Establishment of electron microscopic in situ hybridization for viral nucleic acids

近年来分子生物学的研究进展已经阐明了新病毒的核酸和蛋白质，但其结构仍不清楚。形态结构对了解病毒的基本性质非常重要。为了阐明新发现病毒的结构，近年来我们在以下项目中开发了一种电子显微镜原位杂交（EM-ISH）技术来鉴定病毒核酸。为了建立EM-ISH，我们以EBV BamHl-W为探针，观察EBV感染细胞。在EBV低温包埋切片中建立了EM-ISH的条件和检测系统，并在细胞中发现了病毒前体。低温包埋切片经免疫标记后，用钌红和OsO_4溶液染色获得高对比度图像。用em - ish方法在HIV颗粒核心处发现了核酸。在感染细胞中发现腺病毒的准晶阵列可作为标准样品，为EM-ISH.5的基础条件和检测系统的开发奠定基础。EM-ISH可以检测到每1个或5个腺病毒DNA拷贝的一个标记，并使用PCR制作高度生物素标记的探针。在此过程中，艾滋病病毒的有效标记得到了改进。用免疫电镜观察了博纳病病毒（BDV）的结构和形态发生。EM-ISH正在对BDV进行进一步研究。

The nucleic acids and proteins of new viruses have been elucidated by recent advance of molecular biology, but their structures are still unclear. The morphological structure is very important to know the basic natures of the viruses. To elucidate the structure of the newly found viruses, we have been developing an electron microscopic in situ hybridization (EM-ISH) technique for identifying viral nucleic acids in the following items these years.1.To establish EM-ISH, we observed Epstein-Barr virus (EBV)-infected cells by using EBV BamHl-W as a probe. The conditions and detection system for EM-ISH in EBV were established in lowtemperature embedded sections, and found to be viral precursors in the cells.2.We obtained high contrast images by staining with ruthenium red and OsO_4 solution afterimmunolabeling on the low-temperature embedded sections.3.The nucleic acids in the core of HIV particles were found by EM-ISH.4.The paracrystalline arrays of adenovirus in the infected cells were found to be a standard sample todevelop the fundamental conditions and detection systems for EM-ISH.5.One labeling per one or a fexv copies of adenovirus DNA can be detected by EM-ISH with using PCR to make highly biotin-labeled probes. The efficient labeling for HIV has been improved on the way.6.The structure and morphogenesis of Borna disease virus (BDV) have been found by immunoelectron microscopy. Further studies on BDV are being carried out by EM-ISH.