No description available.

No description available.

No description available.

No description available.

No description available.

Varicella Zoster Virus (VZV) is an a-herpesvirus that causes two related diseases. Primary infection with VZV, causes varicella (chicken pox); the virus then establishes life-long latency in the dorsal root ganglia, and subsequent reactivation causes herpes zoster (shingles). VZV is a ~125kbp DNA virus encoding approximately 70 unique open reading frames, which encode a range of proteins that may be potential targets for the host immune system.

In y2-herpesviruses the balance between lytic infection and reactivation from latency depends entirely on expression of an immediate-early viral gene (ORFSO) encoding the Replication and Transcriptional Activator (Rta) protein. Although studies have elucidated how Rta can activate viral genome leading to a cascade of events necessary for the viral lytic infection, much less is understood about the regulation of Rta expression itself. Nonetheless, extensive work has produced a body of evidence describing several interactions between cellular factors signaling pathways and physiological stimuli, which are able to induce or repress Rta gene expression and hence influence lytic infection cycle as well as reactivation from latency. In this study, the murine model, MHV-68, was used to characterize transcription activity of different domains within the Rta promoter sequence. Initial observations demonstrated that Rta promoter activity is both cell-type dependent and domain-specific. Overall, Rta promoter had a greater basal activity in immunocompetent cells than cells which were unable to respond to type I IFN signaling. Specifically, the transcription activity of a region between 1019 and 391 bp upstream of ORFSO ORF was down regulated when cells are pre-treated with IFN-a/B. Detailed bioinformatics analysis of this domain revealed a series of putative binding sites most closely associated with IFN response elements, including interferon regulatory factors 3 and 7 (IRF3 and IRF7). Site-specific mutation of these viral sequences within the Rta promoter resulted in cell type dependent modulation of Rta promoter. Interestingly, data obtained from different cell types strongly suggested that endogenous IRF7 was involved in upregulation of the promoter in murine B Iymphocytes which was different from non-B cells. Paradoxically, exogenous expression of IRF7 reduced activation of the Rta promoter in fibroblasts with an abrogated type I IFN pathway and in a promoter sequence-specific manner. Although, physical interaction between IRF7 and Rta promoter using EMSA could not be demonstrated further work is required to show how IRF7 may affect Rta promoter and under different cell conditions. Taken together, these findings show for the first time the domain- and cell-type-specific activities of the Rta promoter and the complexities of Rta promoter regulation by type I IFN mediated responses. These findings also give credence to previous work which demonstrated that y- herpesviruses have evolved strategies alongside type I IFN-dependent cellular pathways which modulate their lytic as well as latent (persistent) stages of infection.

Equine herpes virus 1 (EHV-1) causes abortion and neurological disease in horses, largely through the infection of endothelial cells and consequent inflammation which leads to a disruption of the vascular supply to the pregnant uterus and spinal cord. Although many different cell types are susceptible to infection, it is the cell associated viraemia which disseminates virus to these sites of secondary replication. Therefore, in recent years a great deal of research has focused on understanding the binding and entry of EHV-1 to several mammalian cell types. Like other alphaherpes viruses, EHV-1 has been shown to utilise several binding receptors and uptake mechanisms to infect its many permissible cell types. In the current study the physical characteristics of EHV-1 were determined and the mechanism of viral binding and fusion into rabbit kidney 13 cells (RK13s) and resting and activated peripheral blood mononuclear cells (PBMC) was observed. EHV-1 was identified as being 266 nm in diameter and had a zeta potential of -33 mV. Heat inactivated (HI) EHV-1 was shown to co -localise with GM-1 suggesting that it may associate with lipid rafts. Following infection of PBMC collected from horses with homozygous major histocompatibility complex class 1 (MHC-1) serological haplotypes (A2, A3 and A9) withEHV-1 strain RacL11 expressing green fluorescent protein (GFP) in place of glycoprotein 2(.1gp2), no apparent difference in infection was observed. Various fluorescence ii based fusion and endocytosis experiments were carried out using HI EHV- l Ab4 to infect RK13s and equine PBMC to examine the mechanisms involved, but the techniques proved too insensitive. The data suggest that initially, EHV-l associates with rafts and that MHC- l haplotype has no effect on viral infection of PBMCs. It was concluded that heat inactivation may damage viral glycoproteins sufficiently to reduce viral fusion and that future experiments should use live, infectious virus. ii

No description available.

Epstein-Barr virus (EBV) establishes a lifelong latent infection in 95% of the world’s population and is associated with several human cancers. Some of the most abundant viral products in latent infection are the functional EBV-encoded RNAs EBER1 and EBER2. The aim of this thesis was the identification of distinct functions of EBER1 and EBER2 during EBV infection. Epstein-Barr virus bacterial artificial chromosomes with deletion of either EBER1 or EBER2 and corresponding revertant viral genomes were constructed to analyse broad range effects of EBER1 or EBER2 on host cell gene expression. The resulting recombinant viruses were used to infect primary B lymphocytes and establish lymphoblastoid cell lines (LCLs). Microarray expression profiling revealed clear changes in host cell gene expression correlating with EBER expression and significant differences between gene sets regulated with EBER1 and EBER2. Functions of EBER target genes include membrane signalling, regulation of apoptosis and the interferon response. In additional studies, the interaction of EBER1 with ribosomal protein L22 (RPL22), a component of the large ribosomal subunit, was demonstrated in LCL extracts. Using recombinant viruses and EBER expression vectors, the nuclear redistribution of RPL22 by EBER1 was investigated. The delocalisation of RPL22 from nucleoli into the nucleoplasm upon EBV infection was demonstrated in HEK 293, nasopharyngeal carcinoma and gastric carcinoma-derived cell lines. EBER1 was identified as the only viral component necessary for the delocalisation of RPL22. In contrast to the cancer-derived cell lines, LCLs showed a predominantly cytoplasmic expression of RPL22, which was not significantly changed by EBER1. Subsequently, a possible role of RPL22 and EBER1 in p53-dependent stress responses was explored. The data presented in this thesis provide further understanding of the role of EBERs in EBV infection and several of the EBER-regulated genes might be used as markers to elucidate the mechanism of EBER action.