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Producing infectious enterovirus type 71 in a rapid strategy.
Jian-Feng Han1, Rui-Yuan Cao, Xue Tian
1State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Virology Journal
|June 8, 2010
Summary
Researchers developed a rapid method to produce infectious Enterovirus 71 (EV71) from RNA. This technique allows for easy genetic modification of the EV71 genome, aiding in the study of this hand-foot-and-mouth disease virus.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD).
- Recent epidemics linked EV71 to severe brainstem encephalitis with high mortality.
- EV71 has a positive-sense single-stranded RNA genome encoding a polyprotein.
Purpose of the Study:
- To establish a rapid method for producing infectious EV71 from RNA.
- To enable easy genetic engineering of the EV71 genome.
Main Methods:
- Developed a long-distance RT-PCR assay to amplify the full-length EV71 genome cDNA.
- Used specific primers with an SP6 promoter for amplification.
- Transfected in vitro synthesized RNA transcripts into RD cells to rescue the virus.
Main Results:
- Successfully produced infectious EV71 virus from RNA preparations.
- Characterized rescued virus using RT-PCR and indirect fluorescent-antibody (IFA) assay.
- Confirmed rescued virus infectivity in RD cells and neurovirulence in suckling mice.
Conclusions:
- Established a rapid method to produce infectious full-length EV71 cDNA directly from RNA.
- This method facilitates straightforward engineering of specific mutations into the enterovirus genome.
