Assay of lung virus titers in mice. The ps-PA496 was mixed with a 25 kd branched PEI QVDOPh (Sigma, USA) at a nitrogen/phosphorus weight ratio (N/P ratio) of 15 at room temperature for 30 min. For the animal study, 200 μl of the mixture containing 100 μg of ps-PA496 was intravenously injected into 6-week-old female BABL/c mice through the vena caudalis, and 100 μg of PEI-mediated ps-negative was used as a negative control. After 18 h, 50 μl DMEM containing the indicated doses (10 LD50) of virus were instilled into anesthetized mice through the nostrils. In this experiment, mice were anaesthetized with ether. The lungs were harvested to determine virus titers at 24 h post-infection, as described previously [5]. The results are shown as log10 TCID50/ml.
Viral challenge assay in mice. BABL/c mice were injected intravenously with the PEI-mediated ps-negative plasmid or the ps-PA496 plasmid. After 24 h, mice were infected with the virus (10 LD50) and then monitored for mortality.
Statistical analysis. The experimental data were analyzed with t-tests and one-way ANOVA analysis using SPSS 13.0 software. Comparison of survival rate was done by using log-rank test [18]. A probability of p
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