Human herpesvirus (HHV) –6A and HHV-6B are unique among herpesviruses in their ability to integrate into the telomeres of human chromosomes and be inherited in a Mendelian fashion. Based on current data, inherited chromosomally integrated HHV-6 (iciHHV-6) is present in 0.5-2% of the UK population. There is increasing evidence to suggest that iciHHV-6 is not a dead-end for the virus, and that HHV-6 can be excised from the genomes of iciHHV-6-positive individuals. It is hypothesised that this excision occurs from the formation of T-loops between the end of the telomere and the viral telomere like repeats (TLRs). T-loops form naturally in a cell as part of the complex that protects the end of the chromosomes; however, under certain circumstances these can be excised leading to a loss of telomeric repeats from the chromosome and the formation of circular, extra-chromosomal telomeric sequence. There is increasing evidence that excision can lead to reactivation of the virus and potentially cause disease. Our current understanding of the phenotypic associations of iciHHV-6 is based on case-reports and case-control studies of individual diseases. The work presented in this thesis set out to answer a number of questions regarding the phenotypic consequences, and genome dynamics of iciHHV-6. First, the association between both exogenously acquired HHV-6 and iciHHV-6 and classical Hodgkin lymphoma (cHL) was examined in a case-control study. Whilst exogenously acquired HHV-6 was significantly associated with cHL the virus was present at low levels in DNA extracted from cHL tumours. Virus at such low level was concluded as not having a direct role in the pathogenesis of cHL. A case control study of iciHHV-6 and cHL revealed no difference in the prevalence of iciHHV-6 amongst cases and controls, but identified a single iciHHV-6-positive individual who appeared to have four integrated viral genomes. Secondly, iciHHV-6 was examined in the Generation Scotland: Scottish Family Health Study (GS:SFHS) cohort, in a hypothesis generating study. It was revealed the iciHHV-6 was present at a prevalence of 2.7%. Further analysis showed a statistically significant difference in the prevalence of iciHHV-6 between individuals born in Scotland (2.8%) and England (1.8%). Analysis of disease phenotypes revealed potential associations between iciHHV-6 and breast cancer in an unrelated subset of the GS:SFHS cohort. Also confirmed was the recent 3 report of an association between iciHHV-6 and angina pectoris. Analysis of other variables revealed iciHHV-6-positive females had a lower average Mill Hill vocabulary test score than iciHHV-6-negative females; and that iciHHV-6-positivity was associated with participation in fewer years of education. Thirdly, the iciHHV-6 genome in an LCL generated from an iciHHV-6A-positive individual was shown to be dynamic. The gross structure of the HHV-6 genome is a unique (U) region flanked by direct repeats (DR) (DR.U.DR).Analysis using droplet digital PCR (ddPCR), on DNA extracted at various time points of the LCL culture revealed that viral sub-genomic regions were lost in a proportion of cells, which coincided with a reduction in population doublings of the culture. At the point of reduction of viral copy number, an excess of HHV-6 DRs was noted suggesting that only a portion of the viral genome had been lost in these cells. Gradually the viral copy number returned to approximately one copy per cell. It is likely this was caused by an outgrowth of cells where excision had not occurred. This in vitro model demonstrated that whilst excision of iciHHV-6 genomes is possible, it may be accompanied by a reduction in cell viability. Loss of HHV-6 genomic regions was also examined in 59 iciHHV-6-positive individuals and was infrequent. Only six showed some degree of DR loss. Further to this, inheritance of single direct repeats was observed in six individuals and two families in the GS:SFHS cohort. A novel ddPCR and mathematical model was developed to predict the iciHHV-6 genome configuration in samples where atypical U:DR ratios were observed. Through this, we hypothesise that an iciHHV-6-positive individual who had 4 U regions per cell and 5 DR regions per cell, had an integrated HHV-6A genome concatemer with the configuration (DR.U)4.DR, and hypothesised that this arose from integration of a replication intermediate. Finally, phylogenetic analysis of five regions of 26 iciHHV-6 genomes and their counterparts in exogenously acquired HHV-6 genomes was performed. There was a higher degree of divergence between iciHHV-6A and exogenous HHV-6, along with evidence of a common viral ancestor in four iciHHV-6-positive individuals. This divergence was not observed in iciHHV-6B were very little variation was observed between iciHHV-6B and exogenously acquired HHV-6B. These results shed light on the complex relationship between iciHHV-6 and the human host.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:679583 |
| Date | January 2016 |
| Creators | Bell, Adam Joseph |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/7073/ |
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