Immunosuppression-induced Zika virus reactivation causes brain inflammation and behavioral deficits in mice

Abstract

Zika virus (ZIKV) is a neurotropic flavivirus that can persist in several tissues. The late consequences of ZIKV persistence and whether new rounds of active replication can occur, remain unaddressed. Here, we investigated whether neonatally ZIKV-infected mice are susceptible to viral reactivation in adulthood. We found that when ZIKV-infected mice are treated with immunosuppressant drugs, they present increased susceptibility to chemically induced seizures. Levels of subgenomic flavivirus RNAs (sfRNAs) were increased, relative to the amounts of genomic RNAs, in the brains of mice following immunosuppression and were associated with changes in cytokine expression. We investigated the impact of immunosuppression on the testicles and found that ZIKV genomic RNA levels are increased in mice following immunosuppression, which also caused significant testicular damage. These findings suggest that ZIKV can establish new rounds of active replication long after acute stages of disease, so exposed patients should be monitored to ensure complete viral eradication.

Keywords: Immunology; Neuroscience; Virology.

Graphical abstract

Figure 1

Dexamethasone-induced immunosuppression in ZIKV-infected mice increases susceptibility to seizures, with no increase in viral genomic RNA levels in the brain (A) Mice at postnatal day 3 were infected with 10⁶ PFU of ZIKV s.c. or received equal volume of mock medium. When animals reached adulthood, they were daily treated with dexamethasone (DX) or saline (Sal) i.p., for up to ten days. (B) Percentage of lymphocytes in peripheral blood of ZIKV-infected and mock-injected mice before, or after 5 or 10 days of DX treatment. two-way ANOVA followed by Dunnet’s post-hoc resulted in: ∗p = 0.0152 Mock+DX vs. Mock+Sal and #p = 0.0232 ZIKV+DX vs. Mock+Sal at 5 days of treatment. At 10 days of treatment: ∗p = 0.0009 Mock+DX vs. Mock+Sal and #p < 0.0001 ZIKV+DX vs. Mock+Sal. (n = 6 animals/group). (C) Daily assessment of body weight of ZIKV-infected and mock-injected mice treated with Sal or DX (n = 12 Mock+Sal, 15 Mock+DX, 14 ZIKV+Sal, 14 ZIKV+DX). two-way ANOVA followed by Tukey post-hoc, ∗p = 0.0386 and 0.0397 ZIKV+DX vs. Mock+Sal. #p = 0.0062 and 0.0361 ZIKV+DX vs. ZIKV+Sal. (D) Area under the curve (AUC) for body weight measured across treatment of ZIKV-infected and mock-injected mice treated with Sal or DX (n = 12 Mock+Sal, 15 Mock+DX, 14 ZIKV+Sal, 14 ZIKV+DX). Each symbol represents a different experimental subject. One-way ANOVA followed by Tukey post-hoc. ∗p = 0.0399 and ∗∗p = 0.0473. (E) Distance traveled in the open field test by ZIKV-infected or mock-injected mice treated with saline (Sal) or dexamethasone (DX) for 10 days. One-way ANOVA followed by Tukey post-hoc revealed no difference between groups. N = 16 Mock+Sal, 14 Mock+DX, 13 ZIKV+Sal, 10 ZIKV+DX. Each symbol represents a different experimental subject. (F) Time to first pentylenetetrazol-induced seizure shown by ZIKV-infected or mock-injected mice treated with Sal or DX for 10 days. N = 11 Mock+Sal, 12 Mock+DX, 9 ZIKV+Sal, 12 ZIKV+DX. One-way ANOVA followed by Tukey post-hoc revealed no difference between groups. Each symbol represents a different experimental subject. (G) Number of pentylenetetrazol-induced seizures shown by ZIKV-infected or mock-injected mice treated with Sal or DX for 10 days. One-way ANOVA followed by Tukey post-hoc, ∗p = 0.0069, ∗∗p = 0.0039, ∗∗∗p < 0.0001. N = 11 Mock+Sal, 13 Mock+DX, 9 ZIKV+Sal, 13 ZIKV+DX. Each symbol represents a different experimental subject. (H and I) ZIKV genome copies (H; n = 6, 8, 8, and 9 for 0, 2, 5, and 10 days of treatment, respectively) and ZIKV negative RNA strand (I; n = 5, 4, 7, and 4 for 0, 2, 5, and 10 days of treatment, respectively) measured in the brains of mice before, or after 2, 5, or 10 days of DX treatment. One-way ANOVA followed by Tukey post-hoc revealed no difference between groups. Each symbol represents a different experimental subject. Data are expressed as means ± SEM.

Figure 2

Dexamethasone-induced immunosuppression in ZIKV-infected mice leads to increased generation of subgenomic flavivirus RNAs and altered expression of inflammatory mediators in the brain (A) Levels of ZIKV subgenomic flavivirus RNAs (sfRNAs) measured in the brains of infected mice before, or after 5 or 10 days of dexamethasone (DX) treatment. One-way ANOVA followed by Dunnett, ∗p = 0.0171. N = 4, 8, 5, for 0, 5, and 10 days of treatment, respectively. Each symbol represents a different experimental subject. (B) Levels of ZIKV subgenomic flavivirus RNA (sfRNA) measured in the brains of infected mice before or after 10 days of cyclosporine (Cyclo) treatment. Student's t test, ∗p = 0.0408. N = 4 and 8 for 0 and 10 days of treatment, respectively. Each symbol represents a different experimental subject. (C and D) Levels of XRN-1 mRNA were measured in the brains of infected mice treated with Sal or DX (C; n = 5 and 7 for 0 and 10 days of treatment, respectively) and Sal or Cyclo for 10 days (D; n = 5 and 7 for 0 and 10 days of treatment, respectively). Student's t test, #p = 0.0900 and ∗p = 0.033,3, respectively). Each symbol represents a different experimental subject. (E–H) Representative images of Sholl analysis in the brain of mock-injected mice treated with Sal (E) or DX (F), and ZIKV-infected mice treated with Sal (G) or DX (H) for 10 days. Individual cells highlighted in dashed-line rectangles are shown in the right of each representative image. (I) Bar graph represents the number of intersections quantified in 4–6 cells per mouse. One-way ANOVA followed by Tukey ∗p = 0.0004 ZIKV+DX vs. Mock+Sal; ∗∗p = 0.0011 ZIKV+Sal vs. Mock+Sal; ∗∗∗p = 0.0451 ZIKV+DX vs. Mock+DX. Each symbol represents a different experimental subject. (J–O) Levels of IFNβ (J, n = 6 Mock+Sal, 6 Mock+DX, 4 ZIKV+Sal, 6 ZIKV+DX), ISG15 (K, n = 3 Mock+Sal, 6 Mock+DX, 5 ZIKV+Sal, 7 ZIKV+DX), OAS1 (L, n = 3 Mock+Sal, 5 Mock+DX, 5 ZIKV+Sal, 7 ZIKV+DX), IL-1β (M; n = 6 Mock+Sal, 6 Mock+DX,11 ZIKV+Sal, 10 ZIKV+DX), IL-6 (N, n = 6 Mock+Sal, 6 Mock+DX,11 ZIKV+Sal, 12 ZIKV+DX) and TNF-α (O, n = 5 Mock+Sal, 4 Mock+DX, 11 ZIKV+Sal, 11 ZIKV+DX) mRNAs measured in the brains of infected mice treated with Sal or DX for 10 days. Actin was used as endogenous housekeeping gene. One-way ANOVA followed by Tukey, ∗∗p = 0.0138, ∗p = 0.0199 in J, ∗∗p = 0.0011, ∗p = 0.0047 in (M), ∗p < 0.0001, #p = 0.0556 in (N). Data are representative of 2–3 independent experiments with similar results. Data are expressed as means ± SEM.

Figure 3

Dexamethasone-induced immunosuppression in ZIKV-infected mice causes testicular viral replication, morphological changes, and altered expression of steroidogenic pathway enzymes (A and B) ZIKV genome copies measured in the spleen (A, n = 5 ZIKV+Sal, 7 ZIKV+DX) and skeletal muscle (B, n = 5 ZIKV+Sal, 7 ZIKV+DX) of infected mice treated with Sal or DX for 10 days. Each symbol represents a different experimental subject. (C) ZIKV genome copies measured in the testicles of mice before, or after 2, 5 or 10 days of dexamethasone (DX) treatment. Mann-Whitney test, ∗p = 0.0416 and ∗p = 0.0603. N = 5, 6, 7 and 6 for 0, 2, 5 and 10 days of treatment, respectively. Each symbol represents a different experimental subject. (D) Testicle weight of ZIKV-infected mice before or after 2, 5 or 10 days of DX treatment. One-way ANOVA followed by Tukey post-hoc, ∗p = 0.0424. N = 9, 10, 11, and 9 for 0, 2, 5, and 10 days of treatment, respectively. Each symbol represents a different experimental subject. (E–H) Representative images of HE staining performed in testicles of mock-injected mice treated with Sal (E) or DX (F), or ZIKV-infected mice treated with Sal (G) or DX (H) for 10 days. Arrows indicate disrupted epithelium, and arrowheads indicate cells with cytoplasmic inclusions and macrophages/monocytes-similar cells. N = 5 Mock+Sal, 5 Mock+DX, 4 ZIKV+Sal, 6 ZIKV+DX. Scale bar, 50 μm. (I–M) Representative images of TUNEL immunostaining in the testicles of mock-injected mice treated with Sal (I) or DX (J), or ZIKV-infected mice treated with Sal (K) or DX (L) for 10 days (M) Bar graph represents the number of TUNEL-positive cells. One-way ANOVA followed by Tukey ∗p < 0.0001 ZIKV+DX vs. Mock+Sal; ∗∗p = 0.0009 ZIKV+DX vs. Mock+DX; ∗∗∗p = 0.0002 ZIKV+DX vs. ZIKV+Sal. N = 3 Mock+Sal, 6 Mock+DX, 4 ZIKV+Sal, 6 ZIKV+DX. Each symbol represents a different experimental subject. (N–P) Levels of 3β-HSD (N, n = 6/group), CYP11A1 (O, n = 5 ZIKV+Sal, 4 ZIKV+DX) and StAR (P, n = 6 ZIKV+Sal, 5 ZIKV+DX) mRNA detected in the testicles of ZIKV-infected mice treated with Sal or DX for 10 days. Student’s t test, ∗p = 0.0211 in (O), ∗p = 0.0423 in (P). Each symbol represents a different experimental subject. Data are expressed as means ± SEM.