MicroRNAs (miRNAs) have now been identified in a vast array of organisms and a great deal of research has been carried out to elucidate the role they play. The dysregulation of miRNA expression has been implicated in a number of disease states and their importance has been highlighted by the beginning of their utilisation as therapeutics. The focus of this study was to identify the role played by miRNAs encoded by the Marek’s disease vaccine viruses, Marek’s disease virus serotype 2 (MDV-2) and Herpesvirus of turkeys (HVT). In order to better understand the functions of these miRNAs we wanted to identify their targets within the host cell. Using a combination of bioinformatic and biochemical approaches we were able to build up a library of potential targets. Three viral miRNA targets; AKT3, RAP1A and DEK, were further validated using dual-luciferase assays to highlight the exact site of miRNA targeting, and western blots to demonstrate an effect of miRNA targeting on protein abundance. An attempt at using label-free proteomics to observe the viral miRNA mediated changes in the host proteome is also described, however this proved to be unsuccessful. Additionally the function of one particular MDV-2 miRNA, mdv2-miR-M21, was explored in more detail, describing its role as a potential ortholog of the host miRNA; gga-miR-29b. By using the observation that the viral miRNA contained an identical 'seed' region to the host miRNA, we were able to use the data collected from existing studies on miR-29b to search for targets of mdv2-miR-M21. We demonstrated that mdv2-miR-M21 targeted DNMT3B, crucial for epigenetic modification of the genome. The final part of this study aimed to understand the wider context the viral miRNAs played in the viral biology and protective ability of the vaccine viruses. The miRNAs were deleted from the viruses, and then the miRNA-deletion viruses were used to vaccinate birds before challenge with the oncogenic Marek's disease virus serotype 1 (MDV-1), survival rates to the 'wild-type' MDV-2 and HVT vaccine viruses were then compared.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:680425 |
| Date | January 2015 |
| Creators | Popplestone, James Edward |
| Contributors | Lawrie, Charles H. ; Pezzella, Francesco |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:6f43c371-94fd-4410-92dd-82aa6cb9c1a9 |
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