DENV-2 RNA levels increase WT and SK1-/- mice following ic DENV infection.

<p>WT and SK1^-/- mice were ic infected with DENV, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169814#pone.0169814.g001" target="_blank">Fig 1</a>. RNA was isolated from infected mice brain tissues and analysed by real time qRT-PCR for DENV. A. Total DENV-2 RNA increases with time in WT mice, n = 3 at each time point; B. RNA was isolated from infected WT and SK1^-/- mice brain tissues at 3 dpi, n = 8 for each strain; C. RNA was isolated from infected mice brain tissues at the time of humane sacrifice, representing 7 (n = 7), 8 (n = 5), 9 (n = 4) or 14 (n = 1) dpi for WT and 7 (n = 7), 8 (n = 3), 9 (n = 2) or 14 (n = 1) dpi for SK1^-/- mice. Each symbol represents an individual mouse sample. Data represent average PCR values from individual mice. Statistical significance was assessed by unpaired Student <i>t</i>-test.</p

T-cell infiltration in the brain of WT mice following ic infection with DENV-2.

<p>WT mice were ic infected with DENV, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169814#pone.0169814.g001" target="_blank">Fig 1</a> and brain tissue was harvested. <b>A.</b> at 3 and 6 dpi, tissue was fixed and processed for H&E staining. Images are representative of n = 3 mice. Arrows indicate sites of cellular infiltrate; <b>B.</b> at the indicated time point RNA was extracted and CD4 and CD8 mRNA determined by qRT-PCR with n = 3 mice at each time point. Data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. Statistical significance was assessed by unpaired student <i>t</i>-test. * = p < 0.05.</p

Primer sequences used in this study for qRT-PCR.

<p>Primer sequences used in this study for qRT-PCR.</p

Susceptibility of WT and SK1^-/- mice to DENV-2 infection.

<p>3–4 week old C57BL/6 WT (n = 17) and SK1^-/- (n = 13) mice were ic injected with 800 pfu DENV-2 MON601. Mock WT (n = 7) and SK1^-/- (n = 4) control mice were ic injected with vehicle only. Body weight and neurological symptoms were recorded. Body weight is expressed as a percentage of initial body weight. Survival reflects mice that do not show neurological symptoms or >10% of body weight loss. <b>A and B.</b> Comparison of body weight and survival curves of mock with DENV-infected WT mice; <b>C and D.</b> Comparison of body weight and survival curve of mock with DENV-infected SK1^-/- mice; <b>E and F.</b> Comparison of body weight percentage and survival curves of DENV-infected WT and SK1^-/- mice. Data are expressed as mean ± SEM. Statistical analysis of survival curves were determined by long-rank test. * = p < 0.05, ** = p < 0.005.</p

The time course of induction of IFN-β and ISGs in WT mice following ic infection with DENV-2.

<p>WT mice (n = 3 at each time point) were ic infected with DENV, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169814#pone.0169814.g001" target="_blank">Fig 1</a>. At the time point indicated RNA was isolated from infected mice brain tissues and analysed by real time qRT-PCR for <b>A.</b> IFN-β; <b>B.</b> ISGs viperin, Ifi27l2a, IRF7 and CXCL10. Data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. Statistical significance was assessed by unpaired student <i>t</i>-test * = p < 0.05, ** = p < 0.005, *** = p < 0.0005.</p

T-cell infiltration in the brain of WT and SK1^-/- mice following ic infection with DENV-2.

<p>WT and SK1^-/- mice were ic infected with DENV, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169814#pone.0169814.g001" target="_blank">Fig 1</a> and brain tissue harvested at 3 dpi and end stage disease. RNA was isolated from infected mice brain tissues and analysed by real time qRT-PCR for <b>A.</b> CD4 mRNA; <b>B.</b> CD8 mRNA. At 3 dpi n = 8 WT and SK1^-/- DENV-infected, n = 2 WT mock and n = 3 SK1^-/- mock-infected mice. At end stage disease (7–8 dpi) n = 12 WT and n = 10 SK1^-/- DENV-infected, n = 7 WT mock and n = 4 SK1^-/- mock-infected mice. Data points representing non-symptomatic animals (9–14 dpi) are indicated by the half-filled symbols. Statistical analysis has been performed on symptomatic DENV-infected mice only, excluding n = 5 WT and n = 3 SK1^-/- at 9/14 dpi. Data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. Statistical significance was assessed by unpaired student <i>t</i>-test. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005.</p

Induction of IFN-β and ISGs in WT and SK1^-/- mice following ic infection with DENV-2.

<p>WT and SK1^-/- mice were ic infected with DENV, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169814#pone.0169814.g001" target="_blank">Fig 1</a>. RNA was isolated from infected mice brain tissues and analysed by real time qRT-PCR <b>A.</b> at 3 dpi for IFN-β, viperin, Ifi27l2a, and CXCL10. n = 8 WT and SK1^-/- DENV-infected, n = 2 WT mock and n = 3 SK1^-/- mock-infected mice; <b>B.</b> at end stage disease for IFN-β, viperin, Ifi27l2a, and CXCL10. n = 12 WT and n = 10 SK1^-/- DENV-infected, n = 7 WT mock and n = 4 SK1^-/- mock-infected mice. Data points representing non-symptomatic animals (9–14 dpi) are indicated by the half-filled symbols. Statistical analysis has been performed on symptomatic DENV-infected mice only (7–8 dpi), excluding n = 5 WT and n = 3 SK1^-/- at 9/14 dpi. Data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. Statistical significance was assessed by unpaired student <i>t</i>-test. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005.</p

Summary of body weight loss and signs of DENV-induced neurovirulence in WT and SK1^-/- mice and analysed by Fisher’s exact test.

<p>Summary of body weight loss and signs of DENV-induced neurovirulence in WT and SK1^-/- mice and analysed by Fisher’s exact test.</p

Definition of the SK/S1P axis in WT and SK1^-/- mice following ic infection with DENV-2.

<p>WT and SK1^-/- mice were ic infected with DENV, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169814#pone.0169814.g001" target="_blank">Fig 1</a> and at end stage disease, brain tissue was harvested and snap frozen or stored in TRIzol. RNA was extracted from TRIzol and lysates prepared in EB buffer from snap frozen tissue. <b>A.</b> SK1 mRNA was determined by qRT-PCR (left panel) and isoenzyme specific SK1 activity assay (right panel); <b>B.</b> SK2 mRNA was determined by qRT-PCR (left panel) and isoenzyme specific SK2 activity assay (right panel); <b>C.</b> S1P was quantitated in brain lysates by HPLC. n = 11 WT and n = 10 SK1^-/- DENV-infected, n = 7 WT mock and n = 4 SK1^-/- mock-infected mice. PCR data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. SK activity data and S1P quantitation are expressed relative to total protein quantitation. Statistical significance was assessed by unpaired student <i>t</i>-test. ** = p < 0.05, **** = p < 0.00005. ND, not detected. Data points representing non-symptomatic animals (9–14 dpi) are indicated by the half-filled symbols. Statistical analysis has been performed on symptomatic DENV-infected mice only, excluding n = 5 WT and n = 3 SK1^-/- at 9/14 dpi.</p

Viperin mRNA is induced in DENV-2 infected cells.

<p>Cells were infected with DENV-2 (MOI = 1 or MOI = 3 for MDM) and at various time points pi intracellular RNA was extracted and viperin mRNA and DENV −ve strand RNA quantitated by real time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change. Values represent average ± SEM (n = 3). (<b>A</b>) A549; (<b>B</b>) Huh-7; (<b>C</b>) Huh-7.5; (<b>D</b>) MDM. * Significantly different in comparison to 0 h time point, p<0.05.</p