Congenital lymphocytic choriomeningitis virus infection:: An underdiagnosed disease
Congenital lymphocytic choriomeningitis virus infection:: An underdiagnosed disease
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DOI：
10.1097/00006454-199907000-00020
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发表时间：
1999-07-01
影响因子：
3.6
通讯作者：
Eggers, M
中科院分区：
文献类型：
--
作者：
Enders, G;Varho-Göbel, M;Eggers, M
方法。 LCMV 感染的血清学研究是通过间接免疫荧光在载玻片上进行的，其中单层 D6-底特律细胞系持续感染 LCMV，或在接种 10−4 LCMV 阿姆斯特朗株悬浮液 48 小时后单层 Vero 细胞。包括合适的对照。两种细胞系都给出了相似的结果；然而，后一种方法似乎对 IgM 检测更敏感。只有显示受感染细胞细胞质典型颗粒荧光的血清才被视为阳性。后续血清的重复测试再现了之前的 LCMV 特异性滴度，并且仅在±一个稀释步骤的范围内变化。对于所有病例，血清学和病毒学检查均排除了弓形虫、风疹病毒、巨细胞病毒、单纯疱疹病毒、细小病毒 B19 和肠道病毒感染。用于检测 LCMV-RNA 的内部逆转录酶巢式 PCR 方案于 1996 年底建立，仅用于少数储存的血清和脑脊液 (CSF) 样本。羊水和胎儿血液样本无法用于 LCMV-PCR 检测（病例 6）。通过三步免疫酶法对石蜡切片上的 LCMV 抗原进行免疫染色。脱蜡切片在 4℃下用 1/4000 稀释的抗 LCMV 多克隆兔抗血清覆盖过夜。结合的兔抗体用碱性磷酸酶标记的抗兔抗体染色。萘酚 AS-BI 磷酸盐与六氮化新品红（Merck，达姆施塔特，德国）的组合揭示了酶活性。使用天然兔血清作为对照。
Methods. Serologic investigations for LCMV infection were performed by indirect immunofluorescence on slides with monolayers of a D6-Detroit cell line persistently infected with LCMV or with monolayers of Vero cells after 48 h of inoculation with a 10− 4 suspension of LCMV strain Armstrong. Suitable controls were included. Both cell lines gave similar results; however, the latter method seemed to be more sensitive for IgM detection. Only sera showing the typical granular fluorescence of the cytoplasm of the infected cells were regarded as positive. Repeated testing of follow-up sera reproduced the previous LCMV-specific titers and varied only at the range of±one dilution step. For all cases infections by Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, parvovirus B19 and enterovirus infections were excluded by serologic and virologic investigations. An in-house reverse transcriptase-nested PCR protocol for detection of LCMV-RNA was established at the end of 1996 and was used only in few stored serum and cerebrospinal fluid (CSF) samples. Amniotic fluid and fetal blood samples were unavailable for investigation by LCMV-PCR (Case 6). LCMV antigen was immunostained on paraffin sections by a triple step immunoenzymatic method. The deparaffinized sections were overlaid overnight at 4 C with 1/4000 dilution of a polyclonal rabbit antiserum against LCMV. Bound rabbit antibodies were stained with alkaline phosphatase-labeled anti-rabbit antibodies. Enzyme activity was revealed with naphthol AS-BI phosphate in combination with hexazotized new fuchsin (Merck, Darmstadt, Germany). Naive rabbit serum was used as a control.