title: Understanding severe coronavirus disease in humans from the analysis of clinical samples with RNA sequencing
Creators: name: Penrice-Randal, Rebekah
Publisher: name: University of Liverpool Repository
Year: 2021

description: Coronaviruses in humans have been of concern since the emergence of SARS-CoV, MERS-CoV and SARS-CoV-2 over the past two decades. Coronavirus disease in humans can range from asymptomatic to mild or severe where symptomatic disease is associated with fever, cough, and respiratory symptoms. Next generation sequencing and phylogeny studies can provide insight into viral evolution due to the nucleotide polymorphisms which arise due to the inherent error rates. Nanopore sequencing can facilitate these studies rapidly and are therefore a useful public health tool for genome surveillance. MERS-CoV emerged in Saudi Arabia in 2012 and is associated with sporadic outbreaks. To facilitate rapid genomic surveillance of MERS-CoV in Saudi Arabia, a PCR amplification sequencing method compatible with Nanopore was designed. This approach is useful as data derived from this methodology can be used for phylogeny and variant analysis which supports investigation into transmission events and viral evolution. Upon the emergence of SARS-CoV-2 in late 2019, this approach was then repurposed and utilised on a subset of patients from the UK. Alternative viral genome sequencing approaches were then employed to assess the tissue tropism of SARS-CoV-2 in fatal COVID-19 cases, where tropism was widespread, while inflammation was exclusive to pulmonary tissues. To complement the analysis conducted on the tissue of fatal COVID-19 patients, blood from patients at point of care were utilised for blood transcriptomics analysis. Both illumina and nanopore sequencing methodologies were employed to assess differences in transcript abundance in these patients. Transcriptomic profiles from COVID-19 patients were compared to profiles from Influenza patients and healthy controls, in addition to fatal and non-fatal COVID-19 cases. The key finding from this analysis was that immunoglobulin domains transcripts exhibited altered abundance when comparing COVID and Influenza patients, as well as between fatal and non-fatal COVID-19 cases. From this insight, it is hypothesised that an early adaptive immune response is associated with survival, or a delayed adaptive response is associated with fatality. As it is challenging to control variables from patient data, an hACE2 mice model was utilised to explore the host response against Influenza A virus and SARS-CoV-2 as independent and sequential infections using lung tissue. Transcriptomic analysis revealed a sustained cytokine and interferon response in coinfected mice. Transcripts changing in abundance in both human blood and mice lungs were compared to generate a subset of transcripts that may be essential to the response to SARS-CoV-2 infection. In summary, the results described within this thesis provide insights into the novel coronavirus SARS-CoV-2 and COVID-19 disease in humans. Additionally, the outputs of this thesis provide a foundation for further investigation and development of Nanopore sequencing methodologies as a prognostic tool.