Fig 3.

RT-PCR and nucleotide sequencing of the chimeric vAPRRS-EAV2ab34 genome. The P0 viruses harvested from transfected MARC-145 cells were used to inoculate fresh MARC-145, BHK-21, or Vero cells. At 48 h p.i., viral RNA was isolated from the culture supernatants and was used for RT-PCR amplification. (A) Confirmation of the chimeric nature of the vAPRRS-EAV2ab34 genome. RT-PCR was performed with the chimeric primer pair FA12066 (located in vAPRRS ORF1b)–RE10766 (in vEAV030 ORF3) to amplify a 1,139-bp product that is specific for the vAPRRS-EAV2ab34 chimera. The parental viruses were included as negative controls. (B) Sequence analysis of the junction sites in the vAPRRS-EAV2ab34 genome between PRRSV ORF1b and EAV ORF2a and between EAV ORF4 and PRRSV ORF5a. The nucleotide sequences and sequencing traces derived from these upstream and downstream fusion points are shown; the AscI restriction site and inserted EAV sequences are indicated.