Replication

Difficulties in achieving growth of GVs in cell culture have prevented the detailed studies of replication that have been possible with some NPVs. As a result, our understanding of GV replication is based primarily on in vivo studies using electron microscopy. Although there appear to be differences in detail between the various in vivo GV studies, depending on the virus, host and tissue, the overall cytopathology is similar. The infection process initiates when an insect consumes food contaminated with GV granules. The alkaline pH of the lepidopteran midgut promotes the dissociation of the occlusion bodies, with the consequent release of the virus particles into the lumen of the midgut. For many GVs, enhancin protein associated with the granules is believed to catalyze the partial disruption of the peritrophic membrane lining the midgut. This allows the virus particles easier access to the midgut epithelial cells. Virus entry is thought to be by fusion between the viral membrane and the midgut microvilli. The nucleocapsids migrate to the nuclear membrane. There is some evidence to suggest that the nucleo-capsid uncoats at the nuclear pore (TnGV and Estigmene acrea (saltmarsh caterpillar) GV) and/or in the nucleus (Scotogramma trifollii (clover cutworm) GV). Once in the nucleus, there are major differences between the cytopathology of GV and NPV infections. Unlike NPVs, in GV infections the nuclear membrane breaks down before occlusion body formation. There appear to be two phases of nucleocapsid production, one prior to the fragmentation of the nuclear membrane and one postfragmen-tation. The early stages of GV infection resemble the prophase stage of mitosis. There is nuclear 'clearing' as a result of the chromatin and nucleolar complex becoming marginated. The granular and fibrillar components of the nucleolus separate and the nuclear pores become larger. Empty capsids form in the clear area of the nucleus and fill with nucleoprotein; the nucleocapsids appear scattered separately throughout the nucleus (Fig. 3A). The nuclear membrane then fragments, liberating the nucleocapsids into the cytoplasm. The nucleocapsids migrate to the periphery of the cell and align themselves with their conical end perpendicular to the thickened area of the plasma membrane, where budding occurs. While budding is occurring there is further proliferation of nucleocapsids at the virogenic stroma in the portion of the cell once occupied by the nucleus. The progeny nucleo-

Figure 3 (A) Early stages of GV replication. Nucleocapsids throughout the nucleus prior to disintegration of the nuclear membrane. The nuclear membrane is indicated by an arrowhead. Bar = 1 jim. (Reprinted with permission from Winstanley D and Crook NE (1993) J. Gen. Virol. 74: 1599-1609.) (B) Occlusion from one end of the virion. Bar = 100nm. (C) In vitro replication of CpGV. C. pomonella cell late in infection containing occluded virus particles (capsules). Bar = 1 jim.

Figure 3 (A) Early stages of GV replication. Nucleocapsids throughout the nucleus prior to disintegration of the nuclear membrane. The nuclear membrane is indicated by an arrowhead. Bar = 1 jim. (Reprinted with permission from Winstanley D and Crook NE (1993) J. Gen. Virol. 74: 1599-1609.) (B) Occlusion from one end of the virion. Bar = 100nm. (C) In vitro replication of CpGV. C. pomonella cell late in infection containing occluded virus particles (capsules). Bar = 1 jim.

capsids migrate to the periphery of the cell, where they either bud or accumulate in areas rich in vesicular membranes, where envelopment occurs. The nucleocapsids align perpendicularly on membrane structures, where they become enveloped. The deposition of granulin on the virion envelope initiates occlusion, which usually progresses by the crystallization of granulin from one end of the rod-shaped virion (Fig. 3B). During occlusion all stages in viral replication are evident in the cell. The capsules form in the periphery of the cell. In cell culture the cells generally round up and fill with occlusion bodies (Fig. 3C); in vivo cells detach from the basal laminar and from adjacent cells. In infected insects membrane-bound vesicles enclosing occlusion bodies may be observed in some cells, historically named 'boules hyaline'. These may be hemocytes which phagocytose capsules released into the hemolymph. This phenomenon has also been observed in a C. pomonella cell line. Capsules are released from the cells by the rupture of the plasma membrane and accumulate in the center of the fat body, the main site of occlusion body production, and in the hemolymph. As a result, just before death, the infected larva becomes milky/ paler due to the accumulation of capsules. After death the cadaver will melanize and either liquefy or dessicate, depending on the type of GV infection.

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