Study on urea inactivation of Japanese encephalitis virus.

Effect of concentrated urea on JE virus was studied with a special view upon the structure of the virion. Urea concentration less than 2M acted reversibly to destroy the particles at 24 C in a wide range of pH between 7.2 and 10.0. At concentrations of urea higher than 3 M up to 8 M, the particles were disrupted irreversibly with a rapid loss of both infectivity and hemagglutinating activity. As for the action mechanism of 1 M urea on JE virus, it was demonstrated that the first rapid inactivation was completed within 10 minutes, then followed by the second step of slow inactivation. The latter was sensitive to increase of temperature in urea inactivation. Since the second step was diminished when 0.1 M 2-ME was added in the reaction mixture, this reaction might be a reversible one through free exchanging of the hydrogen bonds. Namely, 2-ME blocks free -SH groups which are functioning reversibly for reassociation. However, in the presence of 5 mM cystine-cysteine mixture, this inactivation was reversed apparently and the particle reassociation was proved by centrifugal analysis in sucrose gradient column. When 5 mM cystine-cysteine mixture was contained in a sucrose column, HA activity was recovered in the band of virion by possible reassociation of the smaller particles. From these results, we may conclude, as to the action of 1 M urea on JE virus, that the effect is partly reversible in denaturation of proteins and its structure.