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The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10
Authors:Wang  Min  Yan  Jingjing  Zhu  Liuyao  Wang  Meng  Liu  Lizhen  Yu  Rui  Chen  Ming  Xun  Jingna  Zhang  Yuling  Yi  Zhigang  Zhang  Shuye
Affiliation:1.Shanghai Public Health Clinical Center and Institute of Biomedical Sciences, Fudan University, Shanghai 201508, China
Abstract:Coxsackievirus A10(CVA10) is one of the major etiological agents of hand, foot, and mouth disease. There are no vaccine and antiviral drugs for controlling CVA10 infection. Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals. Here, two infectious clones for the prototype and a Myc-tagged CVA10 were constructed. Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells. Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally. We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively. Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs). Based on CVA10 replicon RNA, SRIPs with either the enterovirus A71(EVA71) capsid or the CVA10 capsid were generated. Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not. Finally, we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib) before the 50-untranslated region(UTR). In summary, reverse genetic tools for prototype strain of CVA10, including both the infectious clone and the SRIPs system, were successfully established. These tools will facilitate the basic and translational study of CVA10.
Keywords:Coxsackievirus A10 (CVA10)   Reverse genetics   Enterovirus   Single round infectious particles (SRIPs)   Replicon
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