Graduate Thesis Or Dissertation

 

Characterization of an inhibitor ("6S") of infectious pancreatic necrosis virus (IPNV) in normal rainbow trout serum (RTS) and its effects on the virus Public Deposited

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  • The characteristics of an inhibitor of infectious pancreatic necrosis virus (IPNV) found in normal rainbow trout serum (RTS) were studied. The serum inhibitor had a molecular weight of approximately 150 kDa and was dependent on divalent cations, either Ca²⁺ or Mg²⁺. It was stable at temperatures up to 50°C and at a pH range between 4-10. The inhibitor directly inactivated the virus and the inhibition level was dependent on cell densities and on the time at which virus was exposed to RTS. The level of virus inhibition by RTS was altered by the cell line in which virus was produced. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines. Most of the salmonid sera tested showed inhibition, while non-salmonid sera did not inhibit IPNV replication. Rainbow trout continuously showed a significant level of inhibition in their serum after 23 weeks post hatch. Three isolates of IPNV were passaged five times in RTG-2 cells with either MEM-10 or MEM-10 with 1% rainbow trout serum and virus from each passage were tested for RTS sensitivity in vitro and virulence in vivo. The mortality level in brook trout fry was highly variable during viral passages, ranging between 30-89%. The RTS sensitivity and virulence were changed during viral passages, and these changes were dependent on cell culture conditions and IPNV isolate used. It was found that an IPNV crayfish isolate passaged in RTG-2 cells with MEM-10 showed significantly increased RTS sensitivity. This was, however, not correlated with decreased virulence. All three isolates showed identical antigenicity patterns with a panel of 11 monoclonal antibodies, irrespective of viral passage conditions. Clones prepared from an IPNV-Jasper (Ja) population which had been twice passed through brook trout were heterogeneous with respect to RTS sensitivity, serotype, and cDNA sequences. Eight percent of clones (4/50) were very sensitive to RTS (Ja-S), as was the parent strain, and eighty four percent of clones (42/50) showed a mid-range of RTS sensitivity. The final eight percent of clones (4/50) were RTS resistant (Ja-R). Enzyme immunodot assay revealed that Ja-S clones and Ja-R clones differed by several epitopes. Ja-S and Ja-R had significant differences in their cDNA sequences for the capsid protein VP2. These two strains shared 80.7% and 86% identity in nucleic acid and in amino acid sequences, respectively.
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