An improved purification procedure yielded bluetongue virus free from any singlestranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 X 106 to 2.8 x 106 daltons, with a total molecular weight estimate of about 1.5 x 107 for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.A striking similarity has been found between the nucleic acid moieties of bluetongue virus (BTV) and reovirus, although the virions differ in size and morphology. Both genomes consist of double-stranded ribonucleic acid (RNA) which has been found, after isolation, to consist of a number of segments of reproducible size. In the case of reovirus, the presence of 10 doublestranded segments was demonstrated in fractionation experiments utilizing polyacrylamide gel electrophoresis (12,20), whereas electron microscopy studies using the Kleinschmidt technique indicated 9 to 11 fragments (15). In addition, a single-stranded, adenine-rich component has been found (1, 11).Previous studies on BTV-RNA indicated the presence of at least three size groups of segments, by the use of sucrose gradient sedimentation for fractionation (16).In the experiments reported here, the presence or absence of a single-stranded RNA component in BTV was investigated, and the double-stranded viral RNA was subjected to further purification and characterization. MATERIALS AND METHODSBuffers. The STE buffer consisted of 0.1 M NaCl, 0.005 M tris(hydroxymethyl)aminomethane (Tris), and 0.001 M ethylenediaminetetraacetic acid (EDTA), pH 7.4; the SSC buffer consisted of 0.15 M NaCl and 0.015 M sodium citrate, pH 7.2.Virus. BTV serotype 10 has been used throughout this work. Methods for the production of virus in BHK-21 cell cultures and for plaque titrations in Lcells have been described (4).Reovirus type 1 was obtained from N. F. Stanley, Melbourne, Australia; grown in BHK-21 cells, and purified according to the method of Bellamy et al.(1).Purification of BTV. The following modifications have been introduced into the procedure described previously (16). After extraction with Tween 80 and ether, sucrose was added to the aqueous phase containing the virus to a final concentration of about 4%, and the salt concentration was adjusted to 0.1 M. It was then layered over 2 ml of a 40% sucrose solution in STE, and the virus was pelleted through this layer for 2 hr at 102,000 X g. The pellets were dissolved in a small volume of 0.002 M Tris-hydrochloride at pH 8.0 and finally purified by centrifugation through a 10 to 30% sucrose gradient in the same buffer at 78,000 X g for 70 min. These modifications are based on the observation that purified BTV form aggregates in 0.1 M buffer, with disaggregation at a salt concentration of 0.002 M. Gradients were fractionated i...