This thesis relates to the work of building a mathematical model of the impact of Rabbit Calicivirus Disease (RCD) on the European Rabbit, Oryctolagus cuniculus, in Australia. After introducing the general biology of rabbits and the immunology of RCD, we build a time-dependent single site model. We construct a single-site population dynamic model with age structure, seasonal birth rate, density dependent regulation of the population size and climatic variability for various regions of Australia. After investigating suitable parameter ranges, we incorporate the disease dynamics through an indirect transmission model based on two different hypotheses which we call the Strong Juvenile hypothesis and the Weak Juvenile hypothesis. These differ in their assumption about Juvenile immunity to the disease. The ecological impact of both hypotheses is tested for both the single site and multiple site (spatial) models. The disease impact is investigated by varying the disease virulence, i.e. a parameter measuring the "strength" of the virus. Subsequently, a multiple site (spatial) model for the Riverina region is built by using the single-site model as building block. Data from Lake Urana is used to parameterize a seasonal emigration rate from each site. Density dependent immigration is added together with a hazard coefficient which rabbits face when leaving one site and trying to become established in another. Acceptance in a new site is regulated by the population density at the entry site. Several spatial configurations of sites are tested and the spatial dynamics of the disease is investigated. Finally, we construct a model to investigate the long term evolution of the disease virus. We postulate the existence of several strains of the disease and trade-offs between disease characteristics. We allow for mutation of the virus and run the model for two contrasting geographical regions of Australia. We compare the results for the different regions and the different hypotheses regarding Juvenile immunity (the Strong Juvenile hypothesis and the Weak Juvenile hypothesis). It is shown, unexpectedly, that intermediate levels of disease virulence are not selected.

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Caliciviruses are a family of single-stranded positive sense RNA viruses with a non- segmented genome. Feline Calicivirus (FCV) causes respiratory disease in cats and noroviruses cause gastroenteritis in humans. More recently, murine norovirus (MNV), a murine form of human norovirus, was described. Although human noroviruses (HuNV) cannot be cultured in vitro, MNV and FCV can, making them excellent models for HuNV. Previous studies of calicivirus entry have shown very different pathways for these two viruses. FCV enters cells via a clathrin-dependent or macropinocytic pathway, but nothing I . . is known about what signalling events ~re involved in this process, where the virus traffics I to post-entry or the effects the virus has on cell metabolism. MNV uses a clathrin- and caveolae- independent pathway that is d~pendent on dynamin, lipid rafts and microtubules. This project aimed at dissecting the early events in FCV and MNV infection: which signalling pathways are involved and what trafficking occurs in the cells. We have used pharmacological inhibitors of signalling pathways (genistein, okadaic acid and wortmannin) and dominant negative inhibitors of proteins, involved in endocytic and signalling pathways as well as markers of endosomal compartments for colocalisation studies. We have now shown that FCV entry is dependent on dynamin, traffics from early to recycling endosomes and is dependent on Pl3-kinases for entry and replication. MNV-1 traffics to the recycling endosome, possibly from the macropinosome, and is dependent on tyrosine kinases and protein phosphatases. We have also begun to investigate the effects of FCV infection on the metabolism of host cells using BiOLOG technology. These experiments showed that in infected cells there is an overall decrease in metabolism, but an increase in glucose uptake. This study has resulted in further elucidation of the early events in infection of both of these viruses which could aid in the future development of antiviral therapies.

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The studies presented in this thesis demonstrate that foot-and-mouth disease virus (FMDV) infection mediated by the integrin ?v?6 takes place through clathrin-dependent endocytosis. Inhibition of clathrin-dependent endocytosis by sucrose treatment or transient expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication. These observations suggest that the low pH within endosomes is required for a post-entry step of infection, most likely capsid uncoating and delivery of viral RNA into the cytosol. FMDV infection occurred in the absence of caveolae and inhibition of lipid-raft dependent endocytosis did not inhibit virus uptake or infection. Using immunofluorescence confocal microscopy, FMDV colocalised with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37°C, virus was rapidly taken up into the cells and colocalised with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). Internalisation of alphavbeta6 and its accumulation in early and recycling endosomes was triggered by virus binding, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes.

Infection of susceptible cells with foot and mouth disease virus (FMDV) results in multiplication of the RNA genome and assembly of mature virions. The entire process of genome replication is completed in a few hours and encompasses many intracellular events. Like other picornaviruses, FMDV uses a peptide primed RNA replication mechanism. The factors that are required to uridylylate each of the three FMDV VPg peptides and the role of the FMDV cis-acting replication element (cre) or 3B Uridylylation Site (bus) in VPg uridylylation have been determined. The native N-terminus of the FMDV 3Dpol enzyme is a pre-requisite for VPg uridylylation in vitro and the effects of mutations in the RNA template are consistent with a slide-back mechanism. The role of the poly(A) tail in uridylylating VPg was insignificant using full-length FMDV RNA transcripts suggesting the possibility of an alternative mechanism of VPg incorporation into negative strand RNA. The optimal RNA sequences required for VPg uridylylation were found to be within the 5' non-coding region (NCR). Furthermore, the results also showed evidence for RNA-RNA interactions between distinct structures from within the 5' NCR that influence VPg uridylylation. The polymerase precursor 3CDpro is also a prerequisite for uridylylation of each of the FMDV VPg peptides. However BCpro alone can substitute for 3 CD, but is much less efficient. It also appeared that the overall charge of the VPg peptides determines their recognition by the FMDV 3Dpol. The RNA binding activity of the 3C was found to be required for its stimulatory effects on VPg uridylylation. Unlike the poliovirus cloverleaf, the FMDV S-fragment (at the 5' end of the genome) does not interact with the FMDV 3CD precursor protein; however it binds specifically to a cellular factor p48. The crude replication complexes (CRCs) isolated from FMDV-infected cells were found to synthesize viral RNA very efficiently and an in vitro RNA replication system developed using these CRCs can be used to study the complete RNA replication events of FMDV.

Foot-and-mouth disease virus (FMDV) causes an acute vesicular disease of domesticated and wild ruminants, and pigs. The virus is highly variable with 7 serotypes and numerous subtypes. A method was developed to sequence the complete genome of the Pan Asia O serotype FMDV that caused the UK 2001 epidemic. The sequence data was used to quantify the genetic diversity of FMDV that arose over the course of this epidemic. 197 nucleotide substitutions were observed at 191 different sites across the genome, with a ratio of non-synonymous to synonymous change of 0.09. It was estimated that the date at which FMDV first infected livestock in the UK was the 7th February 2001 (95% CI Jan 20th – Feb 19th) using coalescent methods, which is in close agreement to that generated on the basis of lesion aging at the first infected premises. The rate of nucleotide substitution during the epidemic was estimated to be 2.26 x 10-5 per site per day (95% CI 1.75-2.80 x 10-5). This was a sufficiently high rate that detailed histories of transmission pathways could be reliably reconstructed and motivated a comprehensive study of genetic changes that arose between infected premises in the Darlington area. This study highlighted the potential of tracing virus transmission between farms using genetic data. A maximum likelihood methodology was proposed to combine epidemiological data (describing the infection profile of individual farms) with the genetic data. Integration of genetic and epidemiological data reduced the number of transmission trees (describing infection transmission within this cluster) that were consistent with the genetic data from 41,472, to 1,728, of which 9 represented 95% of the total likelihood calculated. An average of 4.3 (S.D. 2.1) variant nucleotides within the complete genome, were observed between consecutive farm infections. Difficulties in identifying direct transmission events in this analysis arose mainly as a result of limited data on the extent of genetic variation on a single premises (complicated by variation in animal types, and farm sizes), and the manner in which mutations become fixed within the consensus sequence (i.e. upon replication and selection, and transmission through a ‘bottleneck’). This encouraged an analysis of the minimum mutant frequency of Pan Asia O FMDV from a single lesion sampled from naturally infected hosts which led to an estimate of 3.06 x 10-4 mutations per nucleotide sequenced (this parameter has previously only been measured in cell culture). In addition an experiment was initiated to measure the spontaneous mutation rate of O1BFS FMDV. The relevance of this analysis of the evolution of FMDV during the UK 2001 epidemic was demonstrated during the outbreak of O1BFS FMDV in Surrey in 2007, where complete genome sequencing was used in real time to trace virus spread. Genetic analysis of complete viral genomes generated in real-time revealed a chain of transmission events, predicting undisclosed infected premises, and connecting a second cluster of outbreaks in September to the initial outbreaks in August. Thus, this thesis has pioneered the use of complete genome sequencing for fine scale molecular epidemiology.