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Title: Understanding the early events in Bluetongue virus cell entry
Author: Stevens, Lisa M.
ISNI:       0000 0004 5991 9433
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2016
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Bluetongue virus (BTV) is an arbovirus of the Reoviridae family and causes major economic impact due to the haemorrhagic presentation of Bluetongue disease in ruminants. Target cells of BTV infection are endothelial and immune cells in the mammalian hosts (bovine and ovine) and the midgut and salivary gland cells of the insect host (Culicoides spp.), which is the known vector in the transmission of BTV. Little is known about BTV entry and replication in host derived cells. This thesis investigates the endocytosis mechanisms used by BTV in bovine endothelial (BFA) cells and (KC) cells derived from Culicoides spp. Using a combination of confocal microscopy and flow cytometry, endocytosis pathways were dissected by using pharmacological inhibitors, siRNA and dominant-negative (DN) protein expression which inhibit cellular factors, allowing discrimination between distinct endocytosis pathways. It was found that BTV-1 entry and trafficking to acidic endosomes was relatively slow, consistent with a non-clathrin uptake pathway in BFA cells. During entry, BTV appeared to induce global rearrangements of the actin cytoskeleton and disruption of F-actin filaments with Cytochalasin D inhibited BTV-1 infection by preventing an early step in infection. Furthermore, preliminary evidence suggests that BTV-1 may induce actin rearrangements and uptake by initiating Rho protein signalling. Infection was inhibited by EIPA and IPA-3 which target sodium-proton exchanger 1 and p21 activated kinases respectively, which are cellular factors required for macropinocytosis. Taken together, these results suggest that BTV is utilizing macropinocytosis for entry in BFA cells. Furthermore, expression of DN eps15 and treatment with an inhibitor of CME (Pitstop 2) did not inhibit BTV-1 infection of BFA cells. Interestingly, treatment of KC cells with Pitstop-2 appeared to inhibit BTV-1 infection, indicating that infection may be clathrin dependent in KC cells. Additionally, treatment of BFA and KC cells with Methyl-β-cyclodextrin did not inhibit infection, indicating infection is cholesterol-independent. Treatment of BFA cells with a dynamin inhibitor, Dyngo4a, siRNA targeting dynamin-2 and expression of DN dynamin-2 splice variant ba all inhibited BTV-1 infection and Dyngo4a was shown to inhibit BTV-1 entry. In addition, dynamin inhibitors Dynasore and Dyngo4a also inhibited BTV-1 infection in KC cells. This suggests that the endocytosis pathway used by BTV-1 in BFA and KC cells is dynamin-dependent. This thesis provides evidence that entry of BTV-1 is determined in part by the target cell and uses a dynamin-dependent macropinocytosis-like pathway to enter mammalian cells and CME in insect cells.
Supervisor: Darpel, K. E. ; Jackson, T. Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available