Philosophiae Doctor - PhD / There are approximately 10³¹ bacteriophages in the biosphere, outnumbering bacteria 10:1, hence, the dynamic and specific nature of phage-host interactions exerts significant influence on microbial communities. Bacteriophages also represent the reservoir of the highest known genetic diversity making them a potential source of novel biotechnological products. However, the isolation of novel bacteriophages is limited by the observation that less than 1% of bacterial hosts have been cultured. This study aimed to bypass this problem by developing novel culture independent approaches to improve our ability to isolate novel phage-host pairs. Samples were collected from an abandoned copper prospecting site near the Gobabeb Desert Research and Training Station and a Salt lake located in the Swakopmund region of the Namibian desert. Two approaches were explored in this study namely viral tagging and reverse metaviromics. For viral tagging, fluorescently labelling the environmental phage fraction before challenging the environmental bacterial fraction with tagged phages proved difficult. This was most likely due to the complex interaction of the labelling agent with phages and requires further studies. For the reverse metaviromics approach, total DNA from the environmental phage fractions was extracted, sequenced and analyzed for novel phages. Analysis of the phage diversity showed that the copper site was dominated by tailed viruses as has been shown for other extreme arid environments. However, the saline site was atypical of marine environments, with tailed viruses being the most abundant, suggesting that the diversity present is not only driven by salinity. Using the metaviromic sequence data to guide the selection of potential bacterial hosts, two strategies were employed. In the first, putative hosts were predicted based on similarity of phage sequences to those identified in databases. Media targeting these specific genera were employed, 8 bacterial species were isolated and based on 16S rRNA similarity to the closest known species were identified as Halomonas caseinilytica, Halomonas eurihalina, Halomonas sinaiensis, Idiomarina loihiensis, Marinobacter xestospongiae, Virgibacillus salarius and two Salinivibrio species. The 16S rRNA analysis also suggested that H. sinaiensis, V. salarius and both Salinivibrio species are novel. All 8 isolates were challenged with the environmental phage fraction. A novel phage, SMHB1, was isolated on one of the Salinivibrio spp. and is only the second characterized phage ever described for this genus. SMHB1 is a 32 kb myovirus, with a head diameter of 56 nm, and a tail length of 106 nm. The second approach involved the design of fluorescently labelled probes targeting phages identified from the metaviromic sequence data. In a control E. coli system to detect cloned phage DNA fragments, 87% of the interrogated cells showed significant hybridization of the phage specific probe to the target. The optimized method was applied to a simulated environmental bacterial fraction and a detection limit of 1:100 was observed for the bacteria containing the phage DNA fragment of interest. This study demonstrates the possibility of improving the specificity of isolating phage-host pairs in a culture-independent manner by incorporating sequence data in the experimental design; and contributes to our knowledge of the phage diversity of an understudied extreme environment.

Viral sequence data has great potential for answering questions about the epidemiological dynamics and evolution of viruses. Classical approaches have sought amino acid changes that alter pathogenesis or transmissibility by influencing a virus's ability to enter or replicate within cells. However, this approach rarely recognises the fundamental impact of heterogeneous host contact structures and existing immunological responses on viral transmission. This thesis draws heavily on ecological and immunological concepts to explore the epidemiological dynamics, diversity and evolution of viruses using molecular sequence data. A number of different research approaches and study systems are used in this thesis. I begin by describing a novel polyomavirus in a European badger, and apply phylogenetic techniques to analyze the evolutionary history of the Polyomaviridae. I subsequently describe a large metaviromic study in a population of wild mute swans, for which host demographic data are available. I describe nine new viral species and test whether age and season are associated with differences in abundance and prevalence of different viral taxonomic groups. The study highlights the potential of metaviromics for investigating viral epidemiological dynamics in natural populations. Influenza A viruses of avian origin (AIV) threaten human and animal health. Using phylogeographic methods, I reconstruct the spatial spread of an H5N8 virus at a regional scale, and investigate how bird density and migration shaped this dispersal. Despite the importance of acquisition of humoral immunity to different strains throughout the lifespan of wild birds for epidemiological dynamics, this topic is poorly understood. I assess the accumulation of immune responses to AIV with age in mute swans. I consider how ecological factors, including age-structured immunity, might have affected the epidemiology of an H5N8 outbreak in the population.