The Urabe AM9 mumps vaccine was removed from use in many countries due to an unacceptable incidence of post-vaccination disease. The vaccine was found to be a mixture of closely related viruses, presumably with differing degrees of attenuation. One virus isolated from the vaccine has been shown to be fully attenuated in a rodent model. This virus, Gw7, is characterized by growth in Vero cells (titers 2x106 pfu/ml) but extremely limited growth in human cell lines (<104 pfu/ml), while a virulent Urabe virus, 1004, from a patient with post-vaccination meningitis, grows well (>2x107 pfu/ml) in all the cell lines tested to date. Initial sequencing of the genome of the two viruses has identified genetic differences in 6 of the 7 genes, including the two proteins of the replication complex, the polymerase, L (Large protein), and in the P protein (phosphoprotein). The hypothesis was that the amino acid differences observed in L and P play a role in the differences in attenuation in vivo and growth differences in tissue culture cells.
The first objective was to confirm the sequence differences in the L and P genes of the viruses and the sequencing results confirmed all the reported differences in the L gene between 1004 and Gw7 plus identified another sequence difference in the L gene which had not been reported in the original sequencing; it also showed that there was no sequence difference in the P gene of the two viruses.
The next objective was to compare the polymerase activity of the two viruses. Two methods have been employed to measure the polymerase activity of the viruses; In vivo incorporation of 3H UTP in the presence of Actinomycin D, and transcription of luciferase reporter gene from the Urabe mini-genome construct. For the first assay, the polymerase activity was examined under two different conditions; and in one case, a higher polymerase activity was observed for 1004, but this was not confirmed in the second set of experiments. To compare the polymerase activities of the two viruses with the help of a mini-genome system, the Urabe mini-genome was constructed and the polymerase activity was determined in two ways; using the polymerase complex (L, P and NP proteins) of each virus or the whole viral particles to drive the luciferase expression from the mini-genome. In either case, a significant difference in the polymerase activity of the two viruses was not detected. Thus, it can be concluded that no inherent difference in the polymerase activity between 1004 and Gw7 was observed by the performed experiments in this study.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/27720 |
| Date | January 2008 |
| Creators | Nasheri, Neda |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 97 p. |
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