TABLE 3.
Phenotypic traits of FMDV variants with different passage histories in BHK-21 cell cultures
| FMDV straina | Binding to heparinb | Virulence forc:
|
|||
|---|---|---|---|---|---|
| BHK-21 cells | CHO cells
|
||||
| Wild type | pgsA-745 | pgsD-677 | |||
| C-S8c1 | − | 105 | − | − | − |
| O1K | ++ | 103 | 104 | − | − |
| MARLS | +++ | 102 | 104 | 102 | 104 |
| MARLS/hs−c1 | − | 103 | − | − | − |
| MARLS/hs−c2 | − | 103 | − | − | − |
| MARLS/hs−c3 | − | 102 | − | − | − |
| MARLS/hs−c4 | − | 103 | − | − | − |
| O1K/C-S8c1 | − | 104 | − | − | − |
| O1K/MARLS | +++ | 102 | 102 | 10 | 102 |
| O1K/MARLST2363 | ++ | 102 | 103 | 10 | 102 |
| C-S8c1p100c1 | ++ | 102 | 105 | 103 | 105 |
| C-S8c1p100c10 | ++ | 102 | 105 | 103 | 105 |
| C-S8c1p100RGG | + | 103 | 105 | 104 | 105 |
| R100 | ++ | 103 | − | − | − |
The origin of each FMDV strain is shown in Fig. 1 and is described in Materials and Methods. FMDV C-S8c1p100c1 and C-S8c1p100c10 are two plaque-purified clones derived from C-S8c1 after 100 serial passages in BHK-21 cells; C-S8c1p100RGG is a C-S8c1p100-derived MAR mutant with a substitution at the Arg-Gly-Asp motif; FMDV R100 was rescued after 100 passages of carrier BHK-21 cells persistently infected with C-S8c1. FMDV O1K was obtained by transfection of BHK-21 cells with infectious O1K transcripts derived from plasmid pFMDV-YEP-poly(C) (60). FMDV O1K used was at passage 1.
Binding to heparin was estimated as the ratio of PFU remaining in the supernatant of a viral suspension after incubation with control beads (without heparin) relative to heparin-Sepharose beads. Binding was classified as follows: +, 2 < ratio < 50, where 2 is the limit of positive detection; ++, 50 ≤ ratio < 5 × 102; +++, 5 × 102 ≤ ratio < 5 × 103; −, no detectable binding (ratio, ∼1). Further details on heparin binding assays are given in Materials and Methods.
Minimum number of PFU required for complete cell killing of 104 BHK-21 cells in 24 h or 104 CHO cells in 72 h. Thus, a large number corresponds to low virulence; −, no cytopathic effect in the presence of the following PFU for the different FMDVs: 1 × 106 (C-S8c1), 2 × 105 (O1K), 7 × 106 (MARLS/hs− c1 to c4), 2 × 106 (O1K/C-S8c1), 6 × 105 (R100). Each value represents the average of triplicate determinations.