Norovirus-associated neurological manifestations: summarizing the evidence

Norovirus, a positive-stranded RNA virus, is one of the leading causes of acute gastroenteritis among all age groups worldwide. The neurological manifestations of norovirus are underrecognized, but several wide-spectrum neurological manifestations have been reported among infected individuals in the last few years. Our objective was to summarize the features of norovirus-associated neurological disorders based on the available literature. We used the existing PRISMA consensus statement. Data were collected from PubMed, EMBASE, Web of Science, and Scopus databases up to Jan 30, 2023, using pre‐specified searching strategies. Twenty-one articles were selected for the qualitative synthesis. Among these, seven hundred and seventy-four patients with norovirus-associated neurological manifestations were reported. Most cases were seizure episodes, infection-induced encephalopathy, and immune-driven disorders. However, only a few studies have addressed the pathogenesis of norovirus-related neurological complications. The pathogenesis of these manifestations may be mediated by either neurotropism or aberrant immune-mediated injury, or both, depending on the affected system. Our review could help clinicians to recognize these neurological manifestations better and earlier while deepening the understanding of the pathogenesis of this viral infection.


Introduction
A relatively less known non-enveloped, positive-stranded RNA virus belonging to the Caliciviridae family, norovirus, also known as Norwalk virus, has been considered a potential human foodborne human enteric pathogen since the 1968 outbreak at an elementary school in Norwalk, Ohio (Adler and Zickl 1969;Kapikian et al. 1972). The family comprises five genera, mainly norovirus, sapovirus , lagovirus, nebovirus, and vesivirus. RNA viral particles were confirmed in stool specimens during that outbreak (Kapikian et al. 1972;Robilotti et al. 2015). Initially, the infected individuals manifested nausea, vomiting, low-grade fever, abdominal cramp, lethargy, and, most importantly, severe diarrhea (Adler and Zickl 1969). Since then, worldwide infected cases have been estimated to be around 685 million, among which approximately 200 million infected pediatric cases have been documented (Patel et al. 2008). Substantial morbidity across a wide range of healthcare settings is noted and predominantly among children, estimated to be around 50,000 deaths per year (Patel et al. 2008;Widdowson et al. 2005).
Clinical features of norovirus infection are nausea, vomiting, fever, abdominal pain, and mild self-limited non-bloody diarrhea. Notably, the phrase "stomach flu" was initially used for infected individuals with a low fever and abdominal pain (Kapikian et al. 1972;Robilotti et al. 2015). However, a severe form of this infection is linked to copious diarrhea, which can result in dehydration and occasional death (Kapikian et al. 1972;Robilotti et al. 2015).
Noroviruses can infect brain endothelial cells and increase the expression of matrix metalloproteinases, decreasing the expression of tight junctional proteins and increasing blood-brain barrier permeability (Al-Obaidi et al. 2018). Several wide-spectrum neurological manifestations have been reported among infected individuals in recent years. Our objective was to summarize the norovirus-associated neurological manifestations based on the available literature.

Methods
This review followed the Preferred Reporting for Systematic Review and Meta-Analysis (PRISMA) consensus statement-PROSPERO 2022 CRD42022345256. Studies concerning cases of norovirus infection with confirmed or suspected neurological manifestations were included.

Search strategy
We searched through PubMed, Scopus, Web of Science, Embase, Cochrane Library, and Noro Net databases, which concluded on Jan 30, 2023, using pre-specified search strategies for each database. The search strategy consisted of keywords of relevant medical subject headings and keywords, including "norovirus," "Caliciviridae," "demyelination," "encephalopathy," "encephalitis," "enteric nervous system," "benign convulsions," "meningitis," "meningoencephalitis," "leptomeningitis," "cerebritis," and "brain stem involvement." Sapovirus and vesivirus were also included in our search strategy to capture related articles. We also hand-searched additional norovirus-specific databases using the reference list of the selected studies, relevant journal websites, and renowned preprint servers (medRxiv, bioRxiv, pre-prints.org, and Calcinet) from 2005 to Jan 30, 2023. To decrease publication bias, we invigilated the references of all studies potentially missed in the electrical search. Content experts also searched the gray literature of any relevant articles.

Study selection criteria
All peer-reviewed, preprint (not-peer-reviewed), including cohorts, clinical series, case-control studies, and case reports that met the pre-specified inclusion and exclusion criteria, were included in this study.
Inclusion and exclusion criteria Studies that met the following inclusion criteria were included: (1) studies reporting patients infected with norovirus with or suspected neurological manifestations, (2) studies registering neurological manifestations of norovirus patients, and (3) parallel studies that analyzed the distribution and incidence of neurological disorders in similar Caliciviridae infections, i.e., sapovirus and vesivirus. Only studies that were published in English were considered. Accordingly, we excluded the studies with the following criteria: (1) prior history of neurological disorders; (2) insufficient data and, subsequently, failure to contact the authors; (3) non-clinical research, animal studies and reviews, correspondence, viewpoints, editorials, and commentaries; and (4) duplicate publications. The references of the original articles and reviews identified were manually searched further for any article that had been missed out.

Study selection and evidence synthesis
Before the screening process, teams of three reviewers participated in calibration and screening exercises. One reviewer independently screened the titles and abstracts of all identified citations, and the remaining two verified those and screened papers. One of the other reviewers then retrieved and screened independently the full texts of all citations deemed eligible by the reviewer on the team and analyzed those data. Another reviewer independently verified these extracted full texts for eligibility for analysis and designed the overall study structure. The corresponding and senior author (JBL) resolved disagreements whenever necessary and took final decisions regarding the study. Throughout the screening and data extraction process, the reviewers used piloted forms. In addition to the relevant clinical data, the reviewers also extracted data on the following characteristics: study characteristics (i.e., study identifier, study design, setting, and timeframe); population characteristics; comparator characteristics, outcomes (qualitative and quantitative); clinical factors (definition and measurement methods), measures of association (relative risks, odds ratios, and hazard ratios), reported funding sources and conflict of interests, and study limitations. The Newcastle-Ottawa scale was used to evaluate the study's selection procedure, comparability, and outcomes.

Statistical analyses
Unit discordance among variables was resolved by converting the variables to a standard unit of measurement. A p-value < 0.05 was considered statistically significant but could not be calculated due to insufficient data. A metaanalysis was planned to analyze the association of the demographic findings, symptoms, biochemical and neuroimaging parameters, and outcomes. Still, it was later omitted due to the limited availability of comparable data and significant variability among the included studies.

Ethics
This review was based on the available literature on neurological manifestations among norovirus-infected individuals across all age groups; no animal or human subjects were involved. Henceforth, approval from the ethics committee was not applicable.

Results
The selection procedure was carried out according to the PRISMA consensus statement in Fig. 1. Twenty-one articles were selected for the qualitative synthesis. Among these, seven hundred and seventy-four patients with norovirusassociated neurological manifestations were reported, mainly seizure episodes, infection-induced encephalopathy, and immune-driven disorders (Table 1).
Afebrile infantile seizures with signs of acute gastroenteritis and no other illnesses that cause seizures (e.g., hypoglycemia, electrolyte imbalance, and cerebrospinal fluid abnormalities) are referred to as benign seizures with mild gastroenteritis (Kawano et al. 2007). Norovirus is probably the most common viral pathogen causing benign seizures with mild gastroenteritis (Kim et al. 2018). In a landmark clinical series that included 64 patients infected by norovirus and 101 by rotavirus, norovirus infection was associated with a higher seizure rate in young children than rotavirus infection (19, 29.7% vs. 5, 5%; p < 0.001) (Chen et al. 2009). Only six patients received short-course anticonvulsant therapy, and none of the 19 patients had any neurological sequelae. Compared with rotavirus-associated benign seizures with mild gastroenteritis, those caused by norovirus are less frequent during spring, more frequently seen with vomiting, have a shorter interval from enteric symptom onset to seizure onset, and more frequently show a posterior slowing in electroencephalography (Kim et al. 2018). What seems clear is that young age may be a risk factor for norovirusassociated benign seizures (Kawano et al. 2007;Chen et al. 2009), and long-term neurological sequelae are uncommon.
Kimura et al. first reported a case of norovirus-associated encephalopathy in a 60-year-old female (2010). Similarly, other pediatric patients with norovirus-associated encephalopathy have been reported (Obinata et al. 2010, Tantillo et al. 2021. The common thread among the abovementioned pediatric patients included high-voltage slow waves on electroencephalography without paroxysmal discharges (Obinata et al. 2010); two patients also presented with increased muscle tone and slow pupillary light reflex. Sánchez-Fauquier et al. (2015) reported a patient with encephalitis who presented disorganized cerebral activity in electroencephalography. On the other hand, Yoo et al. (2023) described an infant with norovirus-associated meningoencephalitis who presented with status epilepticus during admission, revealing suppressed pattern activities with continuous right central spike discharge at 3-5-s intervals on electroencephalography and concomitant disseminated intravascular coagulation. A patient with suggestive cerebellitis with mild language delay, gait disturbances, and asymmetric high T2-weighted signal intensity with leptomeningeal enhancement in the right cerebellar folia has also been reported (Chung et al. 2017), as well as another patient with bulbar involvement who presented with diplopia, transient ataxia, bilateral periorbital pain, and stage 4 bilateral papilledema (Yoo et al. 2023).  Miller Fisher syndrome is a rare spectrum of Guillain-Barré syndrome, a broad syndrome encompassing several types of acute immune-mediated polyneuropathies. Both entities are thought to result from an aberrant acute autoimmune response to a previous infection (e.g., Haemophilus influenza, Campylobacter jejuni, cytomegalovirus, or SARS-CoV-2, among others) (Koga et al. 2019, Gutiérrez-Ortiz et al. 2020, suggesting a para-viral or postviral process. Intriguingly, autoimmune demyelinating and neuroinflammatory disorders have also been reported in the context of norovirus infection. Eltayeb and Crowley (2012) first reported an adult case of norovirus-related Guillain-Barré syndrome who presented with general symptoms of norovirus infection, progressive ascending weakness, hyporeflexia, numbness, mild facial weakness, deteriorating respiratory failure, and autonomic dysfunction. Shimizu and Tokuda (2012) reported a case of Miller Fisher syndrome in an adult female who had presented with enteric features of norovirus followed by blurred vision, ataxic gait, pins, and needlelike sensations in the hands bilaterally, progressive ascending weakness, and upward and downward gaze alterations. The two patients showed no significant changes in the brain and spinal MRI; however, neurophysiological studies were consistent with acquired demyelinating polyneuropathy in the patient with Guillain-Barre syndrome. Disorder related to direct myelin injury in the form of transient myelin-sheath edema has also been reported in another case of norovirus infection (Saran et al. 2019). These two cases suggest that norovirus infection should be ruled out in those patients with post-diarrheal immune-driven disorders, especially in endemic territories. Gutierrez-Camus et al. (2022) reported white matter injury in a 2-day-old patient following a norovirus infection. The patient presented abnormal facial and limb movements and generalized tonic-clonic seizure episodes. On the other hand, Nakakubo et al. (2016) reported a patient with norovirus-induced cerebellar infarction associated with an underlying antiphospholipid syndrome in a 6-year-old male. These cases open avenues to understanding the diverse nature of the post-infectious nature of neurological sequelae.
Noteworthy to mention that, apart from the patients with afebrile seizures, who had a good outcome, most who required intravenous immunoglobulin, immunosuppressives, and supportive management showed full recovery (Table 1).

Discussion
This study is the first-ever attempt to explore neurological manifestations in norovirus infection through a systematic review of peer-reviewed data. The current review is important in advancing our knowledge and understanding of norovirus's neurological complications, primarily considered a gastrointestinal virus in adult and pediatric populations. Several neurological manifestations of norovirus infection affecting the central and peripheral nervous systems have been reported (Table 1). Despite this, these complications might be under-recognized. Thus, we have only found seven hundred and seventy-four patients with de novo norovirus-associated neurological disorders in the present review, mainly benign seizure disorders, particularly in young infants. The pathogenesis of norovirus-associated neurological manifestations may be mediated by either neurotropism or aberrant immune-mediated injury, or both, depending on the affected system. Evidence supports an aberrant immunemediated injury. First, there is a gap between the enteric symptom onset and the first neurological symptoms (in the cases of seizures may be a couple of days (Kim et al. 2018), and in the cases of acute immune-mediated polyneuropathies 10 to 14 days) (Shimizu and Tokuda 2012;Eltayeb and Crowley 2012), suggesting a post-infectious autoimmune process. Second, the spectrum of Guillain-Barré syndrome, including Miller Fisher syndrome, is a prototype for post-infectious immune-mediated neuropathy with known infectious triggers (Koga et al. 2019). Third, in the case of post-norovirus Miller Fisher syndrome, antibodies against ganglioside (i.e., GQ-1b) were detected (Eltayeb and Crowley 2012). There is evidence that sialic acid-containing glycosphingolipids (gangliosides) are also ligands for human norovirus (Han et al. 2014). Hence, cross-reactivity and molecular mimicry between norovirus antigenic epitopes and gangliosides, essential in modulating nervous system integrity, notably at the node of Ranvier, may bring immunedriven neuropathy in norovirus-induced Guillain-Barré syndrome and its variants. Fourth, a patient with norovirusassociated encephalopathy showed elevated concentrations of cerebrospinal fluid interleukin-6, interleukin-10, interferon-γ, and tumor necrosis factor-α suggesting that her encephalopathy was related to hypercytokinemia (Obinata et al. 2010). Finally, last but not least, a dramatic response to intravenous immunoglobulin in many cases of norovirus-associated neurological manifestations points towards an underlying immune-driven process (Table 1). Immunotherapy with intravenous immunoglobulin could be used to treat norovirusassociated neurological manifestations. Its efficacy would be much improved if the immune IgG antibodies were collected from patients who have recovered from norovirus infection in the surrounding area to increase the chance of neutralizing the virus.

Authors
On the other hand, direct neurotropism as a pathogenic process did not lie far behind. Specifically, viral RNA of norovirus in the cerebrospinal fluid has been detected in two cases of encephalitis (Gutierrez-Camus et al. 2022, Ito et al. 2006. Studies on immunodeficient mice infected by various murine norovirus have revealed changes in brain histology, suggesting that immunodeficiency may favor neuro-invasion of the norovirus (Haga et al. 2016). Two hypotheses could explain how norovirus could potentially reach the central nervous system. First, genome-wide CRISPR screening and cell line-based in vitro studies suggest that murine norovirus uses members from the CD3000 family (a group of proteins playing vital roles in immune responses), such as CD300ld or CD300lf, as a receptor (Haga et al. 2016). Besides, other members from this family, such as CD300e and CD300f, are homologous to murine CD300ld and CD300lf from the human [Blast] brain (Homological sequence has been found using NCBI blastp; RID-TDPK0RX013) (Fig. 2). Human norovirus could invade the central nervous system through these proteins. Second, there is some evidence that different polymorphisms on different histo-blood group antigens, such as FUT1, FUT2, and FUT3, are linked to high susceptibility toward norovirus infection (Nordgren and Svensson 2019;Ward et al. 2006) (Fig. 2). Human norovirus could cross the blood-brain barrier and bind through these antigens, which are expressed in various regions of the human brain (Fig. 2). Hence, neuro-invasion could alternatively occur through brain endothelial cell-specific blood group antigens.
There are some limitations in the current review. Given the notable asymmetry between the total number of affected cases and reported norovirus-associated neurological disorders, it can be assumed that neurological cases are under-reported. The current review is, however, based on several hundreds of cases, even after an extensive search of available literature. In addition, several available reports do not describe the timeline of events in an organized manner, making interpretation difficult. Laboratory, electroencephalography, and neuroimaging features have also not been detailed in a few cases. In addition, considerable heterogeneity in the available data may be considered a hindrance in advanced analysis. Finally, we have not included non-English articles. Despite these shortcomings, the present organized review will be an introductory guide for clinicians dealing with neurological disorders that appear in norovirus infection.
Only a few studies have addressed the pathogenesis of norovirus-related neurological complications. Hence, further work must be done to understand the mechanisms responsible for these complications. With the growing frequency of such cases, our study could help clinicians recognize these neurological manifestations better and earlier while deepening the understanding of this viral infection.