For assembly of plasmid pWNVsyn-5′TL (containing the 5′ third of the WNV coding sequence) an AscI and BsaI cut fragment of p5′TL-AB was ligated to a BbsI and PacI cut fragment of p5′TL-CD (Fig. 1b). The ligation product was amplified using high fidelity PCR (KOD, Invitrogen). Following digestion with BamHI and XbaI, the fragment was cloned into the low copy plasmid vector pBR322-PL (derived from plasmid pBR322 engineered to contain a matching polylinker between the unique restriction sites EcoR1 and EagI resulting in the partial deletion of the

tetR gene) yielding pWNVsyn-5′TL. This plasmid contains the 3′ two-thirds of the WNV coding sequence and was generated in three steps: (I) An AscI and BsaI cut Selleckchem ABT737 fragment of p3′TL-AB was ligated to a BsaI and PacI cut fragment of p3′TL-CD. This fragment was amplified by high fidelity PCR and integrated into a commercially available pPCRscript vector (Clontech). (II) A BtgZI and AscI cut fragment of p3′TL-EF was ligated to a PacI and BtgZI cut fragment of p3′TL-GH. The resulting 3′TL-EFGH ligation product was amplified by PCR using 5′ and 3′ flanking primers, digested with SpeI and XbaI and integrated in pBR322-PL, leading to plasmid pBR322-3′TL EFGH (Fig. 1b). (III) The final pWNVsyn-3′TL was generated by introduction of the 3′TL-ABCD fragment (derived from pPCRscript3′TL-ABCD) into pBR322-3′TL-EFGH, taking advantage of the unique restriction enzymes SpeI and BamHI (Fig.

1c). All plasmids were amplified in bacterial strain HB101 (Promega) and purified with commercially available systems (Omega and Qiagen). Electroporation of bacterial cells was carried out RNA Synthesis inhibitor using a GenePulser Apparatus (Bio-Rad) with settings of 1.8 kV, 25 μF and 200 Ω. Sequence analysis was carried out using a 3130xl genetic analyzer (ABI) using BigDye Terminator v3.1 cycle sequencing Kit (ABI). these pWNVsyn-3′TL was linearized with XbaI followed by mung bean nuclease digestion to remove single stranded nucleotide overhangs in order to generate the correct

3′ end of the WNV coding sequence. The plasmids pWNVsyn-3′TL and pWNVsyn-5′TL were then digested with SphI and the full-length sequence was generated by ligation (T4 Ligase; New England Biolabs) via the SphI sequence overhangs of the 5′ and 3′ parts. The ligated DNA fragments were extracted with phenol–chloroform twice, precipitated with ethanol and resuspended in nuclease free water. RNA was transcribed at 37 °C for 3 h from ligated template DNA by T7 polymerase transcription, using T7 MEGAscript Kit (Ambion). The integrity of RNA transcripts was analyzed in 1% agarose gels containing 6% formaldehyde. For RNA transfection, subconfluent vaccine-certified Vero cells were collected with trypsin, washed twice in serum free TC Vero Medium (Baxter) and twice in ice-cold PBS buffer. Aliquots of approximately 2 × 107 cells were resuspended in 800 μl of ice-cold PBS, mixed with transcribed RNA and transferred to 0.4 gene pulser cuvettes.