title: Studies on the cellular immune response to feline leukaemia virus
Creators: name: Graham, Elizabeth Mary
Publisher: name: University of Glasgow
Year: 2003
Subjects: subject: Virology, veterinary science, immunology.

description: The aim of this study was to investigate the virus-specific cell-mediated immune responses, particularly virus-specific CD4+ T cells, elicited following oronasal exposure to feline leukaemia virus (FeLV). In Chapter 1, the general features of the retroviridae are outlined; current knowledge on various aspects of FeLV, including epidemiology, pathogenesis and clinical disease is reviewed. General features of the immune responses to retroviridae are described; in addition, the virus-specific immune response to FeLV, and other retroviridae such as human immunodeficiency virus (HIV), Friend retrovirus complex (FV) and bovine leukaemia virus (BLV), is illustrated in detail. The technologies currently available to detect virus-specific CD4+ T cell responses are described and reviewed. This chapter also outlines the aims of this thesis. Chapter 2 describes the development and characterisation of monoclonal and polyclonal antibodies to feline interferon-gamma (flIFN-gamma). These novel reagents were generated as tools to detect virus-specific immune responses using intracellular cytokine staining (ICS), and flow cytometry. The sensitivity of these novel reagents in the detection of both recombinant flIFN-gamma using an enzyme linked immunosorbent assay (ELISA) and Western blots, and the production of intracellular flIFN-gamma in physiologically stimulated cells using flow cytometry, was illustrated. A longitudinal FeLV immunopathogenesis study was conducted to monitor the evolution of virus-specific cell-mediated immune responses, particularly virus- specific CD4+ T cells, following oronasal exposure to FeLV. A group of six specific pathogen free (SPF) cats was exposed oronasally to 5 x 105 focus forming units (ffu) FeLV-A/Glasgow-1 aged sixteen weeks, in the expectation that approximately fifty per cent of cats would recover, and fifty per cent would become persistently infected. However, all six cats became persistently infected. In an attempt to modify the outcome of exposure and improve the chances of recovery, a second group of six SPF cats was exposed to a lower dose of FeLV, 5 x 104 ffu FeLV-A/Glasgow-1, under identical conditions. Five of these cats became persistently infected following oronasal exposure to FeLV, and one cat ostensibly recovered. Although virus was not detected in the peripheral blood from this cat at any time throughout the study, proviral DNA was detected in the bone marrow and lymphoid tissues post mortem, as a latent infection. An account of the virological parameters measured in these cats throughout the study is given in Chapter 3. Sensitive and sophisticated assays, such as the p27 ELISA, virus isolation (VI), and quantitative reverse transcription- polymerase chain reaction (qRT-PCR), were used to detect antigenaemia, viraemia and proviral DNA, respectively, in the peripheral blood throughout the study, and in the lymphoid tissues post mortem. Disease outcomes and proviral DNA loads were correlated with virus-specific CD4+ T cell responses. In Chapter 4, virus-specific cell-mediated immune responses in these cats were investigated using two techniques, the lymphocyte proliferation assay (LPA) and ICS, analysed using flow cytometry. Results obtained in either assay did not correlate with the proviral DNA load; however, the data generated using ICS appeared to be more relevant to the outcome of infection. Latently infected cat E12 failed to develop virus-specific proliferative responses in the peripheral blood; however, the virus-specific upregulation of flIFN-gamma in CD4+ T cells was detected using ICS. Conversely, persistently infected cats generated significant virus-specific proliferative responses in the peripheral blood, and transient low frequencies of virus-specific CD4+ T cell responses were detected using ICS in the critical early weeks following exposure. The bone marrow and lymphoid tissues are important sites of FeLV integration and replication. In Chapter 5, FeLV expression in the post mortem bone marrow and peripheral lymph node (PLN) tissues of these cats is described. In addition, the impact of an active bone marrow infection on haematopoiesis is assessed, using concurrent cytological and haematological analyses of bone marrow and peripheral blood, respectively. Persistently infected cats expressed FeLV in the bone marrow and in PLN tissues; in addition, abnormal haematology and cytology reports were recorded for each of these cats. The latently infected cat did not express FeLV in either the bone marrow or PLN tissues; furthermore, cytology and haematology reports were normal in this cat. In Chapter 6, the results of this thesis are brought together and discussed in the context of current research.