Levels of type I IFN levels in serum of wild type and DKO mice infected with WNV.

<p>Mice were inoculated with 10² PFU of WNV by footpad injection and sacrificed at the indicated times. Type I IFN levels were determined from serum collected on days 1 to 4 after WNV infection by an EMCV bioassay in L929 cells. Data reflect averages of serum samples from 5 to 10 mice per time point and the data are expressed as international units (IU) of IFN-α per ml. The specificity of the assay was confirmed with an anti-IFN-αβR neutralizing antibody (data not shown). Asterisks indicate values that are statistically significant (**, P<0.005, *, P<0.05).</p

The IFN-β response in mDC and MEF is IPS-1-dependent but MyD88-independent.

<p>mDC and MEF generated from wild type, IPS-1^−/− and MyD88^−/−, mice were infected at an MOI of 0.1 and levels of (A, C, and E) IFN-α and (B, D, and F) IFN-β mRNA were quantified by qRT-PCR. Values are an average of duplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001).</p

The IFN-β response in mDC is IRF-1, IRF-8, IFN-α/β-independent but partially IRF-5-dependent.

<p>mDC generated from wild type, (A and B) IRF-1^−/−, IRF-8^−/−, (C and D) IFN-α/βR^−/−, or (E and F) IRF-5^−/− mice were infected at an MOI of 0.1 and levels of IFN-α (A, C, and E) and IFN-β (B, D, and F) mRNA were measured by qRT-PCR. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05) compared to wild type.</p

Survival and Virologic Analysis for Wild-Type and IRF-3^−/− C57BL/6 Mice

<div><p>(A) Eight- to twelve-week-old mice were inoculated with 10² PFU of WNV by footpad injection and followed for mortality for 21 d. Survival differences were statistically significant (<i>n</i> = 20, IRF-3^−/−; and <i>n</i> = 20, wild-type mice; <i>p</i> < 0.0001).</p><p>(B–G) Viral burden in peripheral and CNS tissues after WNV infection. WNV RNA in (B) serum and (C) draining lymph node, and infectious virus in the (D) spleen, (E) kidney, (F) brain, and (G) spinal cord were determined from samples harvested on days 1, 2, 4, 6, 8, and 10 using qRT-PCR (B, C) or viral plaque assay (D–G). Data is shown as viral RNA equivalents or PFU per gram of tissue for ten to 12 mice per time point. For all viral load data, the solid line represents the median PFU per gram at the indicated time point, and the dotted line represents the limit of sensitivity of the assay. Error bars indicate the standard deviations (SD). Asterisks indicate values that are statistically significant (*, <i>p</i> < 0.05; **, <i>p</i> < 0.005; ***, <i>p</i> < 0.0001) compared to wild-type mice.</p></div

The IFN-β response in mDC after TLR stimulation and viral infection.

<p>mDC generated from wild type and DKO mice were (A) stimulated with 50 µg/ml of poly(I∶C) or 2 µg/ml LPS for 24 h or (B) infected with EMCV (MOI 0.1), WNV (MOI 0.1) and CHIK (MOI 1) for 24 h. Levels of IFN-β were measured either by ELISA or qRT-PCR. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, ** P<0.0005) from wild type cells; n.s. indicates differences that were not statistically significant.</p

The Effect of IRF-3 on ISG54, ISG56, RIG-I, and MDA5 Expression in WNV-Infected Cortical Neurons

<p>(A, B) Whole cell lysates were generated at the indicated times from wild-type or IRF-3^−/− primary cortical neurons. These neurons were uninfected (U), infected with WNV (W), pretreated with 100 IU/ml of IFN-β (Pre), or treated with IFN after infection (P/W). (A) ISG54 immunoblot analysis and figure labeling is as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030106#ppat-0030106-g004" target="_blank">Figure 4</a>. (B) Protein levels of IRF-3, RIG-I, MDA5, ISG56, WNV, and GAPDH were examined by immunoblot analysis as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030106#s4" target="_blank">Materials and Methods</a>.</p

IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ.

<p>A. Mφ generated from wild type, IFN-αβR^−/− and DKO mice were infected at an MOI of 0.01 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000607#ppat-1000607-g003" target="_blank">Figure 3</a>. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).</p

WNV Infection and IFN Production in Primary Cortical Neurons

<div><p>(A) Primary cortical neurons generated from wild-type or IRF-3^−/− mice were infected at an MOI of 0.001, and virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments, error bars represent the SD, and asterisks indicate values that are statistically significant (**, <i>p</i> < 0.005).</p><p>(B–D) The induction of IFN-α and IFN-β mRNA in WNV-infected primary cortical neurons. IFN mRNA was analyzed by qRT-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030106#ppat-0030106-g002" target="_blank">Figure 2</a>. (C) IFN-α and (D) IFN-β protein accumulation in supernatants of WNV-infected cortical neurons from wild-type and IRF-3^−/− mice was measured by ELISA.</p><p>(E) The induction of IRF-7 mRNA in WNV-infected primary cortical neurons was analyzed by qRT-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030106#ppat-0030106-g002" target="_blank">Figure 2</a>.</p><p>(B–E) Data are the average of three independent experiments performed in duplicate, and the asterisks indicate statistically significant differences (*, <i>p</i> < 0.05; **, <i>p</i> < 0.005, ***, <i>p</i> < 0.0001).</p></div

The early phase of the type I IFN regulation in MEF partially involves TRAF3 and TBK1 whereas the late phase requires TRAF6.

<p>TRAF3^−/−, TBK1^−/− and TRAF6^−/− MEF were infected at an MOI of 0.1 and levels of (A and C) IFN-α and (B and D) IFN-β mRNA and secreted protein were measured by qRT-PCR and ELISA. Since basal mRNA expression of the IFN-β gene in uninfected TBK1^−/− and TRAF6^−/− MEF was lower than that observed in congenic wild type cells, for these cells only, we compared IFN-β mRNA levels to the wild type MEF. Values are an average of duplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05); n.s. indicates differences that were not statistically significant.</p

Survival and viral burden analysis in mice.

<p>A. Eight to 12 week-old C57BL/6 mice were inoculated with 10² PFU of WNV by footpad injection and followed for mortality for 21 days. Survival differences were statistically significant between immunodeficient and wild type mice (n = 11, IFN-αβR^−/−; n = 20, DKO; and n = 20, wild type mice, P<0.0001). Average survival time between IFN-αβR^−/− (3.5 days) and DKO (6 days) mice was also statistically different (P<0.001). B–G. Viral burden in peripheral and CNS tissues after WNV infection. WNV RNA in (B) serum and (C) draining lymph node, and infectious virus in (D) spleen, (E) kidney, (F) brain and (G) spinal cord were determined from samples harvested on the indicated days using qRT-PCR (B and C) or viral plaque assay (D–G). Data is shown as viral RNA equivalents or PFU per gram of tissue for 10 to 12 mice per time point. For all viral load data, the solid line represents the median PFU per gram at the indicated time point, and the dotted line represents the limit of sensitivity of the assay.</p