Phthorimaea operculella, commonly known as the Potato Tuber Moth, is an economically important agricultural pest worldwide. The baculovirus, Phthorimaea operculella granulovirus (PhoGV) has been considered as a means of control alternative to chemical control because of its host specificity and harmless impact on other organisms and ecosystems. An isolate of PhoGV obtained from a South African PTM population would be beneficial in the production of a biopesticide, which is not yet available. An efficient and cost-effective rearing method would be advantageous for potential commercial production. Commercial table and seed potato plantations and storage facilities located in Patensie, Bathurst, Howick and Ivanhoe were surveyed for PTM infestations. Patensie was the only site where milky discoloured larvae were found, a potential symptom of PhoGV infection. TEM analysis revealed no virus in these samples. Since no virus was found in the field-collected samples, PTM insects were collected to initiate rearing in the laboratory. PTM was raised by three different methods in the laboratory. A cost/benefit analysis, survival rate, fertility and sex ratio were recorded for each rearing method. Rearing method one was deemed unsuccessful for efficient commercial rearing, as survival percentage and fertility were low. Rearing methods two and three had high survival rates and high fertility, and were efficient and less labour intensive than rearing method one. Rearing method three was the most productive technique, but for commercial production rearing method two was considered the most manageable and efficient. The sex ratio was 1:1 for all three cultures. The cost analysis revealed that rearing methods two and three were less expensive than rearing method one because less labour was required to monitor insects. The success of rearing PTM for 19 months will enable these cultures to be up-scaled to a large production facility for mass rearing. Virus was not found in the field surveys or in laboratory cultures, therefore chemical, temperature, humidity and carbon dioxide stressors were used in an attempt to initiate a baculoviral infection. Symptoms were exhibited in larvae subjected to chemical, temperature and humidity treatments, but these were confirmed by TEM analysis not to be a result of PhoGV infection. The success of rearing PTM in the laboratory suggests that the method could be used in the commercial rearing of the insects in a large mass-rearing facility. The data obtained from induction protocols have allowed for better understanding for future induction for PhoGV and other baculoviruses in other insect species. The failure to isolate a South African PhoGV strain for developing a biopesticide against PTM has motivated further studies in obtaining a baculovirus in order for South Africa to develop a commercial product against this pest.

The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.

>Magister Scientiae - MSc / Insect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.

Phosphorylase kinase (EC 2.7.1.38) is a key enzyme involved in the regulation of the glycogenolysis pathway. It catalyzes the Ca²⁺-dependent phosphorylation and activation of the enzyme glycogen phosphorylase to make the active form glycogen phosphorylase. Phosphorylase kinase is composed of 4 subunits with a stoichiometry of (αβγδ)₄. The γ subunit is the catalytic subunit. The regulatory domain (residues 277-387) of γ contains a sequence resembling the sites phosphorylated in known γ substrates with the exception that a valine₃₃₂ occurs at the analogous position of the phosphorylated serine or threonine residue. / Ph. D.

The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.

Baculoviruses are a family of mostly insect-specific viruses with relatively complex infection pathology. The baculovirus genome encodes between 89-181 genes, that are regulated by a complex temporal cascade of gene expression. Although baculoviruses pathogenic to lepidopteran hosts are well characterized, relatively little is known about non-lepidopteran baculoviruses. This thesis provides the genome sequence of a baculovirus pathogenic to the balsam fir sawfly (order: Hymenoptera), the Neodiprion abietis nucleopolyhedrovirus (NeabNPV). Our analyses of the NeabPNV genome indicated that the regulation of NeabNPV early genes by immediate early transactivators may differ from the lepidopteran baculovirus model. Also, through genome sequence analysis, we propose a model for the evolution of sawfly baculoviruses that is mediated by interspersed genome repeats. By selecting key genes transcribed at distinct time points during baculovirus infection as well as quantifying viral DNA in the tissues of infected host larvae, we mapped the progression of NeabNPV infection in vivo. Based on the temporal scale of viral infection, we were able to show that two putative zinc-finger proteins, neab24 and neab52 are expressed in the immediate early and early stages of infection. The hypothesis that the mechanism of early gene regulation in non-lepidopteran baculovirus differs from that of lepidopteran baculoviruses prompted us to investigate promoter elements of 30 baculovirus genomes sequenced to date, in silica. This analysis revealed some sequence motifs may represent promoter elements in a wide range of baculoviruses, and that there may be differences in regulation of transcription between genes of the same temporal class in different baculovirus species.

This work describes the pathology of the baculovirus native to the balsam fir sawfly, Neodiprion abieris nucleopolyhedrovirus (NeabNPV), both in vitro and in vivo. In vitro techniques were initially established through the characterization of Lambdina fiscellaria lugubrosa NPV (LafiNPV-W) in Malacosoma disstria (forest tent caterpillar) and Choristoneura fumiferana (eastern spruce budworm) tissue cultures. The results showed that host cell selection is important for the accurate characterization of viral pathology M. disstria cells infected by LafiNPV-W supported a biased production of extracellular viral progeny and aberrant LafiNPV-W occlusion bodies. C. fumiferana cells, on the other hand, supported production of both the extracellular and occluded phenotypes. The pathology of NeabNPV was studied in vitro using the C. fumiferana cell line and three cell lines derived from the closely related red-headed pine saw-fly, N lecontei. All three sawfly cell lines were non-permissive to NeabNPV, while C. fumiferana was semipermissive and enabled preliminary characterization of early pathology, including early viral gene transcription. Due to only partial success in characterizing NeabNPV infection in vitro, pathology was examined within the native larval host, N. abietis. The first step in characterizing NeabNPV infection in vivo was to define the morphology and ultrastructure of the gut of uninfected N. abietis larvae. The sawfly alimentary canal consisted of cuticle lined foregut and hindgut, which adjoined to an elongated midgut. The epithelial tissue of the midgut was composed of regenerative and digestive columnar cells; the latter possessed a complex ultrastructure that reflexed the cells function in nutrient absorption and digestive enzyme secretion. NeabNPV pathology was only detected in the midgut epithelial cells. A time course of NeabNPV infection enabled the identification of several key cytopathic effects and the correlation of gene expression with specific phases of viral infection. These analyses revealed both differences and similarities between the process of infection and pathology induced by NeabNPV and lepidopteran NPVs and may serve as a different model for baculovirus infection of nonlepidopteran.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 181 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.

No description available.