Exposure of human lung to both pandemic and prototypic influenza virus results in viral infection and replication in alveolar cells.

<p>(A) The lung slices were processed for immunohistochemistry for detection of viral NP using rabbit polyclonal antibody (red). Panels a, b and c show mock (virus dilution buffer), OK/09 and PR8 infection, respectively. Panels d, e and f are corresponding bright-field images that demonstrate that lung architecture is preserved during the experiment. (B) Replication of influenza virus OK/09, OK/06 and PR8 in the human lung organ culture model. Lung slices exposed to virus at 6×10⁶ PFU/ml were cultured for various times and total cellular mRNA was extracted. Quantitative RT-PCR was performed using Oligo dT as the primer for the first strand synthesis. Primers specific for NP were used to examine NP mRNA expression.</p

A time course study showing pandemic OK/09 impairs RIG-I and IFN-β antiviral immune responses compared to OK/06 and PR8 in human lung.

<p>Human lung slices were exposed to 6×10⁶ PFU/ml of PR8 or OK/09 for 4 h, 8 h, 12 h, 16 h, 24 h, and 32 h. Total RNA was then isolated from lung slices. Quantitative RT-PCR was performed using 100 ng sample RNA and SYBR Green (Quanta Biosciences) in a BIO-RAD iCycler IQ instrument (Hercules, CA). Transcript levels of RIG-1 (<i>A</i>) and IFN-β (<i>B</i>) were normalized relative to the constitutively expressed β-actin gene. Data are from 3 separate experiments. *, p<0.05 for PR8 to OK09. #, p<0.05 for OK/06 to OK/09.</p

Pandemic OK/09 induces a diminished antiviral but not proinflammatory cytokine protein response as PR8 in human lung.

<p>For each data point, multiple lung slices were exposed to 6×10⁶ PFU/ml of influenza virus PR8 and OK/09 and allowed to incubate at 37°C, 5% CO₂ for the indicated periods. Virus diluent was used as a negative control, and PMA (100 ng/ml) was used as a positive control. Chemokine and cytokine protein levels were determined by ELISA on lung slice supernatants. Data are expressed as the means ± SEM from three separate lung slice donor experiments. Statistical significance was determined by ANOVA. Means were compared to data from the negative control group.</p

Pandemic OK/09 virus induces a similar TLR7 but diminished NOD2 mRNA expression compared to PR8 virus in human lung.

<p>Lung slices were exposed to 6×10⁶ PFU/ml of PR8 and OK/09 for 8 h and 24 h. Relative end-point RT-PCR was used to determine mRNA expression. Transcript levels of Toll-like receptor (TLR) 3, TLR 7, and nucleotide-binding oligomerization domain 2 (NOD2) were normalized relative to GAPDH mRNA expression. Data are representative of 3 separate experiments.</p

Infectivity of influenza virus and IP-10 induction in macrophages in human lung.

<p>Lung slices were exposed to 6×10⁶ PFU/ml of influenza virus PR8 or OK/09 or virus diluents for 24 h in the presence of brefeldin A (BFA) to enhance the detection of cytokines. Slices were then processed for immunohistochemistry for the detection of the chemokine IP-10 using goat polyclonal antibodies, viral nucleoprotein (NP) using rabbit polyclonal antibody, and macrophages using anti-CD68 monoclonal antibody. Nuclei were stained with SYTOX green. <i>Top</i>: OK/09. <i>Bottom</i>: PR8. <i>A–D</i>: fluorescent images that demonstrate nuclei (<i>A</i>; blue), NP (<i>B</i>; red), IP-10 (<i>C</i>; green), and macrophages (<i>D</i>; cyan). <i>E</i>: bright-field images that demonstrate that lung architecture is preserved during the experiment. <i>F</i>: overlays of the fluorescent images that demonstrate that influenza induces IP-10 in CD68 positive intraalveolar cells, likely alveolar macrophages (arrows). Bars = 100 µm.</p

RIG-I and IP-10 induction in epithelial cells in the human lung.

<p>Lung slices were exposed to 6×10⁶ PFU/ml of influenza virus PR8 or OK/09 or virus diluents for 24 h in the presence of BFA to enhance the detection of cytokines. Slices were then processed for immunohistochemistry for the detection of IP-10 using goat polyclonal antibodies, RIG-I using rabbit polyclonal antibody, and epithelial cells using anti-cytokeratin monoclonal antibody. <i>Top</i>: OK/09. <i>Bottom</i>: PR8. <i>A–D</i>: fluorescent images that demonstrate nuclei (<i>A</i>; blue), RIG-I (<i>B</i>; red), IP-10 (<i>C</i>; green), and epithelial cells (<i>D</i>; cyan). <i>E</i>: bright-field images. <i>F</i>: overlays of the fluorescent images that demonstrate that influenza induces RIG-I in epithelial cells (arrows). Bars = 100 µm.</p

Pandemic OK/09 induces lower RIG-I protein responses than PR8 in human lung.

<p>Human lung slices were exposed to 6×10⁶ PFU/ml of PR8 or OK/09 for 8 h and 24 h. Western blot analysis was used to determine RIG-I protein expression in lung slices. Membranes were probed with anti-RIG-I or anti-GAPDH antibodies. Protein expression of RIG-I was normalized relative to GAPDH. Data are representative of 3 separate experiments.</p