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

Zika leaves lasting impact on brain

Perinatal Zika virus (ZIKV) infection has been associated with brain alterations in newborns. However, whether ZIKV exposure during development has long-term neurological consequences is not completely understood. Nem de Oliveira Souza et al. report that newborn mice infected with ZIKV develop acute brain abnormalities. During adulthood, perinatally infected mice showed persistent viral replication, neuropathological alterations, behavioral impairments, and altered brain excitability. Blocking tumor necrosis factor–α (TNF-α) early after infection prevented this hyperexcitability in mouse brain. The results suggest that anti-inflammatory treatments might be used to prevent the persistent increase in neuronal excitability induced by ZIKV infection in brain tissue.

Abstract

Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor–α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α–mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.

INTRODUCTION

Zika virus (ZIKV) is a flavivirus responsible for a major outbreak in the Americas in 2015 (1). Aedes mosquitoes are considered the main vector for ZIKV transmission, but infections after sexual contact and blood transfusions have also been reported (2, 3). Several studies have shown that ZIKV is highly neurotropic, being especially harmful to the immature nervous system (4). ZIKV has been isolated from brains of newborns with congenital microcephaly, and a causal relationship between in utero viral exposure and severe neurological malformations has been firmly established (5).

Although extensive research has focused on understanding the mechanisms of ZIKV-induced congenital microcephaly, one of the most marked effects reported in ZIKV-infected newborns, it is now known that only 1 to 13% of exposed fetuses show physical malformations at birth (6). However, a spectrum of clinical manifestations frequently appears months to years after birth, both in normocephalic and microcephalic babies exposed to ZIKV during gestation (7). Babies infected during pregnancy and born with normal head size can develop postnatal-onset microcephaly, suggesting that ZIKV has consequences beyond the gestational period (7, 8). Further, motor impairments were reported in normocephalic babies with evidence of congenital ZIKV exposure (7, 9).

Infants who developed ZIKV-induced microcephaly show recurrent seizures, and reports suggest that 60% of normocephalic babies exposed to ZIKV in utero suffer similar symptoms (7, 10, 11). Cortical dysplasia induced by ZIKV has also been described in babies born with normal head size, a finding that is often underdiagnosed and can itself be associated to seizures (7, 12). Thus, it is now clear that focusing only on microcephaly and other physical malformations will lead to an underestimation of the true magnitude of the consequences of the ZIKV epidemics.

Most experimental studies investigating the effects of ZIKV on the developing brain have focused on immunodeficient mice (13, 14) and in vitro organoid cultures (4). Few studies have described the effects of ZIKV infection in the developing brain of immunocompetent rodents (15, 16) and nonhuman primates (1719). Here, we infected newborn wild-type mice systemically with ZIKV and performed a long-term follow-up of the behavioral, neuropathological, and molecular consequences of infection. We report short- and long-term complications after ZIKV infection in mice and show that early tumor necrosis factor–α (TNF-α) neutralization partially prevents these detrimental effects. Our findings unveil the need for special attention to a potential burden of neuropathological complications in children and adolescents after congenital exposure to ZIKV and suggest that early inhibition of neuroinflammation may be a potential treatment to prevent long-term neurological sequelae in ZIKV-infected infants.

RESULTS

ZIKV replicates in neonatal mouse brain and induces weight loss, mortality, and brain atrophy

On postnatal day 3 (P3), Swiss mice were subcutaneously infected with 106 plaque-forming units (PFUs) of ZIKV or mock-infected (Fig. 1A). Because persistent weight loss has been associated with the severity of ZIKV infection in mice (15, 16), mock- or virus-exposed pups were weighed until 66 days post-infection (dpi). From 15 dpi, ZIKV-infected mice had smaller average body weight gain compared to mock-infected animals (Fig. 1B). The calculated area under the curve of weight gain data between 0 and 60 dpi further supports the finding that weight loss is not recovered in adulthood (Fig. 1C). Whereas all mock-infected pups survived up to 66 dpi, 40% mortality was observed in the ZIKV-infected groups, between 9 and 37 dpi (Fig. 1D). Replication in an independent cohort confirmed the presence of a critical period of ZIKV-induced mortality. In control experiments, ZIKV inactivated by ultraviolet (UV) light (iZIKV) induced no mortality or body weight loss in mice (fig. S1, A and B).

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.

Postnatal-onset microcephaly has been reported in fetuses exposed to ZIKV and born with normal head circumference in humans (7, 8). We found marked brain atrophy in ZIKV-infected mice, as demonstrated by the reduced dorsal brain area at 23 dpi when compared to mock-infected mice (Fig. 1E).

We next determined whether peripherally administered ZIKV reached and replicated in the developing brain. ZIKV RNA was detected in the brain as early as 3 dpi and peaked between 6 and 12 dpi (Fig. 1F). After this period, the amount of viral RNA in the brain decreased progressively but remained detectable until 100 dpi (Fig. 1F). The same was found for the negative strand of ZIKV RNA (fig. S1C), indicating a persistent ZIKV replication in the brains of adult mice infected at P3.

Neonatal ZIKV infection induces spontaneous seizures in young mice and increases susceptibility to chemically induced seizures in adult mice

Clinical/epidemiological studies have shown that both microcephalic and normocephalic ZIKV-infected babies exhibit spontaneous seizures at different intervals after birth (1, 7, 8, 11). To determine whether ZIKV-infected mice developed spontaneous seizures, we video-recorded the animals for 1 hour daily at random times of the light cycle starting at 9 dpi. On average, 65% of mice exhibited behavioral seizures at 9 dpi, and this increased to 88% of the pups at 12 dpi (Fig. 2A), coinciding with the peak of viral replication in the brain (see Fig. 1F). The percentage of mice presenting seizures decreased to about 30% by 18 dpi, and no epileptic events were recorded when animals reached adulthood (100 dpi) (Fig. 2A). Seizures in ZIKV-infected mice were not dependent on the time of day of recordings, as percentage of mice with seizures was comparable when using two different recording times (fig. S1D). No seizures were detected in mock-infected mice, and only two mice (from a total of 20; 10%) injected with iZIKV showed seizures in the recording session performed at 9 dpi, but not in subsequent (12 and 18 dpi) sessions (fig. S1E).

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.

For electrophysiological assessment of epileptiform activity induced by ZIKV, mice were implanted with cortical electrodes 9 dpi (fig. S2A). Electrocorticographic (ECG) recordings performed between 10 and 12 dpi showed no signs of epileptiform activity in daily 2-hour-long recording sessions performed in mock- or iZIKV-injected mice, which showed mainly alternating fast and slow low-amplitude waves (Fig. 2B and fig. S2, B to I). Conversely, in ZIKV-infected mice, periods of normal ECG activity (Fig. 2C) were interrupted by spiking activity recorded in both hemispheres (Fig. 2D). After a few minutes, this activity became bilaterally synchronized and was characterized by the presence of polyspike-waves or fast sharp waves that could last up to 7 min (Fig. 2, E to G). The interspersed occurrence of depressed ECG activity within the epileptic discharge was progressively observed (Fig. 2, H to J) and eventually culminated with the interruption of the epileptiform activity and the appearance of postictal ECG depression (Fig. 2K). During the 2-hour-long recording session performed daily, all ZIKV-infected animals (n = 4) presented at least two of these episodes (table S1). Behavioral changes associated with the epileptiform activities included freezing, tremor, and tail erection. Altogether, these results established that our model recapitulates the spontaneous self-resolving seizures during childhood frequently reported after congenital ZIKV exposure (7).

We further examined the susceptibility of adult ZIKV-infected mice to develop seizures upon administration of pentylenetetrazol (PTZ), a γ-aminobutyric acid receptor antagonist widely used as a proconvulsant agent in animal models. We found that both male and female adult (100 to 110 dpi) mice neonatally infected with ZIKV showed decreased latencies to the first seizure and presented more seizure episodes (Fig. 2, L to O) after PTZ treatment than mock-infected mice. The increased sensitivity to pharmacologically induced seizures in adult mice indicates that ZIKV leads to long-term neurochemical imbalances beyond the acute phase of infection.

To evaluate whether these effects were ZIKV-specific or a more general consequence of infection by flaviviruses, we used dengue virus (DENV) as a control. DENV-injected mice showed no substantial seizures when evaluated between 9 and 18 dpi (fig. S3A). As adults, their latency to first seizure after PTZ administration was comparable to mock group (fig. S3, B and C), suggesting that these effects are a specific consequence of early-life infection with a neurotropic flavivirus.

Neonatal ZIKV infection induces motor and cognitive dysfunctions in mice

Several persistent motor dysfunctions are seen in normocephalic babies congenitally exposed to ZIKV (20). Hence, we investigated the impact of early-life ZIKV exposure on the reflexes and behavior of young and adult mice. Because several early-life stressors may have differential impacts on subjects of each sex (21), we evaluated separately the impact of ZIKV infection on male and female mice. No differences were found between ZIKV- and mock-infected pups in time to turn on the negative geotaxis (fig. S4, A and B), righting reflex (fig. S4, C and D), or grasping reflex tests (fig. S4, E and F). However, older ZIKV-infected male and female mice showed decreased muscle strength compared to mock-infected animals in the hindlimb suspension (HLS) test (Fig. 3A and fig. S5A) performed at 9 dpi. Male and female ZIKV-infected mice also showed motor dysfunction in the pole test (15 to 21 dpi) (Fig. 3, B and C, and fig. S5, B and C).

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.

Infections of the central nervous system often lead to significant morbidity and persistent disability (22). To determine whether neonatal infection translated into motor and locomotor alterations in adulthood, animals were subjected to the rotarod and open-field tasks at 85 to 95 dpi. Whereas only male ZIKV-infected mice showed a statistically significant (P = 0.029) decrease in latency to fall in the rotarod test compared to mock-infected animals (Fig. 3D and fig. S5D), mice of both sexes showed impaired locomotor behavior in the open-field task (Fig. 3E and fig. S5E).

We further examined whether early-life exposure to ZIKV induced cognitive deficits in adult life. Both male and female ZIKV-infected mice were unable to distinguish between novel and familiar objects in the novel object recognition (NOR) paradigm tested at 85 to 95 dpi, indicating short-term memory impairment in adult life (Fig. 3F and fig. S5F). These late behavioral effects are specific to infection with a neurotropic flavivirus because DENV-infected mice showed normal performance when evaluated in the rotarod (fig. S3, D and E), open-field (fig. S3, F and G), and NOR (fig. S3, H and I) tasks. Epidemiological and experimental evidence link perinatal infections to the development of neuropsychiatric diseases, including schizophrenia and autism (23), disorders that have, as a common feature, impaired sociability (24). This prompted us to evaluate whether early-life ZIKV infection affects social approach in adult mice (90 to 100 dpi). Although ZIKV infection did not affect preference for exploring the stranger mouse over the empty chamber in male mice (Fig. 3G), female ZIKV-infected mice showed impaired sociability (fig. S5G).

Neonatal ZIKV exposure is persistent in neuropathological alterations in mice

Both cell death and impaired proliferation of neural precursors were shown to underlie ZIKV-induced neuropathology (15, 25). We evaluated the number of proliferating cells in the subgranular zone of the hippocampal dentate gyrus (DG) and in the subventricular zone after ZIKV infection and found no difference in the percentage of Ki67-positive cells compared to mock-infected mice (fig. S6).

To explore the basis for the persistent consequences of early-life ZIKV exposure, we performed neuropathological examination of the brains of mice after the peak of viral replication (23 dpi). Severe brain atrophy with ventriculomegaly was found in 2 of 11 ZIKV-infected animals, whereas no neuropathological abnormalities were found in mock-infected mice (Fig. 4, A to E). ZIKV-infected mice showed multiple necrotic areas, predominantly in the hippocampus, thalamus, striatum, and cortex (Fig. 4, F to H). In the hippocampus, necrosis, dystrophic calcifications with mineral deposition, apoptotic bodies, infiltration of inflammatory cells, and general disruption of the normal cytoarchitecture were observed in all subregions (Fig. 4, J to L). In addition, brains of ZIKV-infected mice showed frequent perivascular lymphocytic cuffings (Fig. 4, M and N). Histological alterations seen in young ZIKV-exposed mice persisted in adulthood; widespread neuronal necrosis with more advanced mineral deposition, especially in the hippocampus, persisted in the brains of 100-day-old ZIKV-infected mice (Fig. 4, O to R). Inflammatory cells and shrunken basophilic neurons were also seen in the brain parenchyma (Fig. 4R).

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).

Neonatal ZIKV exposure increases oxidative stress and inflammation in mouse brain

Neuroinflammation plays an active role in epileptic disorders (26). Compared to brains of mock-infected mice, we found that brains of ZIKV-infected mice showed markedly increased immunoreactivity for glial fibrillary acidic protein (GFAP) and ionized calcium–binding adapter molecule 1 (Iba-1) in proximity of lesions and in the perivascular areas in the CA1 and DG hippocampal regions (Fig. 5, A to J) at 23 dpi. In addition, the peak of viral replication in the brain (12 dpi) was accompanied by increased brain expression of several proinflammatory mediators, including interleukin-6 (IL-6), IL-1β, the murine functional IL-8 homolog keratinocyte chemoattractant (KC), and TNF-α in ZIKV-infected animals when compared to mock-infected mice (Fig. 5, K to N). Brain cytokine expression did not increase in the brain of mice injected with iZIKV (Fig. 5, K to N), indicating that increased expression of these proinflammatory mediators requires active viral replication and not simply the presence of viral particles.

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.

Excessive generation of reactive oxygen species (ROS) has been implicated in the pathology of acute seizures and epilepsy (27). Accordingly, we found that expression of the inducible form of nitric oxide (NO) synthase (iNOS) was increased in the brains of ZIKV-infected mice compared to mock animals (Fig. 5O). Because NO can account for an increased generation of ROS (28), we asked whether ZIKV replication in the brain promoted an increase in ROS generation. In agreement, we found an increased fluorescence of the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein (DCF) diacetate in the brains of ZIKV-infected mice compared to mock-infected mice (Fig. 5P).

TNF-α neutralization prevents seizures in young mice and normalizes susceptibility to chemically induced seizures in adult mice submitted to neonatal ZIKV infection

Both oxidative stress and inflammation have been shown to induce seizures in animal models (29), and pharmacological approaches targeting both processes reduce epileptogenesis (30, 31). Therefore, pups were treated systemically with the antioxidant drug N-acetylcysteine (NAC) or with the TNF-α neutralizing monoclonal antibody infliximab from the day of infection (P3) until seizure evaluation at 12 dpi. Whereas NAC treatment showed no effect on ZIKV-induced seizures, infliximab-treated litters presented a significant (P = 0.0034) decrease in seizures at 12 dpi (Fig. 6A).

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.

We further investigated whether neutralization of TNF-α early after infection translated into reversal of long-term behavioral impairments induced by ZIKV. The latency to first seizure and number of seizures after PTZ administration was higher in infliximab-treated mice (Fig. 6, B to E), when compared to animals that received PBS after infection. On the other hand, infliximab had no effect on the performance of ZIKV-infected mice in the rotarod (Fig. 6, F and G) or NOR (Fig. 6, H and I) tasks. Together, these findings suggest that TNF-α neutralization early after ZIKV infection may prevent development of seizures in young and adult individuals exposed to the virus during the perinatal period.

DISCUSSION

ZIKV isolated from brains of babies after vertical transmission has been associated to microcephaly (12). This appears to be the most extreme consequence of congenital ZIKV exposure; however, a spectrum of neurological abnormalities and dysfunctions is expected to emerge.

Here, we investigated the short- and long-term impacts of ZIKV infection in the developing brain by infecting wild-type mice at P3, a period of rodent brain and immune system development that resembles the second and third trimesters of gestation in humans (32, 33). Studies have reported that normocephalic babies congenitally exposed to ZIKV show decreased head growth beginning several months after birth, which results in postnatal-onset microcephaly (7). In agreement, we here found that mice submitted to early-life (P3) infection by ZIKV show macroscopic brain atrophy after infection (23 dpi).

In agreement with previous reports (15, 34), we found that ZIKV inoculated subcutaneously successfully reaches and replicates in the mouse brain. In addition, our results show that both positive and negative ZIKV RNA strands were still detectable in adult mice (~100 dpi) submitted to early-life viral infection, indicating persistent replication of the virus. These findings provide clinically relevant evidence that brain replication of ZIKV might persist longer after the acute phase of disease.

Seizures have been reported after ZIKV infection in adult patients (35), and studies have shown that 50 to 60% of microcephalic babies due to ZIKV congenital infection develop spontaneous epileptic activity (8, 11). Seizures associated with changes in sleep electroencephalographic patterns were found in these babies, although in some cases, electrographic seizures were identified even in the absence of other clinical manifestations (8, 11). In addition, follow-up studies performed with normocephalic babies exposed in utero to ZIKV suggest that seizures are clinical manifestations in ~50% of babies (7). In agreement, we found a high incidence (~95%) of seizures in neonatal mice exposed to ZIKV. Seizures were detected from 9 dpi and reached higher incidence at 12 dpi, coinciding with the peak of viral RNA detected in the brain. This finding is in agreement with a preliminary report in which the authors showed the presence of self-resolving behavioral seizures in mice after ZIKV infection (16). Here, we further report the presence of spiking activity, polyspike-waves, and fast sharp waves after ZIKV infection in mice. The epileptic activity is likely to be dependent on active viral replication in the brain because seizures were relatively reduced in iZIKV-injected mice. We also found that the number of seizures decreased at 18 dpi and completely disappeared in adult (~100 dpi) mice. These findings suggest that our model recapitulates the proictogenic effect of ZIKV in infants and that spontaneous ZIKV-associated seizures might be reversible as patients grow older. However, the increased PTZ-induced response observed here in ZIKV-infected animals indicates that hyperexcitability might persist long after ZIKV infection is resolved, and further supports that a long-term follow-up of ZIKV-exposed patients should be pursued by public health services, especially in countries affected during the 2015 epidemics.

A number of motor alterations have been reported in babies exposed to ZIKV in utero (20). Moreover, ZIKV has been shown to infect peripheral neurons and spinal cords, causing cell death (25). ZIKV-injected pups showed normal performance in reflex evaluation tests carried out early after infection. These results are in contrast with a previous report of impaired performance in the righting reflex induced by an African ZIKV strain (34), which is much more aggressive to the murine nervous system than the strain used in the present study (36). However, ZIKV-infected mice showed several motor function impairments throughout development and during adulthood, suggesting that ZIKV infection has a long-lasting effect on motor performances.

In addition, our results indicate that ZIKV infection during development affects declarative memory and social behavior in adult animals, suggesting that neuropsychiatric disorders might be a further long-term consequence of perinatal ZIKV exposure. This is in agreement with the association between early-life exposure to several viruses and the development of cognitive impairments (37) and neuropsychiatric disorders (38) later in life. Additional epidemiological studies should address this subject in more detail.

In contrast to previous reports using models of congenital ZIKV transmission (18), we found that the number of Ki67-positive cells did not differ between infected and control mice. This might be explained by the complex brain response induced by neonatal infection, which involves an intense inflammatory response in the brains of mice. It is well known that several aversive conditions (including hypoxia-ischemia, acute seizures, and infections) induce a compensatory increase in neurogenesis in the postnatal brain (39).

To date, a spectrum of neuropathological changes after congenital ZIKV infection, including calcification, necrosis, nerve cell degeneration, microgliosis, hypoplasia, and ventriculomegaly, has been described (40). Neuropathological findings observed in our model are, in several aspects, analogous to those seen in fetuses and newborns with ZIKV congenital syndrome (40). Here, we showed that viral replication was accompanied by increased brain expression of several proinflammatory immune mediators, which is in agreement with the increased levels of proinflammatory cytokines reported in placentas and brains of newborns exposed to ZIKV (9). The importance of brain inflammation in the development of seizures has been demonstrated in several models of infectious diseases (41). Evidence suggests that TNF-α production increases glutamatergic transmission (42), and both seizure frequency and severity are reduced after pharmacological inhibition of TNF-α or deletion of type I TNF-α receptor (26, 31). In agreement, our results showed that blocking TNF-α using the neutralizing monoclonal antibody infliximab shortly after ZIKV infection significantly reduced seizures in young mice. On the other hand, although we also found that ZIKV infection increased ROS generation in mouse brains, treatment with the antioxidant NAC did not prevent seizures. Altogether, these findings support a key role of TNF-α linking inflammation to epilepsy after ZIKV infection.

Experimental evidence supports the repurpose of the U.S. Food and Drug Administration–approved drugs to prevent ZIKV replication, vertical transmission, and even microcephaly in newborns (43). However, strategies to prevent or reverse the long-term behavioral complications associated to infection have never been tested. Our findings show that although early TNF-α neutralization had no effect on ZIKV-induced long-term memory and motor impairments, the treatment was successful in decreasing the susceptibility and number of seizures after chemical stimulation in adult mice neonatally exposed to ZIKV.

There are limitations to this study. First, our model of neonatal infection does not assess the effects of infection at early stages of brain maturation. However, different outcomes are expected depending on the period of fetal development in which exposure occurs (44), and our model only aimed to investigate the long-term impact of ZIKV exposure once it reaches the developing brain. Second, because we found that our treatment schedule with infliximab only partially rescues ZIKV-induced behavioral impairments, future studies will address whether treatment for longer periods can offer full reversal of behavioral and neuropathological damages. Third, it should be noted that although our results suggest an association between early-life ZIKV exposure and the development of neuropsychiatric diseases, we have performed a limited number of tasks to evaluate autism- and schizophrenia-like symptoms in mice. Additional studies should be performed to substantiate an association between these long-term neurological comorbidities. Fourth, our results do not establish whether neonatally ZIKV-infected animals show any improvement in behavioral performance over time because different tasks were performed with animals at different stages after infection.

In conclusion, our findings comprise experimental evidence that ZIKV perinatal exposure is associated to long-term deleterious neuropathological and behavioral consequences. A wide spectrum of neurological and neuropsychiatric manifestations might be expected as a consequence of perinatal ZIKV exposure, especially as an outcome of the 2015 epidemics, which affected a large number of individuals across the Americas. Moreover, our data suggest that TNF-α is a key signaling molecule underlying ZIKV-induced seizures and that patients exposed to the virus during the perinatal period could benefit from the use of TNF-α–blocking strategies. It is now clear that simply monitoring the birth prevalence of congenital microcephaly is an insufficient measure of the burden of ZIKV neuropathology in exposed children and adolescents.

MATERIALS AND METHODS

Study design

The aims of this study were to evaluate the late consequences of ZIKV in the developing brain and to identify potential therapeutic strategies. Sample sizes were chosen according to survival pilot studies. All pups in each litter received the same treatment to avoid cross-contamination, and litters were assigned to a single group by simple randomization. Researchers were blinded to experimental conditions when conducting the experiments and analyzing the results. The Groutt’s test was used to identify potential outliers, which were then excluded from further analyses.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.01 (GraphPad). Data are reported as means ± SEM. Gaussian distribution of data was assessed using the D’Agostino-Pearson test. Body weight curves were compared using two-way ANOVA, followed by Sidak. Data from the object recognition and social approach tests were analyzed by one-sample t test compared to the fixed value of 50. For all other data, either unpaired Student’s t test or one-way ANOVA followed by Tukey test was used. P values are indicated in the corresponding figure legend.

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)

REFERENCES AND NOTES

Acknowledgments: We thank M. Florence, J. Ferreira, and A. C. Rangel. Funding: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (to F.G.D.F., S.T.F., A.T.D.P., I.A.-M., C.P.F., and J.R.C.), Fundação de Amparo à Pesquisa do Estado de São Paulo (to É.A.C.), Institutos Nacionais de Pesquisa–Instituto Nacional de Neurociência Translacional (to F.G.D.F., É.A.C., and S.T.F.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (to P.S.F., G.N., F.G.D.F., É.A.C., S.T.F., A.T.D.P., C.P.F., I.A.-M., and J.R.C.), Institutos Nacionais de Pesquisa–Inovação em Medicamentos e Identificação de Novos Alvos Terapêuticos (to C.P.F.), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (to I.N.O.S., J.V.F., L.F., and D.J.L.L.P.), and Financiadora de Estudos e Projetos (to A.T.D.P.). Author contributions: I.N.d.O.S., P.S.F., G.N., F.G.D.F., S.T.F., A.T.D.P., C.P.F., I.A.-M., and J.R.C. contributed to experimental design. I.N.d.O.S., P.S.F., S.T.F., A.T.D.P., C.P.F., I.A.-M., and J.R.C. analyzed and discussed data. I.N.d.O.S., S.T.F., A.T.D.P., C.P.F., and J.R.C. wrote manuscript. R.L.S.N., I.A.-M., and A.T.D.P. performed viral replication. I.N.d.O.S., P.S.F., J.V.F., J.B.N.-V., and C.O.N. performed experiments in mice. L.F., D.J.L.L.P., and É.A.C. designed, performed, and analyzed electrophysiology. P.S.F., L.C., and C.P.F. performed histological analyses. I.N.d.O.S., P.S.F., R.L.S.N., and J.R.C. performed and analyzed molecular experiements. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data are contained within the manuscript and the Supplementary Materials.
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