Graduate Thesis Or Dissertation
 

Characterization of the Oregon sockeye salmon virus

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  • Characterization of a virus requires that its physical, biochemical and biological properties be determined. In this study the Oregon sockeye salmon virus was characterized. It was isolated in the fall of 1958 from juvenile sockeye salmon, Oncorhynchus nerka, being reared at the Oregon Fish Commission's Willamette River Hatchery. When inoculated into tissue cultures of sockeye embryonic cells the virus produced a definite sequence of cytopathic changes. First there was a thickening of the nuclear membrane accompanied by alteration of the nucleoli. This was followed by changes in the nuclear chromatin and in some cases fragmentation of the nuclei. Finally the cells rounded up and detached from the glass surface. "In vivo" experiments carried out to determine the host range of this virus in salmonids indicated young kokanee salmon, Oncorhynchus nerka were susceptible while juvenile chinook salmon, Oncorhynchus tshawytscha, coho salmon, Oncorhynchus kisutch, and rainbow trout, Salmo gairdnerii were resistant. Histological examination of kokanee salmon infected with the virus indicated the kidney was the only organ showing any appreciable change. Primary cultures of chinook salmon, coho salmon and steelhead trout, Salmo gairdenerli gairdnerii embryonic cells did not become infected upon exposure to this virus. Cell cultures from these species after being maintained for several months in continuous culture were again exposed to the virus. Now the chinook salmon and steelhead trout cells were susceptible to the virus while the coho salmon cultures remained resistant. This was consistent with information of other workers who found that in some cases fish cells carried in cell cultures for long periods of time became susceptible to a wide variety of animal viruses. When the Oregon sockeye salmon virus was inoculated into monolayer cultures of sockeye embryonic cells, easily discernible plaques were formed within seven days. The plaque titration method of quantitating this virus was found to be more precise than the infectivity titration used in this study. It was not used routinely for virus quantitation however because of the large number of cells required. The data from plaque titrations and infectivity titrations made it possible to determine the number of plaque forming units (pfu) per ID₅₀. Results indicated one ID₅₀ was equal to 0.55 pfu. This value was in good agreement with the hypothetical value of 0.69 pfu for one ID₅₀. Attempts to measure active intracellular virus showed the amount to be negligible when compared to the concentration of extracellular virus. This suggested the virus was readily released from infected cells and might not be completely infectious until it passed through the cell membrane. Exposure to ether at a concentration of twenty per cent by volume completely inactivated the virus. This was an indication that the virus particle contained lipid materials which were essential for infection. There was also presumptive evidence that this was an RNA virus since replication was not inhibited by 5-bromodeoxyuridine (an inhibitor of DNA viruses). Experiments carried out at various temperatures showed the virus multiplied most rapidly between 13° C and 18° C. In this temperature range a maximum titer was reached within two days after inoculation. Temperatures outside the optimum range reduced the rate of replication and in some cases the final titer was at least one log lower than in the 13° to 18° range. At a temperature of 23° C no new virus was formed. This was rather surprising since the optimum temperature for cell growth has been reported as 23°C. Before the Oregon sockeye salmon virus could be studied with the electron microscope it was necessary to concentrate and partially purify the virus. Concentration was achieved by centrifugation at 20,000 rpm for one hour. The concentrated material was then placed on a sucrose density gradient which yielded a band of virus material after centrifuging at 23,000 rpm for one hour. This band material was observed with the electron microscope and particles which exhibited evidence of cubic symmetry were observed. Measurement of these particles indicated their size range was between 92 and 145 mμ in diameter. This value was in good agreement with the particle size of the virus as determined with Millipore filters (110 mμ, to 165 mg). Based on the information obtained during this study, the Oregon sockeye salmon virus appears to be closely related to the arbovirus group. This however is only a tentative placement and more information will be needed before the virus can be definitely assigned to a particular group of viruses.
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