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

1. 西尼罗河病毒（WNV）离开血液并侵入中枢神经系统的途径尚不清楚。为了研究西nv如何穿过血脑屏障（BBB），我们在transwell插入物上使用融合人脐静脉内皮细胞（HUVEC）培养物作为体外血脑屏障模型。将WNV 6-LP株和eg101株的结构蛋白基因（C-PrM-E区）克隆到pCMV表达载体中。序列转染具有EGFP表达盒的西尼罗河病毒亚基因组复制子和表达结构蛋白的载体导致病毒样颗粒（vlp）的分泌。VLPs建立了单轮感染而不产生子代，其物理结构与感染性病毒粒子相似。体外血脑屏障模型分别感染含有高致病性6LP株结构蛋白的VLPs （6LP-VLPs）或含有低致病性eg101株结构蛋白的VLPs （egg -VLPs）。感染24小时后，10%的6LP-VLPs穿过体外血脑屏障，而鸡蛋- vlps似乎没有穿过。因此，西尼罗河病毒穿越血脑屏障的能力可能至少部分与病毒毒力相关。HUVEC暴露于6LP菌株后，未改变紧密连接蛋白ZO-1的定位，表明西尼病毒通过血脑屏障不是由紧密连接的破坏引起的。另一方面，氯丙嗪可以抑制6LP菌株对HUVEC的感染，氯丙嗪是一种在质膜上形成网格蛋白包被坑的抑制剂。这些结果提示西尼罗河病毒可能通过网格蛋白介导的内吞途径通过胞吞作用穿过血脑屏障。

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.