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Graduation date: 1995

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 156-178). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 156-178). Also available on the Internet.

Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviridae is the only known virus family that encodes genes with homology to vertebrate and invertebrate fibroblast growth factors (fgfs), key regulators of developmental processes affecting cell growth, differentiation, and motility. The role of viral fgfs during infection is not known. In this study, we investigated gene regulation and function of the Autographa californica M nucleopolyhedrovirus (AcMNPV) fgf during infection of permissive insect cells. We demonstrated that the AcMNPV fgf, vfgf, was transcribed as a 0.6-kb mRNA at early times post infection, but as part of a 1.4-kb bicistronic mRNA at late times. To determine its function, we examined common characteristics between vFGF and other well-characterized FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate chemokinesis of different types of insect cells. We also constructed a recombinant of AcMNPV lacking a functional vfgf and analyzed it in two insect cell lines. The kinetics of budded virus production were similar in the parental and vfgf-deficient viruses in two cell lines and at both high and low multiplicities of infection. In addition, we observed no obvious differences in the viral DNA synthesis and the protein kinetic profiles of cells infected with the mutant and parental viruses. Finally, coinfection of vfgf-containing and -deficient viruses and their passage for several generations did not reveal a consistent growth advantage for either virus. We propose that vFGF is the signal that directs the motility of uninfected tracheal or blood cells to infected tissues, enabling the virus to infect additional cells and spread systemically in the insect host. This proposal may explain a dispensable role for vfgf during virus infection in cell culture; nonetheless, we expect a distinct phenotypic difference between vfgf-deficient and vfgf-containing viruses during infection in the insect host.

Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.

Bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), is an important pest of cruciferous oilseed crops in western Canada. A nuclear polyhedrosis virus, MacoNPV, isolated from M. configurata, has demonstrated as high as 95% infection in field populations of bertha armyworm. MacoNPV isolates from different geographic areas differ in terms of their virulence to bertha armyworm. Restriction endonuclease (REN) fragment analyses show that all of the geographic isolates are closely related viruses but with some distinct REN pattern differences. Most of these geographic isolates are heterogenous mixtures of genotypes. The thesis describes the cloning and physical mapping of the 156.9 kbp genome of the MacoNPV-90/2 geographic isolate, including 112 restriction sites for six common REN, BamHI, EcoRI, HindIII, PstI, SmaI and XhoI. Twenty plaque purified strains of MacoNPV were isolated in a cultured Mamestra brassicae (Mbr) insect cell line. The EcoRI restriction patterns of these pick plaque (pp) strains fell into 10 different categories. In order to investigate the difference among these pp strains, and between these strains and the parental geographic isolates in terms of REN patterns, virulence to insect hosts, and their growth rates in insect cell lines, some of these isolates were selected for bioassays in bertha armyworm larvae and in the Mbr cell line.

Budded virions of AcMNPV can enter a variety of non-host cells, a characteristic likely due to the presence of GP64, an envelope protein found on a small subset of baculoviruses. Results show that AcMNPV's tropism for vertebrate cells can be restricted - a prerequisite for using AcMNPV for targeted in vivo gene delivery - by replacing the gp64 gene with SeF from SeMNPV. Unlike the relatively well characterized GP64 protein, the significance and function of the F homolog (Ac23, a pathogenicity factor), is poorly understood. How Ac23 might contribute to the faster speed of kill was examined by comparing occlusion bodies and occlusion-derived virions (ODV) of Ac23null mutant viruses with control viruses at the ultrastructural level. The results show that Ac23null mutant produces a significantly higher percentage of ODVs with single or lower number of nucleocapsids than controls, suggesting Ac23 may play a role in multicapsid envelopment of ODVs. / xiii, 101 leaves : ill. (some col.) ; 28 cm. --

Thesis (PhD) - Swinburne University of Technology, 2008. / Submitted for the degree of Doctor of Philosophy, Swinburne University of Technology - 2008. Typescript. Includes bibliographical references (p. 195-208).

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 60 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 51-55).