Quasispecies dynamics and treatment outcome during early hepatitis C infection in a cohort of HIV-infected men

Hepatitis C virus (HCV) is emerging as one of the leading causes of morbidity and mortality in individuals infected with HIV and has overtaken AIDS-defining illnesses as a cause of death in HIV patient populations who have access to highly active antiretroviral therapy. For many years, the clonal analysis was the reference method for investigating viral diversity. In this thesis, a next generation sequencing (NGS) approach was developed using 454 pyrosequencing and Illumina-based technology. A sequencing pipeline was developed using two different NGS approaches, nested PCR, and metagenomics. The pipeline was used to study the viral populations in the sera of HCV-infected patients from a unique cohort of 160 HIV-positive patients with early HCV infection. These pipelines resulted in an improved understanding of HCV quasispecies dynamics, especially regarding studying response to treatment. Low viral diversity at baseline correlated with sustained virological response (SVR) while high viral diversity at baseline was associated with treatment failure. The emergence of new viral strains following treatment failure was most commonly associated with emerging dominance of pre-existing minority variants rather than re-infection. In the new era of direct-acting antivirals, next generation sequencing technologies are the most promising tool for identifying minority variants present in the HCV quasispecies populations at baseline. In this cohort, several mutations conferring resistance were detected in genotype 1a treatment-naïve patients. Further research into the impact of baseline HCV variants on SVR rates should be carried out in this population. A clearer understanding of the properties of viral quasispecies would enable clinicians to make improved treatment choices for their patients.

Mixed genotype Hepatitis C virus infections: incidence in Scotland and methods for detection

The diagnosis of mixed genotype hepatitis C virus (HCV) infection is rare and information on incidence in the UK, where genotypes 1a and 3 are the most prevalent, is sparse. Considerable variations in the efficacies of direct-acting antivirals (DAAs) for the HCV genotypes have been documented and the ability of DAAs to treat mixed genotype HCV infections remains unclear, with the possibility that genotype switching may occur. In order to estimate the prevalence of mixed genotype 1a/3 infections in Scotland, a cohort of 512 samples was compiled and then screened using a genotype-specific nested PCR assay. Mixed genotype 1a/3 infections were found in 3.8% of samples tested, with a significantly higher prevalence rate of 6.7% (p<0.05) observed in individuals diagnosed with genotype 3 infections than genotype 1a (0.8%). An analysis of the samples using genotypic-specific qPCR assays found that in two-thirds of samples tested, the minor strain contributed <1% of the total viral load. The potential of deep sequencing methods for the diagnosis of mixed genotype infections was assessed using two pan-genotypic PCR assays compatible with the Illumina MiSeq platform that were developed targeting the E1-E2 and NS5B regions of the virus. The E1-E2 assay detected 75% of the mixed genotype infections, proving to be more sensitive than the NS5B assay which identified only 25% of the mixed infections. Studies of sequence data and linked patient records also identified significantly more neurological disorders in genotype 3 patients. Evidence of distinctive dinucleotide expression within the genotypes was also uncovered. Taken together these findings raise interesting questions about the evolutionary history of the virus and indicate that there is still more to understand about the different genotypes. In an era where clinical medicine is frequently more personalised, the development of diagnostic methods for HCV providing increased patient stratification is increasingly important. This project has shown that sequence-based genotyping methods can be highly discriminatory and informative, and their use should be encouraged in diagnostic laboratories. Mixed genotype infections were challenging to identify and current deep sequencing methods were not as sensitive or cost-effective as Sanger-based approaches in this study. More research is needed to evaluate the clinical prognosis of patients with mixed genotype infection and to develop clinical guidelines on their treatment.

Warfare in the West Highlands and Isles of Scotland, c. 1544-1615

Warfare has long been associated with Scottish Highlanders and Islanders, especially in the period known in Gaelic tradition as ‘Linn nan Creach’ (the ‘Age of Forays’), which followed the forfeiture of the Lordship of the Isles in 1493. The sixteenth century in general is remembered as a particularly tumultuous time within the West Highlands and Isles, characterised by armed conflict on a seemingly unprecedented scale. Relatively little research has been conducted into the nature of warfare however, a gap filled by this thesis through its focus on a series of interconnected themes and in-depth case studies spanning the period c. 1544-1615. It challenges the idea that the sixteenth century and early seventeenth century was a time of endless bloodshed, and explores the rationale behind the distinctive mode of warfare practised in the West Highlands and Isles. The first part of the thesis traces the overall ‘Process of War’. Chapter 1 focuses on the mentality of the social elite in the West Highlands and Isles and demonstrates that warfare was not their raison d'être, but was tied inextricably to chiefs’ prime responsibility of protecting their lands and tenants. Chapter 2 assesses the causation of warfare and reveals that a recurrent catalyst for armed conflict was the assertion of rights to land and inheritance. There were other important causes however, including clan expectation, honour culture, punitive government policies, and the use of proxy warfare by prominent magnates. Chapter 3 takes a fresh approach to the military capacity of the region through analysis of armies and soldiers, and the final thematic chapter tackles the conduct of warfare in the West Highlands and Isles, with analysis of the tactics and strategy of militarised personnel. The second part of this thesis comprises five case studies: the Clanranald, 1544-77; the Colquhouns of Luss and the Lennox, 1592-1603; the MacLeods of Harris and MacDonalds of Sleat, 1594-1601; the Camerons, 1569-1614; and the ‘Islay Rising’, 1614-15. This thesis adopts a unique approach by contextualising the political background of warfare in order to instil a deeper understanding of why early modern Gaelic Scots resorted to bloodshed. Overall, this period was defined by a sharp rise in military activity, followed by an even sharper decline, a trajectory that will be evidenced vividly in the final case study on the ‘Islay Rising’. Although warfare was widespread, it was not unrestrained or continuous, and the traditional image of a region riven by perpetual bloodshed has been greatly exaggerated.