Thesis (MSc (Veterinary Tropical Diseases)--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

No description available.

Much circumstantial evidence has pointed to peripheral blood leukocytes as one site of persistence of Human Cytomegalovirus (HCMV) in healthy carriers. However, the exact population of peripheral blood cells that carry HCMV and to what extent they express HCMV gene products in not known. I have examined the sites of HCMV persistence in the peripheral blood of healthy carriers. Analysis of pure cell populations by the use of the polymerase chain reaction (PCR), sensitive to between 1 and 10 copies of the HCMV genome, showed that the predominant site of persistence was the monocyte. In addition, analysis of healthy seronegative subjects revealed that a significant number (30%) also harbored HCMV. Finally, study of granulocytes demonstrated no evidence of persistent HCMV. Expression of HCMV during persistence was also analyzed, by using reverse transcription PCR (RT-PCR) with a sensitivity of between 1 and 100 infected fibroblasts. RNA from monocytes showed no evidence of polyadenylated immediate early (IE) or late transcripts. In contrast, in vitro differentiated monocyte-derived macrophages (MDM) did show evidence of HCMV gene expression with the class of HCMV genes expressed dependent on the method of differentiation. MDM treated with hydrocortisone (HC) and phorbol 12-myristate 13-acetate, expressed only lEI, but not IE2, glycoprotein B (gB) or phosphoprotein 28 (pp28) transcripts. Whereas, MDM treated with granulocyte-macrophage colony stimulating factor and HC expressed lEI, IE2 and gB, but not pp28 transcripts. In both cases, cocultivation experiments did not show plaques. Therefore, in the healthy carrier, persistence of HCMV in monocytes is independent of HCMV lytic gene expression, but in vitro differentiation of monocytes to MDM induced endogenous HCMV transcription consistent with the known permissivity of in vivo differentiated macrophages to HCMV infection.

No description available.

Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus of domestic sheep and causes the lymphoproliferative disease malignant catarrhal fever (MCF) in susceptible ruminants, including cattle. Sheep are latently infected but do not develop disease. MCF is characterised by proliferation of non-antigen specific cytotoxic large granular lymphocytes which leads to necrosis of infiltrated tissues and death. The molecular basis underlying MCF pathogenesis is poorly understood and it is unknown what controls the differences in the clinical outcome of infection between sheep and cattle, two closely related species. microRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression through targeting of mRNA. A number of herpesviruses have been shown to encode miRNAs that are capable of regulating of both viral and cellular gene expression which can often have an effect on the pathogenesis of the virus. Following RNA seq analysis of an OvHV-2-infected bovine T-cell line (BJ1035) forty-five miRNAs were predicted to be encoded. Eight miRNAs were previously validated by northern blotting, and a further twenty-seven were confirmed using two PCR methods described in this project. It was hypothesised that these virus-encoded miRNAs may differentially target cellular genes in sheep and MCF-susceptible species. Previous work using the technique CLASH (Crosslinking Ligation and Sequencing of Hybrids) identified Delta-like 1 (DLL1), a ligand for Notch signalling, as a potential target of ovhv2-miR-17-2. Initially, differential targeting of DLL1 between sheep and cattle was hypothesised due to differences in the sequence and number of binding sites for ovhv2-miR-17-2. The sheep DLL1 mRNA was shown to be targeted however, due to incorrect annotation of the sheep genome, targeting of DLL1 is likely in both sheep and cattle. One OvHV-2-encoded miRNA, ovhv2-miR-73-1, has partial homology to a mammalian miRNA, miR-216a. Based on this homology it was predicted that ovhv2-miR-73-1 may target Phosphatase and Tensin Homolog (PTEN) and Y Box Binding Protein 1 (YB-1), as they are known targets of miR-216a. A GFP-reporter system was used to demonstrate that despite having similar seed sequences, ovhv2-miR-73-1 does not target PTEN or YB-1. Bioinformatic prediction was used to identify MHC class II genes as potential targets of OvHV-2-encoded miRNAs. Two miRNAs, ovhv2-miR-17-25 and ovhv2-miR-17-9 were shown to target sheep MHC class II genes (DRA and DQB respectively) using a luciferase reporter system. These miRNAs were not predicted to target the equivalent genes in cattle indicating that these genes may be differentially regulated between sheep and cattle. It was also shown that two OvHV-2-encoded miRNAs, ovhv2-miR-17-10 and ovhv2-miR-61-1, target the viral protein Ov2. Ov2 is predicted to contain a basic leucine zipper (bZIP) domain and is therefore likely to be a transcription factor. Other closely related gammaherpesviruses encode proteins that contain bZIP domains and these play major roles in the reactivation of the virus from latency. Immunofluorescence and confocal microscopy was performed to confirm the nuclear localisation of Ov2. RT-qPCRs were performed to investigate whether Ov2 could regulate the expression of any cellular genes. Of the two genes investigated, one of these, Jagged (JAG1), was downregulated in the presence of an Ov2-EGFPN1 construct compared to a control plasmid. JAG1 is another ligand for Notch signalling indicating that the virus may manipulate Notch signalling using multiple methods. Immunoprecipitation and mass spectrometry analysis of an Ov2HA-pcDNA3.1+ construct was performed and a number of potential interacting partners of Ov2 were identified.

No description available.

No description available.

No description available.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

This thesis describes the development, optimisation and use of assays to measure equine herpes virus-specific proliferative and cytotoxic Tlymphocyte (CTL) responses in the blood of horses. Equine T cell blast cells stimulated from peripheral blood mononuclear cells (PBMC) with pokeweed mitogen were found to perform best as targets for CTL in "Cr release assays. CTL induced in vitro with an abortigenic strain of EHV-1 (EHV-1/Ab4) were shown to be antigen specific, genetically restricted and predominantly of the CD4' CD8+ phenotype. Cross-reactive CTL were induced in vitro with live EHV-4 virus, which killed EHV-1 infected blast cells. A proportion of EHV-1 induced CTL were shown to be directed against the immediate early gene products. A proliferative LDA was used to determine whether the frequency of precursor T cells detected before challenge with EHV-1 correlated with immune status. The precursor frequency of antigen-specific T cells increased in 3 out of 4 horses after infection. However, there was no correlation between precursor frequency and outcome of infection. A LDA was developed and used to evaluate the precursor frequencies of EHV-1 and EHV-4 induced CTL after infection with these viruses. Pre-infection CTLp frequencies in susceptible animals were < 1/150,000. CTLp frequencies in animals which were immune to EHV-1 were between 1/10,000 and 1/20,000. To my knowledge this is the first report of the use of LDA techniques in the horse. The development and use of CTL LDA assays have provided new information on CTL responses in horses after EHV-1 and EHV-4 infection.