Cloning of the bluetongue virus L3 gene

Abstract

The genes of the bluetongue virus (BTV) serotype 17 have been cloned into pBR322 by tailing both strands of the double-stranded RNA with polyadenylic acid, transcribing them with reverse transcriptase with an oligodeoxythymidylic acid primer, hybridizing the cDNA products, and completing them into duplex structures with the Klenow fragment of Escherichia coli DNA polymerase. After cloning the double-stranded cDNA molecules into pBR322, the complete sequence of the cloned L3 gene was determined. The clone is 2,772 nucleotides long (1.78 X 10(6) daltons), excluding the 3' polyadenylic acid sequence, and has an open reading frame which codes for a protein of some 901 amino acids (103,412 daltons). This clone can hybridize L3 RNA segments of three other U.S. BTV serotypes, BTV-10, -11, and -13 in addition to -17 but not the equivalent RNA segment of epizootic hemorrhagic disease virus of deer, an orbivirus related to BTV.