Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1

W. C. Wilson From the US Department of Agriculture, Agricultural Research Service, Arthropod-borne Animal Diseases Research Laboratory, PO Box 3965, University Station, Laramie, WY 82071-3965 (Wilson), and University of Alabama, School of Public Health, Birmingham, AL 35294 (Fukusho, Roy).

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A. Fukusho From the US Department of Agriculture, Agricultural Research Service, Arthropod-borne Animal Diseases Research Laboratory, PO Box 3965, University Station, Laramie, WY 82071-3965 (Wilson), and University of Alabama, School of Public Health, Birmingham, AL 35294 (Fukusho, Roy).

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P. Roy From the US Department of Agriculture, Agricultural Research Service, Arthropod-borne Animal Diseases Research Laboratory, PO Box 3965, University Station, Laramie, WY 82071-3965 (Wilson), and University of Alabama, School of Public Health, Birmingham, AL 35294 (Fukusho, Roy).

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Summary

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated rna was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of rna segment 3 of EHDV-1 cross hybridized with the corresponding segment of ehdv serotype 2 by results of cDNA/RNA blot hybridization, but not with rna of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.

Summary

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated rna was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of rna segment 3 of EHDV-1 cross hybridized with the corresponding segment of ehdv serotype 2 by results of cDNA/RNA blot hybridization, but not with rna of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.

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