TABLE 2.
Characterization of recombinant viruses with secondary-site mutations
| Recombinant virus | Mean titer (PFU/ml) ± SDa |
IFAb |
|---|---|---|
| WT | (2.9 ± 1.1) × 106 | +++ |
| R3A+M63L | (6.0 ± 2.1) × 105 | +++ |
| R38A+D40V | (1.1 ± 0.5) × 106 | +++ |
| K242A+E92G | (2.3 ± 0.07) × 104 | ++ |
| K242A+T100K | (1.5 ± 0.2) × 105 | +++ |
| K242A+E92G+T100K | (3.9 ± 0.4) × 105 | +++ |
| K244A+Q145K | ND | + |
| K244A+T237N | (2.5 ± 0.4) × 105 | +++ |
| K244A+Q145K+T237N | (5.0 ± 0.7) × 106 | +++ |
| R250A+T41I | ND | + |
| R250A+E167K | (4.8 ± 1.1) × 102 | + |
| R250A+T41I+E167K | 100 ± 70* | + |
| K274A+I111L | (6.0 ± 0.7) × 104 | ++ |
| K274A+A224V | (4.5 ± 1.4) × 103 | ++ |
| K274A+I111L+A224V | (3.0 ± 0.3) × 105 | +++ |
The mean virus titers at 48 h p.t. were determined by plaque assay from three independent experiments. An asterisk represents the virus titer at 24 h p.t because the titers at 48 h p.t. and 72 h p.t. were below the limit of detection. ND, not detected.
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).