Chuang Ching-Kai, Chiou Shyan-Song, Liang Li-Ching, Chen Wei-June
Department of Public Health and Parasitology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Am J Trop Med Hyg. 2003 Dec;69(6):648-51.
Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin-labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2-3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides.
日本脑炎（JE）是东南亚一种重要的蚊媒病毒性疾病。由于病毒血症短暂且滴度低，通常很难从外周血中分离出JE病毒。设计了一种原位逆转录聚合酶链反应（RT-PCR）方法，用于扩增外周血单个核细胞（PBMC）中JE病毒的基因（包膜）片段，而无需提取RNA。用JE病毒T1P1株（从台湾采集的骚扰阿蚊分离株）感染的幼仓鼠肾-21细胞用4%多聚甲醛固定，并用0.1% Triton X-100通透处理。然后使用地高辛标记的引物在微量管中进行RT-PCR。在小鼠静脉接种JE病毒后的第1天和第3天检测到病毒阳性的PBMC，但在第5天未检测到，这表明在小鼠血液中循环的可检测病毒仅存在2-3天。在检查小鼠脑组织时，直到接种后第3天才检测到病毒RNA。这意味着病毒从外周血迁移到中枢神经系统发生在接种后第3天或之后。该方法的独特之处在于反应可以在试管中进行；与传统的载玻片原位RT-PCR相比，这使其方便、准确且高效。
