Development and characterisation of novel immunodiagnostic reagents for the detection of banana viruses

Vo, Jenny (2013). Development and characterisation of novel immunodiagnostic reagents for the detection of banana viruses PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

Attached Files (Some files may be inaccessible until you login with your UQ eSpace credentials)
Name Description MIMEType Size Downloads
s4075292_corrected_phd_thesis_final.pdf Thesis (fulltext) application/pdf 14.70MB 8
Author Vo, Jenny
Thesis Title Development and characterisation of novel immunodiagnostic reagents for the detection of banana viruses
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Andrew Geering
Ross Barnard
Total pages 205
Language eng
Subjects 060506 Virology
110702 Applied Immunology (incl. Antibody Engineering, Xenotransplantation and T-cell Therapies)
060704 Plant Pathology
Formatted abstract
Banana streak virus (BSV) is the collective name for a cryptic species complex of badnaviruses that occur in almost every banana-growing region of the world. BSV diagnosis is complicated by the fact that endogenous forms of the virus occur in the banana genome, and therefore the most reliable way to detect infection is to test for viral proteins. The capsid protein (CP) is one of the important markers of viral infection and has multiple functions in the virus life cycle. The BSV CP is released from the open reading frame 3 polyprotein through the action of the viral aspartic protease but unlike other classes of protease, it is not possible to predict the cleavage sites by sequence analysis alone. This study identified the virion proteins of two BSV species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) and constructed a 3-D model of the CP of the former using a combination of threading and ab initio modelling by I-TASSER. This study suggested that the BSV CP contains three major domains, the nucleocapsid (NC) domain, the capsid (CA) domain and a small, surface protruding N-terminal domain, (NTD). The NTD of the BSV CP is intrinsically disordered in structure and is likely to have signaling and regulatory functions. This thesis describes the first experimental validation of the structural role of the disordered NTD of BSMYV, and confirmation that it is exposed on the surface of the virion. Functional epitope mapping of the BSMYV-CP by the PepScan approach revealed that immunodominant B-cell epitopes are mainly located in the protruding NTD region. Synthetic peptides that mimic the structure of two identified epitopes were successfully used to induce an antibody response similar to that elicited by purified BSV virions. These antipeptide antibodies recognised both native and denatured antigen, and are BSMYV specific. Information obtained from this study will contribute to development of new diagnostic reagents for BSVs, by better defining virion structure and allowing the possible identification of new BSV serotypes. Another aspect of this study was the generation of recombinant antibody single chain variable fragments (scFvs) to BSMYV, Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV). Recombinant antibody libraries for BSMYV were generated from an immunised chicken, with promising binders selected by phage display. Antibody genes were also cloned and constructed as scFvs from pre-existing anti-BBTV and anti-BBrMV hybridoma cell lines. This research demonstrates the use of a phage-based scFv sandwich ELISA for the detection of banana viruses for the first time, and showed the potential of recombinant antibodies and antipeptide antibodies as new immunodiagnostic reagents for banana virus indexing. An important outcome of this research project has been the development of new strategies for production and characterisation of antibodies to banana viruses. 
Keyword Banana streak virus
Coat protein
Protein structural modeling
Recombinant scFv
Phage display
Epitope mapping
Synthetic peptide
Immunodiagnostic reagents

Citation counts: Google Scholar Search Google Scholar
Created: Mon, 07 Apr 2014, 21:12:57 EST by Miss Jenny Vo on behalf of Scholarly Communication and Digitisation Service