Quantitatively Monitor Enterovirus 71 Infection Through 3Cpro-dependent Real-time Bioluminescence Imaging

Objective: Enterovirus 71?EV71?,a member of Picornavirida,is one of the major pathogens of human hand,foot and mouth disease.EV71 mainly infects children causing severe neurological complications and even death.The pathogenesis of EV71 infection is largely unknown,and no clinically approved vaccine or effective treatment is available so far.Here we described a novel approach to determine EV71 infection by bioluminescence imaging assay.Methods: In this approach,a plasmid-based reporter was constructed to express fusion protein Am NLuc?Q/G?BCLuc which was consisted of a split luciferase and the cleavage sites of the protease 3C^pro of EV71.The cleavage of the fusion protein by 3C^pro would restore the luciferase activity.Results: Our results demonstrated that the Am NLuc?Q/G?BCLuc reporter could be specifically cleaved by 3C^pro and restored the luciferase activity,thus,the EV71 infection in cells and mice was able to quantitatively monitor by luciferase bioluminescence imaging in real-time manner.By this approach,the effect of Raf-1 inhibitor GW5074 and MEK1/2 inhibitor U0126 on the EV71 infection was evaluated.We found that GW5074 could significantly suppress EV71 infection under in vitro and in vivo conditions even more than 50% with the highest dose we used?20 ?M and 0.2 mg/kg?.We further demonstrated that blocking the MAP kinase pathway was related to the anti-EV71 activity of GW5074 with Western blotting.GW5074 might be a pharmaceutical candidate for efficient treatment of EV71 infection.The data from the Am NLuc?Q/G?BCLuc reporter was consistent with Western blotting and histopathology examination.Conclusions: Collectively,this real-time bioluminescence imaging approach can quantitatively monitor the efficacy of anti-EV71 agents,and is useful for the anti-EV71 drug screening and EV71 pathogenesis study.