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/cm3. Infectious Many laboratories have shown that considerable density heterogeneity exists in purified virus preparations. This is easily understood when the particles are deficient or devoid of nucleic acid or are mixed with soluble viral antigens. However, infectious virus of significantly different densities has also been observed, particularly among some of the enveloped viruses such as the arboviruses and the myxoviruses. Two representative arboviruses, Sindbis (4, 7) and Dengue-2 (10), have been characterized with respect to their buoyant densities in CsCl. In each case, considerable density heterogeneity was apparent, and, in some cases, virus specific hemagglutinins and complement-fixing antigens could be resolved from the major band of infectious virus. Sindbis and Semliki Forest virus hemagglutinins are considered to be identical with the virion, which is located in the envelope of the virus (6,8).In this report, we have determined that the buoyant density of the group A arbovirus, Eastern equine encephalitis (EEE), is 1.20 g/cm3 in CsCl. In addition, two other bands of viral material were observed; a light hemagglutinin which had a buoyant density of 1.18 g/cm3 and 1 Present address: Department of Microbiology, University of Georgia, Athens 30601.an infectious virus of a higher density (1.22 to 1.23 g/cm3). These two bands appear to be the salt-induced breakdown products of the EEE virion. MATERIALS AND METHODSVirus anid cell culture. The Louisiana strain of EEE virus, the origin and properties of which were reported by Brown (2), was used in all experiments. The virus was propagated on chick embryo (CE) monolayers and was assayed by the plaque technique described by Zebovitz and Brown (12). Virus was harvested 16 to 20 hr postinfection; titers averaged 4 X 108 to 8 X 108 plaque-forming units (PFU)/ml. Density gradient cenitrifugation. Before density gradient centrifugation, EEE virus was partially purified from 400 to 600 ml of tissue culture fluid that had been decanted from infected cells by adsorption onto AlPO4 gel (9). The virus was eluted from the gel in 0.3 M phosphate buffer (pH 8.0) and was concentrated by centrifugation at 65,000 X g for 60 min. The resulting pellet was allowed to resuspend overnight in borate-saline buffer (pH 9.0) containing 0.1% bovine serum albumin (3). Typically, only 15 to 20% of the total PFU were recovered; however, an overall purification of about 500-fold was obtained (2.0 X 1010 PFU/mg of protein). CsCl was added to 4.5 ml of the partially purified EEE virus to a mean density of 1.21 g/cm3. The virus particles were unstable in the salt when bovine serum 972

A new purification procedure was adopted for Eastern equine encephalitis virus which does not subject the virus to pelleting at any stage. Three- to 4-liter volumes were passed through a diethylaminoethyl cellulose column. The virus-containing fractions were banded on a sucrose cushion and finally concentrated in an isopycnic band in a linear sucrose gradient. This method reduced the volume 1,000-fold with a concomitant increase in viral titer, i.e., better than 90% recovery. Numerous criteria have been used to establish that this viral preparation was essentially free from cellular debris and nonviral material. Physical studies on this purified viral product were initiated. The sedimentation coefficient as determined by band sedimentation was 240 S , the buoyant density in sucrose was 1.18 g/cc, and the diameter of the virus was 54 nm. From the diameter and the buoyant density it was possible to calculate the molecular weight of a spherical particle. In this case, the calculated molecular weight for Eastern equine encephalitis virus was 58 × 10 6 daltons.

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.

No abstract

No abstract