Research ArticleZIKA

Acute and chronic neurological consequences of early-life Zika virus infection in mice

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Science Translational Medicine  06 Jun 2018:
Vol. 10, Issue 444, eaar2749
DOI: 10.1126/scitranslmed.aar2749
  • Fig. 1 ZIKV replicates in neonatal mouse brain and induces weight loss, mortality, and brain atrophy.

    (A) Experimental design consisted of subcutaneous (s.c.) injection of 106 PFU ZIKV or mock medium at P3. qPCR, quantitative polymerase chain reaction. (B) Body weight curves of ZIKV- and mock-infected mice and (C) area under the curve (AUC) obtained from data in (B) (n = 5 mock and n = 4 ZIKV litters). (D) Survival curves from two independent experiments (G1, group 1; G2, group 2) (n = 5 mock and n = 4 ZIKV litters). (E) Representative images of brains from mock- and ZIKV-injected mice (23 dpi). Bar graphs represent dorsal brain area quantification (n = 6 mock and n = 12 ZIKV). (F) Bar graph showing ZIKV mRNA (n = 6 to 7 brains per time point). In (B), *P = 0.0366 and 0.0352 for 39 and 48 dpi, respectively, two-way analysis of variance (ANOVA), followed by Sidak. In (C) and (E), *P = 0.0413 and *P = 0.0003, respectively, Student’s t test, ZIKV versus mock.

  • Fig. 2 Neonatal ZIKV infection induces seizures in young mice and increases susceptibility to chemically induced seizures in adult mice.

    (A) Percentage of ZIKV-infected animals with seizures in each litter (n = 4 litters per time point). (B) Representative electroencephalographic recordings obtained from one mock-infected mouse at 12 dpi. (C to K) Representative electroencephalographic recordings performed in brains of ZIKV-infected mice at 12 dpi (n = 2 mock and n = 4 ZIKV). (L to O) Time to first seizure and number of seizures for male [(L and M); n = 7 mock and n = 5 ZIKV] and female [(N and O); n = 7 mock and n = 6 ZIKV] mice after an intraperitoneal injection of PTZ at 100 to 110 dpi. Data are expressed as means ± SEM. In (L), *P = 0.0155; in (M), *P = 0.0962; in (N), *P = 0.0154; in (O), *P = 0.0008; ZIKV versus mock, Student’s t test. L1 and L2, electrodes in left hemisphere; R1 and R2, electrodes in right hemisphere.

  • Fig. 3 Neonatal ZIKV infection induces motor and cognitive dysfunction in male mice.

    (A) Latency to fall in the HLS test performed in male mice at 9 dpi (n = 4 mock and n = 8 ZIKV). (B and C) Time to turn and time to descend in the pole test performed at 15 to 21 dpi (n = 7 mock and n = 9 ZIKV). (D) Latency to fall in the rotarod at 85 to 95 dpi (n = 10 mock and n = 12 ZIKV). (E) Distance traveled in the open-field test at 85 to 95 dpi (n = 21 mock and n = 14 ZIKV). (F) Percentage of exploration toward familiar (Fam) and novel (New) objects in the NOR test performed at 85 to 95 dpi (n = 12 mock and n = 15 ZIKV). (G) Percentage of exploration of empty cage or cage containing a stranger mouse in the social approach task at 90 to 100 dpi (n = 13 mock and n = 8 ZIKV). Data are expressed as means ± SEM. In (A), *P = 0.029; in (B), *P = 0.0243; in (C), *P = 0.0287; in (D), *P = 0.0149; in (E), *P = 0.0164; ZIKV versus mock, Student’s t test. In (F), *P = 0.0068; in (G), *P = 0.001, one-sample Student’s t test compared to a fixed value of 50.

  • Fig. 4 Neonatal ZIKV exposure causes severe and persistent neuropathological alterations in brains of mice.

    (A to D) Representative images of ventricles (A), hippocampus (B), thalamus (C), striatum, and cortex (D) of mock-infected mice at 23 dpi. (E) Representative image of ventriculomegaly seen in ZIKV-infected mice. (F to H) Representative images of multiple necrotic areas (arrows) in the hippocampus (F), thalamus (G), striatum, and cortex (H) of ZIKV-infected mice. (I and J) Representative images of dystrophic calcifications with mineral deposition (asterisks), apoptotic bodies (white arrows), and inflammatory cells (black arrows) in the brains of ZIKV-infected mice. (J to L) Representative images of complete disruption of normal cytoarchitecture in the cornus ammonis 1 (CA1) (J), CA3 (K), and DG (L) hippocampal subregions of ZIKV-infected mice. (M and N) Representative images of perivascular cuffings (dashed circles) seen in brains of ZIKV-infected mice. (O to R) Representative images of widespread neuronal necrosis with more advanced mineral deposition (asterisks) and disruption of normal cytoarchitecture in CA1 (white arrows) seen in brains of ZIKV-infected mice at 100 dpi. (R) Representative image of a shrunken basophilic neuron called ferruginated neuron (asterisks) seen in brains of ZIKV-infected mice. For all panels, n = 3 mock and n = 11 ZIKV. Scale bars, 450 μm (A, B, E, and F), 300 μm (C, D, G, and H), 50 μm (I, J, O, and P), 35 μm (K to N), and 25 μm (Q and R).

  • Fig. 5 Neonatal ZIKV exposure induces brain inflammation and increased oxidative stress.

    (A to H) Representative images of brain sections immunolabeled for GFAP at 23 dpi in the CA1 (A and B) and DG (C and D) hippocampal regions. (E) Graph shows integrated immunoreactivities for GFAP (optical density) in hippocampus of ZIKV- and mock-infected mice (n = 4 mock and n = 4 ZIKV). (F to I) Representative images of brain sections immunolabeled for Iba-1 at 23 dpi in the CA1 (F and G) and DG (H and I) hippocampal regions. (J) Graph shows integrated immunoreactivities for Iba-1 (optical density) in the hippocampus of ZIKV- and mock-infected mice (n = 4 mock and n = 5 ZIKV). White arrows in insets indicate GFAP or Iba-1 immunostaining. Scale bar, 50 μm. Scale bar (insets), 10 μm. (K to O) Brain mRNA expression of (K) IL-6 (n = 6 mock, n = 6 ZIKV, and n = 3 iZIKV), (L) IL-1β (n = 6 mock, n = 5 ZIKV, and n = 3 iZIKV), (M) KC (n = 6 mock, n = 5 ZIKV, and n = 3 iZIKV), (N) TNF-α (n = 8 mock, n = 6 ZIKV, and n = 3 iZIKV), and (O) iNOS (n = 6 mock, n = 6 ZIKV, and n = 2 iZIKV) in mock-, iZIKV-, and ZIKV-infected animals at 12 dpi. (P) DCF diacetate fluorescence in the brains of mock- and ZIKV-infected animals (n = 4 mock and n = 4 ZIKV). (E and J to P) Data are expressed as means ± SEM. In (E), *P = 0.0176; in (J), *P = 0.0196; in (K), *P = 0.0236; in (L), *P = 0.0008; in (M), *P = 0.0361; in (N), *P = 0.0052; in (O), *P = 0.0187, ZIKV versus mock, one-way ANOVA, followed by Tukey test. In (P), *P = 0.0195 in Student’s t test.

  • Fig. 6 TNF-α neutralization prevents ZIKV-induced seizures in mice.

    (A) Percentage of animals with seizures in each litter at 12 dpi in ZIKV-infected mice treated intraperitoneally with phosphate-buffered saline (PBS) (58 of 67 mice had seizures), NAC (19 of 22 mice had seizures), or infliximab (32 of 72 mice had seizures). No events were observed in 48 mock-infected mice. (B and C) Time to first seizure for male (B) (n = 10 mock + PBS, n = 7 ZIKV + PBS, and n = 7 ZIKV + Inflix) and female (C) (n = 5 mock + PBS, n = 6 ZIKV + PBS, and n = 4 ZIKV + Inflix) mice after an acute intraperitoneal injection of PTZ at 55 to 65 dpi. (D and E) Number of seizures for male (D) (n = 8 mock + PBS, n = 5 ZIKV + PBS, and n = 5 ZIKV + Inflix) and female (E) (n = 6 mock + PBS, n = 7 ZIKV + PBS, and n = 4 ZIKV + Inflix) mice after an intraperitoneal injection of PTZ at 55 to 65 dpi. (F and G) Latency to fall in the rotarod for male (F) (n = 10 mock + PBS, n = 7 ZIKV + PBS, and n = 8 ZIKV + Inflix) and female (G) (n = 9 per group) mice at 55 to 65 dpi. (H and I) Percentage of exploration toward familiar and novel objects in the NOR test performed in male (H) (n = 9 mock + PBS, n = 8 ZIKV + PBS, and n = 16 ZIKV + Inflix) and female (I) (n = 10 mock + PBS, n = 10 ZIKV + PBS, and n = 12 ZIKV + Inflix) mice at 55 to 65 dpi. In (A), *P = 0.0034, one-way ANOVA, followed by Dunnett’s test, ZIKV + Inflix versus ZIKV + PBS; in (B), *P = 0.0209 and **P = 0.0067; in (G), *P = 0.0375, one-way ANOVA, followed by Tukey test; in (H), *P = 0.0125; in (I), *P = 0.0024, one-sample Student’s t test compared to a fixed value of 50.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/444/eaar2749/DC1

    Materials and Methods

    Fig. S1. Late viral brain replication and seizure evaluation in UV-inactivated ZIKV-injected mice.

    Fig. S2. Electrode implantation and electrographic recordings from mock- and UV-inactivated ZIKV-injected mice.

    Fig. S3. DENV does not induce seizures or long-term behavioral impairment in Swiss mice.

    Fig. S4. Neonatal ZIKV infection had no effect on the development of reflexes in newborn mice.

    Fig. S5. Neonatal ZIKV infection induces motor and cognitive dysfunction in female mice.

    Fig. S6. Neonatal ZIKV infection does not affect cellular proliferation in the hippocampus.

    Table S1. Seizure analysis in ZIKV-injected mice.

    Table S2. Raw data of main figures (Excel file provided separately).

    Table S3. Raw data of supplementary figures (Excel file provided separately).

    References (4551)

  • Supplementary Material for:

    Acute and chronic neurological consequences of early-life Zika virus infection in mice

    Isis Nem de Oliveira Souza, Paula S. Frost, Julia V. França, Jéssica B. Nascimento-Viana, Rômulo L. S. Neris, Leandro Freitas, Daniel J. L. L. Pinheiro, Clara O. Nogueira, Gilda Neves, Leila Chimelli, Fernanda G. De Felice, Ésper A. Cavalheiro, Sergio T. Ferreira, Iranaia Assunção-Miranda,* Claudia P. Figueiredo,* Andrea T. Da Poian,* Julia R. Clarke*

    *Corresponding author. Email: juliaclarke{at}pharma.ufrj.br (J.R.C.); iranaiamiranda{at}micro.ufrj.br (I.A.-M.); claufig{at}gmail.com (C.P.F.); dapoian{at}bioqmed.ufrj.br (A.T.D.P.)

    Published 6 June 2018, Sci. Transl. Med. 10, eaar2749 (2018)
    DOI: 10.1126/scitranslmed.aar2749

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Late viral brain replication and seizure evaluation in UV-inactivated ZIKV-injected mice.
    • Fig. S2. Electrode implantation and electrographic recordings from mock- and UV-inactivated ZIKV-injected mice.
    • Fig. S3. DENV does not induce seizures or long-term behavioral impairment in Swiss mice.
    • Fig. S4. Neonatal ZIKV infection had no effect on the development of reflexes in newborn mice.
    • Fig. S5. Neonatal ZIKV infection induces motor and cognitive dysfunction in female mice.
    • Fig. S6. Neonatal ZIKV infection does not affect cellular proliferation in the hippocampus.
    • Table S1. Seizure analysis in ZIKV-injected mice.
    • References (4551)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2. Raw data of main figures (Excel file provided separately).
    • Table S3. Raw data of supplementary figures (Excel file provided separately).

    [Download Tables S2 and S3]

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