Real-time cell analysis of recombinant HMPV strains.

<p>LLC-MK2 monolayers in 96 well-plates were infected with rHMPV at an MOI of 0.01 (a) Output of one real-time cell analysis (RTCA) experiment; data was normalized using mock-infected wells and normalized cell index is plotted. (b) Mean time until cell index is reduced by 50% from 4 independent experiments is plotted. *, p < 0.05 using unpaired, two-tailed Student t-test.</p

Pulmonary cytokine levels of BALB/c mice infected with recombinant HMPV strains.

<p>BALB/c mice were infected with 6x10⁵ TCID₅₀ of rHMPV (as determined by back-titration). On days 3 through 6, four mice per group were euthanized to determine pro-inflammatory cytokine/chemokine levels in the lungs of infected mice. Mock-infected mice are representad as day 0. ***, p < 0.001; **, p < 0.01; * p < 0.05 comparing all other strains to rC-85473.°°°, p < 0.001; °°, p < 0.01; °, p < 0.05 comparing all other strains to rCAN98–75 using Repeated Measures Two-way ANOVA.</p

Cytopathic effects of HMPV strains and recombinant HMPV viruses.

<p>(a) microscopic images of cytopathic effects induced by HMPV infection of LLC-MK2 monolayers. CAN98–75 (B2) induces focal cell-rounding (left) whereas C-85473 (A1) induces multinucleated syncytia (right). Magnification = 10x. (b) Representation of the genomes of the 4 recombinant viruses used in this study; rC-85473 and rCAN98–75 represent the wild-type strains, rC-85473_F represents the chimeric rC-85473 strain in which the F gene has been replaced with that of CAN98–75 and rCAN98–75_F represent the chimeric rCAN98–75 in which the F gene has been replaced with that of C-85473.</p

Lung viral titers and weight loss of BALB/c mice infected with recombinant HMPV strains.

<p>BALB/c mice were infected with 6x10⁵ TCID₅₀ of rHMPV (as determined by backtitration). (a) On days 3 through 6, four mice per group were euthanized to determine pulmonary viral titers. (b) Six mice per group were monitored for weight loss on a daily basis for 14 days. ***, p < 0.001; * p < 0.05 comparing all other strains to rC-85473.°°°, p < 0.001; °, p < 0.05 comparing all other strains to rCAN98–75 using Repeated Measures Two-way ANOVA.</p

Syncytium formation induced by recombinant HMPV strains.

<p>(a) LLC-MK2 monolayers in 24 well-plates were infected with rHMPV at an MOI of 0.01 in quadruplicate. On days 2 through 4 pi, pictures were taken using fluorescent microscopy in 3 random fields (20x magnification) per well and the number of nuclei per GFP-expressing cell was calculated. ***, p < 0.001 comparing all other strains to rC-85473 and °°°, p < 0.001 comparing all other strains to rCAN98–75 using Repeated Measures Two-way ANOVA. (b) An example of the observed difference in syncytium formation between the 4 recombinant strains on day 3 pi.</p

Boxplots of gene expression levels in the lung for <i>BC029578</i> according to genotype groups for SNP rs7667092.

<p>The left y-axis shows the mRNA expression levels for <i>BC029578</i>. The x-axis represents the three genotype groups for SNP rs7667092. The right y-axis shows the proportion of the gene expression variance explained by the SNP (black bar). Each panel represents a different cohort: Laval (n = 406), UBC (n = 287), Groningen (n = 342). The eQTL p-values were 1.8×10⁻⁸, 7.3×10⁻¹⁰ and 6.0×10⁻¹¹, respectively.</p

Clinical characteristics of patients that passed gene expression and genotyping quality control filters.

<p>FEV₁ : forced expiratory volume in 1 second.</p><p>FVC : forced vital capacity.</p><p>[-] = missing value.</p>*<p>pre-BD: pre-bronchodilator.</p

Linkage disequilibrium plot of selected SNPs on the 4q22 locus in the 1000 Genome Project.

<p>The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r²) are indicated in each box. The color of the squares illustrates the strength of pairwise r² values on a black and white scale where black indicates perfect LD (r² = 1) and white indicates perfect equilibrium (r² = 0). The genotypes are from the 1000 Genome Project interim phase 1 release (2010/11/23). Red rectangles are SNPs previously associated with COPD (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070220#pone-0070220-t002" target="_blank">Table 2</a>). Blue rectangles are the most significant eQTL-SNPs for the four regulated genes found on 4q22 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070220#pone-0070220-g001" target="_blank">Figure 1</a>). The other illustrated SNPs were genotyped in our study and in LD (r²>0.5) with COPD SNPs.</p

SNPs associated with COPD in previous GWAS.

<p>SNPs associated with COPD in previous GWAS.</p

Lung eQTLs on 19q13 in the Laval dataset.

<p>Each dot represents an association test between one SNP and one probeset. Only dots above the red line are significant (p<7.1×10⁻⁷). Significant SNPs were regulating the expression levels of <i>ZNF780A</i> in brown, <i>SERTAD3</i> in light blue, <i>NUMBL</i> in orange, <i>EGLN2</i> in dark green, <i>CYP2G1P</i> in dark grey, <i>AXL</i> in yellow, <i>B3GNT8</i> in blue, <i>LOC100505495</i> in red, <i>CEACAM21</i> in green, and <i>CEACAM4</i> in purple. The SNP with the smaller p-value is indicated. SNPs previously associated with COPD are illustrated at the bottom.</p