Graduate Thesis Or Dissertation
 

The Oregon sockeye salmon virus : A. Biophysical biochemical characteristics B. antigenic relationship to two other salmonid viruses

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  • The Oregon sockeye salmon virus (OSV) was isolated from diseased sockeye salmon (Oncorhynchus nerka) fingerlings in 1958 by J. L. Fryer. Experimentation performed prior to the research reported herein indicated that the OSV contained essential lipids, was 100 to 300 mμ in size, and possessed RNA (presumptively identified by 5-bromodeoxyuridine treatment of OSV-infected cell cultures). In the present investigation, OSV was propagated in one of two cell lines derived from the embryonic tissues of either sockeye or chinook (O. tshawytscha) salmon. Infectious cell culture medium was purified by differential centrifugation alone, or in combination with RNase and DNase treatment, and rate-zonal sucrose gradient centrifugation. OSV suspensions had a propensity for forming viral aggregates during differential centrifugation, presumably caused by pelleting virions in the presence of host cell debris and serum proteins. Partially purified virus suspensions were fractionated into four visible bands by rate-zonal or isopycnic sucrose gradient centrifugation. Most virus infectivity was detected in two closely associated bands in the middle of the gradient. Evidence suggested that the more dense, faster sedimenting band contained infectious virus and the other band was composed of incomplete, noninfectious virus particles. The sucrose density of the virus band was determined to be 1.16 g/cm³. The two other visible bands, one above and one below the presumably heterogeneous virus band, were considered to represent nonviral material complexed with varying amounts of virus. Large amounts of virus infectivity were lost either during sucrose gradient centrifugation or when sucrose concentrations were reduced in gradient fractions by dilution and/or dialysis. Purified suspensions of OSV were treated with hot perchloric acid in order to extract viral nucleic acid. RNA and DNA concentrations in the extracts were estimated with the orcinol and diphenylamine tests. Concentrations of RNA were at least 14 times higher than those of DNA. Viral nucleic acid was also extracted from purified ³²P-labeled OSV suspensions by the phenol method at 4°C. Spectral properties of the resulting nucleic acid solutions indicated that they contained relatively large amounts of protein. Viral nuclei acid formed a single band at densities of 1.58 to 1.59 g/cm³ when subjected to isopycnic cesium sulfate gradient centrifugation. Rate-zonal glycerol gradient centrifugation of ³²P-OSV nucleic acid resulted in the formation of a diffuse band of radioactivity with its peak at 26 S and a pronounced shoulder at 37 S. RNase treatment of aliquots from glycerol gradient fractions reduced all trichloroacetic acid-precipitable ³²P-radioactivity by 85 to 97%. Anion exchange chromatography of alkaline-hydrolyzed ³²P-viral nucleic acid was used to determine its base composition. Percentage base compositions were cytidylic acid, 25.8 ± 0.6%; adenylic acid, 23.0 ± 0.8%; uridylic acid, 27.7 ± 0.6%; and guanylic acid, 23.4 ± 0.4%. The fore-mentioned experimental data demonstrate that the OSV virion contains single-stranded RNA. Rabbit-immune sera were produced against OSV, infectious hematopoietic necrosis (IHN) virus, and Sacramento River chinook disease (SRCD) virus. The latter two viruses were isolated from diseased sockeye and chinook salmon, respectively. Differentially centrifuged virus suspensions, containing 5.0 x 10⁸ to 2.0 x 10⁹ TCID₅₀/ml, were injected undiluted or emulsified with Freund's adjuvant into rabbits. The antigenic relationship between OSV, IHN virus, and SRCD virus was investigated using cross plaque neutralization tests with each antiserum versus the three viruses. Fifty percent plaque neutralization end points determined in these tests indicated that all three viruses were antigenically related, with OSV and IHN virus being indistinguishable. Differentially centrifuged, glutaraldehyde-fixed and unfixed suspensions of OSV were stained with phosphotungstic acid and examined with an electron microscope. The most numerous type of particle in fixed preparations was bullet-shaped with average dimensions of 98 x 166 mμ.. However, the most abundant type of particle in unfixed suspensions consisted of two roughly spherical (80 mμ in diameter), closely associated particles. The discrepancy between the appearance of fixed and unfixed OSV suspensions was not experimentally resolved.
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