Response of WT and GKO BALB/c strains to infection with ECTV-WT or ECTV-IFN-γbp^Δ.

<p>Groups of female mice were infected with ECTV-WT or ECTV-IFN-γbp^Δ. Mice were monitored daily for 21 days for disease signs. Data shown are combined results obtained from two separate experiments in which 5–15 mice per strain were used (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s008" target="_blank">S2 Table</a>). <i>P</i> values for survival proportions were obtained by using Kaplan-Meier Log rank statistical test: **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; ****, <i>p</i> < 0.0001. The survival rates of WT mice compared to GKO mice infected with WT or mutant virus are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s005" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s006" target="_blank">S6</a> Figs., respectively and the statistical analysis presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s008" target="_blank">S2 Table</a>.</p

Deficiency in Th2 Cytokine Responses Exacerbate Orthopoxvirus Infection

<div><p>Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbp^Δ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbp^Δ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.</p></div

Numbers of CD4 T cells expressing IFN-γ, IL-4, T-bet or GATA-3.

<p>Mice (n = 5–10/group) were infected with ECTV-WT or ECTV-IFN-γbp^Δ, sacrificed on day 7 p.i. and splenocytes used for intracellular staining for IFN-γ, IL-4, T-bet or GATA-3 without stimulation (A and B) or with PMA + ionomycin stimulation (C and D). Shown are means of absolute numbers of IFN-γ⁺CD4 T cells with no stimulation (A) or PMA + ionomycin stimulation (C), IL-4⁺CD4 T cells with no stimulation (B) or PMA + ionomycin stimulation (D). Also shown are means of absolute numbers of T-bet⁺ and GATA-3⁺ CD4 T cells following ECTV-WT (E) or ECTV-IFN-γbp^Δ (F) infection. Two-way ANOVA followed by Fisher’s LSD test for significance was used. For panel A, numbers of IFN-γ⁺CD4 T cells in WT mice for both viruses were significantly higher (p<0.0001) compared to numbers in all GKO strains. In IL-4^−/− and STAT-6^−/− mice, IFN-γ⁺CD4 T cell numbers generated by ECTV-IFN-γbp^Δ infection were significantly higher (<i>p</i> = 0.0005 and <i>p</i> = 0.0013, respectively) compared to WT virus infection. In IL-13^−/− mice, IFN-γ⁺CD4 T cell numbers generated by WT virus infection were significantly higher (<i>p</i> = 0.0034) compared to ECTV-IFN-γbp^Δ infection. For panel B, numbers of IL-4⁺CD4 T cells in WT mice for both viruses were significantly higher (p<0.01) compared to numbers in all GKO strains. No other significant differences were found. For panel C, numbers of IFN-γ⁺CD4 T cells in WT mice for both viruses were significantly higher (p<0.0001 in ECTV-WT and <i>p</i> < 0.001 in ECTV-IFN-γbp^Δ) compared to numbers in all GKO strains. In IL-4^−/−, STAT-6^−/− IL-4Rα^−/− and IL-13^−/− / IL-4Rα^−/− mice, IFN-γ⁺CD4 T cell numbers generated by ECTV-IFN-γbp^Δ infection were significantly higher compared to WT virus infection. For panel D, numbers of IL-4⁺CD4 T cells in WT mice for ECTV-WT virus was significantly higher (p<0.05) compared to numbers in all GKO strains. In WT mice, IL-4⁺CD4 T cell numbers generated by WT virus infection were significantly higher (<i>p<</i>0.05) compared to ECTV-IFN-γbp^Δ infection. For panel E, T-bet⁺ CD4 T cell numbers were significantly increased (p<0.01) in STAT-6^−/− and IL-13^−/− mice compared to WT animals. For panel F, no significant differences were found between strains. Data shown for panels A and B are from one of two independent experiments with similar results. Data shown for panels C-F are from one experiment. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.0001.</p

Viral load in organs and blood of WT and GKO mice infected with ECTV-WT or ECTV-IFN-γbp^Δ.

<p>Groups of 5 mice were infected with either ECTV-WT or ECTV-IFN-γbp^Δ and sacrificed at day 7 p.i. Viral load was determined by viral plaque assay and expressed as Log₁₀ PFU virus/gram tissue. The dotted line indicates the limit of virus detection by viral plaque assay. Two-way ANOVA followed by Fisher’s LSD test for significance between (i) ECTV-WT titers in WT mice compared with all strains, (ii) ECTV-IFN-γbp^Δ load in WT mice compared with all strains, and (iii) ECTV-WT compared with ECTV-IFN-γbp^Δ load in individual strains (A) liver, (B) spleen, and (C) blood. The detailed statistical analysis of WT and mutant virus titers in each of the 5 GKO mouse strains is presented in S3 (Liver), S4 (Spleen) and S5 (Blood) Tables. Asterisks indicate significant differences in organ titers comparing ECTV-WT or ECTV-IFN-γbp^Δ. ****, p< 0.0001; ***, 0.0001< p <0.001; **, 0.001< p <0.01; * 0.01< p <0.05.</p

CD8 T cell responses to ECTV-WT and ECTV-IFN-γbp^Δ.

<p>Mice (n = 5–10/group) were infected with ECTV-WT or ECTV-IFN-γbp and sacrificed 7 days later to measure splenic CTL activity. Shown is % specific lysis of ⁵¹Cr-labelled, ECTV-infected P815 target cells by splenocytes from (A) ECTV-WT- or (B) ECTV-IFN-γbp^Δ-infected WT and GKO mice. (C) Percent specific lysis of targets by splenocytes at 75:1 effector-to-target ratio. Two-way ANOVA followed by Fisher’s LSD test for significance was used for statistical analysis: (i) CTL response to ECTV-WT vs. ECTV-IFN-γbp^Δ in IL-13^−/− (p < 0.0001) and STAT-6^−/− (p < 0.0001) (ii) CTL response to ECTV-WT in WT vs IL-4^−/− (p = 0.0083), vs STAT-6^−/− (p = 0.0003), vs IL-13^−/− (p = 0.0001), vs IL-13^−/−/IL-4Rα^−/− (p = 0.0037) mice; (iii) CTL response to ECTV-IFN-γbp in WT vs STAT-6^−/− (p = 0.0002); vs IL-13^−/− (p = 0.0478); vs IL-4Rα^−/− (p = 0.0174) and IL-13^−/−/IL-4Rα^−/− (p = 0.000046) mice. ****, p<0.0001. (D—F) Spleen cells from mice infected mice were re-stimulated for 5 h with ECTV-infected P815 cells or H-2^d 8 T cell peptide determinants prior to intracellular IFN-γ staining followed by flow cytometry. Data shown are means of absolute numbers of ECTV-specific IFN-γ⁺ 8 T cells in spleens of ECTV-WT- vs. ECTV-IFN-γbp^<b>Δ</b>-infected mice (D) and determinant-specific IFN-γ⁺ 8 T cells following infection with ECTV-WT (E) or ECTV-IFN-γbp (F) viruses. Two-way ANOVA followed by Fisher’s LSD test for significance was used for statistical analysis. For panels D, E and F, IFN-γ⁺ 8 T cell numbers in WT mice were significantly higher (p<0.0001) than numbers in all GKO strains. Data shown for D-F are representative of one of three independent experiments with similar results. *, <i>p</i>< 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.0001.</p

Immunosuppression with CTX triggers ECTV replication.

<p>(A) Experimental scheme. Groups of 5 WT BALB/c mice were infected with 100 PFU of ECTV-WT or ECTV-IFN-γbp^Δ. Group 1 was sacrificed 80 days p.i. to measure viral load in various tissues, Group 2 was left untreated and Group 3 was treated with CTX. Three weeks later (day 101 p.i.), CTX-treated and untreated mice were sacrificed to measure viral load in organs. (B) ECTV-WT and (C) ECTV-IFN-γbp^Δ titers in organs of untreated or CTX-treated mice at 101 days p.i. BM, bone marrow; AG, adrenal gland. (D) CBA/H mice (n = 5/group) were infected with 100 PFU of ECTV-WT and 80 days p.i., one group was treated with CTX and the second group was left untreated. Shown are ECTV-WT titers in organs of untreated or CTX-treated CBA/H mice at 101 days p.i. For panels B-D, <i>P</i> <0.01 by Mann-Whitney U test for differences between untreated and CTX treated groups. (E) BALB/b.<i>Cmv</i>1^r mice (n = 5) were infected with 10⁵ PFU of ECTV-IFN-α/βbp^Δ and 80 days later treated with CTX 3 times over 15 days and sacrificed at 21 days post commencement of treatment (101 days p.i). (F) C57BL/NCrl (n = 3) and (G) C57BL/6J (n = 4) were infected with 1000 PFU of ECTV-WT and 80 days p.i., treated with CTX 4 times over 20 days. (F) ECTV-WT titers in organs of CTX-treated C57BL/6NCrl mice at 28 days post commencement of treatment (108 days p.i.) (G) Survival of C57BL/6J mice post commencement of treatment with CTX. One mouse that died on day 28 had high titers of virus in liver and spleen whereas no infectivity was detected in the organs of the remaining 3 animals that were sacrificed 35 days post commencement of treatment with CTX.</p

Presence of ECTV-IFN-γbp^Δ in mice that survive infection.

<p>(A) Survival proportions of BALB/c mice infected with 500 PFU ECTV-WT (n = 27) or ECTV-IFN-γbp^Δ (n = 58). <i>P</i><0.0001, Kaplan-Meir log rank statistical test. Survival data combined from 4 separate experiments, with 6–15 mice per experiment. (B) ECTV-IFN-γbp^Δ titers in the indicated organs of BALB/c mice at days 7 and 21 p.i. (C) Lung and (D) spleen titers of ECTV-WT and ECTV-IFN-γbp^Δ. *, <i>P</i><0.05; **, <i>P</i><0.01 in comparing ECTV-WT and ECTV-IFN-γbp^Δ titers at the days p.i. Data shown in (B), (C) and (D) are composite viral loads determined in individual animals from 3 different experiments with groups of 3–6 mice and expressed as means of log₁₀ PFU/gram tissue ± SEM. The limit of virus detection is 2 log₁₀ PFU, shown by the dotted line. (E) ECTV genomes and (F) infectious virus in organs of BALB/c mice 37 days p.i. with ECTV-IFN-γbp^Δ. (G) ECTV genomes in organs of CBA/H mice 37 days p.i. with 500 PFU of ECTV-IFN-γbp^Δ. (H) ECTV genomes in organs of C57BL/6 mice 37 days p.i. with 500 PFU of ECTV-IFN-γbp^Δ. For E, F, G and H, n = 5 mice. The limit of virus genome detection is 10 copies, shown by the dotted line. BM = bone marrow; LN = popliteal lymph node; AG = adrenal gland.</p

The effect of virus-encoded HRM and host resistance loci on virus persistence.

<p>Groups of 5 female BALB/c mice were infected with 100 PFU of WT, IFN-γbp^Δ, IFN-α/βbp^Δ, IL-18bp^Δ, SPI-2^Δ, IFN-γbp^Δ-IL-18bp^Δ (double mutant) IFN-γbp^Δ-IL-18bp^Δ-SPI-2^Δ (triple mutant) ECTV. Separate groups of mice were sacrificed on days 7 and 35 to measure viral load. (A) Viral load in the liver at day 7 p.i. (B) Virus genome copy numbers in the BM at day 35 p.i. (C) Viral genome copy numbers in the BM of BALB/c mice 35 days p.i. with varying doses of ECTV-IFN-α/βbp^Δ or of ECTV-TK^Δ. For (A), (B) and (C), *, <i>P</i><0.05; **, <i>P</i><0.01; ***, P<0.001; ****, <i>P</i><0.0001. (D) Survival of WT (BALB/c, BALB/b) and congenic (BALB/c.<i>Cmv</i>1^r, BALB/b.<i>Cmv</i>1^r) mice infected with 500 PFU ECTV-WT. <i>P</i> values were obtained by Log-rank (Mantel-Cox) test: <i>P</i><0.001 in comparing % survival of BALB/c with BALB/c.<i>Cmv</i>1^r; <i>P</i><0.05 in comparing % survival of BALB/b with BALB/b.<i>Cmv</i>1^r; <i>P</i><0.0001 in comparing % survival of BALB/c with BALB/b; <i>P</i><0.05 in comparing % survival of BALB/c.<i>Cmv</i>1^r with BALB/b. (E) NK cell and (F) CTL responses at day 7 p.i. in WT and congenic mice infected with 100 PFU ECTV-WT. (G) Virus genome copy numbers in the BM at day 35 p.i. in WT and congenic mice infected with 100 PFU ECTV-WT. (H) Virus genome copy numbers in the BM at day 35 p.i. in WT and congenic mice infected with 10⁵ PFU of ECTV-IFN-α/βbp^Δ. The limit of virus detection in A is 2 log₁₀ PFU, shown by a dotted line. The limit of virus genome detection in B, C, G and H is 10 copies, shown by the dotted line. For panels A-C, G and H, <i>P</i> values were obtained by Mann-Whitney U test for the indicated comparisons: *, <i>P</i><0.05, **, <i>P</i><0.01, ***, <i>P</i><0.001 and ****, <i>P</i><0.0001.</p

Antigen-specific CD8 T-cell responses at days 14 and 21 p.i.

<p>Groups of 5 female BALB/c mice were infected with 500 PFU of ECTV-IFN-γbp^Δ and their spleen cells used to measure CD8 T cell responses. (A) <i>Ex vivo</i> cytolytic activity of splenocytes from ECTV-IFN-γbp^Δ infected mice against virus-infected P815 target cells on days indicated p.i. (B) <i>Ex vivo</i> cytolytic activity of splenocytes obtained from ECTV-IFN-γbp^Δ infected mice 21 days p.i. against ECTV-infected (Total) or ECTV peptide determinant-pulsed P815 target cells. ***, <i>P</i><0.001. (C) Percent of ECTV peptide determinant-specific IFN-γ⁺ CD8 T cells. **, <i>P</i><0.01 and ***, <i>P</i><0.001. (D) Numbers of ECTV peptide determinant-specific IFN-γ⁺ CD8 T cells. **, <i>P</i><0.01 and ***, <i>P</i><0.001. (E) Numbers of peptide-MHC class I tetramer⁺ CD8 T cells. **, <i>P</i><0.01 and ****, <i>P</i><0.0001. (F) Percent ECTV-specific (total) IFN-γ⁺ CD8 T cells. (G) Numbers of ECTV-specific (total) IFN-γ⁺ CD8 T cells. **, <i>P</i><0.01. <i>P</i> values for all panels were obtained by Mann-Whitney U test.</p

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice

<div><p>Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.</p></div