FIG 1.
Evaluation of the biological characteristics of the rescued PA-X-deficient viruses. (A) Mutation sites in the PA genes of the PA-X deficient viruses. The red letters represent nucleotides at the mutation sites. (B) Western blotting for PA, PA-X, and β-actin expression. MDCK cells were infected with the indicated virus at an MOI of 10. After about 36 h, whole-cell lysates were subjected to Western blotting. The levels of PA-X expression relative to the expression of the control, β-actin, were determined by densitometry using BandScan software, version 5.0. The data shown are representative of results from three independent experiments and were standardized to the level of r-CK10 (taken as 100%). (C) Renilla luciferase expression in 293T cells cotransfected with the indicated PA or PA-derived mutant plasmid. Cells were transfected with either an empty pcDNA3.1+ vector or a pcDNA3.1+ vector expressing the indicated PA or PA-derived mutant gene together with a Renilla plasmid (200 ng of each plasmid) for 24 to 36 h. The results are means and SD for three independent experiments. **, P < 0.01. (D and E) Mice were infected with 104.0 EID50 of r-CK10, CK-PAX5, or CK-PAX3 (n = 5). Mice were humanely killed when they lost ≥25% of their initial body weight. Shown are the mean weight loss (D) and survival rates (E) of mice infected with the indicated viruses or mock infected with PBS. (F) Determination of the LD50s of the recombinants in mice, chickens, and ducks. Groups of five animals were inoculated intranasally with 100-μl-vial dilutions containing different doses of virus (101.0 to 103.0 EID50 for mice, 101.0 to 105.0 EID50 for chickens, and 102.0 to 105.0 EID50 for mallard ducks). Values are presented as the LD50 (expressed in log10 EID50)/MDT (in days). (G and H) Chickens were infected with 105.0 EID50 of r-CK10, CK-PAX5, or CK-PAX3. Survival rates (G) and MDTs (H) are shown. Data are means and SD. *, P < 0.05.