TABLE 1.
Determination of infectious virions and the NP protein in the culture supernatant of the cells harboring SeV Cl.151-based vectors
All the vectors were installed with the Bsr gene encoding blasticidine S deaminase. Aliquots of 106 of LLCMK2 cells harboring each SeVdp vector were seeded in 90-mm wells with 8 ml of medium. After culturing for 3 days, culture supernatant was recovered and filtered through 0.45-μm cellulose acetate membranes. NP protein was determined by blotting 0.04–20 μl of the supernatant on nitrocellulose membranes as described under “Experimental Procedures.” The supernatant was also incubated with 106 uninfected LLCMK2 cells for 14 h and then cultured in the presence of Bs (10 μg/ml) for 7 days. The numbers of cell colonies resistant to Bs were determined by staining with crystal violet.
| Structural genes | NP protein | Number of Bsr colonies | ||
|---|---|---|---|---|
| ng/day/105 cells | ||||
| Ma | Fa | HNa | 8.75 | > 106 |
| –b | F | HN | 5.36 | 366 |
| M | –b | HN | 7.62 | 2 |
| M | F | –b | 72.05 | 10 |
| M | –b | –b | 2.51c | 0 |
| –b | F | –b | 2.76c | 0 |
| –b | –b | HN | 2.61c | 0 |
| –b | –b | –b | 2.56c | 0 |
a Replication-competent vector.
b Corresponding genes were deleted or replaced with exogenous genes.
c Background caused by spontaneous cell lysis.