Characterization of few polyhedra mutants of Helicoverpa armigera nucleopolyhedrovirus cultivated in vitro

Marcia Regina da Silva Pedrini (2002). Characterization of few polyhedra mutants of Helicoverpa armigera nucleopolyhedrovirus cultivated in vitro PhD Thesis, Department of Chemical Engineering, The University of Queensland.

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Author Marcia Regina da Silva Pedrini
Thesis Title Characterization of few polyhedra mutants of Helicoverpa armigera nucleopolyhedrovirus cultivated in vitro
School, Centre or Institute Department of Chemical Engineering
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Steven Reid
Lars Keld Nielsen
Total pages 157
Language eng
Subjects 0904 Chemical Engineering
Formatted abstract
Naturally occurring insect viruses are a promising means o f intentionally causing disease in insects but they have not competed successfully with synthetic chemicals in the commercial  marketplace. Furthermore, their use for pest control is still restricted. One factor preventing the  development o f baculoviruses as effective biopesticides is concern over production issues. There was evidence that the production process using serial passaging o f baculoviruses in established cell lines may be hindered by the production o f aberrant viruses, which are called few polyhedra  (FP) mutants. FP mutants quickly become the predominant virus type, producing low yields of occlusion bodies with reduced biological activity. Characterization o f these mutants and the study of their dynamics are the main focuses o f this thesis.

This thesis is devoted to the characterization o f H elicoverpa zea cells and wild type H elicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) system. The first study was conducted to determine whether the enhanced stability observed for a plaque purified nucleopolyhedrovirus can be attributed to the cells, media or virus employed. The results indicated that the enhanced stability is most likely due to plaque purification o f the virus, and demonstrated the importance o f performing the plaque purification in the same medium that is used for scale-up o f the biopesticide process.

The FP mutants o f nucleopolyhedroviruses have been correlated with the absence o f the virus 25-kDA protein and with the presence o f D N A mutation in the 25K FP gene, caused by either large insertions o f host D N A or deletions o f viral DNA. This thesis confirmed that the HaSNPV 25K FP gene also undergoes nucleotide changes during serial passaging. One bp insertions in repetitive  sequences and point mutations were observed for HaSNPV and two FP mutants that carry a mutation in  the 25K FP gene were isolated.

Besides the mutations in the 25K FP gene, evidence o f the accumulation o f defective interfering particles (DIPs) could be seen from restriction enzyme (REN) digestion profiles using BamHl and pulsed-field gel electrophoresis (PFGE) analysis o f purified virus DNA. The accumulation of DIPs can account for the decline in budded virus titre after extended serial passaging of wild type HaSNPV-Ac53 (Lua, 2000 and this thesis) and o f plaque-purified FP mutants (this thesis).

Finally, kinetic experiments were performed which confirm that FP mutants produce higher titres and lower OB yields compared to the wild type MP (many polyhedra) isolate. However, there is no evidence that the failure to occlude virions accounts for the increased titres resulting from FP mutants. The higher budded virus production o f FP mutants is the major mechanism for the selection of FP mutants over the wild type MP virus in cell culture.

The parameters obtained during kinetic the experiments were used to model the HaSNPV/ H. zea cells system. This model adequately predicts the population shift from MP to FP virus in cell culture, with good agreement between experimental and simulated data, for both high MOI and low MOI  infections. Such model would be invaluable in helping to design scale-up process for HaSNPV virus.

Keyword characterization of Helicoverpa zea cells
wild type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV)

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Thu, 31 Jul 2014, 11:10:06 EST by Asma Asrar Qureshi on behalf of Scholarly Communication and Digitisation Service