A 3Cpro-dependent bioluminescence imaging assay for in vivo evaluation of anti-enterovirus 71 agents
A 3Cpro-dependent bioluminescence imaging assay for in vivo evaluation of anti-enterovirus 71 agents
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DOI：
10.1016/j.antiviral.2013.11.002
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发表时间：
2014-01-01
影响因子：
7.6
通讯作者：
Zhong, Zhaohua
中科院分区：
文献类型：
--
作者：
Guo, Zhiwei;Zhong, Xiaoyan;Zhong, Zhaohua
肠道病毒71型(EV71)是细小病毒科的一员，是人类手足口病的主要病原体之一。EV71型病毒主要感染儿童，并导致严重的神经系统并发症，甚至死亡。EV71感染的发病机制在很大程度上尚不清楚，到目前为止还没有临床批准的疫苗或有效的治疗方法。在这里，我们描述了一种新的检测EV71的生物发光成像方法。在该方法中，构建了一个表达融合蛋白AMN(Q/G)BC的载体，AMN(Q/G)BC是一种裂解萤火虫荧光素酶突变体，可被EV71蛋白酶3C(PRO)特异性切割。切割后，裂解的融合蛋白恢复荧光素酶活性。我们的试验证实AMN(Q/G)BC被3C(PRO)和EV71特异性切割，并将荧光素酶活性恢复到与3C(PRO)和病毒在细胞和小鼠中的剂量相对应的程度。用该方法评价丝裂原活化蛋白激酶(MAPK)抑制剂GW5074和U0126的抗EV71作用，以验证其在筛选抗EV71药物方面的应用。我们发现，AMN(Q/G)BC报告在体内外条件下有效地监测了GW5074和U0126对EV71感染的抑制作用。AMN(Q/G)BC报道的数据与Western blotting和组织病理学检查结果一致。综上所述，这种实时成像方法可以定量监测抗EV71药物的疗效，对于抗EV71药物的筛选和评价，特别是在体内条件下，具有重要的价值。(C)2013爱思唯尔B.V.保留所有权利。
Enterovirus 71 (EV71), a member of Picornaviridae, is one of the major pathogens of human hand, foot and mouth disease. EV71 mainly infects children and causes severe neurological complications and even death. The pathogenesis of EV71 infection is largely unknown, and no clinically approved vaccine or effective treatment is available to date. Here we described a novel bioluminescence imaging approach for EV71 detection. In this approach, a plasmid-based reporter was constructed to express the fusion protein AmN(Q/G)BC, a split firefly luciferase mutant, which can be specifically cleaved by EV71 protease 3C(pro). Upon cleavage, the splitting fusion protein restores luciferase activity. Our test confirmed that AmN(Q/G)BC was specifically cleaved by 3C(pro) and EV71 and restored the luciferase activity to a degree that corresponds to the 3C(pro) and virus doses in cells and mice. The anti-EV71 effect of GW5074 and U0126, two mitogen-activated protein kinase (MAPK) inhibitors, was evaluated using this approach to validate its application of screening anti-EV71 agents. We found that the AmN(Q/G)BC reporter efficiently monitored the inhibitory effect of GW5074 and U0126 on EV71 infection under in vitro and in vivo conditions. The data from AmN(Q/G)BC reporter were consistent with Western blotting and histopathology examination. Taken together, this real-time imaging approach can quantitatively monitor the efficacy of anti-EV71 agents and is valuable for anti-EV71 drug screening and evaluation, especially, under in vivo conditions. (C) 2013 Elsevier B.V. All rights reserved.