FIG. 1.

Multistep strategy used to assemble full-length cDNA clone of VR-2332. In the top cartoon, the organization of the viral genome is shown, as are the positions of the unique restriction sites used for cloning purposes. The numbers 1a, 1b, and 2 through 7 indicate the PRRSV open reading frames. 5′ indicates the 5′ leader. and 3′ indicates the 3′ nontranslated region. At the 5′ end of the genome, XhoI and AscI restriction sites and a T7 RNA promoter were fused to the genome. The asterisk indicates the transcription start site of T7 RNA polymerase, resulting in the sequence 5′-m⁷G(5′)ppp(5′)G cap analog-TA TGA CGT ATA GGT…3′ as the predicted 5′ terminus of RNA transcribed in vitro with the T7 mMessage Machine kit. Downstream of the 3′ nontranslated region, a poly(A) tail of 38 A's and the restriction sites AclI and XbaI were inserted. The complete viral genome was divided into six fragments flanked by unique restriction sites, represented by the horizontal lines labeled a through f. The length of each fragment is indicated in parentheses below the horizontal lines (in nucleotides). As shown in the bottom cartoon, these fragments were individually cloned into the pOK₁₂ vector in the order indicated by the letters a to f. Prior to viral genome assembly, pOK₁₂ was prepared by inserting a stuffer fragment containing all the unique restriction sites shown in the top cartoon in the XhoI and XbaI sites.