Norovirus (NoV) and Sapovirus (SaV) are major causes of outbreak gastroenteritis worldwide. NoV and SaV are highly infectious, have multiple transmission routes and have a short incubation period, thereby facilitating rapid intercontinental spread of new variants. Consequently, a treatment would be advantageous for controlling them. However, currently little is known about the replication cycle and evolution of human NoV or SaV as neither are culturable. NoV and SaV are RNA viruses of the Caliciviridae family and have great genetic diversity which is thought to facilitate irnmune evasion. Consequently variants of NoV GI1.4 arose in 1996, 2002, 2004 and in 2006 and resulted in pandemics. Therefore, in this study, the role of the two main mechanisms associated with generating viral diversity; recombination, and point mutation were investigated for NoV and SaV. Physiological and kinetic properties of three NoV RdRps (genotypes, Gll.b, Gll.4, Gll.7) and two SaV RdRps (genogroups GI, GII) were also investigated. RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the Calicivirus genus Norovirus (NoV) has led to a rise in the identification of NoV recombinants and they are now reported at high frequency. Despite this no classification system exists for recombinant NoVs As a result, there is duplication in reporting novel recombinants and the precise number of novel NoV recombinant types is unknown. Therefore, in order to elucidate thero!e of recombination in NoV evolution, 121 NoV nucleotide sequences, compiled from the GenBank database and published literature, were analysed for recombination events. NoV recombinants and their recombination breakpoint were identified using three methods: phylogenetic analysis, Simplot analysis and the Maximum Chi-Squared method. In total 19 unique NoV recombinant types were identified in circulation across the globe and they had a common recombination point near the ORF1/2 overlap. Recombination at the ORF1/0RF2 overlap could have important implications in NoV evolution as it enables a virus to swap its antigenic determinates (capsid) and thereby avoid immune clearance in an analogous manner to antigenic shift in influenza virus. This study also examined the role of NoV and SaV replication in generating viral diversity by comparing the physiological, kinetic and biochemical properties of five genotypically distinct RdRps from two different genera of the Caliciviridae. Genetically diverse HuCV RdRps were expressed in Escherichia coli and characterised in an in vitro assay designed for this study. The results indicated that despite high sequence variation between the five enzymes (between 6% and 71% amino acid difference) they shared similar physiological properties. Though there was some variation in their template usage and kinetic properties. SaV was able to perform primer dependent replication on homopolymeric A RNA whereas the NoV RdRps were not. Additionally, NoV RdRps had a higher incorporation rate and were more kinetically efficient than the two SaV RdRps. The incorporation fidelity of the five enzymes was similar (between 2.2x10-5 to 8.9x10-4 ), although interestingly the most prevalent strain, Gll.4, had the lowest fidelity of the caliciviruses. Therefore, suggesting that RdRp fidelity has an important role in NoV evolution. Overall, this study illustrated that NoV and SaV generate genetic diversity in a similar fashion to other RNA viruses, that is, a delicate combination of recombination, point mutation and replication efficiency. Understanding the mechanisms involved in viral replication and genomic diversity of the calicivirus RdRps is essential if a successful control strategy for the human caliciviruses is going to be developed.
| Identifer | oai:union.ndltd.org:ADTP/258345 |
| Date | January 2007 |
| Creators | Bull, Rowena, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Bull Rowena., http://unsworks.unsw.edu.au/copyright |
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