NSP4-Associated Barrier-Centered Neuroimmune Dysregulation in Neonatal Rotavirus Infection

1. Introduction

Rotaviruses are non-enveloped, double-stranded RNA viruses belonging to the family Reoviridae. They exhibit a characteristic wheel-like morphology and possess a triple-layered icosahedral capsid enclosing 11 genome segments that encode six structural (VP1–VP4, VP6, and VP7) and six non-structural (NSP1–NSP6) proteins [1]. Rotavirus infection primarily targets intestinal epithelial cells, where robust expression of non-structural glycoprotein 4 (NSP4) occurs. NSP4 functions as a viral enterotoxin and plays a central role in the induction of secretory diarrhea [1]. Despite the availability of effective vaccines, rotavirus infection remains a major global health burden, accounting for more than 200,000 deaths annually among children under five years of age [2].

Currently, four oral live-attenuated rotavirus vaccines are licensed for administration beginning at ≥6 weeks of age [3]. Although these vaccines have substantially reduced the incidence and severity of rotavirus gastroenteritis, neonates remain vulnerable to infection and its associated complications during the early postnatal period. Notably, increasing clinical evidence that rotavirus infection may be accompanied by neurological manifestations.

Neurological complications associated with rotavirus infection are increasingly recognized and appear to occur more frequently in neonates than in older infants and children, with approximately a twofold higher risk reported in the neonatal population [4]. A wide spectrum of central nervous system (CNS) manifestations has been described, including acute cerebellitis [5], encephalopathy and encephalitis [6,7], acute necrotizing encephalitis [8,9], reversible splenial lesion syndrome [10], and neonatal white matter injury or leukoencephalopathy [11,12,13,14]. In one cohort study, CNS complications were identified in 10 of 59 children hospitalized with rotavirus infection [6]. Furthermore, children aged six years with a history of hospitalization for rotavirus gastroenteritis demonstrated lower motor and cognitive performance compared with age-matched controls [15], suggesting the possibility of subclinical or delayed neurodevelopmental sequelae. Despite these observations, the biological mechanisms linking rotavirus infection to CNS dysfunction remain poorly understood. In particular, whether neurological involvement results from direct viral neuroinvasion or from indirect mechanisms such as systemic inflammation, barrier dysfunction, or host immune responses remains unresolved. Addressing this knowledge gap is essential for understanding rotavirus-associated neurological sequelae, especially during the vulnerable neonatal period.

Several hypotheses have been proposed to explain the mechanisms underlying rotavirus-associated neurological manifestations. Direct viral invasion of the CNS has been suggested in rare cases [16,17]. However, consistent evidence supporting neurotropism of rotavirus remains limited. Alternatively, increasing attention has been directed toward indirect mechanisms, including NSP4, serotonin, cytokines, and second messenger molecules, which may secondarily affect the developing brain [18].

Among rotavirus-derived factors, NSP4 has emerged as a multifunctional viral protein with pleiotropic biological effects extending beyond the intestinal epithelium. In addition to its established enterotoxigenic activity, NSP4 induces intracellular calcium dysregulation, endoplasmic reticulum (ER) stress, and inflammatory signaling pathways in infected cells. These properties indicate that NSP4, or its circulating degradation products, may influence vascular endothelial cells and immune responses at extraintestinal sites.

Notably, the neonatal period is characterized by immature blood–brain barrier (BBB) function and heightened sensitivity to inflammatory stimuli. Under such conditions, excessive innate immune activation and endothelial perturbation may render the developing brain particularly vulnerable to systemic viral insults. Based on these considerations, this review focuses on NSP4-centered mechanisms that may contribute to neuroinflammatory vulnerability through innate immune activation and barrier dysfunction, without necessarily requiring direct viral entry into the CNS.

2. Current Hypotheses and the Need for New Perspectives

Historically, the pathogenesis of CNS complications associated with rotavirus infection has primarily been attributed to direct or indirect mechanisms. Proposed routes of direct involvement include viral access through the BBB, blood–cerebrospinal fluid barrier (BCSFB), circumventricular organs (CVOs), enteric nervous system, and lymphatic pathways. Indirect mechanisms have also been suggested, including the effects of circulating proinflammatory cytokines and activated peripheral leukocytes on the developing brain [18,19]. However, consistent evidence supporting direct neuroinvasion of rotavirus remains limited.

In addition to these mechanisms, several hypotheses have focused on the role of the rotavirus NSP4. NSP4 and its peptide derivatives produced in the gastrointestinal tract have been proposed to act as neuroactive mediators within the enteric nervous system, potentially contributing to CNS manifestations [18]. Innate antiviral immune responses and inflammatory signaling, particularly in neonates, may influence CNS vulnerability. These include the potential effects of NSP4 released from rotavirus-infected monocytes on neuronal function, as well as microglial production of monocyte chemoattractant protein-1 (currently designated C–C motif chemokine ligand 2; CCL2) in the context of neuroinflammation [11]. NSP4 and serotonin activate the enteric nervous system and vagal nerves, contributing to CNS symptoms [18]. Despite these diverse hypotheses, most existing models emphasize either direct viral invasion or generalized immune-mediated injury, and no unifying framework has emerged to explain the characteristic features of rotavirus-associated acute encephalopathy and neonatal white matter injury. This gap highlights the need for renewed perspectives that integrate viral protein biology, host innate immune responses, and developmental vulnerability of the neonatal brain. Accordingly, this review proposes potential NSP4-centered mechanisms that extend beyond classical innate immune activation and may contribute to neuroinflammatory susceptibility without requiring direct viral entry into the CNS.

3. Intestinal Origin of NSP4 and Systemic Immune Activation

Rotavirus infection of intestinal epithelial cells induces the production and release of NSP4, which disrupts intracellular calcium homeostasis and activates innate immune signaling, thereby initiating systemic inflammatory responses, including the production of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor (TNF-α) [1]. Rotavirus infection preferentially targets mature villous tip epithelial cells in the small intestine, leading to epithelial injury and malabsorption [20]. During the acute phase of infection, viral antigens and, in some cases, viral RNA can be detected in the systemic circulation, resulting in antigenemia and transient viremia [21]. NSP4, synthesized and secreted by rotavirus-infected enterocytes, is a multifunctional viral protein that modulates host cellular signaling pathways and contributes to viral replication and disease pathogenesis.

NSP4 activates multiple intracellular signaling cascades, including nuclear factor kappa B (NF-κB), c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase pathways, at least in part through toll-like receptor 2 (TLR2)-dependent mechanisms. Activation of these pathways results in the production of proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α in macrophage-like cell lines through modulation of TLR2-mediated innate immune responses [22]. Through modulation of TLR2-mediated innate immune responses, NSP4 facilitates rotavirus replication and amplifies local inflammatory signaling during infection [22]. Moreover, NSP1 contributes to evasion of the innate interferon response through signal transducer and activator of transcription 1 inhibition [1]. In addition to secretory diarrhea, which is caused by the effect of NSP4-serotonin axis-mediated enteric nervous system activation, osmotic diarrhea due to malabsorption is important in rotavirus gastroenteric symptoms [1]. Specifically, NSP2 induces the degradation of diacylglycerol O-acyltransferase 1, which disrupts lipid metabolism and facilitates viral replication [23]. These findings emphasize the complex interplay of multiple viral proteins in host responses.

The diarrheal phenotype characteristic of rotavirus gastroenteritis is primarily attributed to the biological activity of NSP4 produced by infected intestinal epithelial cells [24,25]. NSP4 disrupts intracellular calcium homeostasis and activates multiple signaling pathways in intestinal epithelial cells, contributing to diarrhea and innate immune activation.

In intestinal enterocytes, elevated intracellular calcium promotes chloride secretion and water efflux into the intestinal lumen, resulting in secretory diarrhea. Accordingly, NSP4 is widely recognized as a viral enterotoxin [1]. In addition to full-length NSP4 [27,28], cleavage products such as NSP4 (112–175) are released from rotavirus-infected epithelial cells in vitro [29]. Secreted NSP4 assembles into oligomeric soluble lipoprotein complexes [28] capable of binding integrin α1 and α2 I domains [30] on the cell surface. Engagement of integrin α2 by extracellular NSP4 increases cytosolic calcium levels in epithelial cell lines through phospholipase C activation and inositol 1,4,5-trisphosphate production [31]. Moreover, NSP4 exposure induces reactive oxygen species generation and chloride secretion in Caco-2 cells [32]. Collectively, both intracellular and extracellular forms of NSP4 disrupt epithelial function, impair ion transport, and damage villous architecture, thereby contributing to diarrhea [33]. Beyond epithelial injury, NSP4 also modulates innate immune gene expression. Transfection of NSP4 into MA104 cells induces upregulation of NF-κB–dependent and innate immune–related genes, including CCL20, C-X-C motif chemokine ligand 8 (IL-8), IL-6, CCL2, and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) [25]. NOD2 is a key regulator of nucleotide-binding domain leucine-rich repeat-containing receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome activation, which can lead to IL-1β production in brain endothelial cells (BECs) [34].

Taken together, these findings establish NSP4 as a potent mediator of epithelial dysfunction and innate immune activation in the intestine. Given the shared signaling pathways between intestinal epithelial cells, vascular endothelial cells, and innate immune cells, it is biologically plausible that NSP4 and its degradation products may exert similar immunomodulatory effects at extraintestinal sites. In neonates, where BBB and choroid plexus barrier functions are immature, such mechanisms may contribute to neuroinflammatory susceptibility. Elucidating whether and how NSP4 interacts with BECs, choroid plexus epithelial cells, and innate immune cells remains a critical area for future investigation. Collectively, these findings support the intestine as the primary source of NSP4-driven systemic immune activation during rotavirus infection.

4. Systemic Dissemination of NSP4 and Barrier-Associated Mechanisms

The following sections outline a set of interconnected and hypothesis-driven mechanisms, derived from established findings in intestinal rotavirus infection, innate immune signaling, and developmental neurobiology. Although direct experimental evidence in the CNS is limited for many of these pathways, they are presented here as biologically plausible models intended to stimulate future experimental investigation rather than to assert definitive pathogenic mechanisms. Following intestinal infection, NSP4 and inflammatory mediators disseminate systemically, where the unique immunological features of neonates may predispose to exaggerated or dysregulated innate immune activation. The proposed inflammatory pathways underlying this pathology are illustrated in Figure 1.

NSP4, a rotaviral enterotoxin, induces calcium dysregulation and inflammatory signaling in intestinal epithelial cells. Circulating NSP4-related factors and cytokines may interact with the BBB, potentially leading to neuroimmune activation. These CNS-related mechanisms remain speculative and require experimental validation. Solid lines indicate mechanisms supported by experimental evidence, primarily in intestinal systems. Dashed lines indicate hypothetical pathways that have not been directly demonstrated in the neonatal central nervous system.

The mechanisms illustrated in this figure are further categorized according to their level of supporting evidence in Table 1 and Table 2.

4.1. NSP4 and NSP4 Fragments

Although direct demonstration of the release of NSP4 and the NSP4 fragment into the systemic circulation of patients with rotavirus gastroenteritis is not known, the presence of anti-NSP4 IgG antibodies in the serum of afflicted children [35] suggests that NSP4, which is synthesized within rotavirus-infected enterocytes, may disseminate into the systemic circulation [19,36]. The cleavage product of NSP4, namely NSP4 112–175 [29], exhibits adjuvant activity [37], presumably via TLR2 [22], whereas the full-length NSP4 [28] is discharged from rotavirus-infected cells. Moreover, the NSP4 protein produced by rotavirus-infected intestinal cells can be secreted from the apical side [27] and interacts with basement membrane components, such as fibronectin and laminin-3, in infected neonatal mice [38]. It could enter the circulation from the tissue beneath the basement membrane [38]. Therefore, it is speculated that free and fibronectin/laminin-β3-bound NSP4 and NSP4 fragments are released from infected enterocytes into the bloodstream. These proteins could then potentially interact with BECs, choroid plexus epithelial cells, and innate immune cells, thereby contributing to inflammatory signaling and cytotoxic stress within the CNS. It is crucial to ascertain whether these disseminated factors can interact with BECs, choroid plexus epithelial cells (CPECs), and innate immune cells, inducing an inflammatory response on the CNS.

4.2. Innate Immune Activation of BECs

NSP4 may directly activate BECs through innate immune pathways, leading to cytokine production and increased BBB permeability in the neonatal period. BECs express TLR2 [39], and the expression of TLR2 and the other innate immune signaling proteins is enhanced in the neonatal mouse brain [40]. We previously hypothesized innate immune activation in severe acute respiratory syndrome coronavirus 2 infection in the neonatal brain under enhanced expression of innate immune molecules [41]. Similarly, we propose the mechanism considering the innate immune response to rotavirus infection in the neonatal brain.

Activation of TLR2 by Pam3Cys and the gram-positive bacterial cell component peptidoglycan-associated lipoprotein induces IL-6 production in in vitro cultures of human endothelial cells [42]. Moreover, IL-6 production by BECs plays a significant role in the TNF-α-dependent generation of reactive oxygen species and the downregulation of adherens and tight junction proteins, ultimately disrupting BBB integrity in experimental models [43]. Similarly, the release of the NSP4 fragment into the circulation may directly interact with BECs, potentially increasing permeability via TLR2.

TLR2 activation in macrophages by NSP4 [22] can trigger the same immune response in circulating monocytes, including IL-6 and TNF-α production. Dendritic cells (DCs) are activated via TLR2, producing IL-6 and TNF-α [44]. Thus, perivascular immature DCs, when activated by NSP4 and its fragment, may result in similar responses within the neonatal brain. Circulating monocytes can interact with perivascular macrophages and, inpathological conditions, replace them [45]. Therefore, it is conceivable that NSP4- and NSP4-fragment-activated monocytes activate BECs, brain perivascular macrophages, and perivascular immature DCs, producing pro-inflammatory cytokines and chemokines. This, in turn, may heighten BBB permeability. NSP4-induced innate immune activation in BECs provides a plausible mechanism for BBB dysfunction in neonatal rotavirus infection.

4.3. Interleukin-8

Children with rotavirus gastroenteritis who exhibit antigenemia and extraintestinal manifestations have elevated serum IL-8 levels [81]. Moreover, neonates affected by rotavirus infection-induced white matter injury display elevated CSF levels of IL-8 [11]. Rotavirus boosts IL-8 gene expression by activating inhibitor of NF-κB kinase in HT-29 cells [82], whereas NSP4 induces IL-8 secretion in TLR2-expressing HEK293 cells [22]. IL-8 plays a crucial role in disrupting the integrity of tight junctions in microvascular endothelial cells by activating the C-X-C chemokine receptor (CXCR)1 and CXCR2 [84]. These observations may suggest that rotavirus infection may initiate IL-8 signaling in BECs and CPECs, ultimately resulting in increased barrier permeability. NSP4 is a multifunctional rotaviral protein that has been shown to disrupt intracellular calcium homeostasis [31], induce ER stress [96], and modulate innate immune signaling in infected cells [22]. These effects are well documented in intestinal epithelial systems and are thought to contribute to rotavirus-induced diarrhea. However, most of these findings are derived from in vitro systems or non-neural tissues, and their direct relevance to the neonatal CNS remains uncertain. Based on these observations, we propose a barrier-centered hypothesis in which circulating NSP4 or NSP4-related signaling molecules may influence the function of endothelial and epithelial barriers, including the BBB and the BCSFB. These interactions may, in turn, alter barrier integrity and promote neuroimmune activation.

Although direct evidence in the neonatal central nervous system remains limited, several potential mechanisms may be considered to explain how NSP4 and rotavirus-associated signaling could influence barrier integrity. These include modulation of tight junction proteins [63,64,65,66,67,69,70,111], activation of TLR2 signaling pathways in the macrophages [22] and choroid plexus [53,54,55], TLR signaling mediated inflammasome activation [88], decreased regulatory T cells/Th17 cells ratio [48], disruption of barrier integrity by IL-8 [11,84] and TLR3 [112,113], induction of ER stress [96,114], and oxidative stress responses [32,115]. However, these mechanisms have not been directly demonstrated in BBB and BCSFB and should therefore be considered hypothetical.

Similarly, possible routes by which rotavirus, NSP4 or its fragments could damage and/or access the CNS—such as plasma membrane permeabilization by NSP4 48–91 [62], integrin interactions [56,71,72,73,75] and receptor-mediated transport [116], or caveolae-dependent transcytosis [57,117]—remain speculative and require experimental validation. However, if NSP4 and NSP4 fragments increase and/or are taken up by BECs and CPECs via the endocytic mechanism, it may be important to determine whether an innate immune response can be induced, as occurs in intestinal epithelial cells [25,34].

Notably, these mechanisms have been primarily characterized in intestinal or non-neural systems, where NSP4-related effects on epithelial barrier function are better established. By contrast, whether similar processes occur at central nervous system barriers, such as the BBB or BCSFB, remains unclear. Therefore, the extension of these mechanisms to the neonatal brain should be regarded as hypothetical and requires further experimental validation.

5. Possible Direct Penetration of Rotavirus into the CNS

Although immune-mediated mechanisms are likely primary, direct viral penetration may occur in a limited subset of cases. Rotavirus RNA has been detected in barrier-associated brain cells [74], but direct viral invasion of the CNS appears to play a limited role compared with barrier-centric inflammatory mechanisms. Although direct viral invasion is unlikely to represent the primary mechanism underlying rotavirus-associated neurological complications, pathological and experimental evidence has suggested that it may occur as a secondary process under specific conditions. Rotavirus causes extraintestinal dissemination in neonatal rat models [118], and viremia is relatively common during rotavirus infection in children [21]. Nevertheless, direct CNS invasion is rare [119].

One proposed mechanism involves CCL2-mediated recruitment of rotavirus-infected monocytes into the brain [11]. The BBB and BCSFB disruption mechanisms may create a permissive environment for such viral entry. Supporting this notion, in children with systemic endothelialitis associated with rotavirus infection, viral RNA has been detected predominantly in BECs, as well as in perivascular mononuclear cells, neurons, and microglia [74]. This distribution suggests that, when direct CNS invasion does occur, the BBB may represent the principal route of entry. While it has not been established that rotavirus infects BECs and CPECs, we hypothesize that rotavirus and NSP4, which are encapsuled within exosomes released into blood stream from infected intestinal epithelial cells, may enter these cells, because some viruses can be transmitted by this mechanism [120]. NSP4 is known to be secreted from rotavirus-infected cells as an extracellular oligomeric lipoprotein capable of binding non-infected cells through cell-surface glycosaminoglycans [28]. In addition, rotavirus components have been reported to associate with extracellular vesicles (EVs) containing exosome markers (such as CD63) [121], and EVs can interact with brain endothelial cells and undergo endocytic/transcytotic trafficking at the BBB [122]. Although there is currently no direct evidence that NSP4 and rotavirus themselves are EV-packaged and delivered to BECs, these findings suggest a plausible mechanism by which circulating NSP4 as well as rotavirus, either in soluble form or potentially in association with EVs, could access the BBB and modulate BEC function. Future studies should determine whether NSP4 and rotavirus are incorporated into EVs released from infected intestinal epithelial cells and whether such EVs are taken up by BECs to alter barrier integrity or inflammatory signaling, and/or penetrate into the CNS.

Importantly, intestinal TLR3 responses in neonates are developmentally limited and do not effectively restrict rotavirus replication [100]. On the contrary, TLR3 expression in the neonatal brain is developmentally increased [101]. TLR3 signaling in the immature brain may be biphasic and context-dependent: transient activation before ischemia may induce tolerance [101,123], whereas sustained activation during ongoing neonatal viral exposure may promote neuroinflammation. Therefore, during neonatal rotavirus infection, insufficient intestinal antiviral control may permit prolonged exposure of the immature brain to rotavirus and viral products including NSP4, where sustained and enhanced TLR3-related signaling in BECs, neurons, and pre-oligodendrocyte-rich white matter may contribute to injury rather than tolerance.

The frequent absence of rotavirus RNA in CSF does not preclude CNS involvement, as endothelial or perivascular localization may trigger neuroimmune responses without detectable viral shedding into CSF. Although direct CNS invasion by rotavirus may occur, available pathological evidence suggests it contributes only modestly to overall disease pathogenesis.

6. BBB and BCSFB as Immunologically Active Interfaces in Neonates

6.1. Barrier-Centered Innate Immune Activation

Rotavirus-associated neuroinflammation is initiated at CNS interfaces (BBB, BCSFB, and CVOs), where perivascular innate immune cells amplify NSP4-driven inflammatory signaling without deep parenchymal viral invasion. Accumulating evidence has suggested that rotavirus-associated neurological complications are not primarily driven by widespread viral invasion of the brain parenchyma but rather by exaggerated innate immune activation at CNS interfaces. In neonates, the BBB, BCSFB, and CVOs constitute immunologically active zones enriched with perivascular macrophages, dendritic cells, and other innate immune sentinels. Rotavirus infection, particularly through the actions of the viral enterotoxin NSP4 and its degradation products, may perturb these barrier-associated cells by engaging pattern recognition receptors and inflammatory signaling pathways. This results in the amplification of cytokine and chemokine cascades, such as IFN-γ, CCL2, high mobility group box 1 (HMGB1), and IL-17, which propagate neuroinflammation without requiring extensive viral replication within neurons or glial cells. Such a barrier-centric model reconciles neuropathological findings showing preferential viral RNA localization in brain endothelial and perivascular cells with clinical observations of encephalopathy occurring in the absence of overt CNS viral burden. In the neonatal brain, where immune regulation and barrier integrity are developmentally immature, this interface-focused inflammatory response may represent a critical mechanism underlying rotavirus-associated neurological vulnerability.

6.2. Barrier-Adjacent Innate Immunity

6.2.1. Perivascular Macrophages

Rotavirus can infect and replicate in pneumocytes and macrophages in the lungs and livers in the neonatal rat [118]. It can target macrophages in the livers of mice with a congenital biliary atresia model and release macrophage inflammatory protein 2 and Cxcl2 which recruits neutrophils [124]. Thus, we hypothesize that rotavirus may infect perivascular macrophages in the neonatal brain and recruit neutrophils. Rotavirus-infected macrophages produce IL-6 and IL-1β through mitochondrial-associated protein signaling [125]. Therefore, if rotavirus crosses the BBB and BCSFB, it may be captured by brain perivascular macrophages, inducing an innate immune-inflammatory response. Moreover, as discussed above, NSP4 and its fragment may induce TLR2-mediated TNF-α production in macrophages. Circulating TLR2-activated monocytes may also activate brain perivascular macrophages.

6.2.2. Perivascular Dendritic Cells

Rotavirus infects neonatal DCs, inducing IL-6 and TNF-α, and sustaining viral replication while also promoting the proliferation of IFN-γ-producing CD4+ T cells [94]. Particularly, immature DCs are more capable of adhering to TNF-α-stimulated BECs and crossing the BBB than mature DCs [126]. Furthermore, CD14+ monocytes migrate across BECs. Activated BECs secrete granulocyte-macrophage colony-stimulating factor and transforming growth factor β, which promote differentiation and maturation of peripheral blood CD14+ monocytes into perivascular DCs [127]. These DCs produce IL-6 and IL-12p70, which promote the development of IFN-γ-producing Th1 cells and IL-17-producing Th17 CD4+ T lymphocytes [127]. Thus, rotavirus infection may activate perivascular DCs in the brain, inducing IFN-γ and IL-17-mediated neuroinflammation, which will be discussed later. However, if rotavirus replicates in brain perivascular macrophages and DCs, then viral genomes may not be detected in the CSF using polymerase chain reaction.

6.2.3. Circumventricular Organs

CVOs have highly permeable, fenestrated capillaries that play an important role in bidirectional communication between peripheral blood and brain parenchyma. The peripheral administration of the TLR2 ligand Pam3CSK4 activates NF-κB to induce cytokine/chemokine production in perivascular macrophages and microglia in the CVOs [128]. Moreover, peripheral TLR2 activation increases c-Fos expression in astrocytes and neurons in the CVOs and adjacent regions [128]. Thus, we propose a mechanism whereby circulating NSP4 and NSP4 fragments reach the CVOs, where they induce neuroinflammation. Furthermore, pretreatment with the TLR4 ligand lipopolysaccharide (LPS) increases TLR2 expression in the CVOs [118], and systemic administration of LPS upregulates Tlr2 mRNA levels in the choroid plexus and activates NF-κB in adjacent microglial cells [129]. In a rotavirus-induced experimental model of biliary atresia, rotavirus disrupts tolerance to physiological levels of LPS, thereby increasing the expression of the TLR4/NF-κB pathway and metalloproteinase-7. Thus, in rotavirus infection, although the NSP4 fragment alone exhibits weak TLR2 activity, TLR2- and TLR4-mediated inflammatory signaling may be activated in the choroid plexus and CVOs, particularly in the presence of LPS.

6.2.4. Junctional Adhesion Molecule-A

Given that astrocytic Junctional adhesion molecule-A (JAM-A) contributes to immune cell trafficking into the CNS by increasing metalloproteinase-2 levels [69], rotavirus binding to JAM-A [63] could activate immune cell entry into the perivascular space. The entry of activated T cells into the brain is also increased by damage to JAM-A [69].

6.3. Amplifiers of Neuroinflammation

6.3.1. Natural Killer Cells

In a low-dose rotavirus-induced congenital biliary atresia model, persistent activation of natural killer (NK) and CD8+ T cells leads to long-term hepatobiliary inflammation, which is associated with enhanced mRNA levels of Th1 cytokines, Ifnγ, Cxcl9, and Cxcl10 [130]. In the rotavirus-associated congenital biliary atresia model, paucity of Tregs in the liver decreases suppressive control on NK cells [131]. Thus, suppressed Treg activity in the brain could activate NK cells, causing enhanced and persistent neuroinflammation.

6.3.2. Interferon-Gamma

Rotavirus increases IFN-γ mRNA expression in peripheral blood mononuclear cells [51], and IFN-γ levels in the CSF of neonates with leukoencephalopathy are high [132]. Rotavirus induces IFN-γ production by infecting neonatal DCs [94]. T cells produce IFN-γ via α2β1 integrin [133], suggesting rotavirus as well as NSP4 may trigger IFN-γ production in BECs-crossing T cells by activating α2β1 integrin signaling. Moreover, NK cells migrate across the BBB and produce IFN-γ by interacting with activated microglia [134]. Furthermore, IFN-γ primes the TLR-mediated inflammatory responses by macrophages [135] and activates TNF-α/NF-κB signaling in microglia [136]. Although IFN-γ and TLR play an important role in enhancing microglial activation [137], IFN-γ plus transforming growth factor β contributes to inflammation and mucosal damage in gastrointestinal inflammation [138]. The same responses may occur in the BBB, BCSFB, and microglia during rotavirus infection, contributing to barrier disruption and enhanced neuroinflammation.

6.3.3. Chemokine Ligand 2

Microglial production in response to rotavirus infection hypothetically increases levels of CCL2 production by microglia, contributing to white matter injury in neonates [11]. The precise mechanism of increased CCL2 levels in the brain and its role remains unclear. TNF-α and IFN-γ increase CCL2 production by BECs [139]. In the intestine, CCL2 recruits inflammatory monocytes via Nod2, which senses pathogens [140]. Therefore, the increased expression of Nod2 in developing mice brains [40] may lead to enhanced peripheral monocyte recruitment in rotavirus infection.

Peripherally stimulated TNF receptor 1-mediated signaling via BECs activates microglia to produce CCL2 [139]. Moreover, IFN-γ activates the interaction of CD40 with CD40 ligand in microglia and CCL2 production [141]. CCR2+monocytes enter the CNS via the choroid plexus, transform into microglia-like cells that promote neuroinflammation in the hypoxic-ischemic injury neonatal mice model [142]. Astrocytes are stimulated by IL-1β and TNF-α to release CCL2 [143], and astrocytes-derived CCL2 also evolves neuroinflammation [144]. Thus, BECs, microglia, and astrocytes may release CCL2, recruiting CCR2+monocytes, which contribute to neuroinflammation.

6.3.4. High Mobility Group Box 1

Elevated levels of high mobility group box 1 (HMGB1) have been reported in the CSF of patients with acute encephalopathy due to viral infection, including rotavirus infection [145]. Rotavirus-infected macrophages in the biliary tract release HMGB1 [146], which is also released through double-stranded RNA-dependent protein kinase-mediated inflammasome activation [147]. Similarly, it seems possible that rotavirus-infected brain perivascular macrophages release HMGB1, contributing to inflammasome activation and neuroinflammation. The elevated IL-1β levels observed in the CSF of patients with acute encephalopathy due to rotavirus infection [148] may support this mechanism.

HMGB1 released from rotavirus-infected macrophages activates NK cells via TLR2/TLR4-mitogen activated protein kinase signaling pathways [146]. IFN-γ-activated macrophages also secrete HMGB1, facilitating transendothelial migration of monocytes [139]. Due to limited NK cell activation in neonatal mice, rotavirus can persistently infect cholangiocytes [146]. The same mechanism may also occur in brain perivascular macrophages, leading to persistent HMGB1 production and NK cell activation. Therefore, rotavirus-infected and IFN-γ-activated macrophages may induce monocyte migration and NK cell activation, contributing to persistent neuroinflammation.

HMGB1 activates astrocytes and induces the production of inflammatory mediators, including cyclooxygenase-2 and chemokines [149]. Thus, we propose the initial activation of brain perivascular macrophages by rotavirus and IFN-γ triggers inflammatory reactions in microglia and astrocytes.

6.3.5. Interleukin-17

Children with rotavirus enteritis have elevated serum levels of IL-17 and circulating Th17 cells [48]. Moreover, IL-17 is higher in the CSF of children with convulsions following gastroenteritis [150]. In the rotavirus-associated congenital biliary atresia model, IL-17-producing γδ T cells infiltrating the periportal area in the liver contribute to inflammation, and anti-IL-17 therapy ameliorated infiltration of inflammatory cells [151]. In the brain, IL-17-producing γδ T cells increase BBB permeability [152]. Therefore, rotavirus may induce IL-17-mediated neuroinflammation in the neonatal brain by inducing γδ T cells around the BBB and BCSFB, as in the neonatal liver. The binding of rotavirus to integrin on BECs may initiate γδ T cells-mediated barrier disruption and neuroinflammation.

Moreover, as discussed above, rotavirus-infected BECs and brain perivascular macrophages may release HMGB1, which upregulates TLR2 on CD14+ monocytes and promotes Th17 cell differentiation [153]. TNF-α and IL-17 facilitate Th17 cells’ adhesion to BECs and transmigration of Th17 cells across the BBB [154]. In addition to decreased Treg/Th17 ratio in the blood in rotavirus infection [48], the transmigration of Tregs across the BBB can be suppressed by IL-6 production by perivascular DCs, macrophages, and BECs. Thus, rotavirus infection may relatively increase Th17 cells’ migration across the BBB than Treg cells, decreasing the Treg/Th17 ratio in the CNS. IL-1β stimulates microglia to produce IL-17 using an autocrine mechanism [155]. IL-17 also causes astrocytes to produce IL-6 and IL-1β [156]. Therefore, microglia and astrocytes may persistently be activated, unless IL-1β and IL-17 are blocked.

6.4. Resident Glial Response

6.4.1. Microglia and Astrocytes

Microglia are activated by TLR2/NF-κB and p38 mitogen activated protein kinase to produce reactive oxygen species (ROS) [157]. In microglia, activating the TLR2/myeloid differentiation primary response protein 88 pathway contributes to neurodegeneration [158]. In astrocytes, TNF-α increases TLR2 expression and nitric oxide release via an NF-κB-dependent pathway, acting in an autocrine and paracrine manner [159]. Thus, TNF-α and ROS-mediated mechanisms in microglia and astrocytes may contribute to neuroinflammation and neuronal damage in rotavirus infection. However, the activity of TNF-α may not be reflected in its concentration in CSF because of its autocrine-paracrine action. Rotavirus-infected epithelial cells release adenosine diphosphate [77]. Extracellular adenosine diphosphate activates the NLRP3 inflammasome in microglia, as well as the NF-κB/IL-6 and IL-1β pathways [160]. Moreover, because viral RNA was detected in microglia in a pathological study [74], TLR3-mediated inflammatory cytokine production may be induced.

7. Neuroimmune Consequences in the Developing Brain

Neonatal rotavirus infection has been associated with neurological complications, including encephalopathy and white matter injury. However, the mechanisms underlying these manifestations remain incompletely understood.

We propose that barrier dysfunction and systemic inflammation may contribute to neuroimmune activation in the developing brain. In this context, NSP4-induced calcium dysregulation [31] and ER stress [115]—well characterized in intestinal cells—may have analogous effects if similar pathways are engaged in barrier-associated or neural cells. However, direct evidence for such effects in the neonatal CNS is currently lacking.

TLR3 recognizes rotavirus double-stranded RNA [112]. TLR3 is developmentally upregulated in the neonatal brain [101], where it regulates neural [161] and oligodendrocyte maturation [113], whereas in the neonatal intestine its expression and antiviral function is impaired [100].

Human neurons induce IL-6, TNF-α, CCL5, and CXCL10 production via TLR3 in response to double-stranded RNA [164]. Moreover, peripheral IL-1β stimulation in mice induces TLR3 upregulation in immature oligodendrocytes [113]. While intestinal TLR3 expressions and function are immature in early life, resulting in insufficient antiviral interferon responses and prolonged viral replication, the developing brain exhibits relatively heightened TLR3 expression and responsiveness. This imbalance allows persistent viral components, including double-stranded RNA and NSP4, to access the systemic circulation and potentially prolong interaction with the immature BBB. Therefore, a key feature of neonatal rotavirus-associated encephalopathy and white matter injury may lie in the developmental mismatch of TLR3 signaling between the intestine and the brain. On the contrary, TLR3 activation has a protective role in hypoxic-ischemic injury in neonates [123]. Thus, the developmentally enhanced expression of TLR2 [40] and TLR3 [101] in the neonatal brain as well as their pleomorphism, may hypothetically modify anti-rotaviral innate immune responses, including interferon, cytokine, and chemokine synthesis, thereby damaging the BBB and neurons in the neonatal brain, by a similar mechanism. Overall, we propose that rotavirus-associated neurological complications may arise from a combination of barrier dysregulation and neuroimmune activation, rather than widespread direct viral invasion. This model remains hypothetical and requires experimental validation.

8. Implications for Prevention and Therapeutic Targeting

Understanding NSP4-mediated barrier dysfunction and innate immune activation suggests a conceptual framework for preventive strategies and therapeutic interventions aimed at reducing neurological sequelae.

9. Research Gaps

Based on this review, we propose a hypothetical model in which NSP4 and NSP4 fragments trigger multiple innate immune responses in the brain. Elucidating the mechanisms of encephalopathy, particularly in neonates, requires understanding the bioactivity and neurotoxicity of NSP4 and its fragments, as well as their effects on neural cells and immune molecule activation in the developing brain. The development of treatments for preventing sequelae is expected to continue in the future.

10. Limitations

This review is inherently limited by its hypothesis-generating nature and by assumptions derived from known biological activities of rotavirus and NSP4. At present, direct experimental evidence establishing a causal relationship between NSP4 and neonatal CNS injury has not been published, and this manuscript does not demonstrate direct neurotoxicity or definitive CNS invasion by NSP4 or its peptide fragments. Furthermore, it remains unclear whether NSP4 or NSP4-derived peptides can directly induce TLR2-mediated inflammatory signaling in BECs, microglia, or astrocytes under physiological conditions. The frequent absence of detectable rotavirus RNA in CSF in clinical cases further complicates the interpretation of CNS involvement and highlights the difficulty of linking peripheral infection to central pathology.

Experimental validation is therefore required to determine whether peripheral administration of NSP4 or its fragments can elicit innate immune activation within the neonatal brain in vivo, and whether such responses are sufficient to disrupt BBB integrity or white matter development. In addition, in silico molecular dynamics analyses may suggest insights into potential interactions between NSP4, BECs, innate immune cells, neurons, astrocytes, and oligodendrocytes; however, such approaches remain predictive and require biological confirmation.

Despite these limitations, this review suggests a conceptual framework that integrates existing clinical, pathological, and molecular observations and aims to generate testable hypotheses for future experimental and translational studies, particularly in the neonatal period.

11. Discussion

The proposed mechanisms and their levels of supporting evidence are summarized in Table 1. This framework integrates findings from intestinal systems, immune signaling studies, and limited CNS-related observations, and distinguishes between mechanisms that are experimentally supported and those that remain indirect or hypothetical in the context of the neonatal brain.

Accumulating evidence from intestinal and other non-neural systems suggests that NSP4 may disrupt epithelial integrity and modulate host responses through multiple pathways, including calcium signaling, cytokine induction, and barrier dysfunction. These findings provide a basis for considering whether NSP4-related factors may enter the circulation and interact with distal tissues. However, direct evidence supporting such systemic effects, particularly in the context of the neonatal CNS, remains limited.

Although several mechanisms may be considered to explain how NSP4 and rotavirus-associated signaling could influence barrier integrity—such as modulation of tight junction proteins, activation of TLR-mediated pathways, induction of ER stress, and oxidative stress responses—these processes have been primarily characterized in intestinal or non-neural systems. Whether similar processes occur at CNS barriers, such as the BBB or BCSFB, remains unclear. Therefore, their relevance to the neonatal brain should be regarded as hypothetical and requires further experimental validation.

Importantly, the neonatal period is characterized by developmental immaturity of barrier systems and heightened sensitivity to inflammatory and metabolic perturbations. In this context, even indirect or transient systemic signals may have disproportionate effects on barrier function and neuroimmune regulation. This raises the possibility that NSP4-associated signaling, even in the absence of direct CNS invasion, could influence brain development through secondary mechanisms involving circulating mediators and barrier-associated immune activation.

Notably, some pathways, such as TLR3 signaling, may exert context-dependent effects, including potentially protective roles in the immature brain, and therefore should not be interpreted as uniformly pathogenic. This highlights the importance of carefully distinguishing between protective and detrimental immune responses in the developing CNS.

The strength and limitations of the proposed mechanisms are further categorized in Table 2. While several pathways, including NSP4-induced calcium signaling and cytokine responses, are supported by experimental data in intestinal or immune systems, their direct relevance to the neonatal CNS remains uncertain. In particular, mechanisms involving BBB permeability changes, infection of brain endothelial cells, and immune cell activation within the CNS are supported only by limited or indirect evidence and should therefore be considered highly speculative. This distinction underscores the importance of separating established biological observations from hypothesis-driven extrapolations.

Importantly, several key experimental questions arise from this framework and should be prioritized in future studies. First, it remains to be determined whether NSP4 or its circulating fragments can directly interact with brain endothelial cells or choroid plexus epithelial cells under physiological conditions. Second, it is essential to clarify whether NSP4-related signaling is sufficient to induce measurable changes in BBB permeability in neonatal models in vivo. Third, the relative contributions of systemic cytokine signaling and neural pathways, including vagal activation, to neuroimmune responses in the developing brain should be quantitatively evaluated. Fourth, the extent to which innate immune activation at CNS interfaces can occur in the absence of direct viral invasion remains unclear and requires experimental validation.

Several limitations should be acknowledged. Most of the mechanistic insights discussed here are derived from non-neural or in vitro systems, and their applicability to the neonatal CNS remains uncertain. In addition, direct experimental evidence linking NSP4 to CNS barrier dysfunction is currently lacking. These limitations emphasize that the present model should be regarded as hypothesis-generating rather than definitive.

In conclusion, this perspective outlines a barrier-centered, hypothesis-driven model linking rotavirus-associated signaling to potential neuroimmune effects in neonates. This framework may suggest a conceptual basis for future mechanistic and translational studies in neonatal viral neurobiology.