Fig. 4.

RT-PCR and nucleotide sequencing of the rescued viruses. For verification if the mutation were retained in the rescued viruses collected from P5 culture supernatant, the target region was amplified as described in the materials and methods. A) The agarose gel electrophoresis of RT-PCR products of v3URAD40 (lane 1), vSURLV (lane 2), and parental virus vAPRRS (lane 4). B) Nucleotide sequence lineup showed that all viruses retained the engineered mutations. The sequence was compared with the parental APRRS (GQ330474), identical sequence displayed as dot (.), while variation was shown the actual nucleotide, natural or artificial deletion denoted as (-). The upstream boxed sequence showed that the vSURLV is homologous with type II APRRS in ORF7, while the downstream box indicated the vSURLV 3′ UTR is identical with type I PRRSV (LV).