Benign convulsions with mild gastroenteritis are among the most common neurological manifestations related to rotavirus infection in children, occurring in approximately 1%–8% of patients with rotavirus gastroenteritis [14,31-34]. Although it has been documented worldwide, the number of reported cases is considerably higher in East Asian countries, particularly Japan, Korea, Taiwan, and China [21-23,35-37]/ This clinical condition is seen in previously healthy children aged 1 month to 6 years (peaking in 1–2-year-olds) and present with afebrile (or febrile) seizures that develop 1–6 days after gastroenteritis onset [34,35]. Some children experience seizures 12–24 hours before or even on the same day as the onset of diarrhea. Patients show neither electrolyte imbalance nor severe dehydration. Although seizures are usually the generalized tonic-clonic type, ictal electroencephalography has almost always demonstrated focal onset followed by secondary generalization [34,38]. Findings on interictal electroen cephalography, cerebrospinal fluid (CSF) analysis, and magnetic resonance imaging (MRI) are almost always normal (Table 1). Some patients show lesions in the splenium of the corpus callosum on diffusion-weighted imaging (DWI) [39]. Rotavirus and norovirus antigens are frequently identified in stool samples from these patients, and rotavirus ribonucleic acid (RNA) or antigens have also been detected in CSF samples in some cases [40-42]. Although patients commonly have multiple seizures, the outcome is favorable, with rare relapse, unimpeded development, and no epilepsy [34].

As mentioned above, rotavirus commonly causes acute encephalopathy and encephalitis in children. In Japan, 2 national surveys were conducted of the epidemiology of pediatric acute encephalopathy during 2007–2010 and 2014–2017 [17,18]. Interestingly, rotavirus was ranked third among the identified pathogens of antecedent infection in acute encephalopathy in both surveys. Influenza virus and human herpes virus 6/7 placed first and second, respectively. In rotavirus-associated encephalopathy, the median age at onset was 2 years in both studies. A recent Swedish prospective cohort study also showed that rotavirus was the third most common pathogen implicated in childhood encephalitis [19]. In 61 of 89 cases (68.5%), the etiologies of encephalitis were established. The common viral etiologies included tick-borne encephalitis virus (n=13), enterovirus (n=10), and rotavirus (n=9).

In general, since viral infection-associated acute encephalopathy and viral encephalitis present with similar brain dysfunction such as acute onset altered consciousness and seizures, it is often difficult to distinguish them from each other; thus, the terms are sometimes used interchangeably in the clinical setting. Encephalitis typically shows inflammation of the brain parenchyma, while acute encephalopathy shows pathologically widespread noninflammatory brain edema that is either cytotoxic (cellular) or vasogenic (vascular) [43]. In principle, although the presence of CSF pleocytosis favors a diagnosis of encephalitis, an absence of CSF pleocytosis in patients with viral encephalitis is not uncommon [44]. On brain MRI, viral encephalitis tends to demonstrate asymmetrical focal or multifocal lesions as seen in herpes simplex virus encephalitis [45], while acute encephalopathy is disposed to show bilaterally symmetrical lesions [16]. Both conditions may reveal normal MRI findings.

Another nationwide survey of rotavirus-associated encephalitis/encephalopathy and sudden unexpected death conducted in Japan between 2009 and 2011 showed that, among a total of 58 cases of rotavirus-associated encephalitis/encephalopathy and 7 cases of sudden unexpected death, 36 patients (62.1%) with encephalitis/encephalopathy had no sequelae, while 15 (25.9%) had neurological sequelae and 7 (12.1%) had fatal outcomes [46]. Pleocytosis was observed in 9 of 40 patients (22.5%). Elevated lactate dehydrogenase levels (>500 IU/L) or acidemia (pH <7.15) were associated with poor prognosis.

Viral infection-associated acute encephalopathy is classified according to the pathogenic virus of antecedent infection, such as rotavirus-associated encephalopathy or influenza-associated encephalopathy. Alternatively, based on clinical manifestations and brain MRI findings, it can be classified as various syndromes, such as acute necrotizing encephalopathy (ANE), acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), mild encephalopathy with a reversible splenial lesion (MERS), hemiconvulsion-hemiplegia-epilepsy syndrome, hemorrhagic shock and encephalopathy syndrome, Reye’s syndrome, and so forth, although unclassified acute encephalopathy is the most common [16]. According to the Japanese national surveys of 2007–2010 and 2014–2017, MERS was the most common syndromic diagnosis of rotavirus-associated encephalopathy (18 cases [45.0%]), followed by AESD (4 cases [10%]) and ANE (1 case [2.5%]) in the first study. The order was the same in the second survey as well: MERS (21 cases [46.7%]), AESD (5 cases [11%]), and ANE (2 cases [4.4%]) [17,18]. A full recovery was observed in 28 (70.0%) and 31 cases (68.9%), while neurologic sequelae were seen in 8 (20.0%) and 13 cases (28.9%) in the first and second survey, respectively. Three (7.5%) and 0 deaths (0%) were reported in the respective surveys. Here we describe the typical findings of MERS, AESD, and ANE.

Rotavirus cerebellitis is characterized by decreased consciousness 1–3 days after the onset of symptoms of gastroenteritis and subsequent mutism [30]. Consciousness clears between days 4 and 12, but mutism is observed after the recovery of consciousness. Mutism may last for up to 20 days. Other possible cerebellar symptoms include slow speech or dysarthria, hypotonia, ataxia, tremors, nystagmus, and dysmetria. MRI, particularly DWI, shows high-signal intensity in the bilateral dentate nuclei/cerebellar white matter in the acute and subacute phases, followed by increased signal intensity in the vermis and cerebellar cortex, and finally cerebellar atrophy 1–2 months after onset (Fig. 1C) [30,62]. Concomitant reversible splenial lesions are often observed during the acute phase [63]. CSF pleocytosis is frequently observed along with normal glucose and protein levels. Despite treatment with corticosteroids and intravenous immunoglobulins, many patients show mild to moderate cerebellar symptoms such as ataxia, dysarthria, tremors, and mental retardation [30].

Neonatal rotavirus-associated leukoencephalopathy typically presents with repetitive or clustered focal or multifocal clonic seizures at around the 5th day after birth accompanied by bilaterally symmetric diffuse white matter and corpus callosum lesions on DWI [64]. We recently demonstrated that neonatal rotavirus-associated leukoencephalopathy was a main cause of fifth-day seizures, which prevailed in European countries and Australia between the 1970s and mid-1980s based on the similarity of clinical features and the annual incidence of an epidemic-like outbreak with abrupt disappearance [65]. Since the first report by our institute in 2014 [64], most cases of neonatal rotavirus-associated leukoencephalopathy have been documented in Korea, particularly in the confined southeast province of Korea, including Ulsan, Busan, and Jinju [66-68]. Between 2008 and 2016, in our institute, rotavirus-associated leukoencephalopathy accounted for 37.2% of neonatal seizures in full-term newborns, which peaked at 70.6% in 2012, gradually decreased, and finally disappeared in 2017. Seizures usually occur in healthy full-term newborns without a history of perinatal asphyxia. There is no known cause of neonatal seizures, such as hypoxic-ischemic encephalopathy or intracranial hemorrhage. Lesions on DWI are observed in symmetric diffuse patterns involving the cerebral white matter and corpus callosum. Lesions on DWI often extend to the internal capsule, external capsule, thalamus, basal ganglia, and brain stem following the white matter tracts (Fig. 1D) [64]. Only a small number of patients have gastrointestinal symptoms, such as diarrhea and vomiting, despite rotavirus infection. Almost all patients had no fever or rashes. Neither rotaviral RNA nor antigen was detected in CSF samples [66,68]. CSF pleocytosis was also not observed. Some patients showed a decrease in cerebral white matter volume and cerebromalacia in the white matter on follow-up MRI taken after 1–3 months [67]. Our previous study demonstrated that 18 of 32 patients (56%) with neonatal rotavirus-associated leukoencephalopathy had delayed performance on the mental and/or motor scales of the Korean Bayley Scales of Infant Development II assessment at a median age of 22 months [69]. The lesion extent on initial DWI was negatively correlated with the mental developmental scale.

Posterior reversible encephalopathy syndrome [70], opsoclonus-myoclonus syndrome [71], and hemiconvulsion-hemiplegia-epilepsy syndrome [72] are reportedly related to rotavirus infection as described in previous case reports.

Rotavirus is a nonenveloped double-stranded RNA virus with a wheel-like icosahedral structure composed of 3 concentric capsids that surround a genome of 11 segments of RNA [1,76]. These RNA segments encode 6 structural viral proteins (VP1–4, VP6, and VP7) and 6 nonstructural proteins (NSP1–NSP6). Among the 6 structural proteins, surface glycoprotein VP7 and spike protein VP4 constitute the outermost layer and are responsible for eliciting an immune response. VP6 constitutes the middle capsid layer; based on the sequence and antigenic differences of VP6, rotavirus is classified into 10 different species (A–J) [77]. Species A rotavirus is the most common cause of infection in children [1]. Rotavirus is further classified into diverse genotypes based on sequence differences in RNA segments 7 and 4, which encode VP7 and VP4, respectively. VP7 and VP4 subtypes determine the G and P genotypes, respectively, in a dual classification system [76].

Rotavirus predominantly infects mature enterocytes at the top and middle of the intestinal villi, leading to the vacuolization of enterocytes [1]. Rotavirus infection and replication in the duodenal mucosa of infants cause shortening and atrophy of the villi, loss of microvilli, mononuclear cell infiltration, distended endoplasmic reticulum, and mitochondrial swelling in enterocytes [78,79]. The suggested mechanisms underlying these findings include virus-mediated apoptosis [80], NSP4-mediated mislocalization of the ZO-1 tight junction protein [81], binding to the basement membrane extracellular matrix proteins laminin subunit-β3 and fibronectin [82], and disruption of normal cellular homeostasis [83]. Previous in vitro studies demonstrated that rotavirus is able to infect neuronal cells, where NSP4 was detected [84,85]. Thus, rotavirus infection may cause a change in cellular structure and function, leading to cell death of neurons as well as enterocytes.

Rotavirus NSP4 is a well-known viral enterotoxin that functions as a viroporin for the disruption of intracellular Ca²⁺ homeostasis through phospholipase C [1]. Viroporins are small virus-encoded ion channel proteins [86]. Rotavirus infection and NSP4 expression alone can increase cytoplasmic Ca²⁺ levels resulting from increased Ca²⁺ leakage from the endoplasmic reticulum via NSP4 viroporin [86,87]. The elevation of cytoplasmic Ca²⁺ levels is critical to the rotavirus replication process, including activation of autophagy, nucleation of rotavirus replication complexes called viroplasms, and assembly of the outer capsid protein VP7 [86,87]. NSP4 can also induce the production of nitric oxide and its metabolites. The overproduction of reactive nitrogen species is known to alter protein structure, thereby inhibiting their normal function, leading to DNA fragmentation and lipid oxidation [88]. One study showed that the treatment of intestinal cells with NSP4 increased inducible nitric oxide synthase mRNA expression [89]. Another study showed that the incubation of human intestinal epithelial cells with purified NSP4 caused NO2/NO3 accumulation in the incubation media [87]. In this study, 46 children with rotavirus infection showed higher urinary NO2/NO3 concentrations compared to their age-matched peers. Previous studies showed that inducible nitric oxide synthase activity depends on intracellular calcium levels and that NSP4 increases intracellular calcium levels. Thus, the authors interpreted that NSP4 increases NO production by elevating intracellular calcium levels. Rotavirus NSP4 can also elicit the production of proinflammatory cytokines, and a recent report demonstrated that NSP4 induces the secretion of proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α together with nitrite from human and murine macrophage-like cell lines via Toll-like receptor 2 [90]. Another study showed that NSP4 induced the time-dependent generation of reactive oxygen species (ROS) in human intestinal epithelial cells [91]. Given that ROS can induce intestinal epithelial cell apoptosis in mice through Fas and Fas-L expression [92], NSP4 may induce cell apoptosis via ROS.

To enter the CNS compartment from the primary site of infection, neurotrophic viruses are known to follow 3 routes: (1) crossing the vascular endothelium; (2) accessing peripheral nerves; and (3) “Trojan horse” entry [93]. Rotavirus is also presumed to exploit the first 2 routes to reach the CNS (Table 2).

In addition to direct viral invasion into the CNS, the entry of various circulating mediators produced during rotavirus infection into the CNS can be considered a possible mechanism of CNS complications. Circulating mediators include proinflammatory cytokines, prostaglandins, ROS, nitric oxide metabolites, excitatory amino acids, and rotavirus antigens such as NSP4 [103]. Systemic inflammation caused by various stimulants, such as viruses, polyinosinic:polycytidylic acid (poly I:C, a mimic of viral double-stranded RNA), cytokines such as IL-1β, IL-6, and TNF-α, and bacteria and lipopolysaccharide, can damage blood-brain barrier integrity [110,111]. Thus, it is presumed that viremia or antigenemia, which often occurs in rotavirus infection as mentioned above, can cause systemic inflammation, thereby inducing a breach of the blood-brain barrier and blood-CSF barrier; eventually, the aforementioned deleterious mediators are able to enter the brain through the breach or the capillaries of the circumventricular organs.

Kawashima et al.112) showed that NO2 and NO3 levels in the serum and CSF of rotavirus gastroenteritis patients with convulsions were significantly higher than those in the control group. This result suggests the possibility of circulating NO metabolites entering the CNS. Host immune responses to rotavirus infection may also cause brain injury. Clinical studies have shown that rotavirus gastroenteritis is associated with increased levels of proinflammatory cytokines, such as IL-1β, IL-6, IL-8, and interferon gamma (IFN-γ) [14,113]. Newborns with rotavirus-associated leukoencephalopathy have significantly higher IL-6, IFN-γ, and monocyte chemoattractant protein-1 levels in the CSF than the control group [114,115]. Furthermore, CSF IL-6 levels correlate positively with white matter lesion extent. A previous report showed elevated PGE2 and PGF2 levels in the plasma and stool of rotavirus-infected children [116]. A clinical study showed that, in 19 children with CNS involvement associated with rotavirus infection, excitatory neurotransmitter levels (glutamate, glycine, and taurine) in the CSF were higher in patients with prolonged seizures accompanied by encephalopathy than in patients with a brief cluster of seizures without encephalopathy [117].

As mentioned earlier, viral encephalitis tends to demonstrate asymmetrical focal lesions on MRI as in herpes simplex virus encephalitis, while rotavirus-associated encephalopathy is generally characterized by bilateral symmetry, the predilection of cerebral white matter including the corpus callosum, and the lesions are well-demarcated, particularly on DWI. Considering these distinct features, it is likely that rotavirus-associated CNS complications may be attributed to the combined effects of CNS entry of various mediators and immune cells as well as virus and viral antigens across blood vessels in the brain rather than virus-induced cytopathic effects that developed after the direct viral infection of neurons and glial cells.

Systemic inflammation caused by a viral infection can induce leukocytes in the blood vessels to transmigrate across the blood-brain barrier, and leukocytes to reach the brain can injure neuronal cells. The presence of CSF pleocytosis in patients with acute rotavirus-associated encephalopathies/encephalitis and cerebellitis indicates the recruitment of leukocytes from circulation into the CNS [30,46]. The migration of leukocytes from the vascular lumen to the infected sites engages sequential adhesive molecular interactions between leukocytes and endothelial cells [118-121]. When viruses infect cells, various chemokines are produced and bind to their cognate chemokine receptors on circulating leukocytes, thereby activating leukocyte integrins, such as very late antigen 4 and lymphocyte function-associated antigen 1, to cluster outside the cell membrane [118]. Besides, vascular endothelial cells enhance the expression of cell adhesion molecules such as vascular cell adhesion molecule 1 and intercellular adhesion molecule 1, which are clustered in the luminal side of vessels after a viral infection [119]. Binding between integrins of circulating leukocytes and cell adhesion molecules on vascular endothelial cells leads to activation of the endothelial cell signaling pathway, which elicits opening of the tight and adherence junctions, and stress fiber formation (endothelial contraction) via actin remodeling [120,121]. These microstructural alterations facilitate passage of the leukocytes through the vascular endothelial cells (diapedesis) and eventual arrival at the inflamed site. Leukocytes that reach the brain can injure the neurons and glial cells.

In future research on the pathophysiology of rotavirus-associated CNS complications, the following should be considered. First, if rotavirus itself, its proteins and RNA, harmful mediators, and immune cells could induce brain lesions, what determines the specific lesion location? Second, what causes bilateral symmetry in brain lesions in almost all rotavirus infection-associated CNS complications? Third, why is cerebral white matter predominantly injured in most rotavirus infection-associated CNS complications instead of cerebral gray matter, as seen in MERS, AESD, and neonatal rotavirus-associated leukoencephalopathy? Finally, what other possible mechanisms are at play in different types of rotavirus infection-associated CNS complications?