Two different types of recombinant viruses each carrying one of two foreign genes, enhanced green fluorescent protein (eGFP) or Cre recombinase, were constructed based on the SFV4 backbone. In the first type of construct the transgene was inserted in the non-structural ORF, between the coding sequences for nsP3 and nsP4, flanked by processing sites recognised by the nsP2 proteinase. In the second type of constructs the 2A sequence from foot-and-mouth disease virus was added to the C-terminus of the foreign gene and this was placed between the capsid and the p62 protein of SFV4 (structural ORF). All recombinant viruses constructed were viable and able to replicate <i>in vitro</i>. eGFP expressing viruses reached titres similar to those of wild-type virus whereas Cre expressing viruses were slightly attenuated. For viruses with the marker gene inserted in the non-structural ORF, western blotting showed that the processing pattern of the non-structural polyprotein was similar to that of SFV4 and verified the expression of both foreign genes. <i>In vivo, </i>following intracerebral inoculation, all viruses caused encephalitis. Viruses expressing the foreign gene as a cleavable component of the structural ORF induced disease slower than SFV4 or viruses carrying the transgene in the replicase ORF. eGFP fluorescence was stronger and occurred later in infection when expressed in the structural ORF than in the replicase ORF. eGFP expression from the replicase ORF marked only recently infected cells; a property useful in pathogenesis studies. eGFP expressing viruses demonstrated the same cell tropism as SFV4 with infection principally of neurons and oligodendrocytes. None of the mice infected intraperitoneally with SFV4 or the recombinant viruses succumbed to infection demonstrating poor neuroinvasiveness. These viruses are likely to be highly valuable for <i>in vivo</i> pathogenesis studies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651072 |
| Date | January 2007 |
| Creators | Fragkoudis, Rennos |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/29767 |
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