Zika virus (ZIKV) is a mosquito-vectored flavivirus, consisting of three structural proteins [capsid (C), pre-membrane/membrane (prM/M), and envelope (E)], seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and a single-stranded RNA genome of positive polarity (Yun and Lee, 2017). ZIKV has been detected in saliva, tears, urine, semen, brain, female genital tract, and testes (Morrison and Diamond, 2017). ZIKV along with the other members of the Flaviviridae family, including West Nile virus (WNV), dengue virus (DENV), hepatitis C virus (HCV), and Japanese encephalitis virus (JEV) possess significant neuroinvasive characteristics and are identified as neurotropic. In recent years, ZIKV received global attention due to the association with more severe neurological manifestations, such as Guillain-Barre syndrome (GBS) in adults (Oehler et al., 2014; Cao-Lormeau et al., 2016; Watrin et al., 2016) as well as microcephaly in infants (Brasil et al., 2016; Schuler-Faccini et al., 2016). Recent studies have demonstrated the neuro-invasiveness, tropism, and virulence of ZIKV (Shao et al., 2016; Costa et al., 2017a; Zhang et al., 2019). Owing to these severe complications, an extensive understanding of ZIKV neuroinvasion mechanisms and the host molecules involved is vital for therapy development.

The entry mechanism of ZIKV into human cells, particularly neural cells, remains poorly understood. Previous studies have demonstrated several ZIKV entry factors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), AXL receptor tyrosine kinase (AXL), TYRO3 protein tyrosine kinase (TYRO3) and though to a lesser extent, T-cell immunoglobulin and mucin domain 1 (TIM-1), mediate entry of ZIKV into human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells (Hamel et al., 2015; Table 1). It is of note that ZIKV infection in primary dermal fibroblast was significantly decreased by RNA inhibitor and neutralizing antibody to AXL (Hamel et al., 2015). Therefore, the AXL receptor appears to play a vital role as the viral entry receptor. High expression of AXL receptor is seen within cells of the developing central nervous system (CNS), such as endothelial cells, microglial, astrocytes, and radial glial cells (Table 1; Nowakowski et al., 2016). A study found that the susceptibility of ZIKV to endothelial cells positively correlated with the cell surface levels of AXL (Liu et al., 2016). Belonging to the group of tyrosine kinase receptors TYRO3, AXL, and MER (TAM) family, AXL acts in apoptotic cells clearance and innate immunity modulation (Rothlin et al., 2007; Lemke and Rothlin, 2008). It is said that ZIKV is attached indirectly to the AXL receptor, mediated by the natural ligand of AXL, growth arrest–specific gene 6 (Gas6) based on the exposure of phosphatidylserine (PS) on viral envelope surface. Astrocytes and microglial cells appear to be the major ZIKV targets, having remained strongly expressed in the developing human cortex even as gestation progress (Meertens et al., 2017).

However, recent findings demonstrated that ZIKV still showed effective invasion, infection, and replication in AXL-depleted cerebral organoids (Wells et al., 2016), neural progenitor cells (NPCs; Wells et al., 2016), and murine models (Wang et al., 2017b; Li et al., 2017c), suggesting that AXL may not serve as the exclusive receptor involved and that its dependency for viral entry may be cell-type or model specific. A further study reported the role of AXL in antagonizing ZIKV-induced activation of type I interferon (IFN) signaling, which facilitates ZIKV infection in astrocytes, instead of being a ZIKV entry receptor (Chen et al., 2018). While active investigations are still required on ZIKV target receptors, other receptor-independent mechanisms should be considered. Exosomes, secreted by most cell types, transport proteins, lipids, and nucleic acids between cells through systemic circulation or paracrine transmission, thereby exerting functional responses in target cells (Ramakrishnaiah et al., 2013). Several studies have shown exosome-mediated intercellular transmission of viruses (Narayanan et al., 2013; Ramakrishnaiah et al., 2013; Zhu et al., 2015). Tunneling nanotubes that connect a number of cell types, including immune and neuronal cells, are also shown to promote viral spread (Sowinski et al., 2008).

Tan et al. (2019) studied the mechanism underlying ZIKV infection in Vero, Huh-7, and induced pluripotent stem cell (iPSC)-derived human NPCs (hNPCs) using cell surface carbohydrates, sialic acid, and being the attachment receptor for several viruses. Although there was no direct involvement with ZIKV attachment, findings suggested that sialic acid could be an important mediator in the internalization of the ZIKV-receptor complex and its depletion significantly reduced infection in NPCs (Tan et al., 2019). Nevertheless, the underlying mechanism remains unclear and requires further research (Tan et al., 2019). Other factors, such as neural cell adhesion molecule 1 (NCAM1) and integrin αVβ5 have been postulated to act as cell-type-specific receptors for ZIKV entry (Srivastava et al., 2020; Wang et al., 2020).

The blood–brain barrier (BBB) comprises endothelial cells strongly adhered via tight junction proteins (TJP), which are associated with pericytes, astrocytes, and microglia (Abbott et al., 2010; Mustafa et al., 2019). The tight junctions (TJ) complexes of transmembrane proteins composed of structural proteins claudins and occludins found on plasma membranes of adjacent brain endothelial cells maintain the integrity of BBB. Several routes of invasion into the CNS have been demonstrated by neurotropic viruses: (Yun and Lee, 2017) transcellular (through cells) transport within endothelial cells through infection or transcytosis, and subsequent release of the virus into the CNS, (Morrison and Diamond, 2017) infected peripheral immune cells such as monocytes enter the CNS through Trojan horse strategy, (Oehler et al., 2014) paracellular (between cells) entry of virus following disruption of the BBB, and destabilization of TJ, (Cao-Lormeau et al., 2016) retrograde axonal transport of virus through peripheral nerves into the CNS, and (Watrin et al., 2016) blood-to-cerebral-spinal-fluid (CSF) translocation (Ayala-Nunez and Gaudin, 2020; Hsieh and St John, 2020; Chen and Li, 2021). Among these, BBB dysfunction is the most common neuroinvasion route for flavivirus due to indirect effects of systemic inflammatory cytokines such as tumor necrosis factor (TNF) and IFN or direct attachment to claudins (Neal, 2014).

It was demonstrated that ZIKV can infect and efficiently replicate in the human brain microvascular endothelial cells (HBMECs) and iPSC-derived BBB models without significantly altering the endothelial barrier integrity and permeability in vitro (Mladinich et al., 2017; Papa et al., 2017; Alimonti et al., 2018). Despite the efficient viral replication, the endothelium displayed tight BBB and its architecture was not extensively perturbed. However, actin cytoskeleton rearrangement was observed upon infection, suggesting possible changes in the morphology of the endothelium (Cle et al., 2020). These findings suggest that endothelial leakage and BBB disruption may not be essential for ZIKV to reach the brain, and ZIKV might utilize other mechanisms to invade the CNS (Figure 1).

Several in vitro studies have demonstrated ZIKV infection and release from both apical and basolateral surfaces of brain endothelial cells without compromising the barrier permeability and integrity (Mladinich et al., 2017; Papa et al., 2017; Alimonti et al., 2018). ZIKV RNA was detected in the lower chamber of the transwell system despite maintaining the endothelial integrity. These studies support that ZIKV may cross the BBB through transcytosis, basolateral virus release, or paracytosis (Figure 1). A charge-based mechanism occurs whereby positively charged ZIKV particles foster attraction with the negatively charged membrane of brain endothelial cells for transcytosis (Nakayama et al., 2021). The secretion of type I and III IFNs and inflammatory cytokines did not affect microvascular endothelial cells (MECs) permeability but contributed to transinfection (Papa et al., 2017). These findings suggest potential drugs that block ZIKV replication or transcytosis can effectively prevent virus extravasation through MEC monolayer. Treatment with Chloroquine (inhibitor of cellular infection) and BFA (exocytosis inhibitor), inhibited virus RNA release and extravasation in a transwell system, indicating active virus replication is required for ZIKV to cross the BBB. On the other hand, treatment with Nystatin (inhibitor of caveolae-mediated transcytosis) impaired extravasation of ZIKV through HBMECs without affecting virus replication, suggesting utilization of transcytosis and/or basolateral release pathways by ZIKV to cross the BBB after infection and activation of endothelial cells (Papa et al., 2017).

The ZIKV transcytosis may be mediated by the major facilitator superfamily domain-containing protein 2 (Mfsd2a; Figure 1). Mfsd2a is selectively expressed on the CNS endothelial cells, and its genetic ablation resulted in increased transcytosis and leaky BBB (Ben-Zvi et al., 2014). It plays a critical role in transporting docosahexaenoic acid (DHA) into CNS (Chen and Li, 2021). The lipids transported by Mfsd2a mediate inhibition of caveolae vesicle formation in CNS endothelial cells and suppress transcytosis through which it maintains the integrity of the BBB. The deficiency of Msfd2a reduced the levels of DHA in the brain, which was accompanied by loss of neuronal cells, cognitive deficits, and microcephaly (Nguyen et al., 2014). ZIKV envelope (E) protein was found to interact with Mfsd2a. ZIKV E promotes polyubiquitination of Mfsd2a and mediates its proteasome-dependent degradation. ZIKV inhibited the levels of Mfsd2a in hBMECs and in neonatal mouse brains. In addition, ZIKV caused a reduction in the Mfsd2a-mediated DHA uptake and supplementation with DHA rescued ZIKV-indued abnormal brain development in mice (Zhou et al., 2019).

A study by Ayala-Nunez et al. (2019) proposed that ZIKV may use the Trojan horse strategy to cross the BBB by infecting the monocytes (Figure 1). The study found ZIKV-infected monocytes in the brain slices from the fetus with microcephaly. It was further demonstrated that ZIKV productively infected human primary monocytes and promoted viral dissemination to cerebral organoids. ZIKV manipulates the adhesive properties of monocytes, particularly CD14 and CD16 monocytes by increasing its expression of surface adhesion molecules (integrins, ICAM3, PECAM1, IQGAP1, catenin, myosins, actinin, KIF5B, vinculin, talin, and filamin A and B). This enables the ZIKV-infected monocytes to have a greater attachment to the blood vessel wall and transmigrate across the endothelium to infect neural cells (Ayala-Nunez et al., 2019). A study showed that HBMECs allowed transmigration of ZIKV-infected human monocyte THP-1 in a transwell system and subsequently infected the astrocytes in the basolateral compartment. As astrocytes play an important role in the maintenance of BBB through direct interaction with endothelial cells in the brains, ZIKV infection of these cells after crossing the endothelial layer may lead to inflammation and alteration of the barrier. In this context, modulation of inflammatory molecules, such as, C-C motif ligand-5 (CCL5/RANTES), C-X-C motif chemokine ligand 10 (CXCL10), and IFN-β was observed in astrocytes (Bramley et al., 2017; Cle et al., 2020). ZIKV infection of monocytes was also observed in macaques, and the infected cells were recruited to tissues, resulting in persistent viral infection (O’Connor et al., 2018). Hence, ZIKV-infected monocytes could represent a carrier for the Trojan horse strategy to invade the CNS.

The Trojan horse transmigration of ZIKV-infected monocytes across BBB depends on two receptors, chemokine receptor 7 (CCR7) and receptor for advanced glycation end (RAGE) expressed on ZIKV-infected monocytes. In a state of inflammation, the natural ligand of CCR7, chemokine ligand 19 (CCL19), and the danger-associated molecular pattern (DAMP) molecule, nuclear high mobility group box 1 (HMGB1) are upregulated. This receptor-ligand interaction induces dysregulation of the endothelium layer associated with disorganization of cadherins and actin fibers, thereby increasing the membrane’s permissiveness (de Carvalho et al., 2019). The process is mediated by the upregulation of C-X-C motif chemokine 12 (CXCL12) in monocyte during ZIKV infection, a key regulator of lymphocytes shifts across BBB into CNS parenchyma as it interacts with C-X-C chemokine receptor type 4 (CXCR4). The accompanying lymphocytes-induced inflammation in brain parenchyma further triggers subsequent downstream activation pathways resulting in increased BBB damage (Panganiban et al., 2020).

The paracellular pathway involving proteasomal degradation mechanism has been proposed as one of the pathways for BBB penetration (Figure 1). A study by Shao et al. showed that ZIKV infection induced altered vasculature and a leaky BBB in developing mouse brain (Shao et al., 2016). An in vitro study using transfected HBMECs (THBMEC) infected with ZIKV demonstrated vascular leakage enhancement by ZIKV through disruption in the cytoskeleton and tight junctional proteins arrangement. This occurs via the upregulation of genes that are involved in the production of proinflammatory cytokines, such as interleukin-6 (IL-6), TNF-α, cell adhesion molecules (CAMs), and growth factors (Ismail et al., 2018), which activates the RhoA/Rho-associated coiled-coil containing protein kinase (ROCK)/phosphorylated myosin light chain (pMLC) signaling effectors, thus intensifying stress fibers production (Chen and Li, 2021). Additionally, matrix metalloproteinases (MMP-2/MMP-9) and the proteasome are stimulated by the RhoA mechanism in the breakdown of TJP (Chen and Li, 2021). Downregulation of TJP zonula occludens-1 (ZO-1), occludin, and Claudin-5 by ZIKV (Leda et al., 2019), affecting their ability for transient phosphorylation and dephosphorylation (Siddiqui et al., 2015), was linked to an increase in trans endothelial permeability and BBB penetration (Argaw et al., 2009). The ZIKV-induced BBB disruption is likely to occur at later stages of the disease (Papa et al., 2017; Leda et al., 2019).

Zika virus infection of HBMECs and in vitro BBB model enhanced expression of type I and III IFNs and induced a significant increase in the release of cytokines [interleukin-1β (IL-1β), interleukin-8 (IL-8), IL-6, and TNF-α], chemokines [C-C motif ligand-2 (CCL2/MCP-1) and CCL5], and CAMs [vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1)]. ZIKV infection of pericytes showed upregulation of chemokines (CCL5 and CXCL10), cytokines [IL-6, IL-8, and interleukin-15 (IL-15)], as well as Toll-like receptor 3 (TLR3; Cle et al., 2020). The modulation of BBB proteins induced by ZIKV infection of endothelial cells and pericytes may trigger recruitment and docking of immune cells to the BBB and potentially lead to immune cell CNS infiltration and neuroinflammation (Papa et al., 2017; Cle et al., 2020). Increased levels of CAM were also observed in ZIKV-infected mouse models as well as in plasma from patients. In a mouse model, BBB permeability was not observed during the early time points, but subtle BBB alteration was detected at later time points, which could be associated with an inflammatory response triggered by viral replication (Papa et al., 2017). A study by Bramley et al. (2017) demonstrated that pre-treatment of a three-dimensional (3D) model of microvascular endothelial cells with TNF-α enhanced virus replication and disorganization of the junctional network, suggesting the potential role of inflammatory response in BBB disruption in vivo.

As a matter of interest, cerebral organoid designs hold promising means in studying the 3D morphological and molecular characterization in ZIKV-induced microcephaly as it is able to recapitulate the human brain environment (Antonucci and Gehrke, 2019). However, this system has limited vascularization required for the prolonged culture period, presenting difficulty in discerning the necrotic core due to ZIKV or suboptimal preparation (Antonucci and Gehrke, 2019; Chen et al., 2019). The lack of BBB affects the ability to emulate the natural neuroimmune response toward ZIKV by infiltrating peripheral immune cells across the BBB (Antonucci and Gehrke, 2019). Therefore, co-culturing or other bioengineering techniques would improve the brain organoid model and provide a clear picture of the relationship between the CNS and peripheral circulatory system (Chen et al., 2019).

On the other hand, ZIKV infection and dissemination along peripheral nerves lack evidence and may not be the primary mechanism; however, it cannot be fully disregarded. Contrary to the BBB endothelial cells, the choroid plexus endothelial cells have fenestrated blood capillaries and are leaky, thereby could provide a pathway for the virus to spread out of the blood and enter into the choroid plexus cisternae. ZIKV can infect the pericytes around the fenestrated capillaries, forming a local amplification site and subsequently transcytosed across the blood-CSF layers (Kim et al., 2020).

Similar to primary microcephaly, ZIKV-related microcephaly was reported to be the neurodevelopmental disruption during the first trimester when cortical neurogenesis is most active (Johansson et al., 2016). ZIKV causes microcephaly by affecting the NPCs through cell-autonomous and non-cell autonomous pathways.

Cell cycle perturbation by ZIKV, in particular the S-phase restriction, provides a favorable cellular environment for ZIKV replication as well as impairs the growth of hNPCs (Hammack et al., 2019). ZIKV causes host DNA breaks, activating the ataxia telangiectasia mutated (ATM)/Chk2 signaling cascade and inhibiting ataxia telangiectasia and rad3+ related (ATR)/Chk1 signaling cascade (Hammack et al., 2019). DNA damage response (DDR) is subsequently activated where DNA repair proteins H2A.X and 53BP1 are phosphorylated and cell cycle regulators proteins cell division cycle 25 A (CDC25A), cyclin A and cyclin E are degraded, resulting in G1/S transition arrest (Hammack et al., 2019). G0/G1 transition arrest was not shown to enhance ZIKV replication, implying a possible need for host DNA replication or damage repair factors for its replication (Hammack et al., 2019). Another in vitro study found that ZIKV envelope proteins restrict G2/M progression and induce apoptosis in neuroendocrine PC12 cells through upregulation of tumor suppressor protein, p53 and cyclin-dependent kinase (CDK) inhibitor (CDKi), p21^Cip1/Waf1 leading to downregulation of G2/M phase regulator, cyclin B1, and augmentation of the proapoptotic pathway with the increased B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax)/Bcl-2 ratio (Liu et al., 2018). In this context, the intrinsic cell death signaling mechanism is triggered by the activation of caspase-9 and caspase-3 (Liu et al., 2018). The attenuation of neural cell proliferation in the ventricular, subventricular, and intermediate zone of the fetal brain causes a significant decrease in intermediate progenitor cells, lateral ventricles size, and cortical surface area (Wu et al., 2016). Additionally, defective cell division due to ZIKV may precede the apoptotic program of neural stem cells; a cellular mechanism used to prevent genomic instability (Vitale et al., 2011; Souza et al., 2016). A number of mitotic dysfunctions were reported, such as extra centrosomes, multipolar spindles, partial segregation of spindle pole, chromosomal aneuploidy, and micronuclei production (Souza et al., 2016).

Recent findings revealed that ZIKV causes mitochondrial stress in human derived-iPSC astrocytes with alteration to both its structure and metabolism to supply the energy demand for viral replication through oxidative phosphorylation (OxPhos) pathway of ATP production (Ledur et al., 2020; Figure 2). Mitochondria being the putative avenue for reactive oxygen species (ROS) production were observed to have an increased level of oxidative stress in infected astrocytes, contributing to the DNA breaks and activation of DDR (Ledur et al., 2020; Figure 2). DDR plays an important role in inducing cell cycle arrest apoptosis (Devhare et al., 2017). The activation of DDR triggered phosphorylation of Histone H2AX (γH2AX) followed by assembly of 53BP1, both of which interact with other DNA repair proteins at the foci of DNA double-stranded break (Ledur et al., 2020; Figure 2). DDR plays an important role in inducing cell cycle arrest apoptosis (Devhare et al., 2017). Intermediate filaments, glial fibrillary acidic protein (GFAP), and Vimentin were elevated in infected astrocytes in vitro and in vivo, corroborating with reactive astrogliosis induced by ZIKV (Ledur et al., 2020). That said, mitochondrial dysfunction has been known to be associated with various neurological disorders, including neurodegenerative diseases, cerebral hypoxia, cerebral ischemia, and other brain injuries (Lopez-Domenech et al., 2016; Alimonti et al., 2018; Ludwig et al., 2018; Zhao et al., 2019). In the context of astrocytes, mitochondrial damage has been shown to cause apoptosis in motor neurons, and axon destruction in GBS (Madigan et al., 2017).

Zika virus is capable of modulating the host endoplasmic reticulum (ER) structure for its replicative benefits (Ropidi et al., 2020; Figure 3). The viral NS4A exploits host reticulon 3.1A to promote ER membrane curvature for viral entry into ER, allowing replication to take place (Ropidi et al., 2020). The budded ZIKV RNA, along with other ZIKV proteins such as NS2B-NS3 and unprocessed C-prM-Env complexes are then assembled and cleaved in adjacent ER by ZIKV NS2A protein and NS2B-NS3 protease, respectively thereby producing new ZIKV virions (Ropidi et al., 2020). However, in the process of ZIKV infection, excessive formation of misfolded proteins overpowers the ER protein-folding capacity, causing ER stress and activation of unfolded protein response (UPR), which ultimately lead to apoptosis (Ropidi et al., 2020; Figure 3). In vitro and in vivo studies have shown an increase in ER stress proteins as well as expression of key molecules of the UPR, such as glucose regulatory protein 78 (GRP78), calreticulin, calnexin, and protein disulfide isomerase (PDI) in infected neural cells (Ropidi et al., 2020), resulting in neurogenesis inhibition and microcephaly (Gladwyn-Ng et al., 2018; Figure 3). In parallel, ZIKV-induced ER stress represses stress granules (SG) assembly, which functions to arrest global translation via eIF2α phosphorylation and SG proteins exploitation (Ropidi et al., 2020; Figure 3). Reticulophagy, a compensatory host innate defense response to engulf viral protein and damaged ER for lysosomal degradation, is inhibited by ZIKV NS2B-NS3 protease (NS2B/3; Ropidi et al., 2020; Figure 3). Besides intrinsic and extrinsic apoptotic cell death, the prolonged ER stress renders paraptosis-like death through extensive cytoplasmic vacuolization in ZIKV-infected cells (Ropidi et al., 2020; Figure 3).

Modulation of the host autophagy mechanism has been demonstrated by flaviviruses, such as DENV (Heaton and Randall, 2010), HCV (Sir et al., 2012), and ZIKV in skin fibroblast (Hamel et al., 2015) to promote viral replication. Recent findings showed that ZIKV induced the autophagosome-specific marker, cytosolic microtubule-associated light chain 3 (LC3), indicating autophagosomes are proviral and provide the avenue for ZIKV replication (Liang et al., 2016). ZIKV NS4A and NS4B have shown to inhibit the AKT-mammalian target of rapamycin (mTOR) signaling, resulting in reduced neurogenesis and increased autophagy in human fetal neural stem cells, therefore elevating viral replication (Liang et al., 2016). Although the precise molecular mechanism by ZIKV on autophagosomes is still elusive, various pathways, such as deregulating the host antiviral innate immunity (Ke and Chen, 2011), upregulating viral RNA translation (Dreux et al., 2009), and modulating host lipid metabolism for viral replication (Heaton and Randall, 2010) have been proposed and would benefit from further exploration.

From the non-cell autonomous pathway aspect, a recent study demonstrated that ZIKV induced neuronal apoptosis on adjacent healthy neurons while maintaining its replication in the infected neurons, potentially via ZIKV’s upregulation of pre-mRNA-processing-splicing factor 8 (Pprf8), an anti-apoptotic factor demonstrated in Picornavirus-infected neurons (Olmo et al., 2017). ZIKV-infected neurons undergo neuroinflammation process, secreting TNF-α, IL-1β, and glutamate (Olmo et al., 2017). These neurotoxic factors activate GluN2B-containing N-methyl-d-aspartate receptor (NMDAR), potentiating Ca2+ influx into the cell, which promotes excitotoxicity and eventually cell death (Olmo et al., 2017).

Cortical development is a highly complex process requiring tightly regulated and finely tuned sequential machinery. Hence, as vascular development is pivotal for organogenesis, the impairment of neurogenesis is demonstrated to be in part, due to vascular abnormalities (Garcez et al., 2018). Defective vasculature and decreased neuronal proliferation were concurrently apparent in the ZIKV-infected mice brain models, particularly in the ventricular zone, indicating the presence of a causal relationship between them (Garcez et al., 2018). These findings are further supported by the significant elevation in anti-angiogenic proteins, Ang1, Ang3, as well as endostatin and pigment epithelium-derived factor (PEDF), albeit to a smaller degree from proteomic analysis (Garcez et al., 2018).

Since the 2015 ZIKV outbreak and its association with congenital abnormalities, tremendous progress has been made in vaccine and antiviral research. More than 25 ZIKV vaccine candidates have been evaluated in nonclinical development and at least 12 are in clinical evaluations. These candidates include DNA, mRNA, viral-vectored, inactivated, and live attenuated vaccines (Barrett, 2018; Poland et al., 2019; Lee et al., 2021). Some of these candidates have demonstrated the ability to prevent viral transmission during pregnancy, as well as fetal malformations and demise in animal models (Richner et al., 2017; Shan et al., 2017; Li et al., 2018). Multiple candidates have been shown to be safe, well-tolerated and immunogenic in humans, with two candidates (VRC5288 and mRNA-1893) having advanced into phase 2 clinical trials (Barrett, 2018; Lee et al., 2021). In addition, therapeutic vaccination for ZIKV has been extensively explored as an alternative to vaccines. A total of 461 monoclonal antibodies (mAbs) that bind to E proteins have been identified, with 70 of them displaying moderate to high neutralizing activities (Wang et al., 2017c). Notably, administration of convalescent serum from a ZIKV-infected patient not only inhibited ZIKV replication but also prevented microcephaly in a mouse model (Wang et al., 2017a). While treatment with a human mAb, ZIKV-117 reduced vertical transmission and improved fetal outcome (Sapparapu et al., 2016). Another study showed that a cocktail of three neutralizing mAbs targeting different domains of the ZIKV E protein completely prevented viremia in non-human primates (NHPs; Magnani et al., 2017).

On the other hand, several approaches have been employed to identify drugs against ZIKV infection. The current search for ZIKV antivirals can be classified based on their mode of action, such as (i) host-directed antivirals, which focus on modulating host cellular processes used for viral life cycle or (ii) direct-acting antivirals, which target viral components. In order to expedite the development of effective antivirals against ZIKV, much focus has been given to drug repurposing or drug re-profiling (Table 2). Recommended therapies for ZIKV infection would require the ability to cross the placenta and BBB, have anti-ZIKV functionality primarily on fetal neural cells and be safe to use during pregnancy.

Following the sudden increase in the number of new-borns with microcephaly during the Brazillian outbreak of ZIKV, numerous studies have been conducted and they verify the causal relationship between ZIKV infection and neurological disorders. ZIKV possesses the ability to cross the placental barrier and BBB, targeting brain cells, particularly in the developing brain. The virus crosses the BBB through (i) transcellular pathway, (ii) monocyte transmigration through Trojan horse pathway, (iii) vascular leakage enhancement, and (iv) disruption of the choroid plexus epithelial layer. Upon crossing the BBB and entering the brain, ZIKV infects various cells in the developing brain, causing neuropathogenesis through (i) cell cycle perturbation, (ii) mitochondrial dysfunction, (iii) ER stress and UTP, (iv) modulation of host autophagy, and (v) neuronal apoptosis. Despite major advances in this field, many important questions remain answered: (i) Why only a small number of fetuses born to infected mothers develop microcephaly? What are the long-term outcomes of infected neonates without detectable abnormalities at birth? (ii) Is an intact BBB permissive to ZIKV infection? Can ZIKV target astrocytes in the adult brain? Will there be any long-term effects in adults infected with ZIKV? and (iii) Since the innate immune responses and mitochondria functions are connected, how does mitochondrial dysfunction upon ZIKV infection affect the cellular immune responses? Therefore, more studies are required to fully understand the mechanisms of cross-talk between ZIKV and the human brain, as well as the host factors involved in utero transmission and neuropathogenesis. In addition, systematic and long-term follow-ups are imperative to determine the unknown long-term neuropathological and behavioral consequences in both new-borns and adults.

Importantly, despite its severe implications and potential future outbreaks, there are still no vaccine or antiviral drugs available against ZIKV. The development of new therapeutic approaches should be one of the priorities for future research. The drug-repurposing strategy offers a promising avenue to identify potential antiviral drugs against ZIKV within a shorter development timeline and known safety. This approach is also particularly appealing for mosquito-borne viruses, which receive less attention from the affluent regions and pharmaceutical companies. A number of host-targeting and virus-targeting agents with potential inhibitory activity against ZIKV have been identified. However, the majority of these drugs have only been tested in vitro. It is also critical to evaluate their efficacy in appropriate animal models to better predict their clinical outcome. In addition, the ability of these drugs to prevent ZIKV CNS invasion needs to be evaluated. Importantly, ZIKV drugs should be clinically safe for use in pregnant women and fetuses.

LT and VB: conceptualization. LT and TK: methodology and writing—original draft preparation. LT, TK, WJ, and VB: writing, review, and editing. VB and WJ: supervision. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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