FIG. 6.

rSDV with an additional subgenomic promoter. (A) An additional transcriptional unit expressing the GFP as a reporter gene was inserted into the SDV genome either upstream or downstream of the structural genes (pGFP-SDV and pSDV-GFP), or three additional transcriptional GFP units were inserted upstream of the structural genes (pGFP₃-SDV). HH, hammerhead ribozyme sequence; pA, poly(A) tail. (B) GFP expression in either rSDV-GFP- or rGFP-SDV-infected BF-2 cells was monitored by UV light microscopy, and typical results for both rSDVs 7 days postinfection are presented. (C) Confirmation by RT-PCR of the three additional transcription units on RNA extracted from infected cells after one passage of the cell supernatant. Lanes: M, DNA molecular weight markers; rGFP-SDV, recombinant SDV containing one additional transcription unit; rGFP₃-SDV, recombinant SDV containing three additional transcription units. The sizes of the RT-PCR products (in nucleotides) are indicated to the left of the gel. The primers used for the RT-PCR are depicted in panel A.