Saudi Cultural Missions Theses & Dissertations
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Item 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 0Item Restricted Investigation of severe coronavirus infection with the host(University of Liverpool, 2023-07) Alruwaili, Muhannad Falah J; Hiscox, Julian A.Over the last two decades, three zoonotic viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2 have emerged and posed significant threats to human health. After the release of the viral genome in the cytoplasm, 16 NSPs are cleaved and released from the ORF1a and ORF1ab essential for forming the replication-transcription complex to replicate the viral genome. Virus replication relies on the interaction with host proteins for the synthesis of the viral genome, and this process could introduce mutations into the viral genome, but the interactome profile and functionality are undetermined. Also, no developed tool for studying mutations, especially recombination, for the MERS-CoV and SARS-CoV-2 allows for sequencing long amplicons. Here, this thesis demonstrated that an amplicon-sequencing approach, named Rapid Sequencing Long Amplicons (RSLAs), combined with Oxford Nanopore technology can generate data from long amplicons from MERS-CoV and SARS-CoV- 2 genomes in clinical samples suitable for indel and recombination studies. Also, this thesis showed that NSP9 and NSP12, the main components of RTC, interacted with host proteins essential for virus replication. The functioning of SARS-CoV-2 NSP9 was dependent on eEF1A1 and MTHFD1, and NSP12 binding to TRiC/CCT complex was critical to the viral replication and NSP12 stability. Furthermore, NSP12 variants exhibited distinct associations with components of a phosphatase complex, including PP2A and STRN3. The virus that carried NSP12L323 demonstrated reduced susceptibility to disruption in PP2A. These findings allow the scientific communities to investigate mutational profiles of coronavirus for a better understanding of any potential evolution of coronavirus in the future using RSLA. They also showed the dependency of SARS-CoV-2 replication on host proteins revealing new targets for therapeutic interventions.15 0Item Restricted Investigation of the roles of MERS-CoV accessory proteins: ORF4a and ORF4b with the host(University of Liverpool, 2024-03-05) Almsaud, Mai; Hiscox, JulianCoronaviruses account for a high burden of respiratory disease in humans, ranging from common colds to severe acute respiratory syndrome. The emergence of severe acute respiratory syndrome (SARS) in 2002, Middle East respiratory syndrome (MERS-CoV) since 2012 and, more recently, SARS-CoV-2 have attracted considerable attention worldwide. The persistence of sporadic MERS-CoV cases, along with the absence of vaccines and antivirals, and the strikingly high morbidity and mortality rate of 36%, underscore the ongoing risk posed by MERS-CoV to global health security. Nevertheless, gaining a comprehensive understanding of the pathogenic significance of MERS-CoV requires further research. As viral replication and infection rely on the host cell machinery for the viral life cycle, studying how cells respond to infection allows us to understand the mechanisms involved in either limiting virus infection or, when control fails, in pathogenesis. In contrast, viruses can encode specific proteins to counteract these host responses. MERS-CoV ORF4a and ORF4b are two such proteins and recognised for their roles in modulating the host immune system, thereby influencing viral pathogenicity. This study investigated the significant roles of MERS-CoV accessory proteins, ORF4a and ORF4b, using two distinct high-throughput methods: a label-free mass spectrometry proteomics approach and a transcriptomic approach based on long-read cDNA-PCR sequencing. The proteomic approach unveiled novel interactions between MERS-CoV ORF4a, ORF4b, and host factors. Complementing the LC-MS/MS analysis, long-read length Nanopore sequencing was employed to explore the transcriptomic landscape influenced by MERS-CoV ORF4b expression. This technique allowed for a comprehensive assessment of isoform usage and mRNA regulation, providing novel insights into the virus hijacking splicing machinery by ORF4b. The investigation of both viral proteins, ORF4a and ORF4b, was extended by comparing their relative strengths in subverting the innate IFN-β response with those of similar proteins encoded by other coronaviruses. This demonstrated the distinct potency of MERS-CoV ORF4a and ORF4b proteins in relation to the severity of MERS disease when compared to other coronaviruses. Collectively, this thesis contributes to the understanding of the interactions that MERS-CoV establishes with its host, highlighting the significance of accessory ORFs in pathogenesis, and emphasising their potential as subjects for ongoing monitoring.39 0Item Restricted Comparative interactomic analysis of MERS-CoV & SARS-CoV-2 proteins in human, bat, and camel cells(Saudi Digital Library, 2023-12-05) Alaa, Abed; Davidson, AndrewThe Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are Betacoronaviruses capable of causing fatal human infections. Both viruses are believed to have emerged from bats via an intermediate host (camels for MERS-CoV, unknown for SARS-CoV-2) into the human population. MERSCoV and SARS-CoV-2 are enveloped positive-sense RNA viruses which encode four structural proteins (envelope (E), nucleocapsid, spike, and membrane (M)). The E and M proteins are involved in virus assembly, budding, envelope formation, and pathogenesis. Finding cellular protein interactors for these viral proteins, conserved across species, will increase our understanding of the coronavirus lifecycle and identify targets for antiviral development. Initial validation of the results for 11 cellular proteins interacting with the MERS-CoV E and/or M proteins by co-immunoprecipitation (co-IP) /Western blot analysis and immunofluorescence co-localisation, confirmed 10 of the interactions. Three cell lines (human HEK293, bat Pteropus alecto PaKiT and Camelus dromedarius Dubca) were used for transient expression of the MERS-CoV and SARS-CoV-2 E and M proteins (FLAG epitope-tagged) followed by co-IP and high-throughput mass spectrometrybased interactomic analysis. Bioinformatic analysis revealed E/M protein interactions with ER, Golgi, mitochondrial and nuclear proteins. There were 32 high-confidence cellular interaction proteins conserved amongst the different cell lines and viruses (p < 0.05, > 0 log 2 fold change compared to the controls). To determine the importance of the 11 cellular proteins interacting with the MERS-CoV E and/or M proteins and 32 cellular proteins conserved across species, in the virus lifecycle, functional validation was done by siRNA depletion in human cells, followed by infection with SARS-CoV-2. An interesting four cellular proteins were shown to be important for SARS-CoV- 2 replication. These interesting proteins include (CERS2, LPCAT1, UBA52, and TM9SF2) that when it was knocked down, succeeded in reducing the SARS-CoV-2 replication. This can be followed by working to find if these proteins can be safely targeted by a drug to be reduced in cells.14 0Item Restricted Mathematical Assessment of the Transmission Dynamics and Control of MERS-CoV and SARS-CoV-2 in the Kingdom of Saudi Arabia(Saudi Digital Library, 2023) Alatawi, Adel; Gumel, AbbaThe Kingdom of Saudi Arabia (KSA), which hosts some of the largest mass gatherings of humans globally every year, has seen the emergence of two coronavirus pandemics, namely the 2012 middle eastern respiratory syndrome (MERS-CoV) and the 2019 SARS-CoV-2 pandemics. This dissertation contributes in providing deeper insight into the transmission dynamics and control of the two diseases in the Kingdom. A model for SARS-CoV-2 transmission dynamics, which incorporates the key features of the disease, was designed first of all. Its disease-free equilibrium was shown, using Lyapunov function theory, to be globally-asymptotically stable when the associated reproduction number is less than one. The model, which has a unique and locally- asymptotically stable endemic equilibrium (for a special case) when the reproduction threshold exceeds one, was fitted using observed data for the KSA. Global sensitivity analysis was carried out to identify the key parameters of the model that have the most influence on the disease burden in the Kingdom. The model was used to assess the population-level impacts of control and mitigation interventions. It was shown that a face mask use strategy, based on using masks of moderate to high efficacy, can lead to the elimination of the pandemic if the coverage in its usage is high enough. A model for the spread of MERS-CoV in the human and camel host populations was also designed, rigorously analysed, and fitted with data. The model was later extended to include the use of intervention measures, notably vaccination of humans and camels and the use of face mask by humans in public or when having frequent closed contacts with camels. The population-level impacts of these interventions, implemented in isolation or in combinations, were assessed. The study showed that focusing intervention resources on containing the MERS-CoV spread in the camel population would be more effective than on containing the spread in humans.41 0