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For the mock group, 25?��l of PBS was used. Body weight, clinical signs of infection, and survival were monitored daily for 14?days and mice with body weight loss greater than 25% of pre-infection values were euthanized humanely. Three mice from each group were sacrificed respectively on days 1, 2, 3, 4, 7 post-infection (d.p.i.). The remainders (7 mice per group) were kept for observation and euthanatized at the end of the experiment (14?d.p.i.). Tissues for viral Lenvatinib cost titrations were removed and kept at ??80?��C until homogenized in a 9 fold volume of cold PBS and clarified by centrifugation. Virus titers of homogenates were determined by plaque assays in MDCK cells and by real-time PCR. Freshly excised tissues of the trachea, lungs, intestines and brains from the mice were buy Fluorouracil preserved in 10% phosphate-buffered formalin. After 24?h' fixation, tissues were dehydrated, embedded in paraffin, and cut into 5-��m-thick sections. For sections from each tissue sample, a standard histochemistry staining procedure with hematoxylin and eosin (Sigma) (H&E) as well as immunohistochemistry (IHC) assay with a mouse anti-NP monoclonal antibody (provided by the National Institute of Diagnostics and Vaccine Development for Infectious Diseases, Xiamen University) was performed in parallel. Goat anti-mouse IgG�Cbiotin conjugate secondary antibody was purchased from Calbiochem. Specific viral antigen was visualized by 3,3��-diaminobenzidaine tetrahydrochloride staining as brown. Nine discrete, non-overlapping fields of view with a magnification of 400�� were observed for Sitaxentan each section and the number of positive viral antigen was calculated. Viral RNA was extracted from CA04 stocks using QIAamp viral RNA minikit (QIAGEN) and cDNAs were synthesized with Uni12 or random primers using Transcriptor High Fidelity cDNA synthesis Kit (Roche). Copy numbers of the influenza M segment were determined on a LightCycler? 480 Real-Time PCR System (Roche) with LightCycler? FastStart DNA MasterPLUS SYBR Green I Kit (Roche) and normalized by the expression of ��-Actin gene. Primers used for the analysis of M gene were M-146F (GACCRATCCTGTCACCTCTGAC) and M-251R (AGGGCATTYTGGACAAAKCGTCTA), while those for ��-Actin gene were Act-1132F (TGGATCAGCAAGCAGGAGTATG) and Act-1188R (GCATTTGCGGTGGACGAT). Statistical analysis was performed by Mean Analysis with PASW Statistics 18 (SPSS Inc., Chicago IL). The probability of a significant difference was computed using ANOVA (analysis of variance). Results were considered significant at P?