Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid

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Abstract
Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization.