Kinetics of native type I IFN production <i>in vivo</i>.

<p>Wild-type (<b>A, E</b>), <i>Ifnar1</i>^-/- (<b>B, F</b>), <i>Ifnb</i>^-/- (<b>C, G</b>) or <i>Irf7</i>^-/- (<b>D, H</b>) mice were treated with 100 μg of pI:C (<b>A-D</b>) or infected with 10² PFU of WNV (<b>E-H</b>) and serum was assayed on the indicated times for IFN activity by VSV CPE bioassay. Samples from three independent sets of pI:C treated mice (3 to 5 per group) were collected at 1, 3 and 6 h post injection. Serum from groups of 3 to 8 mice of each genotype was collected from naïve and WNV infected mice at days 1 to 6 post infection. Each genotype was sampled from at least two (<i>Ifnar1</i>^-/- and <i>Irf7</i>^-/-) or more (wild-type and <i>Ifnb</i>^-/-) independent infections. IFN levels in all samples were determined at least three times. As shown herein, symbols represent samples derived from individual animals at the times indicated and assayed at the same time.</p

Type I IFN blocking mAbs enhance lethality of WNV infection.

<p>Wild-type or <i>Irf7</i>^<i>-/-</i> mice were administered 500 μg of TIF-3C5 or isotype control mAb by intraperitoneal injection one day prior, and two days following subcutaneous infection with 10² PFU of WNV. <b>B.</b> Wild-type or <i>Ifnb</i>^-/- mice received 250 μg of TIF-3C5 or isotype control mAb one day prior, one day following, and three days following WNV infection. <b>C.</b> Wild-type or <i>Ifnb</i>^-/- mice received 250 μg of HDβ-4A7 or isotype control antibody one day prior and two days following WNV infection. <b>D.</b> Wild-type or <i>Ifnb</i>^-/- mice received 250 μg of HDβ-4A7 antibody or isotype control one day prior and three days following WNV infection, as well as 500 μg of TIF-3C5 antibody (or control) one day prior, one day following, and three days following infection. Wild-type and <i>Ifnar1</i>^-/- mice were given 500 μg of MAR1-5A3 or isotype control mAb one day prior, one day following, and three days following infection. Survival was monitored for 21 days. Data represent 9 to 20 mice per group, from 2 or more independent experiments. *, <i>P</i> < 0.05; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001 (log-rank test).</p

Blockade of type I IFN activation of P-STAT1 <i>in vitro</i>.

<p>Titrations of HDβ-4A7 and TIF-3C5 (0.01–10 μg) or MAR1-5A3 (10 μg) were preincubated for 60 min with 3 ng of IFN-β (<b>A, B</b>) or 3.3 ng of IFN-α4 (<b>C, D</b>) and then incubated with L929 cells for 20 min, followed by staining for P-STAT1 and processing by flow cytometry. <b>E</b>. Individual IFN-α subtypes (3.3 ng) were preincubated with 10 μg of mAb and then incubated with L929 cells for 20 min, followed by staining for P-STAT1 and flow cytometric analysis. Data are representative of three or more independent experiments.</p

Inhibition of type I IFN MHC I upregulation.

<p>Type I IFNs (IFN-β, 800 pg; IFN-α1, 40,000 pg; IFN-α4, 4000 pg; IFN-α5, 400 pg; IFN-α11, 1000 pg; or IFN-α13, 1000 pg) were preincubated with 1 to 10 μg of HDβ-4A7 (<b>A, B</b>) or 0.1 to 10 μg of TIF-3C5 (<b>C, D</b>), MAR1-5A3 (10 μg) or control hamster IgG (10 μg) for 30 min then added to fibrosarcoma cells for 72 h and stained for H2-K^b class I MHC antigens by flow cytometry. Plots and histograms are representative of three or more independent assays using either F510 (<b>A, B</b>) or 1969 (<b>C, D</b>) fibrosarcoma cells.</p

Neutralization of type I IFN induced antiviral activity <i>in vitro</i>.

<p>Ten units IFN-β (<b>A</b>) or IFN-α (<b>B</b>) were preincubated for 1 h with indicated mAbs and then added to L929 cells overnight. Ten units/well type I IFN activity used in these assays are as follows: IFN-β, 16 pg; IFN-αA, 22 pg; IFN-α1, 15 pg; IFN-α4, 70 pg; IFN-α5, 20 pg; IFN-α13, 13 pg. IFN-treated cells subsequently were infected with VSV for 48 h and cell viability determined by crystal violet staining and optical density measurements. Results shown are representative of three or more independent experiments.</p

<i>In vitro</i> neutralizing activity of Type I IFN mAbs.

<p>Approximately 20 units of recominant IFN-β (<b>A</b>) or IFN-α1 (<b>B</b>), IFN-α4 (<b>C</b>) or IFN-α13 (<b>D</b>) were preincubated for 1 h with indicated mAbs and then added to L929 cells overnight. Constant levels of Type I IFN used in these assays are as follows: IFN-β, 4.4 ng; IFN-α1, 0.15 ng; IFN-α4, 0.05 ng; IFN-α13, 34 ng. IFN-treated cells subsequently were infected with VSV for 48 h and cell viability determined by crystal violet staining and optical density measurements. Results shown are representative of two independent experiments.</p

Detection of IFN-β and IFN-α in serum following WNV infection.

<p>Serum was collected from wild-type (<b>A-F</b>), <i>Ifnar1</i>^-/- (<b>G-L</b>), <i>Ifnb</i>^-/- (<b>M-R</b>) or <i>Irf7</i>^-/- (<b>S-X</b>) mice infected with 10² PFU of WNV at day 3 (wild-type and <i>Ifnar1</i>^<i>-/-</i>) or day 4 (<i>Ifnb</i>^<i>-/-</i><i>and Irf7</i>^<i>-/-</i>). Volumes of serum containing approximately 5–10 units of IFN activity (as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128636#pone.0128636.g004" target="_blank">Fig 4</a>) were incubated with 10 μg of mAb for 1 h and then titrations were added to L929 cells overnight. IFN-treated cells were infected with VSV and cellular viability assessed after 48 h using crystal violet staining and optical density measurements. Each panel is representative of six independent samples.</p

Detection of IFN-β and IFN-α in serum following pI:C treatment.

<p>Serum was collected from wild-type (<b>A-F</b>), <i>Ifnar1</i>^-/- (<b>G-L</b>), <i>Ifnb</i>^-/- (<b>M-R</b>) or <i>Irf7</i>^-/- (<b>S-X</b>) mice injected with 100 μg of pI:C at 3 to 6 hrs. Volumes of serum containing approximately 5–10 units of IFN activity (as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128636#pone.0128636.g004" target="_blank">Fig 4</a>) were incubated with 10 μg of mAb for 1 h and then titrations were added to L929 cells overnight. IFN-treated cells were infected with VSV and cellular viability assessed after 48 h using crystal violet staining and optical density measurements. Each panel is representative of six independent samples.</p

NK cells specifically control MCMV infection via cytotoxic effector molecules.

<p><b>(A)</b> Splenic viral titers were analyzed by qPCR following a three-day infection with WT MCMV (5000 PFU/mouse) or <b>(B)</b> five-day infection with WT MCMV (20000 PFU/mouse). Statistical analysis performed by comparing values from WT mice with genetically modified or antibody-treated mice. <b>(C)</b> Schematic of the dual-infection system depicting the starting viral mixture and two possible results: Ly49H-dependent control of WT MCMV with resulting selection of Δm157 clones <i>vs</i>. equivalent replication of both MCMV strains due to absence of Ly49H-dependent control of WT MCMV. <b>(D)</b> Splenic viral titers following a five-day infection, as in <b>(B)</b>, with the indicated dose and strains following antibody treatment. <b>(E-F)</b> Splenic viral escape (Δm157) frequency determined five days after a mixed MCMV infection with WT MCMV and Δm157 (20000 PFU/mouse). NK1.1 antibody was administered where indicated 48hrs prior to infection. Data are combined from two independent experiments with individual points representing a single mouse. L = Limit of quantification. I = Input frequency (10% frequency of Δm157). ****<i>p</i> < .0001, ***<i>p</i> < .001, **<i>p</i> < .01, *<i>p</i> < .05, ns = not significant.</p

IL-12 or IFN-I are required for m157-specfic cytotoxicity <i>in vivo</i> during MCMV infection.

<p>Mice were i.p. injected with MCMV (10000 PFU/mouse) and at the indicated timepoint after infection an <i>in vivo</i> cytotoxicity assay was performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005323#ppat.1005323.g006" target="_blank">Fig 6</a>. <b>(A)</b> m157-specific rejection was analyzed at days 1–4 after MCMV infection. <b>(B)</b> Mice were infected with WT or Δm157 MCMV for 3 days, after which m157-specific rejection was analyzed. <b>(C)</b> m157-specific rejection was measured in mice deficient in IFNAR1, IL-12Rβ2, or both was analyzed at 3 days post-infection. Data are representative of three to four independent experiments with individual points representing a single mouse.</p