Molecular and biological characterization of deletion mutants of vaccinia virus.
Item
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Title
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Molecular and biological characterization of deletion mutants of vaccinia virus.
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Identifier
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AAI9000710
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identifier
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9000710
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Creator
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Lai, Alexander Chi-Keung.
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Contributor
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Adviser: Beatriz G.-T. Pogo
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Date
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1989
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology | Biology, Molecular | Health Sciences, Immunology
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Abstract
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Although smallpox has been eradicated, studies with vaccinia virus continue, due to the recent advance in vaccinia recombinants. Several deletion mutants of vaccinia virus were isolated from a persistently infected Friend erythroleukemia cell line after repeated plaque-purification. Two mutants, designated Z4 and Z19, were characterized. The deletion involves the left terminal fragments HindIII C and N, and calculated to be 22 kb (or 12% of the genome) for Z19. The deletions extended into the tandem repeats (TR) of the inverted terminal repeats (ITR). The difference in size of deletions between Z19 and Z4 is probably due to further deletion of sets of TR in Z19. Rearrangements in the right terminal fragment was also found. Phenotype of the deletion mutants is similar to the wild-type (IHD-W strain) when compared in vitro, but is different when compared in vivo. The deletion mutants are highly attenuated, as the LD{dollar}\sb{lcub}50{rcub}{dollar} of the mutants is at least 1000-fold higher than the wild-type. The mutants do not replicate in vivo. Southern blot hybridization of viral DNA with labeled cloned vaccinia growth factor (VGF) gene demonstrated that this gene is absent in the deletion mutants. The inability to replicate in vivo may be the basis for the less efficient induction of protective immunity by the mutants after 3 days or 4 weeks of primary immunization. After 7 and 10 days of immunization, however, 10{dollar}\sp2{dollar} pfu of Z19 elicited protective immunity. Specific cytotoxic T cells (T-CTL) were not induced by the mutants after 7 days of immunization, in contrast to the wild-type, although protective immunity was elicited. Induction of delayed-type hypersensitivity (DTH) was readily demonstrated with both the wild-type and the mutant, suggesting that T-DTH may be associated with protection. Another orthopoxvirus, Indiana virus, was found to have two copies of the VGF gene, one at each of the terminal fragments. It is more virulent than the wild-type IHD-W. Rescue of the deletion mutant Z19 using IHD-W wild-type Hindlll C fragment resulted in isolation of recombinant viruses with the VGF gene and partial recovery of virulence. The results obtained with Indiana and recombinants strongly indicated that the expression of the VGF gene is correlated with virulence. The significance of this study is that it provides information for the design of a safe vaccinia-based recombinant (more attenuated), and of a better protocol for immunization.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
