Density Gradient Centrifugation Studies on Rabies Vinis

NEURATH, A. ROBERT (The Wistar Institute, Philadelphia, Pa.), TADEUSZ J. WIKTOR, AND HILARY KoPROWSKI. Density gradient centrifugation studies on rabies virus. J. Bacteriol. 92:102-106. 1966.-Cesium chloride density gradient centrifugation of rabies virus revealed a heterogeneous population of infectious virus particles, the majority of which showed a density of 1.20 g/ml. From results obtained by rate zonal centrifugation in preformed sucrose gradients, it was possible to calculate a sedimentation coefficient of about 600S for rabies virus. Sedimentation coefficients of about 23S and 10S were calculated for two soluble rabies antigens present in infected tissue-culture fluids, and they showed a density of 1.26 g/ml in cesium chloride solutions. Rabies virus has been characterized as a lipid containing ribonucleic acid (RNA) virus (4, 7, 8) which may belong to the same morphological group as vesicular stomatitis virus, cocalvirus, Egtved virus, and sigmavirus of Drosophila (22). These correspond to the process sequence group 325' of Kilkson (9). It has been suggested that the rabies virus may be a part of the myxovirus group (1). Results contributing to the physical characterization of rabies virus, obtained by density gradient and rate zonal centrifugation techniques, are reported in this communication. MATERIALS AND METHODS Rabies virus. The Pitman Moore (PM) strain of fixed rabies virus received from the National Institutes of Health (NIH, Bethesda, Md.) was used throughout the experiments. The virus was grown in human diploid cell strain (HDCS) for 52 passages and then in hamster kidney fibroblast culture BHK21/C 13 for an additional three passages by a method described elsewhere (21). Infectious tissue-culture medium harvested on the 3rd day was used throughout the experiments. The titer of the virus was 3 X 105 LD50/0.03 ml in 5-week-old Swiss mice inoculated intracerebrally. In some experiments, the tissueculture medium was concentrated (see below) before density gradient centrifugation. Rabies virus antigen. The presence of rabies virus antigen was determined by a fluorometric method recently described (15). Fluorescein isothiocyanate 1 Present address: Wyeth Laboratories, Inc., Radnor, Pa. conjugated antirabies -y-globulin obtained from BBL was used. Cesium chloride equilibrium denisity gradient cetitrifugation. A 0.4-ml amount of virus suspen4ion was layered over 4 ml of a CsCl solution with a 4sity of about 1.22 g/ml adjusted to pH 7 by addition of 1.0 M phosphate buffer to a final concentration of 0.01 M. Centrifugation was performed in a Spinco SW 39 L rotor for 48 hr at 96,000 X g. After the bottom of the tube was punctured, samples were collected in test tubes. A fraction withdrawn from each sample was mixed with a 10-fold amount of 2% calf serum in phosphate-buffered saline and kept at -70 C until assayed for infectivity. The density of the samples was determined refractometrically (20). Rate zonal centrifugationi. A 0.4-ml amount of virus suspension was layered over 25 ml of a preformed sucrose gradient buffered at pH 7.0 with phosphate buffer to a final concentration of 0.01 M. It was then centrifuged in the Spinco SW 25.1 rotor. If the preparation of soluble antigen was centrifuged, 0.3 ml was layered over 4 ml of sucrose gradient solution and centrifuged in the Spinco SW 39 L rotor. Samples were withdrawn after centrifugation and titrated for infectivity and presence of soluble antigens. The concentration of sucrose in the samples was determined refractometrically, and the corresponding values of density and viscosity were found in the Hanidbook of Chemistry an7d Physics, 44th edition. The calculation of sedimentation coefficients (S201,) was calculated according to Martin and Ames (11), but without the use of an internal standard. The time of centrifugation was corrected for the time of acceleration and deceleration. Densities of 1.20 and 1.26 g/ml were used for the calculation of the values of sedimentation coefficients that corresponded to infectious virus and soluble antigens, respectively (see Results). 102 on N ovem er 3, 2017 by gest http/jb.asm .rg/ D ow nladed fom INFECTIOUS PARTICLES IN RABIES VIRUS Protein determination. The concentration of protein in the samples obtained after density gradient centrifugation was determined by the method of Lowry et al. (10).