The viral family Herpesviridae consists of large double stranded DNA viruses including eight species that infect humans with varying pathology from benign rashes to cancerous cell transformation. From three subfamilies, alpha-, beta- and gammaherpes, the alphaherpes contains the genera iltovirus, mardivirus, varicellovirus and simplex, two of which, the human simplex viruses I and 2 (HSV) induce life-long infections that have appeared to have coevolved with their hosts from the origins of our species. Unique features of the simplex genus are latency, tropism in dorsal root ganglia neurons, extraordinary high GC content ranging from 65 to 77%, and nucleosome formation of their genomes within the host's nucleus without integration. Reviewing the basic molecular and genetic characteristics of herpes simplex will introduced in Chapter 1, followed by the introduction of a newly sequenced, de novo assembled and predicatively annotated herpes simplex virus, Leporid Herpes Virus-4 (LHV4). Isolated from a virulent outbreak in domesticated rabbits, LHV4 has the smallest reported simplex virus to date at roughly 125,600 base pairs and presents similar pathology seen in rabbit models infected with HSV. Comparative genomics revealed a high degree of sequence similarity and genome synteny between LHV4 and other simplex viruses. Four genes were not computationally predicted in our annotation and may be absent in the LHV4 genome. The absent proteins correspond to: UL56, ICP34.5, US5 and US12 and have postulated roles in membrane trafficking, neurovirulence, apoptotic control and MHC I presentation respectively. The solved genome structure leads to how this compacted genome functions with the noted absences to produce a similar pathology in rabbits to that of HSV and whether other biological correlates will continue to be found in in vitro and in vivo infection. The inverted repeat regions (IR), duplicated and inverted to simplex virus' two larger blocks of protein-coding regions are described in Chapter 3. The similarities and differences in critical genes from the IR that balance latency and replicative viral cycles are compared. A two-fold reduction in IR content indicates the ability for a simplex virus to maintain infectivity despite this large truncation. The appendix describes the eukaryotic phylogeny of two initiating proteins of the mismatch repair (MMR) pathway. MMR proteins are present in the replicative foci of productive herpes virus infection and this analysis may indicate adaptive pressures involved in both genomic fidelity and host tropism. The emerging era of state-of-the-art genome sequencing and computational power advances this newly characterized herpes virus, along with its model host organism, as excellent candidates for systems interaction, and experimental biology. / Graduation date: 2012

Understanding the transmission dynamics of infectious diseases is important to well-informed public health policy, responsive infection control and individual patient management. The on-going revolution in whole-genome sequencing provides unprecedented resolution for detecting evidence of recent transmission and characterising population-level transmission dynamics. In this thesis, I develop and apply evolutionary approaches to investigating transmission, focusing on three globally important pathogens. Hepatitis C virus (HCV) is a major cause of liver disease affecting 150 million people and killing 350,000 annually. I conducted a meta-analysis of twentieth-century HCV epidemics, finding that the age of the epidemic can be predicted by genetic diversity. Using the coalescent, I fitted classic susceptible-infected (SI), susceptible-infected-susceptible (SIS) and susceptible-infected-recovered (SIR) epidemiological models. Most epidemics showed signatures of SI dynamics, but three, from Argentina, Hong Kong and Thailand, revealed complex SIR dynamics. Norovirus is the leading viral cause of diarrhoea, estimated to cost the NHS around £115 million annually. I analysed whole norovirus genomes via a stochastic transmission model, finding that up to 86% of hospital infection was attributable to transmission from another patient in the hospital. In contrast, the rate of new introductions to hospital by infected patients was extremely low (<0.0001%), underlining the importance of ward management during outbreaks. Campylobacter is the most commonly identified cause of bacterial gastroenteritis worldwide. I developed a zoonotic transmission model based on phylogeography approaches to test whether three strains previously associated with multiple host species were in fact aggregates of strongly host-restricted sub-strains, or genuine generalists. Members of the same strain isolated from different host species were often more closely related than those isolated from the same host species. I estimated 419, 389 and 31 zoonotic transmissions in ST-21, ST-45 and ST-828 respectively, strongly supporting the hypothesis that these strains are adapted to a generalist lifestyle.

Viruses are the most abundant organisms on Earth, yet their collective evolutionary history, biodiversity and functional capacity is not well understood. Viral metagenomics offers a potential means of establishing a more comprehensive view of virus diversity and evolution, as vast amounts of new sequence data becomes available for comparative analysis.Metagenomic DNA from virus-sized particles (smaller than 0.2 microns in diameter) was isolated from approximately 20 liters of sediment obtained from Boiling Springs Lake (BSL) and sequenced. BSL is a large, acidic hot-spring (with a pH of 2.2, and temperatures ranging from 50°C to 96°C) located in Lassen Volcanic National Park, USA. BSL supports a purely microbial ecosystem comprised largely of Archaea and Bacteria, however, the lower temperature regions permit the growth of acid- and thermo-tolerant Eukarya. This distinctive feature of the BSL microbial ecosystem ensures that virus types infecting all domains of life will be present. The metagenomic sequence data was used to characterize the types of viruses present within the microbial ecosystem, to ascertain the extent of genetic diversity and novelty comprising the BSL virus assemblage, and to explore the genomic and structural modalities of virus evolution.Metagenomic surveys of natural virus assemblages, including the survey of BSL, have revealed that the diversity within the virosphere far exceeds what has currently been determined through the detailed study of viruses that are relevant to human health and agriculture. The number of as-yet-uncharacterized virus protein families present in the BSL assemblage was estimated by clustering analysis. Genomic context analysis of the predicted viral protein sequences in the BSL dataset indicates that most of the putative uncharacterized proteins are endemic or unique to BSL, and are largely harbored by known virus types. A comparative metagenomic analysis approach identified a set of conserved, yet uncharacterized BSL protein sequences that are commonly found in other similar and dissimilar environments.New sequence data from metagenomic surveys of natural virus assemblages was also used to better characterize and define known virus protein families, as some of the viruses found in the BSL environment represent distant relatives of well-characterized isolates. By comparing viral genes and protein sequences from these highly divergent species, it is possible to better understand the dynamics of adaptation and evolution in the virosphere. Additionally, as structures of virus proteins continue to be experimentally determined by X-ray crystallography and cryo-electron microscopy, a merger of structural and metagenomic sequence data allows the opportunity to observe the structural dynamics underlying virus protein evolution.Capsid (structural) proteins from two distinct Microviridae strains; a globally ubiquitous and highly sequence-diverse virus family, were identified in, and isolated from the BSL metagenomic DNA sample. These BSL capsid protein sequences, along with several other homologous sequences derived from metagenomic surveys and laboratory isolates, were mapped to the solved structure of a closely related capsid protein from the Spiroplasma phage-4 microvirus. Patterns of amino acid sequence conservation, unveiled by structure-based homology modeling analysis, revealed that the protein sequences within this family exhibit a remarkable level of plasticity, while remaining structurally and functionally congruent.Lateral gene transfer is thought to have had a significant impact on the genomic evolution and adaptation of virus families. Genomic context analysis was also utilized to identify interviral gene transfer within the BSL virus assemblage. An ostensibly rare interviral gene transfer event, having transpired between single-stranded RNA and DNA virus types, was detected in the BSL metagenome. Similar genomes were subsequently detected in other ecosystems around the globe. The discovery of this new virus genome dramatically underscores the scope and importance of genetic mobility and genomic mosaicism as major forces driving the evolution of viruses.The analyses conducted herein demonstrate the many ways in which viral metagenomic sequence data may be utilized to not only evaluate the composition of a natural virus assemblage, but to discover new viral genes, and to better understand the dynamics of both genomic and structural evolution within the virosphere.

The central problem for researchers of HIV-1 evolution is explaining the apparent design of the virus for causing pandemic infection in humans: understanding how HIV-1 spreads is key to halting the pandemic. Current knowledge of how HIV-1 spreads from host to host is based upon experimental observation and indirect inferences informed by theory. The hypothesis of this thesis is that diversity of HIV-1 around the time of transmission is important for viral adaptation to a new human host, rather than intrinsic superiority of particular strains found in infectious fluids from human donor hosts, and that studying recombination is important for understanding this behaviour. To demonstrate the apparent randomness of transmission, I test the null-hypothesis that hard selection accounts for between-host viral divergence in a rare case study of contemporaneous infection. I explain how the experimental data that I have generated and the analyses I have carried out address certain basic assumptions and predictions about HIV-1 transmission and may inform current strategies for vaccine design. Specifically, my approach contributes to the current literature on HIV-1, by investigating an alternative hypothesis to the single virion theory of sexual transmission and by characterizing the role of recombination in a pseudodiploid virus following multiple-infection.