Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264915
Title: Genetic variability of measles virus during propagation in cultured cells
Author: Longhurst, Sharon
ISNI:       0000 0001 3612 5022
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1996
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Abstract:
It has been reported that the phenomenon of biased hypermutation, associated with measles virus recovered from brain tissue, can be reproduced experimentally by propagation of measles virus in cultured neuroblastoma cells. The purpose of the research described in this thesis was to evaluate this claim and establish whether biased hypermutation could be induced in measles virus from an acute infection (ADD-MV), or in an RNA virus not implicated in neural disease such as human respiratory syncytial virus (huRS virus). Accordingly, an isolate of ADD-MV and huRS virus were passaged 10 times in two neuroblastoma cell lines (SK-N-SH and IMR-32), in human lung fibroblasts (MRC-5) and in monkey kidney epithelial (Vero) cells. The M gene sequence of huRS virus passaged 10 times in each cell line was compared with the M gene sequence of virus passaged once in the same cells. No base change was observed in the M gene of huRS virus as a result of passage in these cell lines. A 750 base pair fragment of the M gene of ADD-MV was sequenced. Comparison of the sequence obtained from virus passaged 10 times in Vero cells with that from virus passaged once did not reveal any base changes. The M gene sequence of virus passaged 10 times in SK-N-SH cells contained one base change, at position 222, a uracil to cytosine transition. Two base changes were observed in the M gene of virus passaged 10 times in MRC-5 cells, one at position 222, as described above, and one at position 217, a cytosine to guanine base change. No changes were observed in virus passaged 10 times in IMR-32 cells when compared to that passaged once. These data indicate that neither ADD-MV, nor huRS virus, underwent enhanced mutational events during propagation in cells of neural origin, contrary to observations reported previously by Wong, et al. (1989), which suggested that propagation in neuroblastoma cells induced biased hypermutation. To determine if biased hypermutation occurred at a frequency too low to be detected by cycle sequencing, the M gene of ADD-MV passaged 9 times in IMR-32 cells, generated by PCR, was cloned into the bacteriophage vector M13. Twenty-four clones were sequenced in full. In total 21 base changes were observed, however, no more than 5 changes were seen in any one clone, and there was no directional bias in the mutations observed. However, each clone contained a large deletion at the 3' region of the gene, ranging from 1008 to 622 bases. Thirteen of the clones also contained a 56 base insertion. This insertion was shown to have high identity with human mitochondrial transfer RNA (tRNA). The M gene of Yamagata-1 (SSPE) virus resembles that of acute measles, but contains a number of additional mutations, mainly uracil to cytosine transitions. Propagation of this virus in neuroblastoma cells resulted in additional mutations not observed in virus propagated in Vero cells (Wong et al., 1989). Yamagata-1 virus was passaged in the two neuroblastoma cell lines (SK-N-SH and IMR-32), both with and without propagation in human lung fibroblast cells prior to passage in the neuroblastoma cells. Three M gene clones from passage numbers one and five in both cell lines used for this experiment, were sequenced. Analysis of the M gene sequence of Yamagata-1 virus passaged in each cell line revealed that the virus stock contained a heterogeneous population of M gene mRNA's. Both mutated sequences and sequences identical to the M gene of ADD-MV were obtained. Comparisons of the M gene sequence from virus passaged 5 times in neuroblastoma cells with that passaged once revealed no biased hypermutational events. However, the sequence data determined from two clones appeared to be a chimera consisting of both ADD-MV M gene sequence and Yamagata-1 virus M gene sequence. The recombination point is at position 457, a G-A mutation which is the only base change common to both ADD-MV M gene and Yamagata-1 M gene sequence.
Supervisor: Not available Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.264915  DOI: Not available
Keywords: QR355 Virology
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