STUDIES ON SOME CHEMICAL PROPERTIES OF ARBOR VIRUSES
II. PARTIAL PURIFICATION OF DENGUE FEVER AND SOME OTHER ARBOR VIRUSES
1962 Volume 12 Issue 6 Pages 249-254
Details
1962 Volume 12 Issue 6 Pages 249-254
In the first paper of this serial works, the author reported that crude Japanese B encephalitis virus preparations were successfully purified by techniques of column chromatography with ECTEOLA-Cellulose ion-exchanger or by fluorocarbon deproteinization.
In attempt to develope these findings on Japanese B encephalitis, the present paper describes that some other arbor viruses and poliovirus were also partially purified by cellulose chromatography or fluorocarbon deproteinization.
Results obtained are as follows:
1) The viruses used were dengue fever type 1, Mochizuki strain; yellow fever, strain 17 D; and Western equine encephalitis, Rockefeller Institute stock strain. The viruses were also grown in trypsinized hamster-kidney cell cultures. The concentration of each crude virus in infected fluid was about 105.0 (mouse intracerebral LD50 per 0.02ml) in the case of dengue fever, 104.0 in yellow fever, and 107.0 in Western equine encephalitis respectively.
Poliovirus, strain MEF-1, was used for comparison and grown in HeLa cell culture too. HeLa cells were grown in culture bottles with medium consisting of 2% yeast extract, 0.5% lactalbumin hydrolysate in Earle's balanced salt solution (YLA medium), and 20% bovine serum.
2) Culture fluid harvested from the infected cultures, showing characteristic cellular degenerization, was centrifuged at 3, 000r.p.m./min. for 10 minutes to remove crude debris masses. The supernatant fluid was loaded onto a column containing ECTEOLA-Cellulose and eluted with 0.07M phosphate buffer solution (pH 7.2). Some supernatant fluid was treated with fluorocarbon. In the case of yellow fever and Western equine encephalitis viruses, 100% of the original viruses was practically recovered and 90-93% of the nitrogen contained in the original virus materials was removed. Nitrogen contents of purified virus suspensions, as measured by the micro-Kjeldahl method, ranged mainly from 0.1 to 0.2mg/ml.
