Recombination is an important biological process in a diverse range of viruses, particularly those with single-stranded ribonucleic acid (RNA) genomes including the enteroviruses. Mutations caused by the error-prone RNA-dependent RNA polymerase (RdRp) and the vast population size of these virus populations are evolutionary mechanisms that generate genetic diversity – this allows viruses to survive under changing environmental pressures (e.g. adaptive host immunity). Ribonucleic acid recombination has been identified as another contributing mechanism involved in diversification, by removing interfering or lethal mutations from a virus genome, and by establishing new viruses. Virus RNA recombination is well documented and is identified in several virus families including picornaviruses. However, recombination is a rare event and the study of the molecular mechanisms behind virus recombination is complicated by our ability to isolate and analyse recombinants from a mixed virus population including the parental viruses. The objectives of this study were to firstly devise a method for generating populations of natural recombinant viruses, and secondly, to study the molecular processes that determine where and when recombination occurred in the enteroviral genome. During this project, an in vitro system was developed to allow the recovery of recombinant enteroviruses in the absence of their parental viruses. Two virus RNA molecules containing “lesions” rendering them unable to generate viable virus on their own were co-transfected into mouse L929 cells. The method required two parental virus RNA molecules to be present in a single cell to produce a viable recombinant virus. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and sequencing analysis confirmed the recombinant nature of progeny virus genomes. Experimental data confirmed the effectiveness of the method and provided evidence that recombination occurs in at least two phases. Initial template switching, referring to the transfer of RdRp from one RNA template to another mid-replication, occurred apparently indiscriminately and with the addition of extra virus and non-virus sequence at the junction sites. In the second phase, any additional sequence was lost during subsequent rounds of replication and selection. The approach was expanded to incorporate viruses from different enterovirus species to investigate intra- and interspecies recombination events.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:541149 |
| Date | January 2011 |
| Creators | Lowry, Kym Sheree |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/38168/ |
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