AbstractTMEV, a natural pathogen of the mouse, is a picornavirus that belongs to the Cardiovirus genus. Two major subgroups can be distinguished based on their differences in pathology. The first subgroup includes the neurovirulent GDVII and FA strains which induce an acute and mostly fatal encephalomyelitis. In the few mice that survive, the virus is cleared by the immune system. The second subgroup, also known as the 'TO subgroup' includes the DA en BeAn strains and causes a biphasic disease in genetically susceptible mouse. The early, acute disease is a mild and mostly non fatal encephalomyelitis. The late phase is a chronic, demyelinating disease characterized by the presence of a lifelong persistent infection in mainly the macrophages of the white matter in the spinal cord. The similarities between this late phase and MS, make the TMEV one of the best animal models to study this human disease. The exact mechanism of viral persistence is still unknown, but it is likely the result of a complex interplay between viral determinants and cellular factors.
Two possible hypotheses claim that the TMEV transmission in the DRAW cell line happens either via a vertical or a horizontal mechanism. By determinating the percentage of virally infected cells, these hypotheses could be investigated. Therefore it was necessary to isolate and multiply individual DRAW cells ('cloning'). A total of three different clonal isolation methods were used in the experiments. Contrary to the theoretical calculations, the application of two clonal isolation methods always resulted in the presence of multiple DRAW cells in a well. Problems with the cell counting in the Bürkerchamber could be a possible explanation for this contradiction. The third applied clonal isolation method (based on the formation of a drop which theoretically contains one DRAW cell) was successful in obtaining DRAW clones. These clones were further cultivated and ultimately frozen in at -80°C. A total of 39 DRAW clones were cultivated again. 34 samples of these clones were subjected to a plaque titration 48 hours after renewal of the medium; five were subjected to a plaque titration 24 hours after renewal of the medium. Each investigated sample had a viral titer of 0 pfu/ml. This end result didn't allow a confirmation of one of the two hypotheses. The possibility that the viral replication only starts again after a certain amount of time could be a possible explanation for this remarkable finale result. However, additional experiments in which the DRAW clones are cultivated for a longer period of time than 48 hours are necessary to investigate this possibility.
|Date of Award||2009|
|Supervisor||Raphael Vrijsen (Promotor) & Ellen Merckx (Co-promotor)|