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Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool
Chee Wah Tan1, Han Kang Tee1, Michelle Hui Pheng Lee1
1Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Plos One
|September 13, 2016
Summary
Researchers developed new, efficient reverse genetics tools for Enterovirus A71 (EV-A71), a virus causing hand, foot, and mouth disease. These DNA-based clones simplify viral studies, offering faster and more cost-effective methods for functional gene analysis.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Enterovirus A71 (EV-A71) is a significant pathogen responsible for outbreaks of hand, foot, and mouth disease, with potential for severe neurological complications in children.
- Current reverse genetics tools for EV-A71 rely on T7/SP6 bacteriophage promoter-controlled clones, which are often laborious and costly.
- There is a need for more efficient and accessible reverse genetics systems for studying EV-A71.
Purpose of the Study:
- To develop novel, infectious plasmid DNA-based reverse genetics tools for Enterovirus A71 (EV-A71).
- To establish systems that facilitate rapid functional analysis of EV-A71 genes.
- To improve the efficiency and cost-effectiveness of EV-A71 reverse genetics.
Main Methods:
- Construction and transfection of EV-A71 infectious plasmid DNA under the control of CMV-intermediate early promoter and SV40 transcriptional-termination signal.
- Development of dual-promoter (CMV and T7) infectious plasmids for versatile applications.
- Incorporation of hepatitis delta virus (HDV) ribozyme for precise transcript 3' end generation.
- Evaluation of virus yield and titers following transfection with plasmid DNA or in vitro-transcribed RNA.
Main Results:
- Transfection of CMV-driven EV-A71 infectious DNA yielded high virus titers (6.4 log10PFU/ml), comparable to in vitro-transcribed RNA (5.8 log10PFU/ml).
- Infectious plasmids incorporating reporter genes (eGFP, NanoLuc) produced significant virus titers (4.3 and 5.0 log10 PFU/ml, respectively).
- Dual-promoter plasmids facilitated both direct transfection and in vitro transcription, yielding infectious viral particles.
- HDV ribozyme incorporation enhanced infectious viral production from DNA-launched transcripts, while hammerhead ribozyme improved T7 RNA-derived titers.
Conclusions:
- The developed infectious plasmid DNA-based systems provide rapid and robust reverse genetics tools for EV-A71.
- These novel tools overcome limitations of existing methods, enabling more efficient functional studies of EV-A71.
- The versatility of dual-promoter plasmids and the benefits of specific ribozymes offer significant advantages for EV-A71 research.
