Fig 1.

Assembly of infectious cDNA for the wild-type and mutant NSW2011 viruses by CPEC reaction. A schematic representation of the WNV genome is shown at the top of the figure, with individual genes and untranslated regions (UTR) indicated. Shown are locations of the unique restriction sites with numbers indicating nucleotide positions in the NSW2011 genome (GenBank accession no. JN887352). The primers used to generate cDNA fragments are shown by arrows and labeled by the designations of unique restriction sites. Lines show PCR fragments, with numbers representing their size in base pairs (bp). Asterisks show approximate locations of introduced mutations. The lightly shaded box for the BmgBI-BstEII fragment indicates that this fragment was cloned separately from other fragments in strategy 2. E S156F, PCR fragment contacting the S156F mutation in E protein; NS5 R47G and NS5 V49I, PCR fragments containing the corresponding mutations in the NS5 protein; CMV, cytomegalovirus promoter; HDVr, hepatitis delta virus ribosome; pA, SV40 poly(A) signal.