RNA virus recombination is a key evolutionary mechanism and a driver of genetic diversity. In recent studies using an in vitro “CRE-REP” assay involving replication-compromised parental genomes, recombination was shown to be a biphasic process involving an initial imprecise crossover event which was followed by a resolution process that resulted in the formation of genome-length recombinants (Lowry K. et al 2014). We have extended this study to investigate recombination during dual infection by unmodified parental viruses in the absence of selection. Recombinants were generated by co-infecting HeLa cells with poliovirus type 1 Mahoney and type 3 Leon for 5-hours, followed by RNA extraction, cDNA synthesis, and PCR amplification. Amplified PCR products of both type 1/3 and type 3/1 recombinants were readily detected, cloned individually and sequenced by Sanger sequencing. Within 25 clones sequenced, 18 unique recombination junctions were detected. To get a comprehensive overview of the range of recombination junctions within the virus population the data produced from next generation sequencing of pooled amplified cDNA from dually infected cells was analysed. A bioinformatics pipeline was developed to specifically detect and quantify recombinants within this population. Three types of junctions were identified, precise (i.e. at the same position in both genomes) and imprecise, including both insertions (as seen in the Lowry 2014 study) and deletions. In an analysis of the P2 region of the poliovirus genome, we identified several hundred different precise and imprecise junctions. The data analysis suggests that recombination is a random event; no correlation between the nucleotide base composition or RNA structure near the junctions’ locations of both donor and recipient viral genomes and the recombination frequency was detected. These studies contribute to our understanding of the molecular mechanism of genetic recombination in RNA viruses and suggest ways in which it might be controlled during the development of novel vaccines with reduced recombination potential.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:737675 |
| Date | January 2016 |
| Creators | Alnaji, Fadi |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/98260/ |
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