Hybridohistochemical analysis of latently infected virus

在本项目中，我们重点研究了一种鸡疱疹病毒-马立克氏病病毒(MDV)的潜伏感染。据报道，一些潜伏感染的病毒可能会导致肿瘤和怪物。我们用杂交组织化学方法观察了潜伏感染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。检测下限估计为相同范围的β-肌动蛋白丰度。用同样的方法，我们观察了马立克氏病病毒(MDV)ICP4同源基因的正反义链在感染MDV后3-4周的鸡体内的转录。用多聚甲醛固定石蜡包埋的毛囊上皮(FFE)和肾、肝、神经、脾的淋巴细胞与MDV ICP4的cRNA探针杂交。与淋巴细胞相比，MDV ICP4基因的正义链转录产物在FFE中大量检测到。在一些肾、肝、神经和脾淋巴样细胞中观察到反义转录信号。Northern印迹分析证实这些组织中存在转录本。Northern印迹分析也证实了其丰度和正反义比率。免疫印迹分析也证实了正义基因在FFE、肾脏和肝脏中的表达。因此，淋巴细胞的结果表明，在潜伏期，MDV ICP4的正义和反义转录本在肾、肝和脾的一些淋巴细胞中表达。MDV基因组拷贝数低，未检出DNA。这些数据证实了MD肿瘤细胞中MDV基因组整合到宿主基因组中并具有核小体结构的报道。

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.