A PCR-based protocol for generating West Nile virus replicons.
A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon.
Maeda
Junko
J
Department of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Takagi
Hirotaka
H
Hashimoto
Shingo
S
Kurane
Ichiro
I
Maeda
Akihiko
A
D000818:Animals; D006224:Cricetinae; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis; D014902:West Nile virus / Q000235:genetics*
D012367:RNA, Viral; D014759:Viral Envelope Proteins