Detection of virus shedding in nasal and pharyngeal swabs. (A) Virus shedding in nasal swabs (solid lines) and pharyngeal swabs (dashed lines). The gray trend line joining the average values of the virus titers indicates the A/swine/Alberta/OTH-33-8/2009 isolate; the black trend line indicates the virus isolates from piglets infected with A/Mexico/InDre4487/2009. (B) Detection of viral RNA in nasal swabs (solid lines) and pharyngeal swabs (dashed lines). The gray trend line joins the average copy numbers/ml detected in piglets infected with the A/swine/Alberta/OTH-33-8/2009 isolate; the black line joins the average copy numbers/ml detected in piglets infected with the A/Mexico/InDre4487/2009 isolate. dpi (days p.i.).

Virus and viral-RNA detection in the lower respiratory tract. (A) The black solid trend line indicates RNA detection (copy numbers per 0.1 g of tissues) in the tissue lung homogenates (right apical lobe) from piglets infected with the A/Mexico/InDRE4487/2009 isolate, and the gray line indicates RNA detection in the lung tissue homogenates from a pig infected with the A/swine/Alberta/OTH-33-8/2009 isolate. The black dashed trend line represents the virus isolation from the human isolate and the gray dashed line represents the virus isolation from the lungs of pigs infected with the swine isolate. (B) The black solid trend line indicates RNA detection (copy number per 1 ml) in the BALF from piglets infected with the A/Mexico/InDRE4487/2009 and the gray line indicates RNA detection in the BALF from a pig infected with A/swine/Alberta/OTH-33-8/2009. The black dashed trend line represents the virus isolation from the human isolate; no virus was isolated from BALF of the piglets infected with the A/sw/Alberta/OTH-33-8/2009 isolate.

Phylogenetic trees for H1 and N1 gene sequences. (A) Unrooted nucleotide phylogenetic tree of H1 HA from selected H1N1 and H1N2 influenza A virus isolates of swine, human, and avian origin. The swine viruses were chosen to be representative of current, as well as historical, isolates from North America and Eurasia. The tree is one of a total of 19 equally parsimonious trees that were inferred using maximum parsimony, a close-neighbor interchange search, and 500 bootstrap replicates (bootstrap values are shown on the tree). The scale indicates the number of nucleotide changes. (B) Unrooted nucleotide phylogenetic tree of N1 NA from selected Eurasian and North American swine, human, and avian influenza A virus isolates. The swine isolates are representative of viruses that have been circulating in pigs in Eurasia since the early 1990s. The tree is one of a total of 59 equally parsimonious trees that were inferred using maximum parsimony, a close-neighbor interchange search, and 500 bootstrap replicates (bootstrap values are shown on the tree). The scale indicates the number of nucleotide changes.

Histopathology and IHC detection of the nucleoprotein antigen in lungs. (a) IHC staining in the lung of a piglet inoculated with A/swine/Alberta/OTH-33-8/2009 at 3 days p.i. showing nucleoprotein-positive staining in epithelial cells lining the bronchiole, as well as in macrophages (arrowheads) in the luminal exudates and interstitium. Bar = 50 μm. (b) IHC staining in the lung of a piglet inoculated with A/Mexico/InDRE4487/2009. Specific immunolabeling for influenza A virus nucleoprotein is confined to bronchiolar epithelial cells. The arrow indicates the junction between the bronchiole and alveolus. Bar = 50 μm. (c) Piglet infected with A/swine/Alberta/OTH-33-8/2009, 5 days p.i. Shown is relatively prominent staining of the bronchiolar epithelial cells; the arrowhead indicates an example of staining in macrophages. Bar = 20 μm. (d) Strong prominent immunolabeling for the nucleoprotein in the epithelial cells of bronchioli, with some staining in macrophages (arrowhead) of a piglet infected with the A/Mexico/InDRE4487/2009 isolate, 5 days p.i. Bar = 20 μm. (e) Specific staining for the nucleoprotein in epithelial cells of a small terminal bronchiole and in macrophages in the interstitium (arrowhead) from a piglet infected with the A/swine/Alberta/OTH-33-8/2009 isolate, 7 days p.i. Bar = 50 μm. (f) Serial section of the bronchiole shown in panel e. The small terminal bronchiole is lined by hyperplasic epithelium and surrounded by a moderate number of inflammatory cells. Bar = 50 μm. (g) Small bronchioles with hyperplasic epithelium surrounded by a very intense inflammatory reaction in a piglet infected with A/Mexico/InDRE4487/2009, 7 days p.i. Bar = 50 μm. (h) Only a few individual bronchiolar epithelial cells were positively stained for the nucleoprotein at 13 days p.i. in both human and swine isolates. Shown is an example of the IHC labeling in the lung of a piglet inoculated with the A/Mexico/InDRE4487/2009 isolate. Bar = 20 μm.