All animal research was conducted under the guidance of CDC's Institutional Animal Care and Use Committee and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. 8-10 week old female BALB/c mice (Harlan, Indianapolis, IN) were anesthetized with Avertin [20] (Sigma-Aldrich) and infected intranasally (i.n.) with 50 ml of 10 2 PFU of influenza viruses prepared in phosphate buffered saline (PBS). Avertin was chosen as the anesthetic because it provides consistent mouse infections with the viruses used in these studies. Using the sublethal (10 2 PFU) inoculum, 1918 and Thai/16 virus infected mice survive a prolonged disease course allowing for the measurement of the influx of inflammatory cells into the lung tissue during a full course (,7-9 days) of influenza virus infection. At indicated times post-infection (n = 3 mice per virus group) mice were euthanatized and exsanguinated. Lungs were removed from individual mice without PBS perfusion and included total lung cell counts included cells located in the bronchoalveolar airways. Perfusion was not possible in many cases of HP influenza virus infection due to the presence of microvascular hemorrhage. We obtained similar results when performing these lung cell quantitation assays with or without lung perfusion in LP virus infected mice and therefore did not introduce this variable in our high containment laboratory. Whole lung cell suspensions were prepared in Dulbecco's minimal essential media (DMEM) with 20% fetal calf serum following collagenase-DNase treatment and manual disruption [47] . Red blood cells were removed by lysis buffer treatment (Sigma-Aldrich). Total viable lung cell number was determined for each mouse by trypan blue exclusion on a hemocytometer.