Diagram of the PriMux software workflow.

<p>Diagram of the PriMux software workflow.</p

Text box listing important aspects of PriMux.

<p>Text box listing important aspects of PriMux.</p

Diagram of the PriMux approach.

<p>Diagram of the PriMux approach.</p

Rift Valley Fever (RVF) Degenerate Assay Signatures.

<p>Rift Valley Fever (RVF) Degenerate Assay Signatures.</p

Primer and probe (IO) sequences for RVFV run with all segments in a single input file.

<p>Primer and probe (IO) sequences for RVFV run with all segments in a single input file.</p

Multiplex set of primers and probes to detect all 2863 Dengue virus genomes.

<p>Multiplex set of primers and probes to detect all 2863 Dengue virus genomes.</p

Primer and probe combinations from Table 5, and number and serotypes of Dengue virus genomes detected by each primer/probe combination.

<p>Primer and probe combinations from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034560#pone-0034560-t005" target="_blank">Table 5</a>, and number and serotypes of Dengue virus genomes detected by each primer/probe combination.</p

A subset of the most commonly changed user-specifiable parameters in the options file.

<p>A subset of the most commonly changed user-specifiable parameters in the options file.</p

Multiplexed Digital mRNA Profiling of the Inflammatory Response in the West Nile Swiss Webster Mouse Model

<div><p>Background and purpose</p><p>The ability to track changes in gene expression following viral infection is paramount to understanding viral pathogenesis. This study was undertaken to evaluate the nCounter, a high throughput digital gene expression system, as a means to better understand West Nile virus (WNV) dissemination and the inflammatory response against WNV in the outbred Swiss Webster (SW) mouse model over the course of infection.</p><p>Methodology</p><p>The nCounter Mouse Inflammation gene expression kit containing 179 inflammation related genes was used to analyze gene expression changes in multiple tissues over a nine day course of infection in SW mice following intraperitoneal injection with WNV. Protein expression levels for a subset of these cytokine/chemokine genes were determined using a multiplex protein detection system (BioPlex) and comparisons of protein/RNA expression levels made.</p><p>Results</p><p>Expression analysis of spleen, lung, liver, kidney and brain of SW mice infected with WNV revealed that <i>Cxcl10</i> and <i>Il12b</i> are differentially expressed in all tissues tested except kidney. Data stratification of positively confirmed infected (WNV (+)) versus non-infected (WNV (−) tissues allowed differentiation of the systemic inflammatory gene response from tissue-specific responses arising from WNV infection. Significant (p<0.05) decrease in <i>C3ar1</i> was found in WNV (−) spleen. <i>Il23a</i> was significantly upregulated, while <i>Il10rb</i> was down-regulated in WNV (−) lung. <i>Il3</i> and <i>Mbl2</i> were down-regulated in WNV (−) liver. In WNV (+) livers, <i>Stat1</i>, <i>Tlr2</i>, chemokines <i>Cxcl1</i>, <i>Cxcl3</i>, <i>Cxcl9</i>, <i>Cxcl10</i>, cytokines <i>Il6</i>, <i>Il18</i>, cytokine-related gene <i>Il1r</i> and cytokine agonist <i>Ilrn</i> were significantly upregulated. In WNV (−) brain tissues, <i>Csf2</i> and <i>Cxcl10</i> were significantly upregulated. Similar gene and protein expression kinetics were found for <i>Ccl2</i>, <i>Ccl3</i>, <i>Ccl4 and Ccl5</i> and correlated with the presence of infectious virus. In summary, the utility of the nCounter platform for rapid identification of gene expression changes in SW mice associated with WNV infection was demonstrated.</p></div

Dynamic Protein Expression profiles in various tissues over course of infection.

<p>Heatmap of specific genes in (A) spleen, (B) lung, (C) liver, (D) kidney and (E) brain that had a significant change in protein expression per day over the course of infection. Green shaded boxes represent genes that were upregulated and red shaded boxes represent genes that were downregulated. Fold change for each gene is denoted within each shaded box. Data points represent n = 3 mice per day, p<0.05 consider statistically significant.</p