Graduate Thesis Or Dissertation

 

Molecular study of the cytopathic mechanisms of infectious hematopoietic necrosis virus in vivo and in vitro Public Deposited

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  • Molecular biological approaches were used to study and interfere with the life cycle of infectious hematopoietic necrosis virus (IHNV). These included the control of IHN disease in rainbow trout by genetic immunization or interference in vitro by synthesis of sense and antisense expression of the viral nucleocapsid (N) gene, and in situ localization of the virus in infected fish. The design of DNA vaccines was based upon the fording that fish skeletal muscle cells could express transgenes delivered by direct injection using a needle and syringe. Optimum transgene expression occurred seven days post injection with 25 ug of plasmid DNA. The cytomegalovirus immediate early promoter enhancer was the strongest promoter tested. Transgene expression lasted for at least 115 days post injection. The DNA persisted as an unintegrated and non-replicated plasmid. Vaccination of fish with the IHNV glycoprotein (G) encoding plasmid, pCMV4-G, resulted in a protective immune response. Fish vaccinated with pCMV4-G and an IHNV N encoding plasmid, pCMV4-N, produced a stronger anti-G specific immune response than did fish vaccinated with pCMV4-G. However, the percent survival of fish vaccinated either in combination or with pCMV4-G alone was the same, 86%, still well above the 25-35% survival of fish vaccinated with pCMV4-N or mock vaccinated. A temporal state of intracellular immunity was induced in cells expressing the IHNV N gene in the sense or antisense orientation. The cells were completely resistant to IHNV 3 months after transfection. However, five months after transfection the antisense transfected cells were 99% less susceptible to IHNV plaque formation than control cells, the sense transfected cells were 33% less susceptible. The sites of IHNV infection during the height of a viral epizootic were determined by in situ hybridization using an oligonucleotide complementary to the IHNV N gene. A number of parameters were optimized, the most critical being the amount of probe used and the washing and hybridization stringency. The virus was found systemically. The most conspicuous site of IHNV infection was found in the hematopoietic tissues of the kidney.
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  • 1995
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