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


CONCLUSIONS
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.
Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Republic of Singapore.
Tao
T
Meng
Tanja K
TK
Kiener
Jimmy
J
Kwang
D000818:Animals; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral; D029821:Enterovirus A, Human / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D012318:RNA Polymerase I / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics*
D012367:RNA, Viral; D014765:Viral Vaccines; D012318:RNA Polymerase I