Towards an Understanding of Coronavirus Life Cycle and Host Genetic Polymorphisms in the Duration and Severity of COVID-19

Dixon, Charlotte (2024) Towards an Understanding of Coronavirus Life Cycle and Host Genetic Polymorphisms in the Duration and Severity of COVID-19. Doctoral thesis, University of East Anglia.

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Abstract

The innate immune response is vital in the initial viral infection. Cascades of signalling pathways leading to interferon, proinflammatory cytokine expression and interferon-stimulated gene (ISG) expression detects virus and expresses the necessary genes to destroy the virus. However, single nucleotide polymorphisms (SNPs) in innate immune genes can increase or decrease protein expression and function and subsequently lead to prolonged viral replication and viral spread around the body that may lead to an increased fever through cytokine storms or a more severe infection.

Our laboratory showed that ATG16L1, a protein associated with autophagy, is protective against Influenza A virus (IAV) in mice. When the WD40 domain of ATG16L1 was truncated, mice showed an increase in IAV pathology and mortality. The WD40 domain truncation may also be created in humans through the rs2241880 (T300A) SNP, which results in a caspase 3 cleavage site upstream of the WD40 domain. SNPs in the an;-viral protein IFITM3, rs12252 and rs34481144, have also been shown to increase IAV severity in addition to severity of COVID-19.

SARS-CoV-2 has a unique replication strategy common with other coronaviruses producing subgenomic RNA (sgRNA) when the virus is replicating. Individuals with persistent COVID-19 symptoms, may test positive for SARS-CoV-2 by lateral flow test (LFT) for months after becoming infected. These LFTs detect SARS-CoV-2 antigens and therefore may not be detecting active replicating virus, instead detecting dead virus.

This thesis investigated if detecting N-protein and E-protein negative strand subgenomic RNA (-sgRNA) would be a better indicator of active SARS-CoV-2 replication. Using a chicken coronavirus, infectious bronchitis virus (IBV), replicating in Vero cells as a model, the N-protein -sgRNA was detectable by RT-PCR over a 144hr ;me course of infection, but E-protein -sgRNA was undetectable after 144hrs, indicating that E-protein -sgRNA was decayed faster than N-protein. In human samples, N-protein -sgRNA was shown to persist in freshly obtained nucleic acid samples and historic samples. Similarly, E-protein -sgRNA persisted in fresh samples, implying that neither N-protein or E-protein -sgRNA are good indicators of active replicating SARS-CoV-2. These results show that -sgRNA for coronavirus structural proteins is not only found during replication but persists after assembly of virions and maturation into infectious particles, and release from the cell.

The second goal of this thesis was to investigate if SNPs in ATG16L1 and IFITM3, which have previously been shown in mouse models to determine severity to IAV, are associated with SARS-CoV-2 infection severity. A local Norfolk study and a larger Biobank UK database were interrogated and showed that the rs2241880 ATG16L1 SNP and the rs34481144 IFITM3 SNP were protective against development of severe SARS-CoV-2 infections and specific symptom development. Using cells of differing genotype, this thesis also showed that the rs2241880 ATG16L1 SNP and the rs34481144 IFITM3 SNP showed less Sindbis virus infection. In addition to less Sindbis virus replication, the rs2241880 ATG16L1 SNP and the rs34481144 IFITM3 SNP genotypes in mouse bone marrow-derived macrophages (BMDMs) and human peripheral blood mononuclear cells (PBMCs) showed increased expression of proinflammatory cytokines. Therefore, the rs2241880 ATG ATG16L1 SNP and the rs34481144 IFITM3 SNP may reduce viral infection through increased expression of proinflammatory

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	04 Mar 2025 09:04
Last Modified:	04 Mar 2025 09:04
URI:	https://ueaeprints.uea.ac.uk/id/eprint/98648
DOI:

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