Establishment of electron microscopic in situ hybridization for viral nucleic acids

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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.