Producing infectious enterovirus type 71 in a rapid strategy.
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.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Jian-Feng
JF
Han
Rui-Yuan
RY
Cao
Xue
X
Tian
Man
M
Yu
E-De
ED
Qin
Cheng-Feng
CF
Qin
D000818:Animals; D002460:Cell Line; D029821:Enterovirus A, Human / Q000235:genetics / Q000502:physiology*; D006232:Hand, Foot and Mouth Disease / Q000821:virology*; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication
D012367:RNA, Viral