FIG 3.

Neutrophils were detected in ferret lung sections after infection with influenza A virus (H1N1pdm). Female ferrets (2 per time point) were infected with 10⁶ TCID₅₀s each of the H1N1pdm virus (A/Kentucky/180/2010). (A) The right caudal lung lobe from H1N1pdm virus-infected and mock-infected (1 ferret per time point) ferrets was taken upon euthanasia on days 1, 2, 3, and 7 postinfection and was divided into four sections. Each section was homogenized separately, and cDNA was synthesized for gene expression analysis. Ferret gene-specific primers were developed to cross exons, and RT-PCR was performed to identify the expression of a neutrophil-expressed gene (Csf3r, encoding G-CSFR) relative to the expression of housekeeping controls and to expression in mock-infected animals. Asterisks indicate significant differences between days by nonparametric statistical tests, corrected for multiple comparisons. (B) Other lung lobes (left cranial, right cranial, left caudal, and middle) taken from each ferret were prepared for IHC, and one slice from each was stained for neutrophils (NACE). Neutrophils were quantified in images (taken with a 20× objective) from 3 regions on each of the NACE-stained slides by using ImageJ image analysis software. The number of NACE-positive pixels divided by the number of total tissue-containing pixels is presented as the neutrophil density. The asterisk indicates a significant difference in distribution from that in mock-infected ferrets by nonparametric statistical tests.