Purification, concentration, and inactivation of Venezuelan equine encephalitis virus

Venezuelan equine encephalitis (VEE) virus was purified and concentrated by chromatography of tissue culture supernatant fluids on diethylaminoethyl-cellulose columns. Stepwise gradient elution studies indicated a broad elution pattern for the virus, with recovery occurring from 0.05 to 0.7 m NaCl. Optical density, infectivity, hemagglutination (HA), and complement fixation (CF) assays indicated that complete recovery of input virus in highly purified form was possible. Single-step elution with 0.7 m tris(hydroxymethyl)aminomethane-succinate-salt buffer resulted in a virus volume decrease of 85% with a concomitant increase in infectivity and antigenicity. Recoveries consistently equaled or exceeded 100% of the input preparations. Additional purification of column-recovered virus was obtained by sedimentation of pooled virus eluates on 50% sucrose cushions. Exposure of borate saline and 0.5% histidine suspensions of purified VEE virus preparations to 6 � 10 6 r of gamma radiation resulted in a loss of infectivity for tissue culture and a loss of lethality for weanling and suckling mice. Inactivation was an exponential function of the dosage. In contrast to infectivity, antigencity (HA and CF) of both saline and histidine preparations was retained after irradiation with doses as high as 6 � 10 6 r. Purified and irradiated VEE virus preparations have been successfully used for routine serological tests and are being evaluated as vaccines.