Venter E H, van der Lugt J J, Gerdes G H
Department of Infectious Diseases, Faculty of Veterinary Science, Onderstepoort, South Africa.
Onderstepoort J Vet Res. 1993 Mar;60(1):39-45.
Two radiolabelled complementary DNA (cDNA) probes (1663 bp and 200 bp respectively) were prepared from the genome segment that encodes the non-structural protein 1 (NS1) of bluetongue virus serotype 4 (BTV4). The probes were used to optimize the in situ hybridization (ISH) method on baby hamster kidney-21 (BHK-21) cells and to investigate the use of the technique as a diagnostic procedure. Cells were infected with BTV4 at a multiplicity of infection of 0.5 PFU/cell. An intense cytoplasmic hybridization signal could be detected from 3 hours post-infection onwards, reaching a peak at 17 hours. The ISH procedure has potential use as a diagnostic technique, but will probably find a wider application in pathogenesis studies. An in situ hybridization method was also developed for the detection of BTV RNA in the central nervous system of newborn mice after intracranial inoculation with BTV10. Viral RNA-positive cells were detected from Day 3 onwards, predominantly in areas where the virus caused necrotic encephalitis.
从编码蓝舌病病毒血清型4（BTV4）非结构蛋白1（NS1）的基因组片段制备了两种放射性标记的互补DNA（cDNA）探针（分别为1663 bp和200 bp）。这些探针用于优化在幼仓鼠肾-21（BHK-21）细胞上的原位杂交（ISH）方法，并研究该技术作为诊断程序的用途。细胞以0.5 PFU/细胞的感染复数感染BTV4。感染后3小时起可检测到强烈的细胞质杂交信号，在17小时达到峰值。ISH程序有作为诊断技术的潜在用途，但可能在发病机制研究中有更广泛的应用。还开发了一种原位杂交方法，用于检测新生小鼠经颅内接种BTV10后中枢神经系统中的BTV RNA。从第3天起检测到病毒RNA阳性细胞，主要在病毒引起坏死性脑炎的区域。
