Formalin inactivation studies were carried out on an attenuated strain of Japanese B encephalitis (JBE) virus produced in hamster kidney cell (HKC) cultures. The rate of reaction was found to be roughly proportional to the concentration of formalin. At any formalin concentration the reaction was completed at 37°C in less than one-half the time needed at 30°C. The rate of inactivation was apparently not affected by raising the pH from 7.1 to 8.0, and was independent of whether fluid or total harvest was employed and of the initial virus titer. The course of inactivation deviated from that of a linear relationship between residual log-virus activity and time. The curve had two components: a faster rate for the first few hours, followed by a slower one which was linear at least until interception of the abscissa. A 1:4000 formalin concentration was selected for vaccine preparation as a result of a series of trials indicating several advantages over other concentrations. With Millipore filtration prior to inactivation, a minimal loss of infectivity of about 0.2 log was encountered when using a single membrane of 0.22 µ pore size. Various combinations were tested to find that leading to minimal loss.

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