Analysis of West Nile virus neuroinvasion using in vitro blood-brain barrier models

利用体外血脑屏障模型分析西尼罗河病毒神经侵袭

基本信息

批准号：
18580302
负责人：
KIMURA Takashi
金额：
$ 2.57万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580302/
关键词：
virus central nervous sysytem

项目摘要

1. The pathway used by West Nile virus (WNV) to leave the bloodstream and invade the central nervous system is poorly understood. To investigate how WNV cross the blood-brain barrier (BBB), we used confluent human umbilical vein endothelial cells (HUVEC) cultures on transwell inserts as an in vitro BBB model.2. The structural protein genes (C-PrM-E region) of WNV 6-LP strain and Eg 101 strain were cloned into the pCMV expression vector. Sequential transfection of WNV sub-genomic replicon having an EGFP expression cassette and the vector that expressed the structural proteins led to the secretion of virus-like particles (VLPs). VLPs established a single-round of infection without production of progeny, and the physical structure of the VLPs was similar to that of infectious virions.3. In vitro BBB model was infected with VLPs having structural proteins of high vilulent 6LP strain (6LP-VLPs) or VLPs having structural proteins of low vilurent Eg 101 strain (Eg-VLPs). By 24 h after infection, 10% of 6LP-VLPs had crossed in vitro BBB, whereas no Eg-VLPs seemd to traverse. Thus, the ability of WNV to cross BBB may correlate at least in part with viral virulence. Exposure of HUVEC to 6LP strain did not alter the localization of tight junction protein ZO-1, indicating that the passage of WNV across the BBB is not caused by the disruption of tight junction. On the other hand, infection of HUVEC with 6LP strain was inhibited by Chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. These results suggest the possibility that WNV may cross BBB by transcytosis via a clathrin-mediated endocytic pathway.

1。西尼罗河病毒（WNV）用来离开血液并侵入中枢神经系统的途径知之甚少。为了研究WNV如何穿越血脑屏障（BBB），我们使用了汇合的人脐静脉内皮细胞（HUVEC）在Transwell插入物上作为体外BBB模型。2。 WNV 6-LP菌株和例如101菌株的结构蛋白基因（C-PRM-E区域）被克隆到PCMV表达载体中。具有EGFP表达盒的WNV亚基因组复制子的顺序转染和表达结构蛋白的载体导致病毒样颗粒（VLP）的分泌。 VLP建立了无后代产生的单轮感染，VLP的物理结构与感染性病毒体的物理结构相似。3。体外BBB模型被具有高Vilulent 6LP菌株（6LP-VLP）的结构蛋白（6LP-VLP）或具有低vilurent EG 101菌株（EG-VLP）的结构蛋白的VLP感染。感染后24小时，6LP-VLP中有10％在体外BBB越过，而EG-VLP似乎没有穿越。因此，WNV越过BBB的能力至少可能与病毒毒力相关。 HUVEC暴露于6LP菌株并没有改变紧密连接蛋白ZO-1的定位，这表明WNV通过BBB的通过并不是由于紧密连接的破坏引起的。另一方面，氯丙嗪在质膜上抑制了HUVEC，氯丙嗪的感染受到6LP菌株的抑制作用。这些结果表明，WNV可能通过跨细胞增多症通过网格蛋白介导的内吞途径穿越BBB。