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Open Access Highly Accessed Methodology

RNA polymerase I-driven reverse genetics system for enterovirus 71 and its implications for vaccine production

Tao Meng1, Tanja K Kiener1 and Jimmy Kwang12*

Author Affiliations

1 Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, 117604, Republic of Singapore

2 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore

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Virology Journal 2012, 9:238  doi:10.1186/1743-422X-9-238

Published: 17 October 2012

Abstract

Background

Enterovirus 71 (EV71) is a virus that causes from mild hand, foot and mouth disease (HFMD) to severe neurological complications and deaths in infants and young children. Effective antiviral agents and vaccines against EV71 are not available. However, Vero cell-based chemically inactivated EV71 vaccines could be developed soon based on the success of inactivated polio vaccine. Like poliovirus, EV71 has a positive single-stranded RNA genome of about 7400 nucleotides which contains a single open reading frame (ORF) flanked by conserved and untranslated regions at both the 5 and 3 ends.

Results

The universal amplification of the full length genome of EV71 regardless of its genetic diversity, and the subsequent construction of a human RNA polymerase I-driven reverse genetics (RG) system to produce pure virus stocks in Vero cell within 10 days were described. The rescued viruses were characterized by DNA sequencing, cytopathic effect (CPE) and indirect fluorescent assay (IFA) in comparison with the wild-type viruses. Moreover, the rescued viruses grew to high titers and retained the same immunogenicity as the wild-type viruses.

Conclusion

We have established a simplified method to rescue RG EV71 virus from diverse clinical isolates with detailed genetic information and to prepare virus stocks in only 10 days. This method could accelerate EV71 vaccine development.

Keywords:
Enterovirus 71; Universal RT-PCR; Reverse genetics; Vaccine