Generating West Nile Virus from an Infectious Clone.

WNV infectious clones are valuable tools for elucidating WNV biology. Nevertheless, relatively few infectious WNV clones have been generated because their construction is hampered by the instability of flaviviral genomes. More recently, advances in cloning techniques as well as the development of several two-plasmid WNV infectious clone systems have facilitated the generation of WNV infectious clones. Here we described a protocol for recovering WNV from a two-plasmid system. In this approach, large quantities of these constructs are digested with restriction enzymes to produce complementary restriction sites at the 3' end of the upstream fragment and the 5' end of the downstream fragment. These fragments are then annealed to produce linear template for in vitro transcription to synthesize infectious RNA. The resulting RNA is transfected into cells and after several days WNV is recovered in the culture supernatant. This method can be used to generate virus from infectious clones encoding high- and low-pathogenicity strains of WNV, as well as chimeric virues.