RNA-Seq analysis of DEGs in polarized MФs compared with sham control MФs in PBS post-PRRSV infection.

<p>The heatmaps of 6,624 significant DEGs (left) and numbers of potential signature genes were grouped based on significant up-regulation in only one activation status or co-stimulated in two activation statuses (see table at right, and the supplemented results of DEG statistics in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087613#pone.0087613.s005" target="_blank">Table S2</a>). FDR (false discovery rate) ≤0.001, fold change ≥2 for DEG significant determination. The color scale under the heatmap illustrates the log₂ (fold change) values shown in the heatmap.</p

AMPK-mediated pathways for antiviral regulation.

<p>(<b>A</b>) Heatmap of the subset DEGs in the AMP-kinase pathway, which is critical to control of lipid metabolism, are shown. (<b>B</b>) As illustrated in the pathway, most key genes in the M1 statuses (IFNγ- or LPS-induced) or IFNα-antiviral state (MaV) were differentially regulated, leading to a general suppression of lipid metabolism in contrast to a general increase in the M2-IL4 status. In addition, the illustration of AMPK-mediated pathways in other statuses is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087613#pone.0087613.s003" target="_blank">Figure S3</a> together with the dose-dependent suppression of PRRSV infection by two AMPK-pathway activators. <i>Legend:</i> green-line box, significant suppression; blue-line box, non-significant suppression; red-line box, significant up-regulation; yellow-line box, non-significant up-regulation; and black-line box, non-significant detection. <i>p</i> (FDR) <0.001, fold change ≥2 for significant determination.</p

Epigenetic mechanisms for antiviral regulation.

<p>(<b>A</b>) RNA-Seq analysis of DEGs encoding key enzymes in epigenetic regulation. The heatmaps display family-wide collections of genes encoding DNA/histone methyltransferases, histone deacetylases and histone demethylases. In addition to differential expression analysis, these data also revealed family-wide transcription evidence of most of these porcine epigenetic genes at the mRNA level for the first time. (<b>B</b>) Suppression of PRRSV infection by epigenetic inhibitors at optimized concentrations in MARC-145 cells and porcine MФs. The fluorescent micrographs (inset) show infected cells using a DsRed-labeled PRRSV, whereas the larger bright-field images show cell phenotypes of non-visible cytotoxic effects. The micrographs represent one of three replicates. Summary data are presented below the images.</p

Verification of DEGs at the protein level using a proteomic procedure.

<p>Equal amounts of protein from macrophages at different activation statuses were stained with either red or green fluorescent dyes and co-resolved using a 2D-DIGE procedure (Applied Biomics, Inc., Hayward, CA) to isolate protein spots that significantly increased in macrophages at certain activation statuses and to further identify the isolated proteins by nano LC-MS/MS. Of 16 significantly increased protein spots randomly selected across four activation statuses (black bars) compared with the M0-PBS status (the white bars), 12 (75%) protein spots (WARS, PLEK, RAN, CKB, PLOD3, RNF114, HNRNPU, ATP6V1E1, CANX, MX1, MX2, H2B3A) also showed significant up-regulation at the RNA level, with the other four (SND1, ANXA1, UBE2D3 and MPP5) showing a significant increase only at the protein level. *, FDR ≤0.001 of gene expression and protein ratio ≥2. The number before each gene symbol along the X-axis indicates the protein spot mapped in the gel shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087613#pone.0087613.s002" target="_blank">Figure S2</a>. Gene symbol abbreviations: SND1, staphylococcal nuclease domain-containing protein 1; WARS, tryptophanyl-tRNA synthetase; PLEK, pleckstrin; RAN, Ras-related GTP binding C; CKB, creatine kinase B-type; PLOD3, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3; RNF114, RING finger protein 114; HNRNPU, heterogeneous nuclear ribonucleoprotein U; ANXA1, annexin A1; ATP6V1E1, v-type proton ATPase subunit E 1; UBE2D3, ubiquitin-conjugating enzyme E2 D3; CANX, calnexin; MX, myxovirus resistance gene; H2B3A, histone H2B3A; MPP5, membrane protein palmitoylated 5.</p

Transcriptomic analysis of selected transcription factor (TF) families.

<p>Members of these TF families have been shown to be critical to the regulation of activation status and antiviral activity in murine monocytic cells. Differential expression of TF families of (<b>A</b>) suppressors of cytokine signaling (SOCS), (<b>B</b>) Kruppel-like factors (KLF), and (<b>C</b>) interferon regulatory factor (IRF) in polarized MФs upon PRRSV infection are shown. (<b>D</b>) The differential expression of IRF family was verified with a real-time RT-PCR assay (the Y-axis scale indicating fold change to M0-PBS). The color scale under each heatmap illustrates the midpoint and range of reads per kilobase per million (RPKM) values of listed transcripts.</p

Pathway analysis of DEGs was annotated against the KEGG database.

<p>A <i>p</i>-value and FDR of <0.05 in the two-sided Fisher’s exact test were considered significant. Selected pathway categories are shown along the vertical axis, and the horizontal axis represents the log₁₀ (<i>p</i> value) of these pathways showing the significant difference among cells at different activation statuses.</p

The Wood's(4A) and Extended Wood's(4B) model mean predictions and data-prediction correlations.

<p>The black lines outline the mean model predictions, and the dashed grey lines delimit the 95% prediction confidence intervals. The dotted black lines joined with crosses show the Pearson product-moment correlations between observed data and predicted values.</p

The Wood's and Extended Wood's model parameter's Pearson product-moment correlations.

<p> Upper triangle in bold: Wood's model parameter correlations. Lower triangle: correlations between the Extended Wood's model parameters.</p

Summary of the statistical classifications of the viremia profiles from the PRRSV challenge experiment

<p>% death prior to 21 dpi. Due to facility availability issues trials 7 and 8 had to be terminated at 35 dpi. Overall 17% were rebound, 38% were persistent and the remaining 45% were clearance profiles. Classifications of the viremia profiles are based on the likelihood ratio test comparing the Wood's and the Extended Wood's models. Trial 6 had 48</p

The Wood's model residuals (3A) and the Extended Wood's model residuals (3B).

<p>The red line shows the residual mean and the blue lines delimit two standard deviations from the mean.</p