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Hybridohistochemical analysis of latently infected virus

潜伏感染病毒的杂交组织化学分析

基本信息

批准号：
04454111
负责人：
SATO Fumiaki
金额：
$ 3.2万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

In this project, we focused on the latent infection of a gallid herpesvirus, Marek's disease virus (MDV). It was reported that some latently infected viruses may cause tumors and monsters. We observed the gene expression of the latently infected MDV using hybridohistochemistry. Prior to observation of the expression of a virus gene, we estimated the lower limit of the detection using a digoxigenin-labeled cRNA probe. We used mouse renin as a model. The lower limit of the detection was estimated to be the same range of abundance of beta-actin. Using the same method, we observed transcription of sense and antisense strands of Marek's disease virus (MDV) ICP4 homologue gene in MDV-infected chickens (3-4 weeks postinfection). A cRNA probe of MDV ICP4 was hybridized on paraformaldehyde-fixed and paraffin-embedded sections of feather follicle epithelium (FFE) and some lymphocytes in kidney, liver, nerve and spleen. Sense-strand transcripts of the MDV ICP4 gene were abundantly detected in the FFE compared with lymphocytes. Signals for antisense transcripts were seen in some kidney, liver, nerve and spleen lymphoid cells. Northern blot analysis confirmed the existence of transcripts in these tissues. Abundance and the sense/antisense ratio were also confirmed by Northern blot analysis. Immunoblot analysis also confirmed the expression of sense transcripts in FFE,kidney and liver. Thus, the results from lymphocytes suggested that sense and anti-sense transcripts of MDV ICP4 were expressed in some lymphocytes in the kidney, liver and spleen during the latent period. DNA of MDV genome was not detected because of its low copy umber. These data confirmed the reports on MD tumor cells that MDV genome was integrated in host genome and had a nucleosomal structure.

在本项目中，我们重点研究了一种鸡疱疹病毒马立克病病毒（MDV）的潜伏感染。据报道，一些潜伏感染的病毒可能导致肿瘤和怪物。采用杂交组织化学方法观察潜伏感染的MDV的基因表达。在观察病毒基因的表达之前，我们使用地高辛标记的cRNA探针估计了检测的下限。我们以小鼠肾素为模型。检测的下限估计为β -肌动蛋白丰度的相同范围。用同样的方法观察了马立克病病毒（MDV） ICP4同源基因的正义链和反义链在MDV感染鸡（感染后3-4周）的转录。将MDV ICP4的cRNA探针在多聚甲醛固定和石蜡包埋的肾脏、肝脏、神经和脾脏的羽毛卵泡上皮（FFE）和部分淋巴细胞切片上杂交。与淋巴细胞相比，在FFE中大量检测到MDV ICP4基因的义链转录本。在一些肾、肝、神经和脾淋巴样细胞中可见反义转录物信号。Northern blot分析证实了这些组织中转录本的存在。Northern blot分析也证实了丰度和正义/反义比。免疫印迹分析也证实了FFE、肾脏和肝脏中有sense转录本的表达。因此，淋巴细胞的结果表明，MDV ICP4的正义和反义转录本在潜伏期间在肾、肝和脾的一些淋巴细胞中表达。由于其拷贝数较低，未检测到MDV基因组DNA。这些数据证实了MDV基因组整合到宿主基因组中并具有核小体结构的报道。