MHV-68 is a gammaherpesvirus that is related to Epstein-Barr virus, Kaposi’s sarcoma associated herpesvirus, and Herpesvirus saimiri. The aim of this thesis was to characterize the role of the putative plasmid maintenance protein encoded by ORF73 of MHV-68 in viral latency and pathogenesis. In order to investigate the role of this gene, MHV-68 mutant viruses deficient for ORF73 were constructed and the recombinant viruses were analysed <i>in vitro</i> and <i>in vivo</i>. It was determined that ORF73 was not required for lytic replication of MHV-68 <i>in vitro</i> or<i> in vivo.</i> In contrast, a severe deficit in viral latency <i>in vivo</i> was observed in the ORF73-deficientviruses suggesting that the virus was unable to persist in the host in the absence of ORF73. Furthermore it was shown that this gene product is necessary for the maintenance of latent viral episomes in an <i>in vitro</i> assay utilizing a mouse myeloma B-cell line, NSO, indicating that ORF73 is a critical determinant in gammaherpesvirus latency. In this project I also attempted to rescue the phenotype of the ORF73-deficient virus by insertion of an ORF73-EGFP expression cassette at an ectopic genomic locus. While expression of ORF73 at this site enabled maintenance of viral episomes in NSO cells <i>in vitro, </i>the <i>in vivo</i> latency deficit was not restored. Further <i>in vitro</i> characterization of the ORF73-deficient and ORF73-EGFP rescuant viruses suggests that ORF73 has a role in the regulation of lytic replication. A final aspect of this project involved the potential use of an ORF73-deficient virus as a live-attenuated gammaherpesvirus vaccine. Analysis of vaccinated animals challenged with wild type MHV-68 showed that exposure to the ORF73-deficient virus afforded significant protection against wild type infection and reduced viral latency to a level below the limit of detection. These results indicate that a live-attenuated gammaherpesvirus that cannot persist is an effective vaccine.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:599146 |
| Date | January 2004 |
| Creators | Fowler, P. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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