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关系，应用免疫细胞化学和原位杂交技术对1例单纯疱疹病毒性脑炎患者的脑组织标本进行病毒抗原和RNA的检测。用免疫细胞化学ABC法检测HSV-1抗原，用~ 3 H标记的HSV-1cDNA探针杂交检测RNA，同时用HSV-1感染的Vero细胞作阳性对照。在单纯疱疹病毒性脑炎脑组织冰冻切片中，病毒抗原（VA）阳性细胞在Vero细胞中无杂交信号，也无着色。 关于我们 广泛分布于部分灰质的病灶内，主要位于胞浆内，内含棕色沉积物。这些VA阳性细胞经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阳性细胞还在细胞核上有杂交信号，即VA和RNA阳性细胞，它们弥散分布在VA阳性细胞附近。部分灰质HSV-1感染细胞的定量分析中，病毒抗原阳性细胞占总细胞数的23.2%，VA和RNA阳性细胞及仅RNA阳性细胞分别占13.4%和18.3%。另外，在部分白色组织中，VA和RNA阳性细胞及仅RNA阳性细胞的检出率分别为9.3%和41.7%，但未检出VA阳性细胞。此外，可以认为这些不同阶段的感染细胞是根据许多因素如不同类型的细胞、感染途径和宿主免疫应答而发育的。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