Virus infection and quantification

Mouse-adapted influenza A viruses, A/Puerto Rico/8/34 (PR8; H1N1) provided by Dr. Maryna Eichelberger at the Food and Drug Administration (FDA) and A/California/04/09 (ma2009; H1N1) generated by Dr. Andrew Pekosz from a published sequence [54], were used in these studies. Mice were anesthetized and inoculated intranasally with 30 μl of DMEM (mock) or H1N1 virus (1.78 50% mouse lethal dose (MLD₅₀) for PR8 and 0.4 MLD₅₀ for ma2009). Clinical disease scores for IAV-infected mice were based on four parameters, with one point given for each of the following: dyspnea, piloerection, hunched posture and absence of an escape response. For virus quantification, log₁₀ dilutions of lung homogenates (starting at 10⁻¹) were plated onto a monolayer of MDCK cells in replicates of 6 for 4–6 days. Cells were stained with naphthol blue black (Sigma Aldrich) and scored for cytopathic effects. The 50% tissue culture infectious dose (TCID₅₀) was calculated according to the Reed-Muench method.