Advanced sequencing technologies applied to human cytomegalovirus

Li, Karman Kathy (2024) Advanced sequencing technologies applied to human cytomegalovirus. PhD thesis, University of Glasgow.

Abstract

The betaherpesvirus human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. It is the most common cause of congenital infection in infants and of opportunistic infections in immunocompromised patients worldwide. The large double-stranded DNA genome of HCMV (236 kb) contains several genes that exhibit a high degree of variation among strains within an otherwise highly conserved sequence. These hypervariable genes encode immune escape, tropism or regulatory factors that may affect virulence. Variation arising from these genes and from an evolutionary history of recombination between strains has been hypothesised to be linked to disease severity. To investigate this, the HCMV genome has been scrutinised in detail over the years using a variety of molecular techniques, most looking only at one or a few of these genes at a time. The advent of high-throughput sequencing (HTS) technology 20 years ago then started to enable more in-depth whole-genome analyses. My study extends this field by using both HTS and the more recently developed long-read nanopore technology to determine HCMV genome sequences directly from clinical samples. Firstly, I used an Illumina HTS pipeline to sequence HCMV strains directly from formalin-fixed, paraffin-embedded (FFPE) tissues. FFPE samples are a valuable repository for the study of relatively rare diseases, such as congenital HCMV (cCMV). However, formalin fixation induces DNA fragmentation and cross-linking, making this a challenging sample type for DNA sequencing. I successfully sequenced five whole HCMV genomes from FFPE tissues. Next, I developed a pipeline utilising the single-molecule, long-read sequencer from Oxford Nanopore Technologies (ONT) to sequence HCMV initially from high-titre cellcultured laboratory strains and then from clinical samples with high HCMV loads. Finally, I utilised a direct RNA sequencing protocol with the ONT sequencer to characterise novel HCMV transcripts produced during infection in cell culture, demonstrating the existence of transcript isoforms with multiple splice sites. Overall, my findings demonstrate how advanced sequencing technologies can be used to characterise the genome and transcriptome of a large DNA virus, and will facilitate future studies on HCMV prognostic factors, novel antiviral targets and vaccine development.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR180 Immunology; Q Science > QR Microbiology > QR355 Virology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Davison, Professor Andrew and Orton, Dr. Richard
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84061
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Feb 2024 11:39
• Last Modified: 06 Feb 2024 11:41
• Thesis DOI: 10.5525/gla.thesis.84061
• URI: https://theses.gla.ac.uk/id/eprint/84061