2. ZIKV Infection Is Associated to Late Detrimental Effects to the Developing and Mature Nervous Systems
A spectrum of neuropathological conditions has been reported in newborn babies exposed to ZIKV in uterus, being microcephaly the most severe outcome. Infants who develop ZIKV-induced microcephaly show recurrent seizures [
3], and severely compromised neuropsychomotor development is frequently reported [
4]. Several alterations were described after brain tomography of babies congenitally exposed to ZIKV, including widespread calcifications, cerebral atrophy, brainstem and cerebellar hypoplasia, as well as ventriculomegaly [
5] (
Figure 1A).
Extensive research has focused on unraveling the mechanisms of congenital microcephaly induced by ZIKV [
6,
7,
8]. However, increasing evidence from clinical and experimental studies suggest that even infants considered healthy at birth can develop severe long-term neurological complications as a consequence of exposure. According to US hospital records, 6% of children infected with ZIKV in uterus showed malformations detectable at birth. Importantly, the percentage of infants with ZIKV-associated neurological complications rose to 14% when babies considered normal at birth were followed-up after leaving hospital [
9]. Among the most common symptoms reported were personal-social developmental delays, swallowing difficulties, and a myriad of motor dysfunctions including compromised mobility, muscular hypertonia, dystonic movement, and impaired fine motor skills [
9,
10,
11]. Post-natal onset microcephaly was also reported, and studies suggest that 60% of normocephalic babies exposed to ZIKV in uterus present seizures at some stage of development [
11]. Several alterations were also described in brains of normocephalic ZIKV-exposed babies assessed by neuroimaging tools, including subcortical calcifications, increased frontal cerebrospinal fluid space and ventricle enlargement [
12]. Infants exposed to ZIKV during the epidemic in the American continent are still under their third year of life, making it impossible to predict which of these symptoms may persist and if others may emerge. Animal models are useful tools to investigate the mechanisms of infection and its complications, and whereas most studies have focused on unraveling the mechanisms of ZIKV-induced microcephaly, a few others have provided insight into the mechanisms underlying late ZIKV-induced neurological damage. Using a mouse model of ZIKV infection, our group was able to predict possible long-term alterations in children after a congenital infection. Since the stage of development of the mouse brain at early postnatal period resembles the events taking place in the human brain during the second and third trimester of pregnancy [
13,
14,
15], we performed ZIKV infection shortly after birth (post-natal day 3). Indeed, we found that infected pups presented neuropathological hallmarks that closely resembled those seen in brains of babies after ZIKV congenital infection. Over 90% of mice showed spontaneous seizures during childhood, and intensity of seizures decreased as animals grew into adulthood. Moreover, even though these episodes resolved when animals became adults, mice infected neonatally with ZIKV remained more susceptible to chemically-induced seizures. Besides, ZIKV-infected mice showed motor and cognitive disabilities in adulthood. Similar results were obtained in another study, in which authors demonstrated that early-life infection with an African ZIKV strain results in persistent memory impairments in mice [
16]. These findings were further confirmed in non-human primates infected in uterus or shortly after birth. As adults, these neonatally-infected macaques developed an atypical emotional response [
17] and neuropathological characterization of brains showed reduced white matter volume, ventriculomegaly, and decreased hippocampal growth in ZIKV-infected animals.
Neurological complications following ZIKV infection are not limited to cases of congenital infection [
18]. Mounting clinical evidence show that ZIKV infection in adult patients may result in encephalitis [
18,
19,
20], encephalomyelitis [
18,
20], acute myelitis [
18,
19], chronic inflammatory demyelinating polyneuropathy [
21], and Guillain-Barre syndrome [
19,
22,
23] (
Figure 1B). Episodes of septic shock were also reported, and in rare cases these complications were shown to be fatal [
21,
24]. In addition, ZIKV-associated Guillain–Barre syndrome may, in some cases, lead to development of chronic pain [
18]. One study found that 40% of patients still showed some kind of neurological alteration after 1 year of symptom onset, and after two years some patients had not yet fully recovered [
25]. Collectively, these reports demonstrate that ZIKV infection is also detrimental to the mature nervous system and is associated with a spectrum of neurological syndromes that may culminate in long-term consequences and even mortality. Whether these neurological consequences are a direct effect of ZIKV replication or are secondary to the host immune response still need to be investigated; a question that could lead to important clues on how to prevent long-term consequences of infection. Different research groups have shown beneficial effects of both antiviral and anti-inflammatory strategies in preventing long-term behavioral consequences of infection. These findings are reviewed in the following sections.
Another matter of concern involves the ability of ZIKV to persist in certain organs and tissues, since the late consequences of viral persistence remain unknown. ZIKV usually reaches detectable levels in the serum of patients within 3–10 days after onset of symptoms, shortly returning to undetectable levels [
26]. Even after viral clearance from blood and up to 200 days after infection, detectable levels of ZIKV RNA were found in placenta of pregnant women [
27]. ZIKV was also shown to persists in semen samples up to 93 days after the onset of symptoms, reinforcing the high risk of sexual transmission [
28,
29]. ZIKV RNA was found in brains of babies exposed in utero on average 163 days after infection, and one study described high viral loads after post-mortem analysis of the brain of a 5-month-old infected infant [
5]. The ability of ZIKV to replicate for long periods in specific tissues has also been demonstrated in animal models. In adult non-human primates infected subcutaneously with ZIKV, virus remained in neuronal tissue, cerebrospinal fluid, and lymph-nodes, while in the serum levels were undetectable after only seven days post-infection [
30,
31]. Further evidence of persistent replication in neuronal tissue were found in immunocompetent mice infected during the neonatal period, where ZIKV negative RNA strand was detectable in the brain into adulthood (100 dpi), an indicative of active viral replication [
32]. Altogether, these results provide evidence that ZIKV is capable of persisting in certain tissues long after resolution of symptoms and clearance of virus from blood. Factors that may contribute to viral persistence remain unknown, as well as if new rounds of active viral replication and reoccurrence of symptoms can occur at later stages of life, including moments of compromised immune response or as consequence of other viral infections.
3. What Genetic, Epigenetic, and Environmental Factors May Increase the Risk of ZIKV-Induced Neurological Damage?
Little is known about what factors can contribute to the development of late neurological damage associated to ZIKV infection, and what are the precise mechanisms involved. It is likely that several genetic and environmental factors interplay to increase one’s susceptibility to the development of neurological outcomes (
Figure 1). A comprehensive knowledge of how ZIKV-induced neurological damage is induced in adult and infant patients is crucial to prevention and treatment of such severe sequelae.
ZIKV strains isolated from different geographical regions are derived from two main lineages: African and Southeast Asian [
26]. Even though the African strain is more cytotoxic and neurovirulent in experimental settings [
33,
34], the ZIKV strain responsible for the outbreak in the Americas is genetically closer to strains isolated in French Polynesia in 2013 and belongs to the Southeast Asian subclade [
35]. One study has shown that the contemporary strain (isolated in 2015–2016) carries several nucleotide substitutions when compared to a strain isolated in 2010 in Cambodia. A serine-to-asparagine substitution (S139N) in the Pr domain of the viral precursor membrane protein (prM), was shown to be especially responsible for its increased neurovirulence [
36]. Interestingly, this substitution emerged in the Southeast Asian ZIKV strain shortly before the 2013 outbreak and was maintained in the subsequent epidemics in the Americas [
36].
Mutations in ZIKV genome may also promote host immune system evasion and enhance infectivity of mosquito hosts [
37,
38], which could contribute to increase the geographical spread of the virus. African and Asian populations were highly exposed to ZIKV historically [
39,
40,
41,
42,
43], and it has been speculated that prior contact to the virus may provide protective immunity [
44]. Thus, it is reasonable to speculate that the sudden exposure of an immunologically naïve population, in the American continent, provided unseen absolute numbers of acutely ZIKV-infected patients, favoring the observation of long-term consequences.
Variations on the hosts’ genetic background are determinant to an appropriate immune response, and may thus influence the occurrence of ZIKV-associated neurological complications. Experiments in animal models support this notion, since infection is associated to either mild or severe signs of disease, depending on the genetic characteristics of the host. For example, effective systemic infection in mice requires a limited interferon-mediated immune response, which is obtained either by knocking out specific components of interferon (IFN) pathway [
45,
46,
47] or by using neonatal mice [
32], a stage where interferon-mediated immune response is lacking or insufficient. While type III IFN response is protective against ZIKV infection [
48], type I response is related to miscarriage and growth restriction, especially during mid-gestation [
49]. Several signaling pathways were shown to modulate ZIKV infection and individual differences in their expression may confer variable risk to congenital Zika syndrome (CZS) and other neurological outcomes: TAM receptors (TYRO3, AXL, and MER) are important in mediating ZIKV persistence through type I IFN downregulation [
50,
51,
52,
53,
54,
55]; toll-like receptors 3 (TLR3) increase viral replication and are important mediators of inflammatory responses [
50]; infection triggers autophagy pathways, but their effects on viral replication and immune response are still controversial [
50,
56] (for review see [
57]). In addition, the chances of prior or concomitant infection by other mosquito-borne viruses is high in ZIKV endemic regions and co-infections might modulate the hosts’ immune response either in a protective [
58,
59] or detrimental way [
60,
61,
62,
63,
64]. Experimental evidence suggest that mosquitoes previously infected with Chikungunya virus have enhanced ZIKV infectivity and vector competence [
65].
In line with this, individual responses determined by either genetic or epigenetic backgrounds have been shown to modulate susceptibility of cells in the developing brain to ZIKV. In a recent study, neural stem cells (NSCs) derived from three independent donors were infected in vitro with ZIKV (Mexico isolate). The virus was shown to replicate and induce decreased cell proliferation in all NSCs strains. However, NSCs from one of these donors were resistant to ZIKV-induced decrease in neuronal differentiation. RNA expression patterns changed accordingly: ZIKV-sensitive strains showed pronounced reduction in pathways related to neurogenesis and higher expression in pathways responsible for innate immune response [
66]. In another interesting study, neural progenitor cells (NPC) were induced from salivary cells obtained from twins exposed to ZIKV in uterus [
67]. Even though the study was performed in a small cohort, both concordant (both fetuses showing CZS) and discordant (only one fetus with CZS) twins were analyzed. No unique genetic trait that could explain differential susceptibility of siblings to developing CZS was found, therefore, authors suggest there could be multifactorial inheritance or even epigenetic modifications linked to different responses to viral infection. In line with this, NPCs and neurospheres derived from CZS-fetuses produced more infectious particles when exposed to ZIKV in vitro than those from non-CZS infants. RNA sequencing performed in these cells reported changes in expression patterns, notably upregulation of mammalian target of rapamycin (mTOR) and Wnt pathways, exclusively in affected twins [
67].
Concerning the development of CZS, it can also be speculated that a pregnant woman’s individual response to infection influences the efficiency with which ZIKV crosses the placental barrier. Trophoblasts, placental barrier cells, are permissive to ZIKV only during a narrow time-frame, likely in the first trimester of gestation [
48,
53,
68,
69,
70]. This is probably one of the reasons why infection during this stage of pregnancy is usually associated to the birth of babies with more severe neurological outcomes. Trophoblasts secrete type III IFN [
48,
70], and the amount of type III IFN released could be related to the efficacy of ZIKV infection in these cells. In addition, other events known to limit IFN response, like concomitant viral infections, could enhance ZIKV infectivity. Accordingly, concomitant infection with Herpes Simplex 2 virus (HSV-2) increases susceptibility of trophoblasts to ZIKV [
53]. Alternatively, to direct transport, it is possible that maternal antibody-mediated transcytosis drives ZIKV transportation across the placental barrier. Indeed, experimental evidence has shown that placental ZIKV replication is enhanced by previous infection with Dengue virus [
60,
71]. In addition to trophoblasts, placenta villous macrophages (Hofbauer cells) and fibroblasts are susceptible to ZIKV infection [
68,
72]. When exposed to ZIKV, Hofbauer cells become activated, but the extent of activation and cytokine production is highly variable between individuals. It is thus possible that the intensity of placental immune response influences how much ZIKV actually reach the fetus [
68].
Despite factors specific to host and viral genomes, several other factors can have contributed to the unforeseen scale of ZIKV neurological damage seen in recent years. An interesting study in immunodeficient mice has suggested that sexual transmission of ZIKV favors congenital infection and the development of malformations of fetuses, suggesting that the route of infection may also play a role in the severity of congenital ZIKV syndrome [
73].
As recently reviewed by Barbeito-Andrés and colleagues [
74], environmental stresses emerging from adverse social-economic conditions or other geographical factors can contribute to neurological outcome of infection. In Brazil, although the incidence of ZIKV infection was similar in all regions in 2015, congenital ZIKV syndrome was significantly higher in the northeast region, a severely impoverished region [
75]. When comparing different regions of the northeast of Brazil, epidemiological studies showed that higher incidence of microcephaly was observed in districts with lower developmental index, higher rates of mosquito larvae density, and more inefficient sewage and garbage collection systems [
76]. Impoverished populations are more frequently susceptible to mal-nutrition or inappropriate diet, degrading housing conditions which facilitates mosquito spreading and disease transmission, higher incidence of coinfections and poorer access to health system and family planning, among others. Additional population-based and experimental studies should be performed in order to determine whether and how any of these factors has contributed to determine the higher rate of neurological complications due to ZIKV infection in specific geographical regions.