Genome-scale transcriptomic study of Helicoverpa Zea host cells and H. armigera baculovirus infections in vitro

Nguyen, Hoang Quan (2013). Genome-scale transcriptomic study of Helicoverpa Zea host cells and H. armigera baculovirus infections in vitro PhD Thesis, Australian Institute For Bioengineering and Nanotechnology, The University of Queensland.

       
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Author Nguyen, Hoang Quan
Thesis Title Genome-scale transcriptomic study of Helicoverpa Zea host cells and H. armigera baculovirus infections in vitro
Formatted title
Genome-scale transcriptomic study of Helicoverpa Zea host cells and H. armigera baculovirus infections in vitro
School, Centre or Institute Australian Institute For Bioengineering and Nanotechnology
Institution The University of Queensland
Publication date 2013-07
Thesis type PhD Thesis
Supervisor Steven Reid
Total pages 149
Total colour pages 24
Total black and white pages 125
Language eng
Subjects 100302 Bioprocessing, Bioproduction and Bioproducts
060405 Gene Expression (incl. Microarray and other genome-wide approaches)
110804 Medical Virology
Formatted abstract
Baculovirus-insect cell technologies are rapidly developing for the production of eukaryotic proteins, veterinary and human vaccines, delivery vectors for gene-therapy‚ and biopesticides. The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely related Helicoverpa zea and H. armigera caterpillars. Biological control using baculoviruses is practiced at a moderate level worldwide. However, cell-virus interactions make baculovirus-based manufacturing processes inherently complex. This study uses a genome-scale transcriptomic approach to investigate the interactions at the mRNA levels of a H. zea cell line and a H. armigera virus with the ultimate aim of identifying genes important for high productivity of in vitro production of baculoviruses using insect cell culture.

While many baculovirus genomes, including that for H. armigera, have been sequenced, none of the Heliothine insect genomes are available. In this study, I sequenced, assembled and functionally annotated 29,586 H. zea transcripts that had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes (Chapter 2).

The sequences were used to construct a microarray, which was applied to simultaneously study expression kinetics of 24,206 H. zea insect transcripts and 134 H. armigera genes at 0, 12, 18, 24 and 48 hours post infection (h.p.i) (Chapter 3). This study represents the first genome-scale study of the interactions between a group II alpha baculovirus (HearNPV) and its permissive insect host (H. zea).The use of a species-specific microarray, based on coding sequences obtained from RNA-sequencing, provided a comprehensive means of elucidating the genome-scale expression kinetics of both host and virus genes at different infection stages, and their effect on the formation of progeny virions and occlusion bodies (OB). This study provided insights on the systemic process of virus production in infected cells and identified a large number of host genes that are important for virus propagation in insect cell culture.

To further identify genes that contribute to increased productivity, three systems that produced different cell specific occlusion body yields were investigated (Chapter 5). The first system used two clones‚ which were selected among 250 clones isolated from a parental H. zea cell population so that the two clones display the highest difference in OB/cell yield (Chapter 4). The second system compared infections in three media‚ varying in nutrition content from poor to intermediate to rich. In addition‚ two infections at a low and a high cell density that had different OB/cell yields due to the cell density effect were also compared. Different responses to infection, which were defined as expression changes between samples at 18 h.p.i and 0 h.p.i‚ were compared among the three infection setups above. Specific host genes important for different virus propagation processes were identified. The high consistency of 14 genes that were up-regulated in 11 different infection conditions suggested that the expression changes were not a random process, but rather a result of controlled regulation between the virus and insect.

Finally‚ to validate candidate genes‚ which are identified by transcriptomic studies as being important for increasing production yields of baculovirus-insect systems, the virus genome and/or host insect genome needs to be readily engineered. The availability of methods that are effective for knockdown, knockout, and insertion of host or virus genes can strongly facilitate the process of validating conclusions drawn from transcriptomic studies. These methods were evaluated in Chapter 6.
Keyword Baculovirus
Transcriptomics
Microarray
Next generation sequencing
HearNPV
Helicoverpa
Genome
System biology
Virus-host interaction

 
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Created: Fri, 13 Dec 2013, 09:51:54 EST by Mr Hoang Quan Nguyen on behalf of Scholarly Communication and Digitisation Service