PA-X reduces expression of Ifnb1 and Ifna4 mRNA in the lungs of infected mice. Wild-type mice were infected with 50,000 pfu of PR8 WT, PR8 FS or mock infected via the intranasal route. Mice were weighed every 24 h and sacrificed 48 h.p.i. (A) Weight loss in mice infected with PR8 WT and PR8 FS. Data are represented as mean weight loss from 13 (mock), 40 (PR8 WT) and 39 (PR8 FS) mice per group ± s.e.m., pooled from 11 independent experiments using two independently prepared virus stocks. (B) Levels of Ifnb1 and Ifna4 mRNA in the lung were measured by qRT-PCR. Data are from 26 (mock) and 40–47 (infected) mice per group and are represented as expression relative to the housekeeping gene Gapdh ± s.e.m., pooled from 15 independent experiments. (C) The proportion of NP+ cells in the lung was quantified by flow cytometry. Exemplary FACS plots are shown on the left. Data are represented as percentage of single, live cells ± s.e.m and are from 3 (mock), 7 (PR8 WT) and 5 (PR8 FS) mice per group, pooled from two independent experiments (right). (D) Levels of the IAV M RNA were measured by qRT-PCR. Data are from 17 (mock), 37 (PR8 WT) and 35 (PR8 FS) mice per group ± s.e.m. and are pooled from nine independent experiments. (E) Viral loads in the right lungs of infected mice were determined using a TCID50 assay. Data are from 16 (mock; none detected) and 26 (infected) mice per group ± s.e.m. and are pooled from six independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns not significant; unpaired Student’s t-test.