Virology Journal (Jun 2010)

Producing infectious enterovirus type 71 in a rapid strategy

  • Qin E-De,
  • Yu Man,
  • Tian Xue,
  • Cao Rui-Yuan,
  • Han Jian-Feng,
  • Qin Cheng-Feng

DOI
https://doi.org/10.1186/1743-422X-7-116
Journal volume & issue
Vol. 7, no. 1
p. 116

Abstract

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Abstract Background Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF), which is flanked by untranslated regions at both the 5' and 3' ends. Results A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA) assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice. Conclusions Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method.