Browsing by Subject "Host-virus relationships"
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Item Comparative replication of Autographa californica nuclear polyhedrosis virus in abortive and productive infections of insect cell lines(Texas Tech University, 1993-05) Demirbag, ZihniNuclear polyhedrosis viruses (NPVs) are enveloped, rod-shaped insect viruses containing a circular double-strand DNA genome (Family: Baculoviridae). NPV genes are expressed according to a temporally regulated program consisting of four phases: immediate early, delayed-early, late and very late. With the exception of the immediate-early genes, which do not require viral factors for expression, each phase is triggered by genes of a previous class. The NPVs have become important gene expression vectors and are being developed as biopesticides. The ideal NPV would have high virulence for a broad range of pests and yet be harmless for beneficial insects and nontarget organisms. However, before such strains can be developed by genetic engineering approaches, it is necessary to understand the molecular mechanisms of NPV specificity and virulence. Our laboratory has developed a tissue culture model system for the study of NPV host specificity. This model is based on earlier observations that Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) does not produce polyhedral inclusion bodies (PIBs) in Bombyx mori (BM) cells, but 90% of the cells contain viral antigen. This constitutes the nonpermissive or abortive component of our model. The permissive or productive component is AcMNPV-infected Spodopterafrugiperda (SF) cells. Previous studies in our laboratory have shown that AcMNPV initiates infection in BM cells and produces unique cytopathic effect (CPE). The CPE was characterized as large "sac-like bodies" at the cell periphery and elongated structures referred to as "protrusions." PIBs were not observed. SDS-PAGE analyses showed that some viral proteins are synthesized in infected BM cells. In addition, novel proteins, not present in the permissive (SF) cells, are also observed. In the present study, I examined additional aspects of die differential replication of AcMNPV in SF and BM cell lines. I designed this study to see if viral particles of any type are formed in BM cells and whether restriction of virus replication is due to a block at the transcription level. I also tested for replication of viral DNA. Electron microscopy data showed that both cell lines displayed nuclear hypertrophy and virogenic stroma upon infection; however, BM cells did not contain polyhedral inclusion bodies or virions. Nucleocapsids were detected in approximately one of every five cells and were defective. Viral DNA replicated in BM cells but more slowly and at a much lower level than in SF cells. Northem blot analysis showed that: (a) AcMNPV immediate-early genes (IE-1 and pe-38) were transcribed at normal levels at early times post infection in both cell lines; however, in BM cells they were transcribed at higher than expected levels at late times post infection; (b) a delayed-early gene (dnapol) and a late gene which codes for the major component of the nucleocapsid (ccp) were transcribed at relatively low levels in the abortive infection; and (c) a very late gene (polh), responsible for the matrix of PIBs, was transcribed in BM cells at barely detectable levels. The results strongly suggest that delayed-early, but not immediate-early, genes require host factor(s) for expression, I propose that virus replication in BM cells is primarily restricted between the immediate-early and delayed-early stages and that the block occurs at the transcriptional level; other aspects of sub-optimal gene expression and particle assembly in this abortive infection appear to be consequences of the primary block.Item Gene expression in Autographa californica nuclear polyhedrosis virus infections exhibiting host specific modulation of occlusion body formation(Texas Tech University, 1989-08) Reinisch, Alesia JNuclear polyhedrosis viruses (NPVs) are enveloped, rod-shaped insect viruses containing a circular DNA genome (family: Baculoviridae). These viruses, which are being developed as biopesticides, are transmitted via polyhedral inclusion bodies (PIBs) in which viruses are embedded. In order to genetically engineer safe viral pesticides, it is necessary to understand the molecular basis of NPV host specificity and virulence. This research deals with a host range model (System III) in which Autographa californica multicapsid NPV (AcMNPV) replicates permissively in Spodoptera frugiperda (SF) cells but semipermissively in Bombyx mori (BM) cells. Early in infection, both cell lines displayed similar nuclear hypertrophy, but subsequent stages of cytopathic effect (CPE) were very different. PIBs were produced in SF but not BM cells. Instead, BM cells exhibited a unique CPE characterized by large sac-like bodies at the cell periphery. Virus growth kinetics experiments showed that infectious progeny virus was not produced in BM cells. Because PIBs were not found in the BM infection, gel electrophoresis was performed to determine whether polyhedrin, the major constituent protein of the PIB, was synthesized. Polyhedrin was not produced. The synthesis of another major late viral protein known as plO was also blocked. Thirteen other infected cell-specific polypeptides (ICSPs) were not detected in the semipermissive infection. Most of these appeared to be late ICSPs. Six ICSPs were found only in infected BM cells. Eight ICSPs were produced in both SF and BM infections but at different rates in each cell line. Inhibition of host proteins synthesis occurred in SF cells, but there was no clear evidence for host inhibition in BM cells. System III demonstrates a strong host range restriction which is manifested late in the BM infection. Additional research on the detailed mechanisms of the restriction should lead to the identification of viral DNA sequences responsible for host specificity and pave the way for the engineering of safe pesticides.Item Studies with semi-permissive infections of nuclear polyhedrosis viruses: evidence for intracellular restriction of host-range(Texas Tech University, 1983-08) Carpenter, William MichaelNuclear polyhedrosis viruses (Family: Baculoviridae) show great promise in agricultural pest control applications. However, in order for these viruses to be used most effectively, basic questions regarding the molecular basis of their replication, evolution, and host-specificity must be addressed. This thesis is concerned with delineating mechanisms of nuclear polyhedrosis virus host-specificity. An in vitro model was used to compare the importance of membrane-level and intracellular host-range mechanisms for nuclear polyhedrosis viruses. This model was composed of two systems: Autographa califomica NPV (Acal NPV) , strain Gl (Gerwig, 1981), which at low multiplicity produces semi-permissive infections in Spodoptera frugiperda cells, formed system I of the model. System II was composed of semi-permissive, S_. frugiperda NPV (SF NPV) infections in Trichoplusia ni cells. Transfection studies with Acal NPV Gl (System 1) showed that naked DNA does not enhance this semi-permissive infection. In addition, electron microscopy revealed that 50 percent of these infections produced progeny virus, but not polyhedral inclusion bodies, indicating that there is a host-range block in this system between virion morphogenesis and PIB formation. Infection kinetic studies with system II revealed that progeny virus was produced, but the yield was 700-fold less than positive controls. However, electron microscopy showed that the progeny virus was not enveloped, and was found in only five percent of infected TN cells; the remaining 95 percent of the cells contained forms of cytopathic effect such as virogenic stroma, but no virions. This suggests a host-range block at, or prior to, virion morphogenesis. These data strongly indicate intracellular, and perhaps intranuclear sites of host-range restriction for the above model infection systems. Further experiments based on these data should eventually lead to a better understanding of the genetic and biochemical mechanisms of host-range systems for the NPV. Once these mechanisms are understood, highly effective and specific strains of these viruses can be manufactured for the purpose of controlling agriculturally important pests.Item Studies with Semi-Permissive Infections of Nuclear Polyhedrosis Viruses: Evidence for Intracellular Restriction of Host-Range(Texas Tech University, 1983-08) Carpenter, William MichaelNuclear polyhedrosis viruses (Family: Baculoviridae) show great promise in agricultural pest control applications. However, in order for these viruses to be used most effectively, basic questions regarding the molecular basis of their replication, evolution, and host-specificity must be addressed. This thesis is concerned with delineating mechanisms of nuclear polyhedrosis virus host-specificity. An 2 ^ vitro model was used to compare the importance of membrane-level and intracellular host-range mechanisms for nuclear polyhedrosis viruses. This model was composed of two systems: Autographa califomica NPV (Acal NPV) , strain Gl (Gerwig, 1981), which at low multiplicity produces semi-permissive infections in Spodoptera frugiperda cells, formed system I of the model. System II was composed of semi-permissive, infections in. frugiperda NPV (SF NPV) infections in Trichoplusia ni cells. Transfection studies with Acal NPV Gl (System 1) showed that naked DNA does not enhance this semi-permissive infection. In addition, electron microscopy revealed that 50 percent of these infections produced progeny virus, but not polyhedral inclusion bodies, indicating that there is a host-range block in this system between virion morphogenesis and PIE formation. Infection kinetic studies with system II revealed that progeny virus was produced, but the yield was 700-fold less than positive controls. However, electron microscopy showed that the progeny virus was not enveloped, and was found in only five percent of infected TN cells; the remaining 95 percent of the cells contained forms of cytopathic effect such as virogenic stroma, but no virions. This suggests a host-range block at, or prior to, virion morphogenesis. These data strongly indicate intracellular, and perhaps intranuclear sites of host-range restriction for the above model infection systems. Further experiments based on these data should eventually lead to a better understanding of the genetic and biochemical mechanisms of host-range systems for the NPV. Once these mechanisms are understood, highly effective and specific strains of these viruses can be manufactured for the purpose of controlling agriculturally important pests.