Plasmids.

To engineer the recombinant PR8 virus NS and M split segments (NSs and Ms, respectively), overlapping PCR and standard molecular biology techniques were used to introduce the modifications into the ambisense pDZ-NS or pDZ-M viral rescue plasmid (26). The modified plasmids, named pDZ-NSs and pDZ-Ms, respectively, contain the NS1 (NS segment) or M1 (M segment) open reading frames (ORFs) without stop codons or splice acceptor sites, followed by the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site (ATNFSLLKQAGDVEENPGP) and the entire sequence of the NEP (NS segment) or M2 (M segment) ORF (27, 29). The sequences of the modified pDZ-NSs and -Ms plasmids were confirmed by sequencing, and the plasmids were used for virus rescue.