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Title: The generation characterization and exploitation of recombinant sendai virus (SeV) as a novel virus vector.
Author: Touzelet, Oliver
ISNI:       0000 0001 3535 6259
Awarding Body: Queens University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2008
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Reverse genetics technology has facilitated the genetic manipulation of many nonsegmented negative strand RNA viruses (NNSV), including Sendai virus (SeV). It has provided the means to exploit SeV as a vector for vaccines or gene therapy, via insertion of extra-numeral transcription units (ENTU) encoding heterologous genes. SeV contains six contiguous genes flanked by the leader (Ld) and trailer (Tr) promoter sequences (3 'Ld-N-P-M-F-HN-L-Tr 5 '). Characterisation of an infectious clone containing the entire antigenome of SeV confirmed that it contained all elements necessary for recombinant (r)SeV rescue. We inserted a heterologous gene encoding respiratory syncytial virus (RSV) fusion (F) protein between the Nand P genes of SeV. A recombinant virus (rSeV/RSV F) was successfully rescued and expressed functional RSV F protein. Importantly, it induced protective immunity in a BALB/c mouse model, demonstrating its vaccine potential against RSV. A common consequence of ENTU insertion is growth attenuation. Therefore, we hypothesised that a rSey containing a bicistronic gene, in which the second cistron encodes a heterologous gene, would circumvent this limitation. To address this, we used a 9nucleotide sequence with known Internal Ribosome Entry Site (IRES) activity. Furthermore, . multiple linked repeats of this IRES increased the expression efficiency of the second cistron. We inserted the Renilla luciferase (rLlle) ORF, preceded by 1, 3 or 7 synthetic IRES copies within the SeV N gene 5' untranslated region. We successfully rescued the corresponding rSeVs, thereby confirming the feasibility of generating bicistronic NNSVs. We confirmed luciferase expression in infected cells and that the number of IRES copies influenced expression efficiency. However, luciferase activity was invariably lower than that from rSeV expressing rLuc from an ENTU. Importantly, and in contrast to rSeV ENTU constructs, our data demonstrated that bicistronic rSeVs were not growth attenuated. Therefore, our approach offers a novel way to express heterologous genes from NNSVs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available