Browsing by Subject "Reovirus"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Cellular determinants of reovirus entry and infection(2012-10) Schulz, Wade LorenEfficient entry is a critical determinant of reovirus oncolysis. While a model for the entry of reovirus virions has been described, the method by which partially uncoated intermediate subviral particles (ISVPs) enter host cells remains unknown. Biochemical studies have suggested that ISVPs can directly penetrate the plasma membrane since these particles can cause membrane damage and hemolysis at high multiplicities of infection. We used a combination of techniques to show that these particles can use endocytosis to enter and productively infect host cells. We found that uptake of reovirus virions and ISVPs was inhibited in cells treated with a small molecule inhibitor of dynasore. Analysis of reovirus replication in cells treated with genistein and in cells expressing dominantnegative caveolin-1 revealed that ISVPs use dynamin-dependent caveolar endocytosis to enter host cells. These studies also showed that reovirus virions were able to take advantage of caveolar endocytosis to infect cells. Because many viruses have recently been found to enter cells through dynamindependent and –independent endocytic pathways, we also assessed the role of cholesterol in reovirus infection. The growth of reovirus virions, but not ISVPs, was inhibited in cells treated with methyl-β-cyclodextrin, which extracts membrane cholesterol. We also wanted to learn whether reovirus entry had cell type-specific differences. Analysis of reovirus infection in a rat embryo fibroblast (CREF) and Ras-transformed CREF cell line suggested that reovirus particles lose the ability to use clathrin-mediated endocytosis in CREF cells following Ras transformation. Reovirus can also infect cells that lack caveolae. To assess whether reovirus used different endocytic pathways to infect cells devoid of caveolae, we analyzed reovirus entry and infection in polarized Caco-2 cells. While reovirus infection of these cells was dynamin-independent, particle internalization occurred through both dynamin-dependent and –independent pathways. Visualization of particle uptake showed that particles internalized through dynamin-dependent endocytosis were transcytosed to the basolateral surface. These findings have demonstrated that reovirus can use a diverse set of endocytic pathways to enter and infect cells. The data presented in this thesis show that reovirus entry is a complex process that can vary in different cell types. In addition, it has revealed that particles internalized into various endocytic pathways are trafficked to different destinations in polarized epithelial cells.Item Turkey arthritis reovirus: pathogenesis and immune response(2015-01) Sharaf Eldin, Tamer Ahmed IbrahimIn 2011, turkey reoviruses were isolated from tendons and synovial fluids of >15-week-old lame turkeys displaying swollen joints and occasionally ruptured leg tendons in Midwest, USA. These reoviruses were tentatively called turkey arthritis reoviruses (TARV) to differentiate them from reoviruses isolated from intestinal contents and feces of turkeys namely turkey enteric reoviruses (TERV). TARV were found to be genetically distinct from chicken arthritis reoviruses (CARV). Five experiments were conducted to test the pathogenicity of TARV in turkeys and in chickens and to compare it with that of TERV and CARV. Additionally, this work investigated the virus pathogenesis and cytokine immune responses. TARV showed unique capability to induce significantly higher tenosynovitis scores in turkeys as compared with TERV and CARV which induced minimal scores. Clinical lameness was first displayed at 8 weeks of age in TARV-inoculated turkeys at 1 week of age. Lameness in infected group reached approximately 50% at 16 weeks of age. TARV did not induce any lesions in chickens via intratracheal or oral route. TARV inoculation via footpad route induced tenosynovitis in chickens at 2 and 3 weeks PI with no clinical lameness. In pathogenesis study, TARV displayed the greatest replication in intestines and bursa of Fabricius than in leg tendons of turkeys. Viral infection mediated effective antiviral cytokines immune response that limited virus replication in the intestines. Furthermore, viral infection mediated a significantly elevated T helper-1(Th1) cytokine response in intestines and tendons and minimal Th2 and Th17 cytokine response during the early stage (2 weeks) of infection. This work established an experimental model to study TARV which provides early end points that are indicative of disease pathogenicity. Additionally this work developed a new grading system for histologic tenosynovitis which can be used in a wide variety of experimental models. For lameness evaluation in turkeys, this work developed a grading system for gait scores. In summary, this work showed unique pathogenicity of the newly isolated TARV and added significant knowledge to TARV pathogenesis and immune response using the newly established reproducible experimental model and the newly developed grading systems for evaluation of tenosynovitis and clinical lameness.