A Novel Bacterium-Free Method for Generation of Flavivirus Infectious DNA by Circular Polymerase Extension Reaction Allows Accurate Recapitulation of Viral Heterogeneity

A novel bacterium-free approach for rapid assembly of flavivirus infectious cDNAs using circular polymerase extension reaction was applied to generate infectious cDNA for the virulent New South Wales isolate of the Kunjin strain of West Nile virus (KUNV) that recently emerged in Australia. Recovered virus recapitulated the genetic heterogeneity present in the original isolate. The approach was utilized to generate viral mutants with designed phenotypic properties and to identify E protein glycosylation as one of the virulence determinants.