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Defining latent EBV load in B lymphocytes: Application to multiple sclerosis

Sheahan, Jordana (2017) Defining latent EBV load in B lymphocytes: Application to multiple sclerosis. Honours thesis, Murdoch University.

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Multiple Sclerosis (MS) is a complex autoimmune and neurodegenerative disease affecting more than 1 in 1000 people. The disease is characterized by T and B lymphocytes attacking the central nervous system, leading to demyelination. This can lead to symptoms affecting the motor function and can result in permanent disability. Epstein-Barr virus (EBV) has been implicated in MS with universal presence and elevated EBV antibody titres in MS patients. The role of B lymphocytes has become of major interest with beneficial effects of B lymphocyte-depleting agents and studies demonstrating increased autoreactive memory-B lymphocytes in MS patients. While EBV is known to reside in B lymphocytes, due to the low EBV copy number during latency it is unknown in which B lymphocyte subset EBV is residing in and if the quantity of EBV infected lymphocytes differs between B lymphocyte subsets in MS patients and healthy controls. The use of a specific and sensitive method to quantitate EBV latency in B lymphocyte subsets is yet to be determined and is the focus of this research study. Three independent methods were compared: Quantitative polymerase chain reaction (qPCR), digital droplet polymerase chain reaction (ddPCR) and flow cytometry coupled with fluorescent in situ hybridization (Flow-FISH) targeting EBV Nuclear Antigen-1 (EBNA-1) and EBER (small viral RNAs), respectively. We have successfully quantified EBNA-1 and EBER in healthy control samples with the techniques qPCR and flow-FISH, respectively. However, as a result of the sequence homology between EBNA-1 and genomic DNA, ddPCR was determined to be too non-specific as it did not quantify target EBNA-1. Phenotyping latently EBV infected lymphocytes could be used in the future as a biomarker for MS diagnosis and to target B lymphocyte depletion therapy more specifically to EBV infected or reactive lymphocytes, limiting side effects and improving patient quality of life.

Item Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
Institute for Immunology and Infectious Diseases
Supervisor(s): Tschochner, Monika and Redwood, Alec
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