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Construction and characterization of an infectious cDNA clone of enterovirus 71: a rapid method for rescuing infectious virus based on stable cells expressing T7 polymerase

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Abstract

Enterovirus 71 (EV71) is a causative agent of hand, foot and, mouth disease (HFMD) in young children. It is valuable for virologists to develop a fast method to rescue infectious virus from a viral cDNA clone. Here, we report a method for rapid rescue of infectious EV71 by using cells expressing T7 polymerase. The full-length EV71 genome was amplified in one step by long-distance PCR with a T7 promoter at the 5ʹ end, and the T7 polymerase gene was cloned into a lentivirus vector for construction of a stable cell line expressing T7 polymerase. The infectious virus was rapidly and efficiently rescued by single transfection of cells with the infectious cDNA clone. Further experiments showed that the rescued virus had characteristics similar to those of the parental virus. This method circumvented the difficulty in performing in vitro transcription of a long linear DNA to obtain high-quality RNA. The construction of the viral cDNA clone and the fast rescue of the infectious virus will greatly benefit future investigations.

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Acknowledgements

The authors would like to thank Professor Cheng Tong of Xiamen University for providing the plasmid of pAR3126. This work was supported by the National Natural Science Foundation of China (no. 32070184 and no. 31570156).

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Authors and Affiliations

  1. Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China

    Meixian Fu, Jinjin Bai, Zhangmei Chang, Xin Zhou & Jian-Er Long

  2. Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, 138 Yixueyuan R., Shanghai, 200032, China

    Shuang Gao & Jian-Er Long

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  1. Meixian Fu
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  2. Jinjin Bai
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  3. Shuang Gao
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  4. Zhangmei Chang
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  5. Xin Zhou
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  6. Jian-Er Long
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Correspondence to Jian-Er Long.

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Fu, M., Bai, J., Gao, S. et al. Construction and characterization of an infectious cDNA clone of enterovirus 71: a rapid method for rescuing infectious virus based on stable cells expressing T7 polymerase. Arch Virol 166, 627–632 (2021). https://doi.org/10.1007/s00705-020-04940-9

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  • DOI: https://doi.org/10.1007/s00705-020-04940-9

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