Impaired clearance of viral replication sustains IFNβ production. Total leukocytes were harvested from naïve or virus-infected mice at day 7 post-infection. (A) RNA was extracted from total leukocytes, Gr1 + CD11b + sorted cells and Gr1-CD11b- sorted cells. Expression of IFNβ was measured by RT-QPCR. The mRNA relative folds were determined by normalizing the level of each group to the corresponding GAPDH level and then to total leukocytes from naïve mice (mean ± SEM). Experiment (n = 3-6 mice per group) was performed twice and one representative is shown. (B) Lungs were harvested from virus infected mice at the time points indicated and the virus load was measured by plaque assays (n = 3 mice per group; mean ± SEM). (C) Protein lysates of lungs were harvested from infected mice (n = 3 mice per group) on day 7 and expression of influenza NP was detected by western blotting; β-actin expression served as the internal control. This is a representative result from three repeated experiments. (D) Total leukocytes were harvested from PR8-infected mice at day 7 post-infection. Expression of influenza NP in Ly6ChighCCR2+ cells was detected by flow cytometry. This is a representative result from three repeated experiments. (E) Body weight changes of PBS- and Oseltamivir-treated mice were monitored at day 0, 3 and 6 post-infection (mean ± SEM). (F) Leukocytes were harvested from PBS- and Oseltamivir-treated mice at day 6 post-infection. Cells were stained with Abs against Gr1, CD11b, Ly6C and CCR2, and then CCR2+ monocytes were analyzed by flow cytometry. The numbers of CCR2+ inflammatory monocytes were calculated in each group. These data are a composite of two independent experiments (n = 4 mice per group, mean ± SEM; *P < 0.05).