title: A PCR-based protocol for generating West Nile virus replicons.
abstract: 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.
authors.affiliation: Department of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Maeda
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Takagi
authors.forename: Shingo
authors.initials: S
authors.lastname: Hashimoto
authors.forename: Ichiro
authors.initials: I
authors.lastname: Kurane
authors.forename: Akihiko
authors.initials: A
authors.lastname: Maeda
mesh_terms: 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*
chemical_list: D012367:RNA, Viral; D014759:Viral Envelope Proteins