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/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

Biological, Physical, and Chemical Properties of Eastern Equine Encephalitis Virus I. Purification and Physical Properties

“…The density of the infectious virus particles increased to a value of 1.20 g/cc. A radioactivity peak (3H-uridine) was located at a density of 1.22 g/cc with a shoulder at 1.20 g/cc which confirms the results of Aaslestad et al (1). Tubes 7 through 21 of the CsCl gradient were pooled, dialyzed, and then centrifuged on a second 30 to 50%O sucrose gradient.…”

Inactivated Eastern Equine Encephalomyelitis Vaccines Prepared in Monolayer and Concentrated Suspension Chick Embryo Cultures

“…2). These results are in accord with those of Aalestad et al on the fractionation by CsCl of the Louisiana strain of EEE virus (1). Although there were differences in the density peaks of infectious virus and HA particles, there were significant titratable virus and HA activity over a broad range of fractions.…”

Purification and Physical Properties of Eastern Equine Encephalitis Virus

“…Hoffman, unpublisled data. 6 where N = Avogadro's number, D -diameter of the virus measured in cm, and p -the density of the virus. A basic difficulty with this formula is the assumption that the buoyant density can be substituted for the actual density.…”