The invasion of novel host species by pathogens is one of the primary causes of pandemics and emerging diseases. Such host shifts are difficult to anticipate, in part because we lack an understanding of the barriers that prevent maintenance of pathogens by multiple species. Rabies virus provides an ideal model system in which to study these barriers, as well as the mechanisms used by host-shifting pathogens to overcome them. This virus appears to able to infect all mammals, but paradoxically is maintained in species-specific transmission cycles. These maintenance hosts are almost exclusively found within the Carnivora (carnivores) and Chiroptera (bats), and were established by numerous host shifts within and between these taxonomic orders. However, it remains unclear which – if any – adaptations are required when switching to a new host species, nor is it known why the virus is incapable of utilising multiple host species despite evidence of relatively frequent spill-overs between maintenance host species. This thesis describes investigations of the determinants of rabies virus host shifts across multiple scales of organisation, which aim to understand the host-specialisation of this virus. A meta-analysis of within- and cross-species infection studies shows evidence for a reduction in effective dose as the body temperature difference between the original and inoculated species increases. This affects the duration of the incubation period, suggesting adaptation of infectivity may be involved. Crucially, previous studies have linked temperature differences to infectivity differences in cell culture, and to the process of cell entry in particular. These data further show evidence for phylogenetic clustering of incubation period durations, which may be linked to clustering of sensitivity among inoculated host species. This in turn may help explain why only some carnivore and bat species are maintenance hosts of rabies virus. At the molecular level, whole genome sequencing of samples from a system in which rabies virus has made an unusual host shift to bovids suggests involvement of a single amino acid change in the acetylcholine receptor-binding site of the glycoprotein. Phylogenetic analyses involving these and other sequences shows evidence for distinct maintenance cycles in three host species in the same region, despite numerous spill-over infections between species. The involvement of the glycoprotein in this host shift further points to a role for infectivity changes in host adaptation. At the cellular level, the bovid-associated viruses show increased infectivity to a bovid cell line compared to canid-associated viruses. The bovid-associated viruses also show decreased infectivity to a canid cell line representing the original host species. Combined, these results point to a new model for the host adaptation of rabies virus. In this model, the infectivity of viruses is decreased at temperatures to which they are not adapted. This allows the emergence of genotypes with increased infectivity in the novel host species, which can be achieved through relatively minor genomic alterations. Because these adaptations are detrimental in the original host, they may combine with ecological differences between hosts to create the single-species maintenance cycles observed. Identifying the mechanisms constraining maintenance host range will allow better predictions of which host shifts are likely to be successful. In particular, the effect of body temperature difference found here is shown to explain a large part of the previously observed effect of phylogenetic distance between host species in reducing the frequency of rabies virus host shifts. The phylogenetic clustering of host sensitivity meanwhile, may explain how host shifts between very divergent hosts are possible.

Protection against viruses is provided by the host innate, adaptive and cellular intrinsic immunities. Cellular intrinsic immunity, or intrinsic defence, against herpesviruses is in part enacted by components that constitute nuclear substructures known as ND10. ND10 (also called PML nuclear bodies after its major organising component) are dynamic nuclear domains that contain various cellular proteins, including PML itself, Sp100, hDaxx and ATRX. In the early stages of infection, herpesvirus genomes and sites of immediate early transcription become associated with ND10 and their components. Representative herpesviruses such as an alphaherpesvirus HSV-1 and a betaherpesvirus HCMV encode for strong transcriptional activators, namely the Immediate Early protein ICP0 and the tegument protein pp71, respectively. These proteins are known to counteract cellular intrinsic defence mechanisms. In the context of HSV-1 and HCMV infections, the major ND10 component PML has been identified as an important constituent of cellular intrinsic defence. In addition, a number of research studies have demonstrated that Sp100 and hDaxx contribute to these processes during infection with HSV-1 and HCMV, respectively. The general hypothesis of the present study implies that cellular chromatin-associated factors within ND10 may act to repress viral gene expression. The two representative ND10 components present a particular interest for the current investigation based on the facts that: (i) hDaxx is a transcriptional co-repressor; (ii) ATRX is a chromatin-remodelling enzyme; and (iii) ATRX and hDaxx interact with each other to form a chromatin-remodelling complex with repressive properties. The purpose of the present study was therefore to investigate the roles of these two proteins in HSV-1 and HCMV infection. By using virus mutants incapable of efficient stimulation of Immediate Early gene expression (ICP0-null HSV-1 and pp71-null HCMV) it was possible to analyse the contribution of ATRX and hDaxx to the repression mechanism that occurs in the absence of these viral transactivators. An RNA interference approach was utilised for generating cell lines depleted of ATRX or hDaxx in order to assess their roles in viral infectivity. In addition, cell lines reconstituted with wt hDaxx, and ATRX-interaction or SUMO-interaction deficient hDaxx mutants were constructed in order to study the contribution of these functional elements to the role of hDaxx in the repression of ICP0-null mutant HSV-1. The key findings presented in the current study can be summarised as follows: (1) ATRX contributes to the intrinsic resistance against HCMV infection, and this mechanism is strongly counteracted by viral pp71; (2) A chromatin-remodelling complex formed between ATRX and hDaxx contributes to the efficient repression of ICP0-null HSV-1 genomes, thereby constituting a part of anti-HSV-1 intrinsic cellular defence. ICP0 counteracts this process, and the possible mechanisms of ICP0 action are proposed. These data provide the first evidence for the role of ATRX in viral infection and in addition demonstrate a role for hDaxx in the regulation of HSV-1 infection. The strong indication that ATRX and hDaxx act as a complex opens a possibility of chromatin-dependent repression of ICP0-null HSV-1 genomes. Whether ATRX and hDaxx contribute to the repression of pp71-null HCMV genomes as a complex is yet to be established. In summary, the conclusion of the studies presented in this thesis suggests regulatory roles of ND10-localised chromatin remodelling proteins ATRX and hDaxx in cellular anti-herpesvirus intrinsic resistance mechanisms.

Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a single bacterial pathogen, Mycobacterium tuberculosis. Current TB chemotherapy remains useful in treating susceptible M. tuberculosis strains, however, the emergence of MDR-TB and XDR-TB demand the development of new drugs. Enzymes involved in mycobactin biosynthesis, low molecular weight iron chelators, do not have mammalian homologues; therefore they are considered potential targets for the development of new anti-TB drugs. The aims of this study were to identify potential inhibitors and to investigate the function of the mbtG and AmbtE and AMbtF genes during mycobactin biosynthesis. The full length of mbtB and the ArCP domain were successfully cloned and post-translationally modified by MtaA, a broad phosphopantetheinyl transferase from Stigmatella aurantiaca, using Escherichia coli. Inhibitors identified by virtual screening as well as 13 chemically synthesised PAS analogues were initially investigated in whole-cell assay against Mycobacterium bovis BCG Pasteur. Seven of these compounds had interesting growth inhibition under ironsufficient conditions. The mbtA gene was cloned and expressed as soluble protein using Mycobacterium smegmatis mc2155. Preliminary in vitro MbtA assays provided hints of its activity, although, the KM for SAL and ATP have not been determined yet. The mbtG and ambtE genes have been cloned and expressed in E. coli to further investigate their biochemical function in mycobactin biosynthesis.

The work described in this thesis identifies some properties of the major polypeptides of pneumonia virus of mice (PVM) and of turkey rhinotracheitis (TRT) virus. The PVM glycoproteins have been studied in particular detail while the results obtained with TRT virus provide a preliminary description of the polypeptides of this virus. Twelve major PVM specific polypeptides designated L, G1, G2, F1, N, 39K, 35K, M, 20K, 19K, 16K and 12K were identified. In addition PVM specific polypeptides designated 25K, 24K, 23K, 18K and 17K were sometimes detected. Monoclonal antibodies directed against the G1/G2, 39K and M polypeptides were produced. The a~ility of a monoclonal antibody to precipitate G1 and G2 suggested that these two glycosylated proteins were related and this was confirmed by tryptic peptide mapping. G2 was shown to be derived from G1 in pulse chase experiments and a similar relationship between two higher mobility polypeptides synthesized in the presence of tunicamycin was observed. The G protein may have a precursor since G1 did not appear immediately following a pulse labelling. The precursor could not however be identified. An additional minor glycosylated polypeptide of 42K was found to be related to the G protein. The F1 protein appeared to be poorly glycosylated and a difference in mobility of the polypeptide synthesized in the presence of tunicamycin did not appear to be directly due to a lack of N-linked oligosaccharides. The polypeptide migrated more slowly under non-reducing conditions but no evidence of a small disulphide bonded polypeptide was found in contrast to the situation with other paramyxoviruses. This polypeptide appeared to be the major PVM protein expressed on the cell surface and was associated with G1 and G2 as the major protein in a particulate fraction of the infected cell supernatant. Tentative relationships were suggested between the 39K, 35K and 25K polypeptides, the M and 24K polypeptides and the 20K and 19K polypeptides. This together with the observation that the 12K polypeptide was not a primary gene product suggested that there may be about 11 PVM polypeptides. The N or 39K and the 20K or 19K polypeptides were observed to be phosphorylated. Twelve possible TRT virus specific polypeptides of 150K, 129K, 95K, 83K, 57K, 45K, 38K, 35K, 3DK, 23K, 19K and 15K were identified. The 150K, 95K, 83K, 57K, 45K and 15K polypeptides were glycosylated with the latter three polypeptides showing a similar relationship to the F1,2, F1 and F2 polypeptides of paramyxoviruses. A broad glycosylated band designated the 31K polypeptide was identified that was similar to a smeared band observed on prolonged exposure of immunoprecipitates of PVM polypeptides labelled with [3H]-glucosamine. The 35K and 19K polypeptides were observed to be phosphorylated. PVM may be more closely related to RS virus than TRT virus since anti-PVM serum irnmunoprecipitated the RS virus N polypeptide but not any TRT virus polypeptides. The PVM 39K polypeptide and the RS virus P protein were recognised by a monoclonal antibody providing further evidence of a relationship between PVM and RS virus.

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.

The requirements for prompt diagnosis of foot-and-mouth disease (FMD) during outbreaks, and the need to establish robust laboratory testing capacity within FMD-endemic countries, have motivated the development of point-of-care tests (POCTs) to support current diagnostic strategies. Despite numerous publications detailing the design of platforms and assays for this purpose, the majority have only been evaluated in laboratory settings, using protocols incompatible for use in challenging environments. To address this gap, this thesis describes the development of an end-to-end molecular toolbox for the detection and characterisation of FMD virus (FMDV) RNA in decentralised settings. A critical review and multiway comparison of seven assay formats and 11 sample preparation methods revealed that reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time reverse transcription PCR (rRT-PCR) POCT-formats exhibited comparable analytical and diagnostic sensitivity to their laboratory-based equivalents. Additionally, reagent lyophilisation provided a solution for cold chain and storage considerations, whilst not compromising assay performance. Both assays were compatible with simple sample preparation methods, removing the requirement for nucleic acid extraction. For example, dilution of samples in nuclease-free water enabled FMDV RNA to be detected in multiple sample types (epithelial tissue suspensions, serum, oesophageal-pharyngeal fluid and lesion swabs), from as early as one day post infection. Notably, when the robust field-ready protocols were deployed into challenging low-resource laboratory and field-settings within East Africa, POCT results (rRT-PCR = 144; RT-LAMP = 145) were consistent with clinical observations and a reference rRT-PCR, with FMDV detected from acutely infected as well as convalescent cattle. Furthermore, transitioning of East Africa-specific FMDV-typing rRT-PCR assays (for serotypes O, A, Southern African Territories [SAT] 1 and SAT 2) into a multiplex POCT-format enabled rapid identification of FMDV serotype in situ, confirming active outbreaks of both O and A. This thesis also describes the development of GoPrime, a novel real-time PCR (rPCR) primer/probe validation tool. By parameterising GoPrime with experimental data, collected to investigate the effects of primer/probe-template mismatches on cycle threshold and limit of detection, it was possible to quantitatively predict the performance of rPCR assays in silico. The work of this thesis supports the deployment of molecular POCTs into non-specialised, resource-limited and challenging settings for simple, highly sensitive and rapid detection and/or characterisation of FMDV.