title: A 3C(pro)-dependent bioluminescence imaging assay for in vivo evaluation of anti-enterovirus 71 agents.
abstract: 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.
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhong
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Lexun
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Tianying
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhai
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Tong
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Yelu
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Pan
authors.affiliation: Department of Microbiology, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Yihong
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Si
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors.forename: Fengmin
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, Harbin Medical University, Harbin 150081, China. Electronic address: zhaowr@ems.hrbmu.edu.cn.
authors.forename: Wenran
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin 150081, China. Electronic address: zhongzh@hrbmu.edu.cn.
authors.forename: Zhaohua
authors.initials: Z
authors.lastname: Zhong
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification / Q000494:pharmacology*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D029821:Enterovirus A, Human / Q000187:drug effects*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008813:Mice, Inbred ICR; D061848:Optical Imaging / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D014764:Viral Proteins; D049409:Luciferases, Firefly; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
keywords: 3C protease; Enterovirus 71; In vivo imaging; Split luciferase