Characterization of Bluetongue Virus Ribonucleic Acid

Abstract

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...

Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of Bluetongue virus with that of vaccinia virus: conservation of RNA polymerase genes from diverse species

“…terminal sequences of the BTV RNA segments (17) were identified in the DNA clones. Based on the data, the double-stranded Li RNA is estimated to have a mass of 2.7 x 106 Daltons in agreement with previous reports (18).…”

Recombinant virus vaccine for bluetongue disease in sheep

“…BTV challenge and clinical reaction index. All the sheep were challenged by subcutaneous injection with 1 ml of infective sheep blood containing virulent BTV-10 (South African strain) at day 75, i.e., 33 days after the final immunization of sheep. The clinical reactions were monitored from 3 to 14 days postchallenge.…”

“…In the United States, although five BTV serotypes have been identified (BTV-2, -10, -11, -13, and -17), a modified live vaccine is only available for The 10 segments of the double-stranded RNA genome of BTV are located in the core of the virus particle. This core contains two major (VP3 and VP7) and three minor protein species (VP1, VP4, and VP6) and is surrounded by an outer capsid consisting of two major proteins, VP2 and VP5 (9, 18,32,33). It has been demonstrated both in vivo (using intertypic reassortment viruses) (14) and in vitro (by translation of each RNA segment) (19) that BTV RNA segment 2 codes for VP2.…”

VP2‐segment Sequence Analysis of Some Isolates of Bluetongue Virus Recovered in the Mediterranean Basin During the 1998–2003 Outbreak

“…BTV is the prototype virus of the genus Orbivirus in the Reoviridae family (de Mattos et al, 1994a). The viral genome consists of 10 double-stranded RNA segments, which encode seven structural (VP1-VP7) and four non-structural (NS1, NS2, NS3 and NS3A) proteins (Roy, 1989;Verwoerd et al, 1970). The structural proteins, VP1, VP3, VP4, VP6 and VP7, represent the inner coat proteins and form the viral core (Huismans et al, 1987, Verwoerd et al, 1972.…”