A ribonucleic acid (RNA)-dependent RNA polymerase has been demonstrated in Kern Canyon virus (KCV) particles. The RNA product of the KCV polymerase hybridizes to KCV viral RNA. The properties of this viral enzyme have been characterized and compared with those of vesicular stomatitis virus (VSV). RNA polymerases from both viruses require similar conditions of temperature, pH, and detergent and magnesium concentrations for maximal synthesis of RNA. The RNA polymerase contained in the virion of KCV was more dependent on the presence of a sulfhydryl agent than was the VSV enzyme. Under optimal conditions, the specific activity of the VSV polymerase is about twenty-five times as great as that of KCV. for VSV were in the range of 2 X 109 plaque-forming 726 Vol. 7, No. 6

Ionizing radiation, f-propiolactone, and acetylethyleneimine were compared for their ability as virus-inactivating agents for the preparation of rabies vaccine. Each agent reduced viral infectivity exponentially; ionizing radiation also destroyed viral hemagglutinin. The vaccine prepared by ionizing radiation was equal or superior to that prepared by f3-propiolactone in its ability to protect mice from rabies infection. The acetylethyleneimine-treated vaccine was a less potent immunogen.

A bacterium isolated from soil was found to oxidize 2-methylalanine to 65% of the theoretical value by an adaptive enzyme system. Manometric experiments with whole cells indicated that acetone but not isopropylamine was an intermediate. Cellular extracts produced carbon dioxide from 2-methylalanine when pyruvate was added to the reaction mixture. Dialysis stimulated the 2-methylalanine metabolizing system, providing pyridoxal phosphate and pyruvate were supplied. A study of the stoichiometry of this pyridoxal phosphate-dependent reaction indicated that acetone and carbon dioxide were principal products. The formation of carbon dioxide from 2-methylalanine seemed dependent upon transfer of the amino group to pyridoxal phosphate. Other 2-methyl amino acids were found to be decarboxylated by this system, thus indicating a lack of absolute specificity.

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