A reverse genetics system for enterovirus D68 using human RNA polymerase I
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Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5′-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.
KeywordsHuman enterovirus D68 Pol I promoter Reverse genetic Minireplicon Infectious cDNA clone
This work was supported by the National Key research and Development Program of China (Grant No. 2017YFA0205102), the National Science Foundation of China (Grant No. 31270201), and the seed foundation of Tianjin University (Grant No. 2014XRX-0026). We thank Xiaofang Yu for critical reagents.
Conceived and designed the experiments: MP and TW. Performed the experiments: MP. Analyzed the data: MP. Contributed reagents/materials/analysis tools: MP, SG, ZZ, KZ, SL, ZW, and TW. Wrote the paper: MP and TW.
Compliance with ethical standards
Conflict of interest
No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication.
Research involving human and animal participants
This article does not contain any studies with human participants or animals performed by any of the authors.