Growth of viruses in primary macrophages.

<p>Primary macrophages were harvested from the lungs of healthy BALB/c mice through tissue digestion (A) and developed from human peripheral blood monocytes (B) and infected <i>in vitro</i> (MOI = 0.1) with influenza viruses as described (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat-1000115-t001" target="_blank">Table 1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#s4" target="_blank">Methods</a>). Mouse lung macrophages were grown in 12 well plates and infected with viruses in duplicate and the supernatants sampled for virus growth. Human macrophages were grown in 6 well plates and also infected in duplicate. Virus titers were determined from supernatants in duplicate by plaque assay on MDCK cells. Graphs are representative of results obtained from three independent infection experiments. ˆ <i>p</i><0.05 between Thai/16 and 1918, SP/83 and TX/91 viruses 72 hrs p.i (A). * <i>p</i><0.05 between HP Thai/16 and 1918, and LP SP/83 and TX/91 viruses 48 hrs p.i. (B). ^†<i>p</i><0.05 between the 1918 virus and other viruses 2 hours post- infection (B).</p

Viruses used in this study.

†<p>Abbreviations used in the text: TX/91, 1918, SP/83, Thai/16</p>*<p>Fifty-percent mouse lethal dose (log10) titers.</p>‡<p>NL = Not mouse lethal <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat.1000115-Maines1" target="_blank">[18]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat.1000115-Tumpey4" target="_blank">[21]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat.1000115-Tumpey5" target="_blank">[22]</a>.</p>ˆ<p>Generated by reverse genetics <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat.1000115-Tumpey4" target="_blank">[21]</a>.</p

Mouse lung immune cell population dynamics during influenza virus infection.

<p>BALB/c mice were infected intra-nasally with 102 PFU influenza viruses (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat-1000115-t001" target="_blank">Table 1</a>). Lung immune cell populations were analyzed by flow cytometry from single cell suspensions from three mice per time point, per virus group.</p>‡<p>Numbers of immune cell sub-populations are represented here as percent of the total leukocytes measured in these assays. Standard deviations are shown in parenthesis where available. PBS group % populations represented are daily averages.</p>*<p>p<0.05 between HP (1918, Thai/16) and LP (TX/91,SP/83) infection groups.</p>ˆ<p>p<0.05 between 1918 and TX/91 infection groups.</p>±<p>p<0.05 between Thai/16 and SP/83 infection groups.</p>†<p>Not analyzed.</p

Cytokine response from infected human macrophages.

<p>Primary macrophages were developed from human peripheral blood monocytes in 6 well plates and infected <i>in vitro</i> (MOI = 0.1) with influenza viruses in duplicate as described (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#s4" target="_blank">Methods</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat-1000115-t001" target="_blank">Table 1</a>). Supernatants were sampled 48 hours post-infection and cytokines measured by Bioplex Protein Array assay (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#s4" target="_blank">Methods</a>). Macrophages were also treated with bacterial lipopolysaccharide (LPS, 100 ng) and Poly I/C (100 ng) to serve as positive inducers. * <i>p</i><0.05 between H5N1 and H1N1 viruses.</p

Virus replication from primary lung macrophages and dendritic cells cultured <i>ex vivo</i>.

<p>BALB/c mice were infected intranasally with 10² PFU of the indicated viruses. Three days post-inoculation, lungs were removed from 2–3 mice per virus group. Cell suspensions were prepared from pooled samples and macrophages (A) and dendritic cells (B) were isolated by CD11b+ ad CD11c+ MACS column purification, respectively. Supernatants from cultures were sampled over time to measure virus production. Virus was titered in duplicate by plaque assay.</p

Lung cytokine response.

<p>Cytokine levels from infected lungs (n = 3 mice per virus group, days 1 and 4 post-inoculation (p.i.)) were measured individually and in duplicate by the Bioplex Protein Array system lungs. Baseline cytokine levels from PBS inoculated mice (mock) are shown as a dashed line in each cytokine graph. Bars represent means of 3 mice from each infection group±standard deviation (SD). Protein levels of IL-6 in Thai/16 infected animals exceeded the y scale shown (indicated by a dash//, the concentration in Thai/16 infected mice was 11.7 ng/ml). ˆ <i>p</i><0.05 between 1918 and TX/91 viruses, * <i>p</i><0.05 between 1918 and Thai/16 (HP) virus groups and SP/83 and TX/91 (LP).</p

Lung virus titers.

<p>Female BALB/c mice were infected intranasally with 10² PFU of influenza viruses and lungs were harvested for virus titration at various times post-inoculation. Lungs were homogenized in 1 ml of PBS and virus titers determined by plaque assay (+ TPCK trypsin 1 µg/ml) on MDCK cells in duplicate (n = 3 mice per time point). * <i>p</i><0.05 between 1918 and Thai/16 infected lungs and TX/91 and SP/83 infected lungs.</p

Growth of viruses in primary dendritic cells.

<p>Primary dendritic cells were isolated from the lungs of healthy BALB/c mice and developed from human monocytes as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#s4" target="_blank">Methods</a> and infected <i>in vitro</i> with LP and HP influenza viruses (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000115#ppat-1000115-t001" target="_blank">Table 1</a>). Growth of H5N1 and H1N1 viruses in mouse lung macrophages (A, MOI = 0.1) and in human monocyte-derived dendritic cells (B, MOI = 1.0). (ˆ <i>p</i><0.05 between HP Thai/16 and 1918 viruses and LP SP/83 and TX/91 viruses. * <i>p</i><0.05 between Thai/16 and 1918, SP/83 and TX/91 viruses.) Graphs are representative of results obtained from three independent experiments.</p

Incidence of viral replication in ferrets following 10⁶ EID₅₀ ocular inoculation.

a<p>Limit of virus detection in nasal wash (NW) and rectal swab (RS) was 10^1.5 EID₅₀/ml, conjunctival wash (CW) was 10^0.8 EID₅₀/ml.</p>b<p>Titer of ferrets with positive virus isolation expressed as log₁₀ EID₅₀/ml ± standard deviation.</p>c<p>ND, not detected.</p

Comparison of influenza virus recovery in conjunctival wash samples following ocular inoculation of ferrets.

<p>Ferrets were inoculated by the ocular route with 10⁶ EID₅₀/ml of each virus shown. Viral titers were measured in conjunctival washes (CW) collected on indicated days following serial titration in eggs; endpoint titers are expressed as mean log₁₀ EID₅₀/ml plus standard deviation (left y-axis and bars). Relative viral RNA copy number in conjunctival washes was determined by real-time PCR using a universal M1 primer and extrapolated using a standard curve based on samples of known virus (right y-axis and lines). The limit of virus detection was 10^1.5 EID₅₀/ml. †, no ferrets survived until day 9 p.i. R-squared values are shown for those viruses where a statistically significant (p<0.05) correlation between viral titer and viral RNA copy number exists. NS, not significant.</p