(A) Eight to twelve week old mice were inoculated with 10² PFU of WNV by footpad injection and followed for mortality for 21 days. Survival differences were statistically significant (n = 24, IRF-1^-/-; n = 20, wild type mice; P<0.0001). 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, 3, 4, 5, 6, and 8 using qRT-PCR (B and C) or viral plaque assay (D-G) as indicated. Data are shown as viral RNA equivalents or PFU per gram of tissue for 7 to 10 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 (*, P<0.05; ***, P<0.0001) compared to wild type mice.

A-B. MEF (A) or Mφ (B) generated from wild type or IRF-1^-/- mice were infected, 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, error bars represent the SD, and asterisks indicate differences that are statistically significant relative to wild type mice (*, P<0.05; **, P<0.005). C-F. The induction of IFN-α (C-D) or IFN-β (E-F) mRNA in WNV-infected MEF (C, E) or Mφ (D, F) was analyzed by qRT-PCR. The data are the average of at least three independent experiments performed in triplicate, error bars represent the SD, and asterisks indicate differences that are statistically significant relative to wild type mice (*, P<0.05).

A-B. Wild type and IRF-1^-/- mice were inoculated with 10² PFU of WNV-NY by footpad injection and serum samples collected on days 5 and 8 after subcutaneous WNV infection were assayed by ELISA for WNV-E specific (A) IgM and (B) IgG. Titers are expressed as a scatter plot of the reciprocal serum dilution that was three standard deviations above background (endpoint titer). All IgM and IgG titers were not statistically significant from wild type mice (P > 0.1). C. Neutralizing antibody titers in wild type and IRF-1^-/- mice from serum collected at day 8 after infection. Filled circles reflect samples from individual mice and reflect the serum dilution that inhibits 50% of infection of Vero cells as judged by a focus reduction neutralization assay. No statistical difference (P > 0.3) was observed.

A. Assessment of CD8⁺ T cell avidity. Ex vivo restimulation of CD8⁺ T cells from wild type or IRF-1^-/- mice was performed over the indicated range of NS4b peptide concentrations. Cells were stained for CD3, CD8b, and intracellular IFN-γ, and analyzed by flow cytometry. The data are normalized relative to the peak percentage of cells expressing IFN-γ (for wild type (∼5%) or IRF-1^-/- (∼30%)) after incubation with NS4b peptide (10^-7 M). Data were generated from two independent experiments with 6 to 8 wild type or IRF-1^-/- mice in total. B. Kinetics of CD8⁺ T cell induction. Wild type or IRF-1^-/- CD8⁺ T cells from WNV-infected mice were harvested at day 8 and restimulated ex vivo with NS4b peptide (10^-7 M) for the indicated times. Cells were stained for CD3, CD8b, and intracellular IFN-γ, and analyzed by flow cytometry. The data are normalized relative to the peak percentage of cells expressing IFN-γ (for wild type (∼5%) or IRF-1^-/- (30%) after incubation with NS4b peptide (6 hour time point). Data were generated two independent experiments with 6 to 8 wild type or IRF-1^-/- mice in total. C. Treg analysis in WNV-infected wild type or IRF-1^-/- mice. At day 8, spleens were harvested and analyzed for the number of Treg using antibodies to CD4, CD25, and FoxP3. Data are displayed as a scatterplot and reflect six individual wild type or IRF-1^-/- mice from two independent experiments. Asterisks indicate values that are statistically different.

A. Assessment of CD8⁺ T cell proliferative capacity at day 8 after WNV infection. CD8⁺ T cells from wild type or IRF-1^-/- mice were stained for CD8b, intracellular GrB and intranuclear Ki-67, and analyzed by flow cytometry. Representative flow cytometry dot plots gated on CD8b⁺ cells, showing the percentage of antigen-specific proliferating CD8b⁺GrB⁺Ki-67⁺ cells. Graphical summary of the percentage and total number of CD8b⁺GrB⁺Ki-67⁺ T cells. A scatterplot of the data is shown and was generated from two independent experiments with 5 wild type or IRF-1^-/- mice. B. In vitro proliferative capacity of purified naïve CD8⁺ T cells. Representative flow cytometry profiles of carboxyfluorescein diacetate dilution from CD8b⁺ T cells stimulated with plate bound anti-CD3e (1 µM) and anti-CD28 (1 µM) at three time points. The data are representative of one individual mouse per group and the experiment was repeated three independent times with two mice per group each time.

A. Mice were inoculated with 10² PFU of WNV by footpad injection and sacrificed at 2 days post infection. After harvesting of the draining inguinal lymph node, IFN-α, IFN-β, SOCS-1, and SOCS-3 mRNA levels were measured by quantitative RT-PCR and compared to mock-infected animals. B. Type I IFN levels were determined from serum collected on days 1 through 5 after WNV infection of wild type or IRF-1^-/- mice by an EMCV bioassay in L929 cells. Data reflect averages of serum samples from 3 to 7 mice per time point and the data are expressed as international units (IU) of type I IFN 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<0.001; *, P<0.05).

A-B.The antiviral effects of exogenous IFN-β or IFN-γ in Mφ. Wild type or IRF-1^-/- Mφ were pretreated overnight with the indicated amount of (A) IFN-β (1 to 100 IU/ml) or (B) IFN-γ (1 to 100 IU/ml). Subsequently, cells were infected with WNV at an MOI of 0.01 and one-day later supernatants were harvested and titrated for viral infection. The data are expressed as the log₁₀ fold inhibition relative to the no IFN treatment control. Asterisks indicate differences that are statistically significant (*, P<0.05). C-E. The antiviral effect of IRF-1 in macrophages is independent of IFN-γ expression. C. The H22 MAb efficiently neutralizes the antiviral effect of exogenous IFN-γ treatment. Mφ were pretreated with the indicated concentrations of IFN-γ in the presence of H22 anti-IFN-γ or an isotype control MAb. Subsequently, cells were infected with WNV at an MOI of 0.01 and one-day later supernatants were harvested and titrated for viral infection. The data are expressed as the log₁₀ fold inhibition relative to the no IFN treatment control. (D) Wild type or (E) IRF-1^-/- Mφ were infected at an MOI of 0.01 in the presence of H22 anti-IFN-γ or an isotype control MAb. Virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments, and error bars represent the SD. Differences were not statistically significant.

A-B. The total percentage (A) and number (B) of CD4⁺ and CD8⁺ T cells were obtained from naïve mice and determined by flow cytometry. A scatterplot of the data is shown and was generated from three to six wild type or IRF-1^-/- mice. The bar indicates average values and asterisks denote differences that are statistically significant (P<0.05). C-H Wild type and IRF-1^-/- mice were inoculated with 10² PFU of WNV by footpad injection and spleens were harvested on day 8. C-D. The total percentage (C) and number (D) of CD8⁺ T cells were determined by flow cytometry. A scatterplot of the data is shown and was generated from 12 to 14 wild type or IRF-1^-/- mice. The bar indicates average values and asterisks denote differences that are statistically significant (P<0.05). E-F. The total percentage (E) and number (F) of splenic CD8⁺ T cells expressing GrB. Cells were harvested directly from dissected spleens and analyzed by flow cytometry (K, top dot plots). A scatterplot of the data is shown and was generated from 12 to 14 wild type or IRF-1^-/- mice. The bar indicates average values and asterisks denote differences that are statistically significant (P<0.05). G-H. Splenocytes were stimulated ex vivo with D^b-restricted NS4b peptide, stained for CD3 and CD8b, and intracellular IFN-γ, and analyzed by flow cytometry. The primary data is shown as dot plots of gated CD3⁺CD8⁺ cells (K, bottom dot plot) and (G) a summary of the percentage and (H) total number of CD3⁺CD8⁺ T cells positive for intracellular IFN-γ after peptide restimulation. A scatterplot of the data is shown and was generated from 12 to 14 wild type or IRF-1^-/- mice. Note, the data in panel G is presented with the background IFN-γ staining subtracted: wild type CD8⁺ IFN-γ⁺ background of 0.1% (n = 14 mice) and IRF-1^-/- CD8⁺ IFN-γ⁺ background of 0.2% (n = 12). The bar indicates average values and asterisks denote differences that are statistically significant (P<0.05). I-J. The total percentage (I) and number (J) of splenic CD8⁺ T cells co-expressing GrB and D^b-NS4b peptide as judged by tetramer staining and flow cytometry. A scatterplot of the data from three independent experiments is shown and was generated from 9 to 10 wild type or IRF-1^-/- mice. The bar indicates average values and asterisks denote differences that are statistically significant (P<0.05).

A-B. Equal numbers of naïve donor splenic CD8⁺ T cells from wild type SJL (CD45.1) or IRF-1^-/- (CD45.2) mice were harvested, purified by negative selection, and transferred (2×10⁶ cells) into recipient IRF-1^+/+ RAG1^-/- congenic mice. Mice were infected with WNV and eight days later, (A) splenocytes were harvested, and the number and percentage of CD45.1⁺ and CD45.2⁺ CD8⁺ T cells were enumerated. (B) Splenocytes were stimulated ex vivo with D^b-restricted NS4B peptide, stained for CD3 and CD8b, and intracellularly for IFN-γ and GrB, and analyzed by flow cytometry. Results are the average of experiments with five mice. C-E. Naïve donor splenic CD8⁺ T cells from wild type SJL (CD45.1) mice were harvested, purified by negative selection, and transferred (1×10⁶ cells) into recipient CD45.2⁺ IRF-1^-/- mice. Animals were infected with WNV and eight days later, splenocytes were harvested. (C) Flow cytometry dot plots showing percentage of donor (SJL (CD45.1)) and host (CD45.2⁺ IRF-1^-/-) CD8⁺ T cells that are positive for GrB or IFN-γ after ex vivo peptide restimulation. D-E. Graphical summary of the percentage and total number of IFN-γ⁺ or GrB⁺ SJL (CD45.1) CD8⁺ T cells after transfer into CD45.2⁺ IRF-1^-/- mice and ex vivo peptide stimulation. A scatterplot of the data is shown and was generated from 5 wild type or IRF-1^-/- mice. Note, the percentage and total number of IFN-γ⁺ or GrB⁺ wild type and IRF-1^-/- CD8⁺ T cells in unperturbed yet WNV-infected mice are shown for comparison, some of which were performed in parallel. The bar indicates average values and asterisks denote differences that are statistically significant (P<0.05).

Wild type and IRF-1^-/- mice were inoculated with 10² PFU of WNV by footpad injection, brains were harvested on day 8, and leukocytes were isolated after percoll gradient centrifugation. A-B The percentage and number of WNV-specific (Db-NS4b peptide tetramer⁺) CD8⁺ T cells was evaluated. The data are an average of experiments from 6 wild type and 5 IRF-1^-/- mice. Asterisks indicate differences that are statistically significant. C. Representative flow cytometry profiles are shown of CD8 and WNV-specific tetramer staining of brain leukocytes from wild type and IRF-1^-/- mice. D. In vivo cytolysis assay. At day 8 after infection, CFDA-labeled peptide-pulsed CD45.1 naive target cells (splenocytes from B6.SJL mice) were transferred to wild type or IRF-1^-/- (CD45.2) WNV-infected or naïve mice (n = 6 to 12 per group from two independent experiments), and six hours later, mice were sacrificed and splenocytes analyzed for the ratio of peptide pulsed to non-pulsed cells by interrogating cells in the CD45.1 gate. The percentage of target cell killing in vivo was calculated by determining the ratio of peptide-pulsed versus unpulsed cells for each mouse, and by normalizing to that seen in naive mice. E. WNV titers from the brains (day 6 post infection) of wild type mice after adoptive transfer of wild type or IRF-1^-/- WNV primed NS4b-specific CD8⁺ T cells (2×10⁵). CD8⁺ T cells were purified by negative selection and transferred 48 hours after infection. The data are an average of experiments from 6 to 9 recipient mice. Asterisks indicate differences that are statistically significant. F. CD8⁺ T cell-mediated control of WNV infection in cortical neurons. Cortical neurons from embryonic wild type mice were dissected and cultured. After infection with WNV at an MOI of 0.001 for one hour and extensive washing, positively-selected WNV-primed CD8⁺ T cells from wild type or IRF-1^-/- mice were added at a 10∶1 E∶T ratio. Supernatants were harvested 48 hours later and the viral titer was measured by focus forming assay. Asterisks indicate differences that are statistically significant.