West Nile Virus (WNV) forms part of the Japanese encephalitis serocomplex in the genus Flavivirus, family Flaviviridae. This enveloped positive single-stranded RNA (+ssRNA ) virus is the etiological agent of West Nile fever, and in more severe cases WNV neuroinvasive disease, in both humans and animals. WNV is distributed worldwide and is phylogenetically classified into five distinct lineages. The WNV genome is ~11 Kb in length and encodes a single open reading frame (ORF) that is post-translationally cleaved into three structural proteins and seven non-structural proteins. In this study, two contemporary and two historic South African WNV strains were genetically characterised as lineage 2 strains based on complete genome sequences. Genetic change as a result of passage number and propagation system was quantified on both the consensus genome- and quasispecies level. A lack of variation was observed amongst the consensus genome sequences of WNV strains subject to changes in propagation system from BHK-21 cell culture to mouse brain and vice versa. In contrast, variation amongst the latter was observed on the quasispecies level. Genome-wide single nucleotide polymorphism (SNP) profiles as well as full-length haplotype sequences reconstructed from ultra deep sequence data indicated that high levels of quasispecies diversity persists, particularly in the capsid gene region, during changes in propagation environment. The changes in frequency of variants were consistent throughout isolates propagated in different systems. The increased variation in the capsid gene region may result from selective pressures brought about by differences in host cell type between propagation systems. This study is the first to demonstrate quasispecies dynamics resulting from changes in propagation system of a lineage 2 WNV based on the reconstruction of full-length haplotype sequences from ultra deep sequence data. The approach demonstrates a cost-effective alternative to the estimation of viral population structure in light of viral evolutionary dynamics, which may in turn be assessed by the single plasmid reverse genetic system designed in this study. Although early attempts at rescuing an infectious WNV clone were unsuccessful, the system shows promise in the application of future studies concerning vaccine and diagnostic development, virulence studies and disease control. / Dissertation (MSc)--University of Pretoria, 2013. / gm2013 / Zoology and Entomology / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/32944 |
| Date | 12 December 2013 |
| Creators | Kortenhoeven, Cornell |
| Contributors | Bastos, Armanda D.S., Abolnik, Celia |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2013 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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