Ovine herpesvirus 2 (OvHV-2) is a gamma herpesvirus and is the causative agent of lymphoproliferative disease – sheep-associated malignant catarrhal fever in susceptible ruminants, including cattle. Sheep become persistently infected but do not show apparent clinical infection. MCF is characterized by marked T cell hyperplasia and proliferation of unrestricted cytotoxic large granular lymphocytes (LGLs) which leads to necrosis of infiltrated tissues and generally causes death of the host. Little is known about the underlying molecular basis of MCF pathogenesis or what controls the differences in clinical outcome of infection in two closely-related host species. MicroRNAs (miRNAs) constitute a large family of small, ~22nt, noncoding RNA molecules that regulate gene expression by targeting messenger RNAs post-transcriptianally in eukaryotes and viruses. Herpesvirus encoded miRNAs have been shown to play a role in regulating viral and cellular processes including cell cycle and may have a role in pathogenesis. OvHV-2 has also been found to encode for at least 46 OvHV-2 miRNAs in an immortalized bovine LGL cell line. 23 of these miRNAs have also been validated by northern blot analysis and RT qPCR. It was hypothesised that these OvHV-2 miRNAs may regulate viral and cellular genes expression and may play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 miRNAs have functional targets within viral and host cell genes. Bio-informatic analysis has predicted several targets for these OvHV2 miRNAs in the 5’ and 3’ UTRs of several virus genes. Luciferase inhibition assay confirmed that out of 13 selected predicted targets, three (two targets ORF73 and one within ORF50) were positive and functional. A fourth predicted target was also found functional (ORF20), but its functionality could not be confirmed by knocking out the target site. A newly developed technique Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was also used to identify miRNAs bound targets within cattle and sheep genome. High throughput sequencing and analysis of the hybrid data revealed many target genes. Four of those targeted genes, were validated by luciferase inhibition assays and three were found to be targeted by OvHV-2 miRNAs. This study gives the first evidence of viral miRNAs bound to their targets in cattle and sheep cells, by a highly sensitive technique-CLASH and provides a tool for studying differences in pathogenesis of two closely-related host species.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:646685 |
| Date | January 2014 |
| Creators | Riaz, Aayesha |
| Contributors | Dalziel, Robert; Hopkins, John |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10061 |
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