Wilson W C, Fukusho A, Roy P
US Department of Agriculture, Agricultural Research Service, Laramie, WY 82071-3965.
Am J Vet Res. 1990 Jun;51(6):855-60.
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.
已制备出1型流行性出血性疾病病毒（EHDV-1）基因片段2和3的潜在诊断性互补DNA（cDNA）克隆。分离出EHDV-1的各个片段，用氢氧化甲基汞变性，然后进行聚腺苷酸化。将聚腺苷酸化的RNA进行逆转录并自杂交形成双链结构，修复不完全末端。然后使用互补加尾法将所得产物克隆到质粒载体pBR322中。分离出两个克隆，一个来自片段2（E1-2-10），另一个来自片段3（E1-3-16），进行菌落纯化，并通过cDNA/RNA印迹杂交和核酸内切酶限制分析进行鉴定。通过cDNA/RNA印迹杂交结果，EHDV-1 RNA片段3的cDNA克隆与2型EHDV的相应片段交叉杂交，但与美国分离的蓝舌病毒血清型的RNA不杂交。对17株EHDV野外毒株进行cDNA/RNA斑点印迹杂交分析后，发现片段2克隆具有血清型特异性，而片段3克隆具有血清群特异性。
