Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CsCl

Abstract

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm 3 . Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm 3 and a lesser amount banded at 1.22 to 1.23 g/cm 3 . Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either 32 PO 4 or 3 H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260 S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm 3 ) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm 3 ), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.