Concentration and purification studies of western equine encephalitis virus.

Attempts to purify and concentrate Western equine encephalitis (WEE) virus have been hindered since it is relatively sensitive to pH changes and to many organic solvents. The virus was stable between pH 6.0 and 8.0 and rapid inactivation occurred outside this range. Complete inactivation of infectivity resulted when virus was treated with ether, chloroform, methanol, and ethanol. Carbon tetrachloride and trifluorotrichloroethane extraction removed 37% and 43% total nitrogen from the virus suspension while 70-90% of the initial virus was recovered. A WEE virus preparation with a “purity†of approximately 94% relative to decrease in measureable nitrogen was obtained utilizing protamine sulfate precipitation in conjunction with low and high speed centrifugation. A difference in the response of WEE virus mutants SP-6 and LP-7 to protamine sulfate was observed. LP-7 virus infectivity was reduced by 55% and, in contrast, the infectivity of the SP-6 virus was not decreased under the same conditions. A partial and rapid purification of crude WEE virus suspensions was accomplished using G-25, G-50, and G-200 Sephadex. Between 93 and 99% of the virus activity could be recovered. Gel filtration was especially useful for the separation of virus from smaller amino nitrogen containing contaminants. Elution chromatography on calcium phosphate afforded another method for the purification of this virus. Many of the contaminating components present in crude virus suspension failed to absorb a 0.001 M phosphate concentrations and were separated from the infective virus. The virus was eluted from the adsorbent using increased phosphate concentrations. WEE virus was concentrated by precipitation with 40% ammonium sulfate at pH 7.0. Sixty-two per cent of the virus activity was recoverable from the resulting pellet. An aqueous two-phase system composed of sodium dextran sulfate-polyethylene glycol (NaDS-PEG) was used to concentrate large volumes of virus. The phase system was constructed so that a concentration of 100 times was accomplished in one step. Yields of virus activity of 50-95% were routinely recovered from the NaDS-rich bottom phase. The majority of the NaDS and approximately 90% of the total nitrogen were precipitated from the bottom phase with KC1 while most of the virus activity remained in the supernatant. Gel filtration of virus prepared by this procedure resulted in considerable inactivation. Fifty-four percent of the virus introduced into a G-50 Sephadex column was recovered while only 17 to 20% was recoverable after filtration through G-200 Sephadex columns. Considerable purification of WEE virus was effected by rate zonal centrifugation in a sucrose gradient. A virus band was collected which contained 69% of the virus applied to the gradient. In cesium chloride density gradients WEE virus populations appeared to be comprised of particles with different densities. This heterogeneity did not appear to be an artifact of the techniques employed. Studies of two plaque mutant viruses failed to indicate differences in buoyant density. Peak infectivity of both viruses occurred in fractions from the gradient which corresponded to density of 1.195 g cm-3. Studies of virus stock propagated in a variety of host cell types suggested that virus density differed according to the cell type used for replication. Infectivity peak densities of virus propagated in primary chick embryo, monkey and hamster kidney cell occurred at 1.195 g of 1.22 and increased to 1.235 g cm-3 upon second passage.