Saudi Cultural Missions Theses & Dissertations
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Item Restricted Optimisation of Respiratory Syncytial Virus (RSV) whole genome sequencing of subtypes A and B(University of Nottingham, 2024-07) Alamoudi, Nouran Abubaker; King, Barnabas; Mcclure, PatrickRespiratory Syncytial Virus (RSV) is a major cause of lower respiratory tract infections, causing significant morbidity and mortality rates worldwide. RSV mainly affects infants, children under five years old, and older or immunocompromised adults. Establishing a cohesive method for the detection and identification of RSV could significantly improve monitoring and surveillance of RSV cases globally. The present study focused on optimising previously developed 400 base pair tiled-amplicon schemes for the whole genome sequencing of RSV subtypes A and B. We have developed novel ~1200 base pair primer schemes to increase sensitivity and produce a cost-efficient method for RSV genome sequencing. Samples were chosen between 2014 to 2024 and sequenced using Oxford Nanopore Technology’s PromethION device to acquire high- throughput genome data of both RSV subtypes. Our results revealed high potential for the optimised RSV A scheme, with all but two amplicons achieving genomic coverage depth of > 1000 reads per amplicon. However, the developed RSV B schemes generated insufficient results for most of RSV B samples. Moreover, the samples tested for RSV A displayed greater strain variability after phylogenetic analysis. In contrast, RSV B samples were of closely related strains and showed less genomic diversity. Acquiring high-throughput genomic data for RSV can hugely influence our understanding of the diversity of RSV, in addition to its evolutionary and seasonality patterns, which subsequently assists in the management of this virus on a global range. Furthermore, constituting a unified method for RSV sequencing largely impacts the implementation of successful surveillance systems for RSV infections.4 0Item Restricted Using a high-throughput sequencing approach to investigate the effect of ribavirin treatment on the human respiratory syncytial virus genome and its impact on host cells(University of Liverpool, 2023-12) Shawli, Ghada; Hiscox, JulianHuman respiratory syncytial virus (HRSV) infection is the most common cause of lower respiratory tract infections in infants and children worldwide. It can affect individuals of all ages, particularly those with compromised or weakened immune systems. By the age of two, nearly everyone has been infected with HRSV, with symptoms ranging from mild cold-like illness to severe bronchiolitis or pneumonia. Unfortunately, there is currently no safe and effective antiviral treatment available for HRSV infection. Ribavirin (RBV), a guanosine analogue, is the only drug approved for the treatment of severe HRSV lower respiratory tract infections. However, its use is associated with concerns due to its toxicity. RBV can act as a mutagen because its triazole carboxamide pseudobase can base pair with both uracil and cytosine, depending on the orientation of the amide group, leading to increased mutation rates in the viral genome. Understanding both the direct effects of RBV on HRSV infection and its indirect effects on host cells could therefore be valuable for advancing therapeutic strategies. This thesis focused on investigating the antiviral effects of RBV on both HRSV infected and uninfected A549 cells using high-throughput sequencing methods, including both short-read and long-read sequencing platforms. The first approach quantified the increased mutation rates between HRSV infected and RBV treated HRSV conditions using short-read Illumina sequencing. To date, the mutagenic effect of RBV on the HRSV genome in A549 cells has not been tested. A significant increase in transition mutations was observed in the presence of RBV treatment. Complementing the Illumina sequencing results, long-read direct RNA sequencing was employed to detect ribavirin triphosphate as a single molecule incorporated into the viral genome. Using xPore software, the differences in signal intensity between synthetic and natural nucleotides was calculated. Only kmers with NNGNN or NNANN were included in this analysis; eleven positions were identified where ribavirin triphosphate may have been incorporated into the viral genome during RNA synthesis. Interestingly, the counts of NNANN kmers were four-fold higher than those of NNGNN kmers, suggesting that RBV preferentially mimics adenine (A) nucleotides. Next, the incorporation of RBV during the polyadenylation process was investigated by examining the difference in the poly(A) tail length between HRSV and HRSV_RBV conditions using Nanopolish software. A significant reduction in poly(A) tail of some HRSV transcripts treated with ribavirin was found at 9 h and 24 h post infection. This long-read sequencing data provides further insight into ribavirin triphosphate incorporation and its potential use as a substrate by viral polymerase during polyadenylation. Finally, the effect of RBV on the host cellular processes during infection was examined through differential gene expression analysis to identify up and down regulated genes in infected cells or uninfected cells in the presence or absence of the RBV treatment. Overall, these findings improve our understanding of ribavirin’s antiviral mechanism against HRSV in A549 cells, particularly its direct inhibitory effect by increasing the mutation rate within the viral genome and its indirect effects on host cells, which might be crucial for inhibiting the viral replication.4 0Item Restricted Investigating the impact of coronavirus infections on the host respiratory microbiome(University of Liverpool, 2024-05) Alrezaihi, Abdulrahman; Hiscox, JulianCoronaviruses, including the recent SARS-CoV-2, present a complex challenge for public health due to their ability to cause illnesses ranging from asymptomatic infection to patients suffering severe symptoms and death. This thesis employed advanced sequencing methods to identify and study different coronaviruses, from seasonal varieties to MERS-CoV and SARS- CoV-2, and their interactions with the host microbiome in both human clinical samples and animal models. This was underpinned by Oxford Nanopore long-read sequencing combined with modifications of sequence-independent single primer amplification (SISPA). This research aims were to improve the ability to detect coronaviruses, particularly in samples with low viral loads, by modifying different enrichment methods that were designed to detect SARS-CoV-2, identify lineages, and simultaneously define the nasal microbiome. The study revealed changes in the microbiome of patients with coronavirus infections, showing how the virus and the nasal microbiome interact. The data indicated that certain bacterial populations were more predominant in specific groups of patients with COVID-19 or MERS, potentially affecting the course of their illness. The investigation showed that dysbiosis, or microbial imbalance, was significant within coronavirus infections and occurred irrespective of the virus type (MERS-CoV or SARS-CoV-2) and mortality outcome. The investigation compared these patterns in human patients with those observed in a mouse model of SARS-CoV-2 that recapitulated severe disease. This study specifically identified differences in the microbiome between the upper and lower respiratory tracts. Examining the lower respiratory tract in humans is not common as this involves invasive sampling, and use of samples from intensive care patients facilitated this research. This comparison clarified how SARS- CoV-2 affected various parts of the respiratory system and showed that changes in viral load affected the microbiome composition within these distinct areas. Furthermore, the study defined how the microbiome changed throughout the infection, particularly in patients with severe illness who required intensive care.38 0