Graduate Thesis Or Dissertation
 

Characterization of the infection cycle of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus in Lymantria dispar cells

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  • 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.
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