The studies presented in this thesis describe the identification of novel capsid motifs involved in cell culture adaptation of FMDV A/Iran/87 A-. Field isolates of foot-and-mouth disease virus (FMDV) use integrins as receptors due to the presence of a conserved integrin-binding RGD motif located on the G-H loop of VP1, whereas cell-culture adapted variants can use other receptors such as heparan sulphate (HS). FMDV A/Iran/87 A- was isolated from a vaccine stock and has a major deletion within the VP1 G-H loop. This virus is unable to bind to integrins (as it lacks the RGD), and lacks the known HS contact residues. Sequence comparison identified a limited number of surface exposed residue changes, including a LEK to SAR tri-peptide at VP2 78-80 and a KE to EK di-peptide at VP2 130-131. Reverse genetics was used to investigate a functional role for these motifs in infection where it was found that VP2 80 (R) and 131 (K) were critical for infectivity. Mutagenesis was then used to introduce these motifs in to another type-A virus whilst simultaneously abrogating integrin-binding. Infectious virus could only be recovered when both the SAR and EK motifs were present suggesting that both the SAR and EK motifs are essential for infection, most likely by forming a novel receptor attachment site. Using immunofluorescence microscopy, cell surface-bound A/Iran/87 A- was rapidly internalised (within 5 mins), and co-localised with markers of early endosomes. This was characteristic of clathrin-mediated endocytosis and this conclusion was supported by observations that entry was inhibited by clathrin- and dynamin-inhibitors. However A/Iran/87 A- also co-localised with caveolin-1, a marker of caveolae, suggesting it may use more than one entry pathway. Taken together, this research may pave the way for future studies into rationally designed vaccine-viruses.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:635568 |
| Date | January 2015 |
| Creators | Chamberlain, Kyle |
| Contributors | Jackson, Terry; Roberts, Lisa |
| Publisher | University of Surrey |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://epubs.surrey.ac.uk/807097/ |
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