Geographic variation in prevalence of seropositivity to A(H1N1)pdm09 in 2009 (circles) and the proportion of the population reporting A(H1N1)pdm09 vaccination (squares) during the same time period, by state with 95% confidence intervals.

<p>Values are age–standardized to the US population.</p

Prevalence of Seropositivity to Pandemic Influenza A/H1N1 Virus in the United States following the 2009 Pandemic

<div><h3>Background</h3><p>2009 pandemic influenza A/H1N1 (A(H1N1)pdm09) was first detected in the United States in April 2009 and resulted in a global pandemic. We conducted a serologic survey to estimate the cumulative incidence of A(H1N1)pdm09 through the end of 2009 when pandemic activity had waned in the United States.</p> <h3>Methods</h3><p>We conducted a pair of cross sectional serologic surveys before and after the spring/fall waves of the pandemic for evidence of seropositivity (titer ≥40) using the hemagglutination inhibition (HI) assay. We tested a baseline sample of 1,142 serum specimens from the 2007–2008 National Health and Nutrition Examination Survey (NHANES), and 2,759 serum specimens submitted for routine screening to clinical diagnostic laboratories from ten representative sites.</p> <h3>Results</h3><p>The age-adjusted prevalence of seropositivity to A(H1N1)pdm09 by year-end 2009 was 36.9% (95%CI: 31.7–42.2%). After adjusting for baseline cross-reactive antibody, pandemic vaccination coverage and the sensitivity/specificity of the HI assay, we estimate that 20.2% (95%CI: 10.1–28.3%) of the population was infected with A(H1N1)pdm09 by December 2009, including 53.3% (95%CI: 39.0–67.1%) of children aged 5–17 years.</p> <h3>Conclusions</h3><p>By December 2009, approximately one-fifth of the US population, or 61.9 million persons, may have been infected with A(H1N1)pdm09, including around half of school-aged children.</p> </div

Comparison of the reverse cumulative distribution of HI titers between baseline and 2009 serum specimens, by age group.

<p>Comparison of the reverse cumulative distribution of HI titers between baseline and 2009 serum specimens, by age group.</p

Vaccination and adjusted estimates of the increase in seropositivity from baseline to December 2009 due to natural infection with A(H1N1) pdm2009.

<p>SE = Standard error, CI = Confidence interval.</p>a<p>Age-standardized to the US population.</p>b<p>Adjusting for overlap from vaccination among people already infected with A(H1N1)pdm09. See methods for description of adjustment.</p>c<p>After adjusting for vaccination and assuming the following HI test characteristics: Sensitivity = 75%, Specificity = 97% (for ages <65) or 94% (for ages 65+).</p

Overall prevalence of seropositivity to A(H1N1)pdm09 at baseline and December 2009, by age group.

a<p>Estimates were weighted using the adjusted sample weight for this sample, WTH1N1.</p>b<p>Assuming no pre-existing seropositivity at baseline.</p>c<p>Age-standardized to the U.S. Census Bureau population estimates as of July 1, 2009 by age groups in the table.</p

National trends in influenza–like illness (ILI) (solid line)^a and coverage with A(H1N1)pdm09 vaccine (dashed line)^b in the United States, and the period of sera collection.

<p>^a Source: U.S. Outpatient Influenza-like Illness Surveillance Network (ILINet). ^b Source: CDC estimates from combined Behavioral Risk Factors Surveillance System (BRFSS) and National 2009 H1N1 Flu Survey (NHFS) data.</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

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

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

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