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Title: Manipulating cell lines with CRISPR/Cas9 to investigate Foot and Mouth Disease Virus infection
Author: Fletcher, Sarah Louise
ISNI:       0000 0004 7969 4470
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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With the world population expected to reach 9.7 billion by 2050; food security is a growing concern. As animal protein is one of the most accessible proteins available to people in the developing world, reducing disease outbreaks in livestock is vital. Currently, genomic selection of quantitative trait loci (QTLs) associated with resistance to certain diseases can allow farmers to make an informed choice of which animals to breed from. Unfortunately, appropriate QTLs have not been identified for many diseases and, where they are available, selecting for disease resistance could lead to a loss in productivity in other areas such as milk yield or carcass weight. Recent advances in gene editing technologies can be employed to make specific changes to the DNA of prime breeding animals to introduce existing or novel disease resistance alleles without negatively impacting on the overall productivity of the animal. Although QTLs can be useful in narrowing down specific targets to edit, our knowledge of host-pathogen interactions can also be beneficial in finding ways of increasing disease-resistance where QTLs have not been identified within the population. Foot and Mouth Disease (FMD) is an economically important disease that costs between £5-16 billion globally each year. Eradicated from North America and the majority of Europe, the causative agent of the disease - FMD Virus or FMDV - remains widespread throughout Asia, Africa, and South America. A member of the Picornavirus family, FMDV is a small, single-stranded, positive-sense, unencapsulated RNA virus that is both highly infectious and severely pathogenic. Typical symptoms of infection are mastitis and the development of lesions that the disease is named after within the oral cavity and between the toes of cloven-hooved mammals such as cattle and pigs. There are currently two methods used to control the spread of FMDV; vaccination and mass culling of infected animals. Currently available vaccines are unable to provide cross-protection between the 7 FMDV serotypes, making them ineffective without having a large-scale co-ordinated vaccination strategy in place. Although effective, culling at the scale required to achieve eradication is extremely costly, for example, culls carried out by the UK during the 2001 FMDV outbreak led to the destruction of over 6 million animals and cost £8 billion. In order to control and eventually eradicate FMDV, better control strategies are required. Significant international effort is being spent on the development of new vaccines that offer cross-serotype protection. As an alternative, genome editors could potentially be used to generate disease resistance alleles as has been recently described for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). However, in order to apply this strategy to FMDV, we first need to better understand the numerous host-virus interactions involved throughout the replication cycle so that we can identify candidate genes for editing. FMDV is known to bind RGD-binding integrins such as integrin αVβ6 in order to enter the cell via clathrin-mediated endocytosis. This project aimed to investigate whether integrin αVβ6 from different species would make cells more, or less, susceptible to RGD-binding of FMDV peptides. A human colon adenocarcinoma cell line that is not susceptible to FMDV (SW480 cells) was utilised in this study. Full-length cDNAs encoding both integrin αV and integrin β6 from species considered to be either naturally infected, experimentally infected or resistant to the virus, were synthesised for cloning into lentiviral vectors. To prevent the formation of chimeric integrin αVβ6, CRISPR-Cas9 was used in an attempt to knock out the human integrin αV subunit from SW480 cells. Due to issues with both deletion of the human integrin αV subunit as well as full-length synthesis of the genes, these experiments were not completed. The body of work carried out to identify candidate gene variants for analysis forms the basis for future work in an area that could provide valuable insight into the mechanisms of FMDV entry into the cell. In order to further investigate entry, a cross-species sequence homology study was carried out to identify potential sequence variants in integrin αV and integrin β6 that could impact on virus binding or entry. CRISPR-Cas9 reagents were designed and optimised in order to engineer allelic variants from other species into an endothelial porcine kidney cell line (PK-15). Four cell lines were successfully engineered and genotyped prior to being analysed for their ability to bind RGD-peptides from FMDV. However, a full challenge study with the virus will be necessary to test the impact of these edits on FMDV entry. Lab strains of FMDV at the Pirbright Institute in the UK have a limited ability to infect PK-15 cells whereas FMDV replicates efficiently within the interferon-incompetent Baby Hamster Kidney cell line (BHK-21). A 700 kb deletion of the entire Type I Interferon gene cluster (IFN GC) was carried out in PK-15 cells to investigate if the susceptibility of BHK-21 cells compared to PK-15 cells was due to the lack of a Type I IFN response during FMDV infection in vitro. 3/515 (0.006 %) clones were genotyped as being homozygous for the deletion. Testing whether the cells were more susceptible to interferon-sensitive viruses compared to WT PK-15 cells, they were challenged with two strains of Influenza A Virus that are either IFN sensitive (NS1 R38A/K41A) or not (PR8). Plaque assays showed that the NS1 R38A/K41A virus infected the Type I IFN GC-/- more readily when compared to WT cells whereas the PR8 virus showed no significant difference. These results suggest that these cells are more susceptible to interferon sensitive-viruses and that they could potentially be serially infected with viruses in order to obtain attenuated strains.
Supervisor: Whitelaw, Bruce ; Lillico, Simon Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Foot and Mouth Disease Virus ; FMDV ; CRISPR-Cas9 ; host anti-viral responses ; quantitative trait loci ; disease resistance alleles ; integrin aVß6