title: Investigation of how host and viral genes contribute to the pathogenicity of avian oncogenic viruses
Creators: name: Look, Toby James
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: subject: CRISPR
subject: Avian
subject: virus
subject: Oncogenicity
subject: Mareks disease
subject: Avian Leukosis Virus
subject: Gene editing
subject: CRISPR Screens
subject: GecKO screen
subject: Avian oncogenic viruses
subject: Chickens

description: The avian oncogenic viruses include retroviruses such as Avian Leukosis virus (ALV) and herpesvirus Marek’s disease virus (MDV) which together are major contributors to cancer in chickens worldwide. Both threaten the stability of poultry farming, causing significant economic losses and welfare implications. Increasing our knowledge of these diseases is critical to control ALV and MDV in poultry, allowing increased productivity and sustainable growth in the industry.The causative agent, of Marek’s disease, is MDV serotype-1 (Gallid herpesvirus 2, GaHV-2), a double-stranded DNA virus belonging to the family Herpesviridae. In non-vaccinated poultry, MDV infects 100% of immunologically naïve chickens and continues to evolve with increased virulence, making vaccination programmes more challenging. MDV infects CD4+TCRαβ+T-cells culminating in visceral lymphomas, neural lesions and paralysis, leading to death several days later. ALVs of the genus alpharetrovirus, are globally endemic in domestic chicken flocks and also replicate in related species. Lymphoid leukosis occurs in infected poultry.This B-cell lymphoma originates in the bursa of Fabricius and metastasizes to other internal organs. In industry, tumour mortality commonly causes a loss of 1-2% of birds with occasional losses &gt;20%. CRISPR-Cas9 gene editing was quickly established in many labs and opportunities to apply the basic CRISPR-Cas9 approach to a diverse array of biological problems followed. These include the investigation of genephenotype correlations in a single cell but also led to the ability to screen entire genomes for specific phenotypes. We utilised both single-cell and genome-wide screen editing techniques to investigate pathogenic determinates in MDV and ALV. To investigate which genes of the MDV-1 vaccine strain CVI988 are essential or non-essential to virus growth and spread in cell culture, I designed and optimised an arrayed CRISPR-Cas9 screen to target all viral open reading frames. crRNA/tracRNA transfection of fluorescent CVI988 infected DF1s enabled systematic analysis of gene essentiality across the whole MDV genome. Two identified genes as essential to viral replication and spread in DF1 cell culture were validated using CRISPR-Cas9 genome editing, providing further evidence that of the reliability of the assay and the requirement of MDV-19 and MDV-20 for viral replication and spread in DF1 cells. Furthermore, the non-essential genes identified maybe useful targets for recombinant virus generation. In the future, this methodology can be applied to other viruses. 4MDV infection commonly results in latently infected T-cells, culminating in Tcell lymphomas. We aimed to investigate the essential host genes in a transformed chicken T-cell line (HP8 cells) using a Genome-Wide CRISPR Knockout (GeCKO) screen. We transduced a library of sgRNAs into HP8 cells and selected using the puromycin resistance marker, taking cell samples for up to 25 days. After removing genes that are considered essential in chickens, using previous chicken screen data and HP8 transcriptomic data to remove non-expressed genes, we shortlisted 339 hit genes that are required for the transformed phenotype of HP8 cells. The hits showed a significant mitochondrial signature; therefore, I investigated the use of mitochondrial inhibitors, metformin, chloramphenicol and A1210477. My data shows mitochondrial function is critical for the growth of HP8 cells and when mitochondria are perturbed, apoptosis increases, and MDV lytic genes are upregulated, suggesting a mechanism for viral reactivation upon mitochondrial stress. Furthermore, the gene IRF4 was shown to be critical for the proliferation of HP8 cells.ALV subgroups are characterised by possessing unique entry receptors to the host cells. Two of the most pathogenically important ALVs, ALV-A (RCAS-A) and ALV-J (RCAS-J) were investigated using a CRISPR screen. Cas9-expressing DF1 cells were transduced with a library of sgRNAs, then infected with a green fluorescent protein (GFP) tagged RCAS-A or RCAS-J virus and sorted for non-GFP expression. The resulting cells were compared to an untreated control population, identifying genes that prevent RCAS-A/Jinfection and/or replication. Significant enrichment of guide RNAs targeting the respective receptors of each virus was observed. However, no other nonessential host genes appeared critical to infection with both RCAS-A and RCAS-J viruses. gga-miR-21 is an oncogenic miRNA in chicken cells. It was proposed that the MDV oncoprotein Meq transactivates gga-miR-21, contributing to the oncogenicity of MDV. We deployed CRISPR-Cas9 gene editing to edit the gga-miR-21 promoter in HP8 cells and measured gga-miR-21 by RT-qPCR. Our results show that in HP8 cells, gga-miR-21 plays no significant role in the oncogenicity of HP8 cells. Additionally, Meq has been reported to bind the gga-miR-21 promoter causing its upregulation. Through knockout of Meq expression and overexpression, no correlation was seen between Meq and gga-miR-21 in HP8 cells. This suggests the gga-miR-21 interplay is not conserved across transformed MDV lymphocytes and while it may play a contributing role in some cell types it is not a critical mechanism in the oncogenic nature of MDV latently infected T-Cells.