TABLE 1.
Characterization of each mutation
| Mutation | Location in VP1 | Mean titer (PFU/ml) ± SDa | IFAb | Passage that displayed CPEc | Revertantd |
|---|---|---|---|---|---|
| WT | (1.2 ± 0.1) × 106 | +++ | P0 | NA | |
| R3A | N terminus | (1.2 ± 0.6) × 102* | + | P3 | VP1 M63L |
| R18A | N terminus | (1.4 ± 0.4) × 105 | +++ | P0 | NA |
| R38A | N terminus | (1.2 ± 1.0) × 102 | ++ | P2 | VP1 D40V |
| K43A | N terminus | (1.8 ± 0.2) × 104 | ++ | P2 | Reverted to WT |
| R67A | N terminus | ND | − | ND | ND |
| R86A | N terminus | (5.3 ± 2.5) × 102* | + | P2 | Reverted to WT |
| R120A | α3 | ND | − | P3 | Reverted to WT |
| R121A | α3 | ND | − | ND | ND |
| K122A | α3 | ND | − | ND | ND |
| R130A | βD | ND | − | ND | ND |
| K162A | EF loop | (1.5 ± 0.2) × 106 | +++ | P0 | NA |
| R166A | EF loop | ND | − | ND | ND |
| K182A | βF | ND | − | ND | ND |
| K215A | GH loop | (3.6 ± 0.6) × 102* | − | P3 | ND |
| K218A | GH loop | (1.7 ± 0.1) × 106 | +++ | P0 | NA |
| R236A | βH | ND | − | ND | ND |
| K242A | HI loop | ND | − | P5 | VP1 E92G, VP1 T100K |
| K244A | HI loop | ND | − | P8 | VP1 Q145K, VP1 T237N |
| R250A | βI | ND | − | P11 | VP1 T41I, VP1 E167K |
| R254A | βI | ND | − | ND | ND |
| K256A | βI | ND | − | ND | ND |
| R259A | βI | ND | − | ND | ND |
| R264A | C terminus | ND | − | ND | ND |
| R267A | C terminus | ND | − | ND | ND |
| K274A | C terminus | ND | − | P7 | VP1 I111L, VP1 A224V |
| K285A | C terminus | (2.0 ± 0.8) × 106 | +++ | P0 | NA |
| R291A | C terminus | ND | − | ND | ND |
The mean virus titers at 48 h p.t. were determined by plaque assay from three independent experiments. An asterisk represents the titer at 72 h p.t because the titer at 48 h.p.t was below the limit of detection. ND, not detected.
For each mutant, IFA was repeated at least three times, and the positive rate was calculated from five random views under a microscope using Image-Pro Plus software. The positive rate for the WT was set as strong (+++) and mutant levels as strong (+++) (IFA positive rate ≥66.7% of that of the WT), ++ (medium) (IFA positive rate between 33.3% and 66.7% of that of the WT), + (weak) (IFA positive rate between 5% and 33.3% of that of the WT), and negative (−) (IFA positive rate less than 5% of that of the WT).
Defective mutants were subjected to revertant analysis by continuous culturing on Vero cells up to 20 rounds. CPE was monitored under a microscope. ND, the mutant did not display CPE by P20.
NA, not applicable; ND, not detected.