A 3Cpro-dependent bioluminescence imaging assay for in vivo evaluation of anti-enterovirus 71 agents

• A 3Cpro-dependent split luciferase imaging assay was established for EV71 detection.
• This real-time imaging assay can specifically and quantitatively monitor EV71 infection.
• This approach could efficiently evaluate the efficacy of anti-EV71 agents in vitro and in vivo.
• This imaging approach is valuable for anti-EV71 drug screening and evaluation.

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 3Cpro. Upon cleavage, the splitting fusion protein restores luciferase activity. Our test confirmed that AmN(Q/G)BC was specifically cleaved by 3Cpro and EV71 and restored the luciferase activity to a degree that corresponds to the 3Cpro 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.