The Baculoviridae is a family of DNA viruses which are obligate pathogens for a variety of insects. Baculovirus strains came to the attention of researchers because they provided a nonchemical alternative for the biological control of insect pests of agriculture and forestry (Ignoffo, 1968); however, recently they have been studied for their potential as vectors for the expression of foreign genes (Jasny, 1988). Baculoviruses are divided into three subgroups designated A, B, and C. Subgroup A, the most studied, consists of nuclear polyhedrosis viruses (NPVs) in which many virions are embedded in crystals called polyhedra. This group can be further subdivided into two morphological types (SNPV and MNPV). The SNPV feature single enveloped nucleocapsids which are occluded, whereas the MNPV contain one or more nucleocapsids within an envelope which is then occluded. Subgroup B, known as the granulosis viruses (GVs), occlude single virions in the crystalline matrix made of the protein granulin. Subgroup C, the nonoccluded baculoviruses (NOB), do not have an occlusion body surrounding the virions (for review, see Granados, 1980). After the initial infection, the virus progeny may exhibit one of two phenotypes. After encapsidation the virion may leave the nucleus and acquire an envelope by budding through the host plasma membrane. This budded form (BV) goes on to infect cells and spread the virus within the original host. As the infection proceeds with time the viral DNA can become encapsidated, enveloped and occluded within the nucleus. This polyhedra-derived virus (PDV) form spreads the infection from insect to insect. The following report is a study of the sequence of events that occur during the infection of Lymantria dispar cells in culture by the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV), each of which was examined at three multiplicities of infection (5, 10, and 100). During the time course of infection (0 to120 h), DNA was isolated from infected cell lysates and Southern blot anaylsis demonstrated that viral DNA is first observable at 12-18 h p.i. for this system. The total production of BV and its rate of production were measured by titration of infected cell supernatants and both results show the maximum increase of BV to be during the period of 24-36 h p.i. Light microscopy revealed the presence of polyhedra by 48 h p.i. Western blot analysis was used to examine the time course of the expression of the three viral-induced proteins (gp64, p39-capsid, and polyhedrin) each of which represents a temporal phase of baculovirus gene expression within the infection cycle. The m.o.i. appeared to have little effect on the timing of all of the events studied (DNA synthesis, BV production, PIB detection, and protein synthesis). However, the magnitude of such events as early DNA synthesis and gp64 expression, and the later levels of PIB production appeared to correspond to the m.o.i. / Graduation date: 1991

Thesis (Ph.D.) -- James Cook University, 2003. / Typescript (photocopy) Bibliography: leaves [149]-164.

Graduation date: 1996

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 227-245). Also available on the World Wide Web.

Regulation of expression of three genes in the polyhedron envelope protein (PEP) gene region of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was examined. These genes include open reading frame (ORF) 1 (encoding p21), ORF 2 (encoding gp16), and ORF 3 (encoding the polyhedron envelope protein). The effect of elimination of the late promoter elements of each ORF or both ORFs 1 and 2 on ORF 3 expression was examined by using an ORF 3 promoter-CAT gene fusion. The data indicated that the ORF 3 promoter was essential for the expression of the PEP. Destruction of ORF 1 caused no effect whereas destruction of ORF 2 promoter resulted in a 29% increase in CAT activity. To characterize the role of ORF 1 and 2 in the viral life cycle and the location of the proteins in virions and infected cells, antisera were produced against these proteins. The 21 kDa protein was present in both purified budded and occluded virions as demonstrated by Western blot analysis. Immunoelectron microscopy showed that the 21 kDa protein was a capsid-associated protein in both phenotypes. The ORF 2 gene encodes a 12 kDa protein that is N-glycosylated, migrates at a MW of 16 kDa, and is not present in budded or occluded virions. Immunoelectron microscopy indicated that gp16 is associated with lamellar-like membranous structures in close association with the nuclear membrane. It was also found associated with envelopes of virions that had budded from the nucleus into the cytoplasm. A gene that reportedly has a similar role to ORF 3 (polyhedron envelope protein) has been described in Autographa californica MNPV. This gene encodes a protein called the spheroidin-like protein (SLP) because of its sequence similarity to the spheroidin inclusion protein of the Choristoneura biennis entomopox virus. The gene was located, sequenced, transcriptionally mapped in OpMNPV and an antiserum was produced against a fusion protein containing most of the SLP ORF. Immunoelectron microscopy showed that the protein was concentrated in cytoplasmic inclusion bodies and was not associated with the polyhedron envelope structure in OpMNPV. It was found to be associated with polyhedra of AcMNPV, but no specific association with the polyhedron envelope was found. The role of the PEP and the p10 protein in polyhedron morphogenesis was examined using deletion mutants of OpMNPV and immunoelectron microscopy. The p10 deletion mutant produced polyhedra with patchy and poorly attached polyhedron envelopes, suggesting p10 has a direct or indirect role in the proper formation of the polyhedron envelope. The PEP deletion mutant showed that PEP was an essential component in the formation of the polyhedron envelope. The mutant with both p10 and PEP deleted had polyhedra that showed a distinct cubic morphology. These data suggest that these two proteins may affect polyhedra morphology. / Graduation date: 1993

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 134-148).

Efficacy of Baculovirus heliothis against larvae of the tobacco budworm was studied under laboratory, greenhouse, and field conditions. Dosage-mortality studies using a diet surface inoculation technique resulted in LC₅₀ values of less than 2 PIB/mm² for 1- to 5-day-old larvae. Onset of mortality was delayed in older larvae, and a greater quantity of inoculum was needed to produce the same mortality level as larvae matured. Length of the incubation period was shortened by increasing the dose. In laboratory and greenhouse studies, mortality of neonates was enhanced by the addition of commercial feeding stimulants. A cottonseed-base adjuvant was more effective than either virus alone or virus mixed with soybean flour. The value of the bait was particularly apparent when larvae were held for short durations on virus-treated terminals. Water extracts of fresh and dried garbanzo beans were shown to be highly attractive to tobacco budworm larvae, suggesting their potential for use as a feeding stimulant. Both bean treatments performed as well as the commercial cottonseed adjuvant. Virus-water extracts of garbanzo bean leaves and pods were no more effective in producing larval mortality than virus in water alone. Although addition of a feeding stimulant significantly extended activity of virus residues on cotton terminals bioassayed with H. virescens in the laboratory, the combination did not improve efficacy when larvae were allowed to feed on treated plants in the field. It may be that the effect of bait on young larvae was overridden by high temperatures or light intensities in the upper plant canopy. Time of application studies directed at the egg-stage showed that larvae are capable of ingesting lethal quantities of the pathogen while chewing out of treated eggs. Applications should coincide as closely as possible with egg hatch to maximize infection. Following hatching, there was a consistent decline in effectiveness as treatments were delayed. No significant effects on longevity or fecundity resulted from the feeding of virus to adults in a sucrose solution. Transovum transmission of virus to progeny was inefficient, regardless of the dose administered.

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Typescript (photocopy). / Digitized by Kansas Correctional Industries