results were obtained for the amino acid sequences translated from nucleotide sequence data. Thus, it may be necessary to include components from a number of prototype HCMV strains when formulating a subunit vaccine. Concatenation of sequence data, followed by phylogenetic analysis served to provide a unique identity for HCMV strains, and characterise relationships between subtypes. Such data might be useful where correlations are made between virus subtype and the pathogenesis of virus infection. Functional analyses were performed with variant virus strains to determine the biological implications of mutations observed within neutralising determinants of HCMV. The reactivity of a panel of monoclonal antibodies and human sera was markedly altered against a number of the variant strains, and in one case neutralising activity of antibodies was enhanced by mutations. These findings suggest that strain specific differences may contribute to the pathogenicity of HCMV, and it is therefore important to consider sequence heterogeneity within neutralising determinants of the envelope glycoproteins of HCMV when designing subunit vaccines or therapeutic agents such as human monoclonal antibodies

This thesis reports on the infection by Trichuris muris of resistant (8ALB/c) and susceptible (AKR) strains of mice, here used as a model to determine the histological aspects of host-parasite interaction. The resistant BALB/c strain rejected the parasites after Day 14 post-infection. Sequential stages in the infection of the caecum were studied by LM; SEM and TEM. The structure of the crypts were examined in all stages from Day 0 (control) to adult stage (45 Days) of post-infection of AKR mice and from Day 0 (control) to Day 14 post-infection of BALB/c mice. The caecum tract is made up of four distinctive layers, the mucosa; the submucosa; the muscularis externa and the serosa. The mucosa is lined by columnar epithelium which is folded into numerous Crypts of Lieberkuhn. The crypts contain mucosal mast cells, enterocytes and goblet cells. The lamina propria contains some different cell types such as fibroblasts, and cells of the immune system. The early larvae were present in the caecal mucosa within the enterocytes at the base of the crypts and coiled towards the lumen. As the larvae developed, there was a corresponding increase in damage to the epithelium. In the late stages, as the worms matured in AKR mice, they expanded out of the crypt towards the lumen where they were visible near the surface. The worms were always surrounded by distorted syncytial tissue of the mucosa which forms a tunnel. In cross section, the pharyngeal region of the larvae showed an enormously elongated capillary-like oesophagus surrounded by an oesophageal gland (stichosome) which contains the stichocytes. The stichosome of the larvae at Day 14 post-infection of AKR mice contains two set of stichocytes, whilst in BALB/c mice the stichosome contains only one set of stichocytes. Also the larvae at Day 14 post-infection in BALB/c mice did not form tortuous tunnels on the surface of the mucosa. This is probably because the larvae in BALB/c mice do not develop as far as those in AKR mice. The cuticle of the larvae is a two-layered structure which appeared similar in both AKR and BALB/c mice. At Day 14 post-infection of both AKR & BALB/c mice, the larvae exhibited similar moulting activity. The oesophageal region of the adult worm within the syncytial tunnel is covered with a three-layered cuticle. The oesophageal region of the adult worm within the syncytial tunnel is covered with a three-layered cuticle. The oesophageal region of the larvae at Day 14 post-infection of both AKR & BALB/c contain a single cuticular pore area, the bacillary band which was at an early stage of its formation. The bacillary band of the adult faces the main body of the host's tissue. The bacillary band is an area of cuticular pores, each with underlying hypodermal gland cells and is a highly specialised region of the worm, probably with a secretory activity. The area beneath the cuticular pore forms the pore chamber. The cell membrane of the gland cells beneath the pore chamber is highly enfolded to form the lamellar apparatus. The adult stichosome is made up of a long column of 15-25 stichocytes. Cytoplasmic components of the stichocytes include granules, rough endoplasmic reticulum, lipid droplets, glycogen, mitochondria, Golgi complex and canalicular trees. The possible role of secretory activity in the bacillary band has obvious implications in initiating a host immune response and was investigated by EM immunocytochemistry in order to detect the binding sites of antibodies to Emuris specific proteins. The rabbit polyclonal IgG showed strong reactivity with the cuticle, stichocytes, bacillary band, and the host tissue. The polyclonal anti-human IFN-gamma also showed strong potential in detecting antigens in the cuticle, stichocyte and bacillary band, but it was not particularly effective in detecting antigens in the host tissue. The polyclonal antimurine IFN-gamma demonstrated weak staining in the worm tissue, although it had a stronger staining in the host tissue. Rat monoclonal IgM showed a relative potential in staining the cuticle, bacillary band, stichocyte, and host tissue. This thesis provides new knowledge about the early and late stages of post-infection. It also provides new knowledge about the possible secretory function of the bacillary band in invoking an Immune response.

The aim of this study was to incriminate the vector (s) of visceral leishmaniasis in Northern Pakistan. Two contrasting disease foci were studied- Azad Jammu & Kashmir (AJK) and Northern Areas (NA) through monthly sampling. Studies on the systematics defined morphologically variable species of the subgenera Phlebolomus (Larrousslus) and P. (Adlerius). Morphological characters of all the species belonging to these subgenera were intensively studied. The species belonging to the subgenus Phlebotoinus (Larroussius) were identified as P. major, P.keshishiani, P.kandelakii burneyi and P. spA. and those belonging to subgenus Alderius as P.hindustanicus and P. salangensis. Species belonging to other subgenera. P (Phlebotomus) papatasi, P (Paraphlebtomus) sergentli and P (Paraphlebtomus) alexandri were easily identified. The longitudinal entomological studies were carried out in Bagh district (Rehra village) in AJK and Chilas district (Hudur village, 1200m and Thor village, 1700m) in Northern Areas between April 1991 and November 1991 and in the same months in 1992. Sampling was done using CDC-light traps, sticky-paper traps and mouth aspirators. In addition, a general survey was also undertaken at higher altitudes in Northern Areas and AJK. A total of 9656 Phlebotomus sandflies were collected (8797 during the longitudinal studies and 859 during the general survey). Nine species of Phlebotomus were found,Phlebotomus (A) salangensis for the first time and a new species P. (Larroussius) sp. A. The species composition and relative abundance of species differed within and between the two areas. In Northern Areas P. papatasi (35.75%) followed by P. sergenti (19%) were dominant at the lower altitude of Hudur village but P. keshishiani (29.66%) was most abundant at the higher altitude of Thor village, whereas in AJK, P. hindustanicus (64.62%) was predominant. Species differed in their seasonal abundance. In Northern Areas P. papatasi and P. sergenti showed peak activity in June and July and P. keshishiani in August. In AJK P.hindustanicus showed a clear peak in June preceding the monsoon. The activity was found to be positively correlated with temperature in Northern Areas and negatively correlated with rainfall in AJK. The dominant anthropophilic species included P. papatasi at lower altitudes (Hudur village), P.keshishiani at higher altitudes (Thor village) in Northern Areas and P.hindustanicus in AJK. Most biting occurred around midnight between -2200-2400 hours with a second peak between 0400-0600 hours in Northern Areas. None of the sandflies were found biting human volunteers in AJK. Blood-meal analysis revealed that the dominant species P.papatasi, P. sergenti, P. keshishiani and P. hindustanicus have a range of hosts mainly human, bovines and dogs. Monthly sampling with dog baited traps also showed P. keshishiani and P. hindustanicus as the dominant species attracted to the potential reservoir host (dog). Females of P.keshishiani (754), P. salangensis (36), P. alexandri (15) and P.hindustanicus (301) were dissected for natural infections with Leishmania promastigotes and squash blots probed by Leishmania infantum specific DNA probe (LUCA D2 200 BP). Only P.hindustanicus was found to be naturally infected with Leishinania infantum, (parasites identified by DNA hybridization) in AJK. This species is the principal vector involved in the transmission of VL in AJK. From ecological studies it is concluded that the transmission of visceral leishmaniasis takes place at higher altitudes in Northern Areas and that P. keshishiani is the vector.

Campylobacter jejuni is the leading cause of foodborne gastroenteritis worldwide, yet despite the organism's prevalence, relatively little is known about the mechanisms of pathogenesis. This is mainly due to the lack of a convenient small animal model of infection combined with certain inherent weaknesses with widely used in vitro models. The aim was to develop two improved models to study C. jejuni interactions with intestinal epithelial cells. The Ex Vivo Organ Culture (EVOC) model involves co-culturing C. jejuni with human intestinal biopsies. C. jejuni 11168H and 81-176 wild-type strains were demonstrated to induce the secretion of human beta-defensins 2 and 3 (hBD-2 and hBD-3). Furthermore, the supernatants of infected biopsies were demonstrated to contain significantly higher levels of IL-l~, IL-6, IL-12 and IL-23 compared to uninfected controls. Experiments using 11168H flaA and neuBl mutants demonstrated that the induced defensin response was not due to host recognition of either flagellin or the terminal sialic acid residue of C. jejuni illS. The Vertical Diffusion Chamber (VDC) model involves co-culturing C. jejuni with polarised human intestinal epithelial cells (lECs) with micro aerobic conditions at the apical surface and aerobic conditions at the basolateral surface. Survival and integrity of the IECs under these conditions over 24 hours was demonstrated. Co- culture experiments under these conditions resulted in an increase in both C. jejuni interaction with and invasion of IECs. This was mirrored by an increased, polarised host innate immune response. Transcriptional analysis of aerobically and microaerobically co-cultured C. jejuni 11168H identified several genes that may playa role in these increased interactions. The levels of interaction and invasion of defined C. jejuni 11168H mutants with Caco-2 cells were analysed to identify bacterial factors that contribute to these increased interactions. Both 11168HflaA and rpoN mutants exhibited lower levels of interaction and invasion than the wild-type strain, suggesting the involvement of bacterial motility in the increased interactions under micro aerobic conditions. The reduction in this increased interaction phenotype was more pronounced at shorter co-incubation times, suggesting that motility is particularly important during the early phases of interaction. The development of these two model systems should allow· future 3 experiments to more accurately investigate host-pathogen interactions during C. jejuni infection of the human intestinal tract

Malaria /HIV co-infection is a major challenge to public health in developing countries and yet potential drug-drug interactions between antimalarial and antiviral regimens have not been adequately investigated in people with both HIV and Plasmodium falciparum infections. Earlier studies on the use of artemether-lumefantrine (AL) in Nigeria have neither addressed its use in HIV-positive subjects nor in asymptomatic-malaria infection. The present study investigated associations between drug resistant P. falciparum and the use of medication for HIV management, drug-drug interactions between artemether-lumefantrine and antiretroviral drugs (ARV) and the molecular markers of artemether-lumefantrine and other antimalarial drugs. Results of the study revealed an elevated day 7 lumefantrine concentrations in HIV subjects on nevirapine treatment compared to their HIV-negative counterparts. Associations between elevated day 7 levels of lumefantrine and the persistent parasitaemia could not be evaluated due to inadequate power. Genetic analysis by DNA sequence of P. falciparum isolates revealed strong selection for the pfmdr1codon86N allele among all treated individuals. This polymorphism is a strong indicator of AL treatment failure or slow clearance in vivo. There was a 72.6% prevalence of the pfcrt76T mutations in the population and this was observed to be higher in the HIV-positive subjects. Three new mutations F73S, S97L and G165R were detected on the pfmdr1 gene and the first case S436F mutation on the pfdhps gene to be reported in Nigeria. The dhpsK540E and dhfrI164L mutations, associated with high-level resistance to sulfadoxine-pyrimethamine (SP) were not observed in our small sample size. The study also revealed that HIV-positive subjects were more likely to harbour parasites, at a higher density, before and after treatment. Improvement of the immune status of HIV-infected patients was suggested by the increase of CD4 cell count level in about 68% of the HIV-positive patients. This is a preliminary study and first of its kind to investigate drug-drug interactions between ARVs and the antimalarial drug AL in HIV-positive patients co-infected with P. falciparum in relation to parasite clearance. The findings of the study are very important but more work is urgently needed with a larger sample size to confirm these findings.

In Ethiopia, malaria transmission is seasonal and epidemic-prone, with both Plasmodium falciparum and Plasmodium vivax being endemic. Such spatial and temporal clustering of malaria only serves to underscore the importance of regularly collecting up-to-date malaria surveillance data to inform decision-making in malaria control and improve responsiveness to potential epidemics. This thesis compares indicators and strategies used for the monitoring and surveillance of malaria in Ethiopia. Cross-sectional school-based surveys were conducted throughout Oromia Regional State, generating data on malaria prevalence by microscopy, risk factors for infection and intervention use. Filter paper blood samples collected during these school surveys were subsequently tested to determine exposure to malaria based on presence of anti-Plasmodium antibodies, and Bayesian geostatistical modelling was employed to predict P. falciparum and P. vivax seroprevalence across Oromia. In southern Ethiopia, a schoolbased syndromic surveillance system was piloted, exploring the utility of school absenteeism as a complementary indicator of malaria epidemics at community level. Finally, findings from the school surveys, measured and modelled seroprevalence, as well as data from the national Malaria Indicator Survey in 2011 were compared with spatially congruent estimates of malaria incidence collected from health facilities and to modelled parasite rate from the Malaria Atlas Project. Findings from this thesis demonstrate the limitations of microscopy as a primary indicator of malaria infection in cross-sectional surveys in areas of very low transmission. The work highlights the potential of serological indicators of Plasmodium exposure for inclusion in periodic large-scale malaria monitoring activities and develops a first ever geostatistical risk map based on serological indictors. This was supported by comparative analysis of a range of survey and modelling indicators against estimates of incidence from passive surveillance, indicating the inadequacy of cross-sectional surveys estimating population parasitaemia to reflect the spatial extent and temporal variability of transmission. The piloted syndromic surveillance system indicates that monitoring school absenteeism has potential as a complementary epidemic alert system, operating alongside the existing system at health posts, but is limited by low school enrolment in the piloted setting. The findings of this thesis indicate that existing periodic monitoring strategies and tools are insufficient to fully describe the extent of malaria in settings where Plasmodium transmission is spatially and temporally variable. Modifications to monitoring strategies are recommended, including incorporation of serological indicators and spatial modelling.

Burkholderia pseudomallei, the causative agent of melioidosis, has evolved multiple strategies to facilitate survival in the environment and can cause serious disease in the human host. The lipoprotein BPSL3147 (VacJ) was previously shown to be important in the growth and survival of B. pseudomallei in an in vivo mouse model and a VacJ transposon mutant was highly attenuated. This work has focused on elucidating the role of VacJ as a virulence determinant in B. pseudomallei. The gene was characterized using bioinformatic and genetic techniques, utilizing comparisons with B. thailandensis to study the in vivo and in vitro roles. In this study a rationally defined B. pseudomallei VacJ deletion mutant was constructed, verified and evaluated. The VacJ mutant was able to colonize mice organs during the initial infection phase, but was unable to sustain the infection. In in vitro assays the VacJ mutant did not display any defect in early steps of the intracellular lifecycle. However, VacJ appears to play a contributory role to human serum resistance, as evidenced by the serum susceptibility of an acapsular B. pseudomallei ΔBPSL3147 mutant and B. thailandensis VacJ mutants. Taken together, VacJ contributes to virulence by affecting the outer membrane of B. pseudomallei and B. thailandensis affecting serum resistance sensitivity. The B. pseudomallei VacJ mutant was also investigated for potential as a live attenuated vaccine and displayed partial protection against a lethal challenge in an acute intranasal mice infection model.

This study investigates the evolution of fluoroquinolone resistance in Burkholderia cepacia and assesses fitness of clinical isolates of the B. cepacia complex. B. cepacia was chosen as a clinically relevant model of antibiotic resistance because these bacteria cause chronic infections in cystic fibrosis patients, are highly resistant to killing by many antimicrobials and consequently require long term antibiotic treatment. Fluoroquinolones are a widely used class of antimicrobials, increasingly used in medical and veterinary practice. A method was optimised and used to determine the rate of mutation occurring in topoisomerase genes that confer resistance to fluoroquinolones. The fitness cost associated with fluoroquinolone resistance mutations was assessed as a measure of the stability of resistance in the bacterial population. Clinical isolates were assessed for hypermutability using mutation to fluoroquinolone resistance as a selective tool. In Gram-negative bacteria resistance to fluoroquinolones occurs via three major mechanisms drug efflux, reduced permeability and target alteration. The spectrum of fluoroquinolone resistance mutations occurring in vitro, the rate at which they arise, and the fitness costs of characterised topoisomerase mutations was investigated, using models relevant to transmission of the Burkholderia cepacia complex. Previous studies have shown that single point mutations in DNA gyrase, conferring resistance, have no or low cost. Only double mutations in gyrA and parC conferred a fitness cost. Second step mutations occur at a faster rate than first step mutations. Mutation in gyrA, therefore, may predispose the genome to mutation in topoisomerase genes.

HCMV is a near-ubiquitous p-herpesvirus that asymptomatically infects the majority of immunocompetent adults. However, in the immunodeficient or the immunosuppressed, HCMV becomes a major cause of morbidity. Solid organ transplant recipients require immunosuppressive regimens prior to organ transplantation the resultant reduction in their T-cell responses leads to an increased risk of uncontrolled HCMV replication, which left unchecked is associated with HCMV disease. In this thesis we observed fluctuations of HCMV-specific CD4 and CD8 T-cell populations in 20 renal transplant patients during the six months following transplant using flow cytometry. The frequencies of these populations were related to incidences of viraemia. Patients suffering viraemic episodes post-transplant had significantly decreased HCMV-specific CD8+ cell responses when compared to individuals who remained HCMV PCR negative and a link was established between the absolute numbers of HCMV-specific CD4 cells and the size of the HCMV-specific CD8 response. Temporal increases in virus-specific CD8 responses were also observed, which corresponded with decreases in the incidences of HCMV viraemia after immunosuppressive regimens were reduced. Further work focused on the emergence of CTL-escape mutants in individuals who, despite having detectable HCMV epitope-specific CD8 responses failed to prevent viraemic episodes. Although no mutations were detected in the patients analysed, a substitution analysis was performed on a defined epitope to determine the possible effects of any substitutions on MHC I molecules binding and recognition by CTLs. Results showed a variety of substitutions could abrogate IFNy production by CTLs whilst binding and stabilising MHC I complexes. I also demonstrated for the first time that individuals suffering from common variable immunodeficiency (CVID) - who lack the capacity to mount their own B-cell responses, had detectable HCMV-specific T-cell responses and did not suffer symptomatic HCMV infection, indicating that control of HCMV replication could be mediated by the T-cell arm of the adaptive immune response.

Human herpesvirus 6 (HHV-6) was discovered in 1986 in patients with lymphoproliferative diseases and it has a predominant tropism for CD4+ T cells in vitro and in vivo. The virus can be divided into two variants: HHV-6A and 6B, based on the differences in biological properties and DNA sequences. HHV-6B has been shown to be the causative agent of exanthem subitum while HHV-6A has no clear association with any disease yet. The genome for both variants has been defined and each encodes just over 100 open readings frames (ORFs). However, there is limited knowledge regarding the functions and transcription kinetics of most ORFs. This thesis discusses the development of DNA microarrays for HHV-6 and the application of the arrays to characterise HHV-6B gene expression in the SupT1 cell line. The expression pattern of individual viral genes over a 60h time course (<1 replication cycle) was profiled. Viral genes were further classified into three kinetic groups: immediately-early (IE), early (E), and late (L), according to their transcriptional activity in the absence of de novo protein synthesis or DNA replication. In addition, HHV-6 presents an atypical stage in the herpesvirus life cycle in which the viral genome is integrated into host chromosomes. The prevalence of HHV-6 integration was estimated to be between 0.21% to 3%. An individual harbouring integrated HHV-6 was previously identified. The molecular biology and gene expression of this integrated HHV-6 DNA were characterised. Expression of viral genes belonging to all three kinetic classes (IE, E, and L) was detected in vitro and ex vivo. The data strongly suggest that the chromosomal HHV-6 sequence is transcriptionally active and the implications of this are discussed.