Abstract
Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3’ ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71.
Highlights
Enterovirus A71 (EV-A71) is the main causative agent of hand, foot and mouth disease (HFMD) in children
Based on the knowledge previously gained from constructing a T7/SP6-driven EV-A71 infectious clone [21], we sought to establish a convenient DNA-launched infectious cDNA clone driven by the CMV promoter for transient expression of EV-A71 genomic RNA
DNA-launched infectious clones have been reported for a number of viruses, including dengue virus [27,28], Japanese encephalitis virus [28,29] and chikungunya virus [22]
Summary
Enterovirus A71 (EV-A71) is the main causative agent of hand, foot and mouth disease (HFMD) in children. The clinical symptoms of HFMD are fever, rash on hands and feet, and oral ulcers [1]. Unlike other enteroviruses that cause HFMD, EV-A71 infections are associated with severe neurological complications such as aseptic meningitis, brainstem encephalitis and acute flaccid paralysis [1,2,3]. Along with poliovirus, EV-A71 is a neurotropic enterovirus of great public health concern. No vaccine or antiviral is available to prevent or treat EV-A71 infection [4].
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