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dc.contributor.advisorFleming , Stephen
dc.contributor.advisorMercer , Andrew
dc.contributor.authorAl Daif, Basheer Ali H
dc.date.available2020-09-07T03:15:16Z
dc.date.copyright2020
dc.identifier.citationAl Daif, B. A. H. (2020). ORFV virus manipulation of type I interferon expression (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/10272en
dc.identifier.urihttp://hdl.handle.net/10523/10272
dc.description.abstractOrf virus (ORFV) is the type species of the Parapoxvirus genus that belongs to the Poxviridae family. ORFV is an epitheliotropic virus inducing cutaneous pustular skin lesions in sheep and goats and is transmissible to humans. Type I interferons (IFN) are critical in the host defence against viruses. They induce hundreds of interferon stimulated genes (ISGs) many of which have an antiviral role. The aim of this study was to investigate whether ORFV modulates the type I IFN response. Previous studies suggested a role for the ORFV gene ORF116 in modulating the ISG response using a recombinant ORF116 deletion mutant. These preliminary findings were validated in this study by constructing an ORF116 revertant recombinant virus. The approach taken was to generate a revertant virus in which the ORF116 gene was re-inserted at the same locus as the deleted ORF116 gene in the knockout virus by homologous recombination and then infecting HeLa cells with the two recombinant viruses and the wt strain and studying specific changes in ISG expression by qRT-PCR. The data suggested that ORFV was either directly modulating the expression of about 10 ISGs involved in the anti-viral response or indirectly modulating ISG expression by inhibiting IFN production or both. The ability of ORFV to modulate type I IFN production in several cell types was undertaken to investigate whether ORFV could inhibit IFN-β via dsRNA and dsDNA dependant signalling pathways. HEK293 cells were utilized to model an RNA sensing system. It has been shown that these cells respond to poly(dA:dT), that is converted to dsRNA via RNA polymerase III and poly(I:C) and produce high fold IFN-β expression of RIG-I-dependent signalling as confirmed by siRNA knock-down of RIG-I and RNA polymerase III. HEK293 cells are permissive for ORFV replication and ORFV caused a potent inhibition of IFN-β mRNA when stimulated with poly(dA:dT) or poly(I:C) in a dose-dependent manner. THP-1 cells and human dermal fibroblasts (hNDF) were used to investigate the effects of ORFV on a cytoplasmic DNA sensing system. These cells respond to transfected poly(dA:dT) or poly(I:C) by producing high levels of IFN-β expression in a dose-dependent manner. Both cells are permissive for ORFV early gene synthesis and only hNDFs are permissive for ORFV replication. ORFV infection resulted in strong inhibition of IFN-β in hNDFs treated with either poly(I:C) or poly(dA:dT). The IFN signalling pathway in hNDFs was characterized by RNA interference. siRNA knock-down of STING, a critical adaptor for cytosolic DNA sensing, resulted in a reduction in IFN-β expression in cells stimulated with poly(dA:dT) and this inhibition was further enhanced when both STING and RIG-I were knocked down. Although it was clearly shown that IFN induction was via DNA sensing, cytosolic DNA sensing has yet to be confirmed. Preliminary findings suggest that the two early genes ORF116 and ORF020 expressed from plasmid vectors are potentially involved in antagonising IFN expression. Overall, this study has shown for first time the ability of ORFV to counteract type I IFN expression by antagonising dsRNA and dsDNA activated IFN signalling pathways. This study suggests that ORFV early genes are involved in ISG modulation and IFN-β inhibition.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectparapoxvirus
dc.subjectORFV
dc.subjectinterferon
dc.subjectnucleic acid detection
dc.subjectimmune evasion
dc.titleORFV virus manipulation of type I interferon expression
dc.typeThesis
dc.date.updated2020-09-07T00:11:01Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
otago.evidence.presentYes
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