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No description available.

No description available.

No description available.

No description available.

No description available.

Human metapneumovirus (hMPV) is a newly described respiratory virus belonging to the Paramyxoviridae family, first identified in respiratory samples of ·children with acute respiratory tract infection (ARTI). Since its discovery hMPV has been associated with ARTI worldwide, however, important questions remain as to the contribution ofhMPV to respiratory illness, and its impact on public health. Extensive surveillance ofhMPV within different populations of the United Kingdom (UK) demonstrates it is an important cause of ART! in the elderly, and influenza like illness ~ (ILl) in people of all ages in the community. Furthermore it is a frequent cause of hospitalisation in young children. Recombinant baculovirus expressed hMPV nucleocapsid protein (N) proved to be a useful source of antigen for the development of an hMPV specific ELISA. Analysis of age stratified sera from the UK indicates that a majority of children are infected by the age of 6 years with primary infections occurring throughout infancy. Virtually all adults have detectable levels ofhMPV antibody; however, reinfection with hMPV is common, raising questions concerning acquisition and duration of immunity. Sequence analysis of the attachment glycoprotein (0), shows a high degree of nucleotide and amino acid variation and extensive glycosylation. Frequent nucleotide insertions or deletions result in frame shift mutations which can drastically alter the appearance of the protein and often results in premature termination. Antibody recognition of hMPV 0 is likely to be highly strain specific and dependent on the extent of glycosylation, suggesting an involvement of 0 in immune evasion. Phylogenetic analysis of hMPV 0 gene sequence shows that whilst a large degree of variation exists within this gene, strains circulating within the UK are genetically similar to strains circulating elsewhere in the world, and similar strains circulate throughout different years and populations within the UK.

This thesis describes human immunodeficiency virus type 1 (HIV-1) infection in Kenyan infants. Paediatric HIV-1 infection causes rapid disease progression in the absence of antiretroviral (ARV) therapy. Afri can cohorts have reported the highest rates of mortality, from 20-50% at 2 years oflife. Understanding the pathogenesis in HIV-1 infected children is important to the design of prevention, treatment. and vaccination strategies. A cohort of 476 Kenyan children born to HIV-1 infected women was studied longitudinally from the time of birth to 24 months. Despite the provision of ARVs to prevent mother-to-child transmission, i 9.4% of infants became infected with HIV-1. Infant HIV-1 infection resulted in persistently high levels of viraemia and rapid CD4 depletion. Cumulative mortality at 2 years was 54%. Peak and set-point HIV-1 viral load, and CD4% at 6 months were predictors of 2-year mortality. Co-infection with cytomegalovirus (CMV) before the age of 1 month was also associated with increased;isk of death. Infants with HIV-1 infection had poorly contained CMV viraemia in comparison with HIV-1 exposed uninfected controls. Multicolour flow cytometry was used to describe the phenotype of T cells during primary viral infection. Both CMV and mv-1 infection resulted in dynamic redistribution of T cell populations. High frequencies of activated, apoptotic vulnerable, differentiating CD8 T cells were observed concurrent to acute infection with either HIV-l or CMV. Co-infection with both viruses resulted in even more profound changes in cellular phenotype. CD4 T cell phenotype was also affected by acute viral infection, but at a much lower magni1ude than observed in the CD8 subset. HIV-1 specific CD8 T cells were studied in a subset of infants using IFN-y ELISpot assays and tetramer staining. Very high frequencies of HIV-1 specific CD8 T c~lls were identified with tetramer staining, and these cells resembled adult T cell responses in magnitude and phenotype. ELISpot assays revealed weak responses in infants less than 6 months old that increased with age. These data suggest that HIV-1 specific CD8 T cell responses can be generated during acute infection in infants, but IFN-y production is lowercompared to adult cells. Reduced functional capacity may explain the inability of infant T cell responses to contain HIV-1 viral load.

Hepatitis C virus (HCV) is the main cause of viral hepatitis in the UK and leads to chronic liver disease in many infected individuals. There is a substantial burden on diagnostic laboratories to provide rapid, cost-effective tests for monitoring HCV infection. Commercial assays are expensive and so the development of validated in-house methods is beneficial. This thesis describes the development and implementation of rapid and inexpensive real-time PCR assays for HCV quantitation and genotyping to support clinical practice. Additionally, the development of methods for defining HCV isolates at the subtype level, important in epidemiological and transmission studies, is described. These assays were utilised in a study on spontaneous HCV clearance, the results of which suggest that HCV type 1 infection and younger age at infection are factors which are associated with spontaneous viral clearance. Chronic HCV infection is linked to oxidative stress with numerous deleterious cellular effects. Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA making it an ideal marker to assess the overall level of cellular DNA damage - including deletions and mutations. This thesis illustrates the development of real-time PCR assays to detect and quantify two major mtDNA deletions. The D-Ioop region of mtDNA is particularly prone to damage - with two well recognised 'hotspots of mutation'. The creation of an RSCA (heteroduplexbased) method using capillary electrophoresis, to detect and quantify damage in this region, is described. To evaluate the clinical utility of these assays, a study of mtDNA damage in patients with liver disease was undertaken. The aim of this study was to identify whether chronic HCV infection results in increased levels of mtDNA damage compared to other liver pathologies. Low levels of mtDNA deletions were detected in the majority of liver biopsy specimens and there was no correlation between liver aetiology and quantity of deletions. The RSCA method identified numerous D-Ioop mtDNA species within the liver tissue of several individuals. There was no correlation between liver aetiology and the presence of multiple D-Ioop species.