Graduate Thesis Or Dissertation
 

A molecular study of viral proteins in the pathogenesis of infectious hematopoietic necrosis virus

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  • The role of viral proteins in the pathogenesis of infectious hematopoietic necrosis virus (IHNV) was studied at the molecular level. The expression of the viral genes at the protein and RNA level, and their cellular localization, were characterized to further our understanding of viral pathogenesis. The pathogenic effect of individual viral proteins was also investigated and a method for detecting viral RNA in infected fish tissues was developed. The polarity of transcription was confirmed in terms of the relative amounts of each viral protein. Also, cells treated with glycosylation inhibitors did not exhibit cytopathic effect, demonstrating that a functioning host glycosylation system is necessary for viral replication. These studies also revealed a previously undescribed non-glycosylated protein, S, which appeared to be virus-encoded. The expression of the nonvirion protein (NV), was also detected in infected kidney tissues. The location of M2 and NV in the cell was found to be the nucleus and cytoplasm. The expression of the NV gene was further analyzed at the level of transcription and the regulation signals for IHNV transcription were investigated. Unique transcriptional initiation and terminational signals for the fish lyssa-like rhabdoviruses were identified. The transcriptional initiation signal, 3'-CGUG-5', was distinctly different from that of the other rhabdoviruses, 3'-UUGU-5'. The role of the M2 and NV proteins in viral pathogenesis was investigated by transient expression of these proteins individually in cultured fish cells. The M2 protein alone resulted in inhibition of host-directed gene expression at the level of transcription and induction of nuclear fragmentation. The NV protein was not involved in the regulation of the host gene expression, but was involved in another type of cytopathic effect characterized as cell rounding. This is the first biological function attributed to the NV protein. A PCR method was developed for detecting IHNV N-specific RNA in formalin-fixed, paraffin-embedded fish tissues. The method is sensitive and specific. The technique is capable of detecting viral RNA in samples that have been remained at room temperature in 10% buffered formalin for over 2 years.
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