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<p>Propagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5&rsquo / and 3&rsquo / ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.</p>

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When expressed in Spodoptera frugiperda cells using recombinant baculoviruses, the HIV - 1 gag gene product, p55, self assembles to form immature HIV core like particles that are secreted into the culture medium. Using this system, the gag open reading frame was progressively truncated from the carboxy terminus, and each deleted Gag protein examined for its ability to produce core like particles. Deletions that removed the distal region of the Gag nucleocapsid domain, including both Cys - His motifs thought to function as RNA capture signals, did not disrupt particle formation. Analysis of the truncation mutants by north - western blot with a radiolabelled HIV - 1 RNA encapsidation signal, confirmed that only precursors with the Cys - His motif present were able to bind the probe. It was concluded that HIV - 1 Gag particle formation per se does not require RNA encapsidation. This finding clarifies uncertainties proposed previously regarding the role of RNA encapsidation in particle assembly. Further deletion mutants led to the delineation of the carboxy terminal boundary of a Gag assembly domain. Properties of the SIV env encoded TM glycoprotein were also explored. Previous reports have found that the cytoplasmic tail of this membrane spanning protein was preferentially deleted in vivo when SIV was grown in a variety of cultured human cells. However, when propagated in cells of simian origin, the TM glycoprotein cytoplasmic tail was retained. Analysis of the function of this domain was addressed by expression of both the truncated and the full length proteins in insect cells using recombinant baculoviruses, coupled with a study of their biochemical characteristics. A role for the cytoplasmic tail was identified in the post - translational modification and localization of the glycoprotein. The use of the Gag particles in designing vaccines was investigated. Acting as non - infectious carriers of immunogenic antigens, the particles represent a safe and efficient means for presentation of epitopes for eliciting a protective immune response. Two possible approaches for loading the particles with foreign antigen were examined. In the first, the deleted portion of Gag was replaced with a sequence encoding the foreign antigen. The second method involved co - expression of Gag with another recombinant virus that expressed a foreign antigen on the surface of the cell. Both methods were found to be feasible, although limitations were identified when addition of the fusion protein promoted excess degradation of the Gag precursor.

Copies of author's previously published articles inserted. / Bibliography: leaves 128-143. / xi, 143, [44] leaves, [36] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the development of serological and nucleic acid based diagnostic methods for pea-seed borne mosaic virus (PSbMV), the isolation of specific effects on infected pea plants, the collection and biological comparison of new PSbMV isolates from Pakistan, the cloning and sequencing of specific parts of the genome of selected isolates, nucleotide and amino acid sequence comparisons between selected isolates, and the development of a ribonuclease protection assay (RPA) for identifying genomic differences among the PSbMV isolates. It is the first comparison of a range of geographically different isolates of PSbMV on the basis of both biological and molecular properties. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1999