RESEARCH ON MECHANISNS OF HSV-1 LATENT INFECTION BY IN SITU HYBRIDIZATION

为了阐明单纯疱疹病毒（HSV-1）与中枢神经系统组织的相关性，我们采用免疫细胞化学和原位杂交技术检测了一例单纯疱疹病毒性脑炎患者脑组织切片的病毒抗原和RNA。在本研究中，采用Haase AT等人报道的杂交方案，对病毒RNA和抗原进行了少量修改。切片先免疫细胞染色，ABC法检测HSV-1抗原，然后与^ 3h标记的HSV-1 cDNA探针杂交，检测载玻片乙酰化后的RNA，以防止同位素对载玻片的非特异性结合。HSV-1感染的Vero细胞作为阳性对照显示抗原和RNA。然而，在未感染的Vero细胞中未观察到杂交信号和颜色沉积。在单纯性脑炎的冷冻脑组织切片中，病毒抗原（VA）阳性细胞…More s，主要位于细胞质中，含有棕色沉积物，广泛分布于部分灰质病变。VA阳性细胞形态学鉴定为肿胀的神经元、胶质细胞和内皮细胞。此外，部分VA阳性细胞在细胞核上也有杂交信号，即VA和RNA阳性细胞弥散分布在VA阳性细胞附近。此外，只有胞核上有大量银粒的RNA阳性细胞主要分布在VA阳性细胞附近和白质。在部分灰质感染HSV-1细胞的定量分析中，病毒抗原阳性细胞占总细胞的23.2%。VA细胞和RNA细胞及仅RNA细胞的检出率分别为13.4%和18.3%。此外，部分白质VA和RNA细胞及仅RNA细胞分别占9.3%和41.7%，未检出VA阳性细胞。结论：单纯疱疹病毒（HSV）感染由脑组织组成的带病毒细胞，并表现出病毒感染阶段，即；VA阳性细胞，VA和Rna阳性细胞和仅Rna阳性细胞。此外，可以认为这些不同阶段的感染细胞是根据不同种类的细胞、感染途径和宿主免疫反应等因素发展而成的。少

In order to elucidate the correlation between herpes simplex virus(HSV-1) and the central nervous system tissue, we performed the detection of viral antigens and RNA in the brain tissue sections from a patient with herpes simplex virus encephalitis using immunocytochemistry and in situ hybridization.In the present study the hybridization protocol reported by Haase AT et al. were applied for the detection of viral RNA and antigens with a few modifications.The sections were first immunocytocehmically stained to detect HSV-1 antigens by ABC method, and then hybridized with ^3H-labelled HSV-1 cDNA probe for the detection of RNA after the acetylation of sildes for the prevention of nonspecific bibdings of isotope to slides.HSV-1 infacted Vero cells as positive control demonstrated both antigens and RNA. However, no hybridization signals and color deposits were observed in uninfacted Vero cells.In frozen brain tissue sections of herpes simplex encephalitis the viral antigen(VA) positive cell … More s, containg brown-colored deposits located primarily in the cytoplasm, were widely distributed throughout the lesions of a part of gray matter. Such VA positive cells were morphologically identified to be swollen neurons, glial cells and endothelial cells. In addition, some of VA positive cells have also hybridization signals over the nuclei, namely VA and RNA positive cells which were diffusely located colse to VA positive cells. Moreover, only RNA postive cells with numerous silver grains over the nuclei were predominantly distributed at the neighborhood of VA positive cells and at the white matter.In quantitative analysis of HSV-1 infecgted cells of a part of gray matter, cells positive with viral antigen were counted 23.2% of total cells.Both VA and RNA cells and only RNA cells were detected 13.4% and 18.3% respectively. In addition, in a part of white matter VA and RNA cells and only RNA cells were found 9.3% and 41.7% although there were was not detected VA positive cells.We concluded that the herpes simplex virus (HSV) infects the virious kind of cells composed of brian tissues and shows the virious stages of infection, namely; VA positive cells, VA and Rna positive cells and only RNA positive cells. Moreover, it may be considered that these various stages of infacted cells are developed according to many factors such as different kind of cells, infactious routes and host immune responses. Less