Expression of the outer capsid protein, VP2, from a full length cDNA clone of genome segment 2 of bluetongue serotype 1 from South Africa, using both Sp6 and vaccinia expression systems and a comparison of the nucleic acid sequence of this segment with those of other serotypes.
Genome segment 2 of bluetongue virus serotype 1 from South AfricA (BTV-1SA) was purified from a preparation of all ten dsRNA segments. This dsRNA was used as a template to make a full-length DNA copy of segment 2, which was then cloned into pUC19. The cDNA insert was transferred into a bacterial expression vector (pGEM; PROMEGA) and, by means of in vitro transcription and translation systems, used to synthesise a polypeptide of similar size to VP2 (as analyzed by PAGE). The cDNA insert was also transferred into a vaccinia virus vector using homologous recombination. The resulting recombinant virus when transfected into TK- cells produced a protein that co-migrated with VP2 of bluetongue virus. Immunoprecipitation of these polypeptides, synthesised by in vitro and in vivo techniques, using BTV-1SA antisera, confirmed that they were virus specific. Nucleotide sequence analysis of the cDNA demonstrated that genome segment 2 is 2940 base pairs in length. The positive sense (+ ve) RNA strand contains an open reading frame, coding for a polypeptide of 961 amino acids, which is flanked by 3' and 5' terminal non-coding regions of 37 and 17 nucleotides, respectively. Comparison with published data shows that genome segment 2 of BTV-1SA is identical in these characteristics to segment 2 of BTV-1 from Australia (BTV-1AUS) but differs from isolates of the five American serotypes of BTV (BTV-2, -10, -11, -13 and -17). However, there is a higher level of homology, in both the nucleotide and the amino acid sequence of genome segment 2 and protein VP2 respectively, between the two isolates of BTV-1 and the American isolate of BTV-2, than there is between BTV-2 and the other American serotypes. The significance of this similarity is discussed.
Wade-Evans
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AM
AFRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, U.K.
Mertens
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PP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics*; D002213:Capsid / Q000235:genetics* / Q000276:immunology; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D007120:Immunochemistry; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000235:genetics; D012087:Reoviridae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D012703:Serotyping; D014616:Vaccinia virus / Q000235:genetics
D036022:Capsid Proteins; D004279:DNA, Viral; D012330:RNA, Double-Stranded; C442520:VP2 protein, Bluetongue virus; D004247:DNA