title: A PCR-based protocol for the generation of a recombinant West Nile virus.
abstract: Viral reverse genetics, particularly infectious cloning, is a valuable tool with applications to many areas of viral research including the generation of vaccine candidates. However, this technology is sometimes insufficient for the construction cDNA clones as the genome sequences and/or encoding proteins of some viral agents may be toxic to the host cells used for cloning. To circumvent this problem, we developed a polymerase chain reaction (PCR)-based protocol for generating a complete West Nile virus (WNV) cDNA. The fragmented cDNAs were synthesized from WNV RNA by reverse transcription-PCR, and subsequently cloned into plasmids for use as templates for WNV cDNA synthesis. The fragmented cDNAs were amplified and assembled by PCR to generate a full-length WNV cDNA. Using this cDNA as a template, WNV RNA was synthesized in vitro and transfected into mammalian cells. We also examined the generation of a mutant recombinant WNV containing a site-directed mutation within the viral genome sequence. Here, we discuss the possibility of developing a method for the generation of recombinant WNVs.
authors.affiliation: Department of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. amaeda@vetmed.hokudai.ac.jp
authors.forename: Akihiko
authors.initials: A
authors.lastname: Maeda
authors.forename: Ryo
authors.initials: R
authors.lastname: Murata
authors.forename: Minoru
authors.initials: M
authors.lastname: Akiyama
authors.forename: Ikuo
authors.initials: I
authors.lastname: Takashima
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kariwa
authors.forename: Tomomasa
authors.initials: T
authors.lastname: Watanabe
authors.forename: Ichiro
authors.initials: I
authors.lastname: Kurane
authors.forename: Junko
authors.initials: J
authors.lastname: Maeda
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D014162:Transfection; D014773:Virology / Q000379:methods*; D014902:West Nile virus / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral