Severe acute encephalopathy/encephalitis (AE) associated with SARS-CoV-2 has been increasingly reported since the emergence of the Omicron variant. Several pediatric cases have shown the development of acute fulminant cerebral edema (AFCE) or hemorrhagic shock encephalopathy syndrome (HSES), which are linked to high morbidity and mortality. However, the underlying pathogenic mechanisms remain unclear. We performed single-cell RNA sequencing of peripheral blood mononuclear cells from a pediatric patient with SARS-CoV-2-associated AE presenting with AFCE/HSES and compared the data with those from two patients with mild AE, one patient with febrile seizures due to non-SARS-CoV-2 pathogens, and publicly available pediatric COVID-19 datasets without neurological complications. During the acute phase, we observed a prominent expansion of B-cell populations, including distinct activated B-cell clusters. Cell-cell communication analysis identified macrophage migration inhibitory factor signaling, although it was not specific to SARS-CoV-2-associated AE. Notably, heat shock protein genes, particularly HSPA1A and HSPB1, were selectively upregulated across multiple immune cell types only in severe SARS-CoV-2-associated AE. Enzyme-linked immunosorbent assay confirmed significantly elevated plasma and serum protein levels of HSPA1A and HSPB1 during the acute phase. These findings highlight HSPA1A and HSPB1 as potential biomarkers of severe SARS-CoV-2-associated AE and suggest a pathogenic possible role for stress-response pathways.

Background Congenital cytomegalovirus (CMV) infection is a leading cause of neurodevelopmental disabilities. Although oral valganciclovir (VGCV) treatment has shown short-term benefits, long-term outcomes beyond 3 years remain unclear. Objective To evaluate 4-year neurodevelopmental outcomes in infants with symptomatic congenital CMV (SCCMV) disease treated with VGCV and identify predictors of adverse outcomes. Methods This prospective follow-up study (VGCV-2) included 24 infants with SCCMV disease who received oral VGCV (16 mg/kg, twice daily for 6 months). Neurodevelopmental assessments were performed at 1, 2, 3, and 4 years of age using the Kyoto Scale of Psychological Development (KSPD). The primary outcome was developmental delay (developmental quotient [DQ] < 70) at 4 years. Secondary outcomes included diagnoses of neurodevelopmental disorders and results of autism spectrum disorder (ASD) screening. Results Twenty-one participants (87.5%) completed the 4-year follow-up. Developmental delay was evident in 28.6% (6/21) of patients at 4 years. Neurodevelopmental disorders, including intellectual disability (28.6%), ASD (19.0%), and cerebral palsy (14.3%), were diagnosed in 42.9% (9/21) of patients. A poorer best-ear hearing assessment at baseline was a significant predictor of developmental delay. Shorter body length, smaller head circumference at birth, and poorer baseline hearing were significantly associated with the diagnosis of neurodevelopmental disorders. Early positive Modified Checklist for Autism in Toddlers screening at 2 and 3 years strongly predicted the diagnosis of neurodevelopmental disorders. Conclusion Despite VGCV treatment, substantial neurodevelopmental impairment persisted for 4 years in children with SCCMV disease. Early clinical markers can help identify high-risk infants who require intensive developmental support. (Clinical trial registration: jRCT2051190075).

Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is an intractable form of epilepsy involving the hippocampus, and temporal lobectomy remains an effective treatment. Human herpesvirus 6B (HHV-6B) establishes latency in the hippocampus and may contribute to MTLE-HS pathogenesis by altering host gene expression; however, transcriptomic data from healthy controls remain limited. This study investigated the role of HHV-6B to MTLE-HS pathogenesis by analyzing gene expression in resected hippocampal tissues. Samples were collected from 12 to 43 HHV-6 DNA-positive and -negative patients, respectively, and three controls. RNA sequencing was performed on eight representative samples, followed by RT-qPCR validation of nine selected genes in 58 samples. RNA sequencing identified 600 differentially expressed genes (210 upregulated, 390 downregulated) between HHV-6B-positive MTLE-HS and controls. Pathway enrichment analysis revealed involvement of synaptic signaling and inflammatory responses, with prostaglandin biosynthesis specifically upregulated in HHV-6B-positive tissues. Two genes were significantly upregulated in HHV-6B-positive compared with HHV-6B-negative samples. RT-qPCR confirmed elevated cholesterol 25-hydroxylase and interleukin 1 beta expression in HHV-6 DNA-positive samples (both p = 0.031). These findings suggest that HHV-6B may contribute to MTLE-HS pathogenesis by modulating the expression of host inflammatory genes, supporting a role for neuroinflammation and the potential benefits of anti-inflammatory therapies.

Fulminant myocarditis (FM) is a rare but life-threatening pediatric condition that can rapidly progress to cardiogenic shock and fatal arrhythmia. Prognostic biomarkers in FM are essential for optimizing treatment strategies. Although inflammatory cytokines have been associated with the pathogenesis of FM, their prognostic value remains unclear. This study aimed to identify mortality-associated markers by integrating cytokine profiles and clinical variables through a machine learning approach. We retrospectively analyzed 21 pediatric FM cases from two tertiary centers (2012–2022). At admission, 37 cytokines and 14 clinical parameters were assessed. Partial least squares discriminant analysis was employed to identify prognostic features, with variable importance in projection scores quantifying their contribution. Model performance was evaluated using repeated stratified 3-fold cross-validation with 100 repeats. For each repeat, predictions obtained from the three folds were averaged at the sample level, and performance metrics were computed from these fold-averaged predictions. Statistical significance was determined via the Benjamini–Hochberg method at a false discovery rate of 0.05. Of the 51 features analyzed, 18 emerged as key predictors, 15 cytokines and 3 clinical parameters, with variable importance in projection (VIP) scores above 1.0. Seven cytokines (TNF-α, MIP-1α, M-CSF, IL-8, IL-6, IL-15, and IP-10) were both statistically significant and highly important. TNF-α had the highest VIP score among all predictors. Three clinical parameters (CK-MB, pH, and lactate) were also linked to poor outcomes. The model performed robustly, with an area under the receiver operating characteristic curve (AUROC) of 0.912, an area under the precision–recall curve (AUPRC) of 0.874, 83.8% accuracy, 71.0% sensitivity, and 90.1% specificity. Furthermore, the cytokine-enriched model showed higher predictive performance than the clinical-only model across accuracy, AUROC, and AUPRC. Analysis of cytokine profiles using machine learning may identify biomarkers associated with mortality risk in pediatric FM. TNF-α emerged as a key cytokine associated with mortality, supporting its potential role as a prognostic biomarker. Importantly, cytokine markers added prognostic value beyond routine clinical variables.

Lambl’s excrescences (LEs) are filiform extensions arising on the closing surface of cardiac valves, typically occurring at coaptation lines of left-sided valves. LEs are more commonly reported in adults and pediatric cases remain rare, with no previously documented cases in Japan. A 9-year-old Japanese boy initially presented with a fever and sore throat but developed right-sided neck pain several days later. Blood tests showed a white blood cell count of 33,900/mL and C-reactive protein concentration of 9.95 mg/dL. Contrast-enhanced computed tomography showed a right peritonsillar abscess, and cefotaxime and clindamycin were administered. The patient’s symptoms persisted for 4 days. Therefore, he was transferred to our hospital. Transthoracic echocardiography showed trivial aortic regurgitation and a 4.4×1.0-mm filamentous structure attached to the aortic valve. The patient improved with antibiotic treatment alone, which was continued because infective endocarditis was suspected. However, transesophageal echocardiography showed no shape-like vegetations, papillary fibroelastomas, or thrombi, and repeated blood cultures were all negative, which led to a diagnosis of LEs. After completing a 3-week antibiotic course, the patient was discharged and remained asymptomatic with no structural changes in the LE in 2 years. To the best of our knowledge, this is the first report of a pediatric case of LEs associated with aortic regurgitation in Japan, highlighting that LEs should be considered in children with aortic regurgitation.

Zika virus infection is primarily transmitted by mosquitoes and is generally a self-limiting, mild disease. However, infection during pregnancy can cause severe fetal abnormalities, such as microcephaly, and in adults, the condition may occasionally lead to neurological complications, including Guillain-Barré syndrome. A large outbreak occurred in South America between 2015 and 2016 and has become a major global public health concern. Diagnosis involved reverse transcription-polymerase chain reaction and serological testing. As currently no specific antiviral treatments or vaccines are available, public health interventions, such as mosquito bite prevention and advising pregnant women to avoid traveling to affected regions, are essential.

Kawasaki disease (KD) is a systemic vasculitis in young children, and early diagnosis remains challenging when clinical features are incomplete or overlap with those of other febrile illnesses. Because electrocardiography (ECG) is noninvasive and widely available, we investigated whether ECG-derived features could help distinguish complete KD from pediatric patients with fevers. We conducted a single-center retrospective study of hospitalized febrile children aged 1-8 years who underwent digital 12-lead ECG recording during the initial evaluation at a hospital. Five amplitude features and six timing features extracted from the ECG were used to develop a logistic regression model to distinguish between complete KD and other febrile illnesses. The model succeeded in the discrimination between KD and non-KD groups. The prediction performance was not strongly correlated with the age and body temperature. Wave amplitudes and RR interval were suggested as the important features for the discrimination. These findings suggest that ECG-derived features may provide adjunctive information for distinguishing complete KD from other febrile illnesses. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by JSPS KAKENHI Grant Number JP26K00592, Suzuken Memorial Foundation, Research Foundation for the Electrotechnology of Chubu, and Aichi Health Promotion Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the institutional ethics committee of Fujita Health University (approval number: HM25-281). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data supporting the findings of this study are not publicly available because they include potentially identifiable clinical and electrocardiographic data from pediatric patients. Data are available upon reasonable request and approval of institutional ethics committee. Analysis codes used in this study will be available.

Viral infections, particularly rhinoviruses (RVs) and respiratory syncytial virus (RSV), are major causes of pediatric acute asthma exacerbations. The FilmArray Respiratory Panel 2.1 (FARP2.1) is an automated multiplex PCR tool that is being increasingly used in clinical settings; however, its application in asthma exacerbations has not been studied yet. From April 2022 to March 2025, children aged between 6 months and 5 years who had been hospitalized for asthma exacerbations were enrolled ("asthma group," n = 67), alongside children with respiratory infections without wheezing within the same age range ("non-asthma group," n = 207). Nasopharyngeal swabs were performed in all, tested with FARP2.1, and all detected pathogens and clinical features were compared between the two groups. RV/enterovirus (EV) prevalence was significantly higher in the asthma group (82.1%) than in the non-asthma group (28.5%). RSV prevalence was similar in both groups (19.4% vs. 30.0%), while parainfluenza virus was more frequent in the non-asthma group (11.9% vs. 24.2%). The peaks in asthma hospitalizations coincided with RV/EV detection peaks during the autumn. These results highlight that RV/EV was significantly associated with pediatric asthma exacerbations, frequently occurring. Although typically a cause of the common cold, RV/EV may represent an important trigger of asthma exacerbation in children.

BACKGROUND:Buckwheat allergy carries a high risk of anaphylaxis. Although the oral food challenge (OFC) is the diagnostic gold standard, it can provoke severe reactions, leading many patients to adopt elimination diets. Conventional markers, including buckwheat-specific IgE (Bw-sIgE) and skin prick testing, have limited accuracy. The IgE crosslinking-induced luciferase expression (EXiLE) assay is a serum-based in vitro cell-activation test that has shown high accuracy for other food allergies. We evaluated its utility for buckwheat allergy. METHODS:This multicenter observational study in Japan enrolled participants with buckwheat-specific IgE ≥ 0.35 UA/mL (n = 25), categorized as buckwheat allergy (BwA) or non-buckwheat allergy (non-BwA) based on OFC results. The EXiLE assay used crude buckwheat antigen at 0.1-1000 ng/mL. Diagnostic performance was compared using receiver operating characteristic (ROC) curves for EXiLE, Bw-sIgE, and the Bw-sIgE/total IgE ratio. RESULTS:Among 25 participants, the EXiLE assay demonstrated superior diagnostic performance compared to Bw-sIgE and the Bw-sIgE/total IgE ratio. The area under the ROC curve (AUC) was 0.96 at 10 ng/mL (1 ng/mL, 0.94), exceeding Bw-sIgE (0.68) and the Bw-sIgE/total IgE ratio (0.72). At optimal cutoffs, EXiLE achieved sensitivity 0.92, specificity 0.92, and LR+ 11.1 (versus LR+ 3.2 for Bw-sIgE and 2.5 for the IgE ratio), indicating fewer false positives and better overall accuracy. CONCLUSION:The EXiLE assay provides high diagnostic accuracy for IgE-mediated buckwheat allergy and, as a serum-based in vitro assay, may reduce reliance on OFC and help avoid unnecessary elimination diets.

BACKGROUND:Biliary atresia (BA) is a severe infantile hepatobiliary disorder of unknown etiology. Perinatal rotavirus (RV) infection has been implicated in animal models of BA; however, supporting human data remains limited. The study investigated the serological evidence of recent RV infection in infants with BA using RV-specific immunoglobulin (Ig)-A, a marker of primary infection unaffected by maternal antibodies. METHODS:Serum samples from 17 infants with BA and 30 age-matched controls without gastrointestinal symptoms or prior RV vaccination were retrospectively analyzed. Anti-RV-IgA titers were measured by enzyme-linked immunosorbent assay using purified WA-strain virions. Cytomegalovirus (CMV)-IgM and Epstein-Barr virus (EBV)-viral capsid antigen (VCA)-IgM levels were assessed using commercial enzyme immunoassays. RESULTS:RV-IgA was detected in 70.6% (12/17) of the patients with BA versus 3.4% (1/29) of the controls (p < 0.001). RV-IgA titers were significantly higher in the BA group (median: interquartile range 28.0:26.0-210.0) than in the control group (23.5:22.0-24.8) (p = 0.004). Among patients diagnosed with BA after 14 days of age, 84.6% (11/13) were RV-IgA-positive. CMV-IgM was detected in three patients in the BA group and one individual in the control group, while EBV-VCA-IgM was negative in BA patients and positive in two controls; neither difference was statistically significant. CONCLUSIONS:The study findings support the potential association between RV infection and BA pathogenesis. However, the lack of an epidemiological reduction in BA following the introduction of the RV vaccine warrants caution in other studies. Further prospective multicenter studies are required to elucidate the causal role of RV infection in BA development.

Congenital protein C deficiency is a rare autosomal recessive disorder that predisposes patients to severe thrombosis due to markedly reduced protein C activity. Although primarily associated with venous events, an increased risk of arterial thrombosis is also recognized, particularly in severe cases. We report the case of a 17-year-old male patient with compound heterozygous protein C deficiency who developed venous thromboembolism (VTE) after coronavirus disease 2019 (COVID-19) and subsequently experienced splenic infarction despite anticoagulant therapy. Diagnosed shortly after birth following neonatal purpura fulminans, he had been maintained on long-term warfarin therapy. The patient presented with fever and cough and was diagnosed with COVID-19 by antigen testing. Soon after symptom onset, he developed right thigh pain with rapidly expanding ecchymosis, leading to a diagnosis of recurrent VTE. Intravenous heparin and fresh frozen plasma (FFP) were initiated. Although his symptoms and coagulation markers improved by day 10, he developed sudden abdominal pain on day 11, and a contrast-enhanced CT scan revealed a partial splenic infarction. Anticoagulation was intensified, and he subsequently recovered without sequelae. COVID-19 can precipitate both venous and arterial thrombosis through endothelial injury and inflammation. This case illustrates that thrombotic events may occur even after apparent clinical and biochemical improvement, emphasizing the need for careful monitoring and individualized anticoagulation strategies in patients with protein C deficiency during and after COVID-19.

A 77-year-old woman was being treated for rheumatoid arthritis with prednisolone, methotrexate, and iguratimod. The patient was administered a live-attenuated varicella vaccine (Oka strain) for herpes zoster prevention by a primary care physician. Four weeks later, a vesicular rash appeared on the body. The patient was treated with amenamevir, and the rash improved. Based on the pathological findings and viral genotyping, she was diagnosed with a vaccine-induced disseminated varicella-zoster virus infection. For herpes zoster vaccination, caution is warranted as some medical questionnaires for vaccination do not include glucocorticoid use or immunosuppressive therapy, and some primary care physicians administer live vaccines only.

BACKGROUND:Human herpesvirus 6B (HHV-6B) infection causes clinically distinct diseases in primary infection or viral reactivation. Primary infection manifests as exanthem subitum (ES), a common childhood febrile exanthem; whereas reactivation is associated with drug reaction with eosinophilia and systemic symptoms (DRESS), a severe cutaneous adverse reaction. The immune mechanisms underlying these divergent outcomes remain poorly understood. METHODS:We performed single-cell RNA sequencing of peripheral blood mononuclear cells from patients with ES and DRESS. Comparative analysis of immune cell populations and transcriptional programs during the acute and recovery phases revealed distinct immune interaction networks. RESULTS:Primary HHV-6B infection was characterized by marked expansion of proliferating natural killer (NK) cells, which expressed cytotoxic effector genes including GNLY and NKG7. Cell-cell communication analysis predicted strong communication between proliferating NK cells and inflammatory monocytes during acute ES. In contrast, HHV-6B reactivation in DRESS was associated with expansion of proliferating CD4+ T cells with inflammatory transcriptional signatures that showed predicted communication with CD14+ monocytes. Despite these differences, monocytes occupied a prominent position within the predicted interaction networks in both conditions. Complementary protein-level analysis identified CXCL10 as a shared systemic inflammatory marker in active HHV-6B infection. CONCLUSIONS:HHV-6B infection exhibits distinct immune interaction networks depending on the clinical context, characterized by proliferating NK cell-monocyte interactions during primary infection and proliferating CD4+ T cell-monocyte interactions during viral reactivation.

Live-attenuated rotavirus (RV) vaccines are the most effective interventions for preventing RV gastroenteritis (RVGE) in young children. However, the molecular basis of attenuation remains not well understood. Here, we describe a compact but comprehensive strategy to identify RV virulence determinants by combining low-passage bidirectional selection, sequence analysis, and segment-level phenotype testing via a reverse genetics infectious system. Using the virulent murine RV strain EW, virulence was quantified by diarrhea severity/duration and body-weight gain. Serial passaging in cell culture selected an attenuated population, which regained virulence after passaging in suckling mice. Sequence comparison of the virulent and attenuated EW populations revealed only seven amino acid differences. We summarized literature describing attenuation/virulence-associated mutations in various RV group A (RVA) strains and found previous findings identical or similar to four of the seven mutations: NSP4-T45M, VP4-S470L, VP4-T612A, and VP7-T75P. Virulent- and attenuated-type EW variants of VP2, VP4, VP7, and NSP4 were introduced individually, or as NSP4/VP7 or VP4/VP7 pairs, into a simian SA11-L2 backbone using an 11-plasmid reverse genetics system. Phenotyping of rescued viruses consistently linked cell-culture-adapted VP4 to enhanced replication in vitro and reduced virulence in suckling mice. In vivo passaging strongly favored VP4 residue S470 over cell-culture-selected L470. More generally, our findings (i) underscore VP4 and VP7 as key determinants of EW virulence, (ii) provide a practical framework for identifying driver mutations underlying RVA attenuation, and (iii) highlight attenuation-associated substitutions shared across diverse RVAs.

Hemophagocytic lymphohistiocytosis (HLH) is a severe, rapidly progressive disease. While viral infection is considered a common etiology of pediatric HLH, specific causative viruses other than the Epstein-Barr virus (EBV) have been rarely identified. This study utilized metagenomic next-generation sequencing (NGS) to identify potential causative pathogens in plasma samples from 17 pediatric patients with suspected HLH. Additionally, one case each of confirmed EBV- and cytomegalovirus (CMV)-associated HLH was analyzed for methodological validation. Plasma cell-free RNA (cfRNA) profiling was performed using NGS data to assess the host transcriptome response. Significant viral reads of human herpesvirus-6B, human herpesvirus-7, and Hubei reo-like virus (HRLV) 14 were detected using metagenomic NGS in one patient each. Plasma cfRNA profiles from five patients with viral infection (including EBV and CMV) were compared to those of 14 patients without viral infection. By comparing the two patient groups, 1053 differentially expressed genes were identified. The gene ontology (GO) term of "adaptive immune response" (GO: 0002250) was significantly enriched among upregulated genes in the virus-positive group. Furthermore, an isolated cluster consisting specifically of mitochondrial RNAs, was identified in the upregulated genes of the virus-positive group. Using metagenomic NGS, several candidate viral pathogens were identified in patients with suspected infection-related HLH. The viral genome of HRLV 14, previously undetected in human clinical samples, was identified in one patient. The results from plasma cfRNA profiling suggest that mitochondrial RNAs may reflect the underlying pathogenesis of virus-associated HLH and have potential utility as disease biomarkers.

BACKGROUND:Endogenous human herpesvirus 6 (eHHV-6), in which the entire viral genome is integrated into human chromosomes, is present in approximately 1% of the population and has been associated with various clinical conditions, including neurological disorders. However, its biological significance remains unclear due to the lack of appropriate experimental models. METHODS:We established a patient-derived induced pluripotent stem cell (iPSC)-based tissue culture model using lymphoblastoid cell lines from individuals with eHHV-6B. iPSCs retaining the integrated viral genome were generated and subsequently differentiated into neural stem cells (NSCs). Viral gene expression was evaluated by RT-qPCR under basal conditions and following chemical stimulation. RESULTS:The integrated HHV-6B genome was transcriptionally silent in iPSCs but exhibited spontaneous low-level expression of the immediate-early gene U90 and the late gene U100 in NSCs. Chemical stimulation further enhanced U90 expression, whereas induction of U100 did not reach statistical significance. These findings indicate preferential activation of early viral transcriptional programs in neural lineage cells. CONCLUSIONS:Neural lineage cells provide a permissive environment for expression of eHHV-6B transcripts. This patient-derived iPSC-based model provides a platform for investigating the biological significance of neural lineage-specific eHHV-6 transcripts and may facilitate interpretation of HHV-6 DNA detection in patients with eHHV-6 by enabling studies of cell type-dependent viral transcription.

BACKGROUND:Rotavirus vaccination effectively prevents severe rotavirus gastroenteritis; however, administration during neonatal hospitalization is often avoided because of theoretical concerns regarding vaccine-virus transmission. Data on the safety of in-hospital rotavirus vaccination in neonatal step-down care settings remain limited. METHODS:We conducted a 1-year prospective cohort study in a Japanese growing care unit, a step-down neonatal unit comparable to Level II-III neonatal intensive care units in the United States. Hospitalized infants were monitored for adverse events and vaccine-strain shedding after administration of monovalent rotavirus vaccine (RV1). Stool samples were collected weekly and analyzed using RV1 strain-specific real-time quantitative reverse transcription polymerase chain reaction targeting the NSP2 gene. Routine contact precautions, including gown and glove use for all patient care activities and environmental cleaning, were consistently implemented. RESULTS:Among 237 infants included in the analysis, 15 received a total of 19 doses of RV1 during hospitalization. RV1 vaccine-strain RNA was detected in 26 of 38 postvaccination stool samples (68.4%). No RV1 strain RNA was detected in unvaccinated infants or in samples collected before vaccination. No serious adverse events were observed, and no evidence of horizontal transmission was identified. Six vaccinated infants exceeded the upper age limit for vaccine initiation at discharge and would have missed vaccination opportunities without in-hospital vaccination. CONCLUSIONS:RV1 vaccination was not associated with detectable transmission or serious adverse events in a neonatal step-down care setting under routine contact precautions, supporting its potential safety and role in preventing missed vaccination opportunities among high-risk infants.

Background. Exanthem subitum (ES), a benign febrile exanthematous disease, is caused by primary human betaherpesvirus 6B (HHV-6B) infection. It may cause neurological complications, including complex febrile seizures (cFS), acute encephalopathy with biphasic seizures, and late reduced diffusion (AESD). cFS resolves spontaneously; however, AESD can pose severe sequelae. We aimed to elucidate AESD pathogenesis using a proteomic analysis. Methods. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), serum and cerebrospinal fluid (CSF) protein profiles were compared between patients with AESD and those with cFS (n = 3 or 4 per group). Metascape was used for enrichment analysis, and the selected proteins were validated using a large sample via enzyme-linked immunosorbent assay (ELISA). Results. A total of 698 proteins were identified across all serum and CSF samples using LC-MS/MS. Nineteen serum proteins were differentially expressed in AESD and cFS during the acute phase. The glycolytic pathway was upregulated in AESD. Myristoylated alanine-rich C kinase substrate (MARCKS) and Golgi membrane protein 1 (GOLM1) were selected for validation using ELISA. Both proteins were upregulated during the acute phase (n = 11) compared with the convalescent phase (n = 21) in AESD (MARCKS, P = .016; GOLM1, P < .001). MARCKS during the acute phase was also upregulated in AESD compared with that in uncomplicated ES (n = 15) (P = .015). In CSF, 38 proteins were differentially expressed between AESD and cFS during the acute phase. Cholesteryl ester transfer protein in the CSF of patients with AESD was upregulated; however, this could not be validated using ELISA. Conclusions. Glycolysis and MARCKS pathways might be involved in HHV-6B-associated AESD pathogenesis.

Acute encephalopathy/encephalitis (AE) associated with SARS-CoV-2 has been increasingly reported since the emergence of the Omicron variant. Some patients developed acute fulminant cerebral edema (AFCE) or hemorrhagic shock encephalopathy syndrome (HSES) and had poor prognoses; however, the underlying pathogenesis of these conditions remains unclear. In this study, we performed single-cell RNA sequencing (scRNA-seq) on a patient with SARS-CoV-2-associated AE diagnosed with AFCE/HSES and compared the findings with those from patients with mild AE and febrile seizures caused by other pathogens. We also compared these cases with pediatric patients with COVID-19 without neurological complications. Four pediatric patients were enrolled: one with SARS-CoV-2-associated AFCE/HSES, two with mild AE of different etiologies (influenza A and Epstein–Barr virus), and one with febrile seizures associated with HHV-6. Peripheral blood mononuclear cell (PBMC) samples were collected from each patient during the acute and convalescent phases. Single-cell RNA libraries were prepared using the 10x Chromium platform. The sequences were analyzed using Cell Ranger and Seurat. Publicly available scRNA-seq PBMC data for pediatric patients with COVID-19 were used for comparison. A total of 109,940 cells were analyzed in this study. In the acute phase of SARS-CoV-2-associated AFCE/HSES, the B-cell fraction was markedly increased. Enrichment analysis of upregulated genes in B cells showed activation of pathways associated with protein stabilization (GO:0050821), with particularly significant upregulation of HSPA1A and HSPB1. The gene expression levels of these heat shock proteins were low during the convalescent phases of SARS-CoV-2-associated AFCE/HSES, mild AE, and COVID-19. ELISA measurements of HSPA1A and HSPB1 in plasma and serum of the three AE cases revealed a marked increase during the SARS-CoV-2-associated AFCE/HSES acute phase. Our findings suggest that HSPA1A and HSPB1 are potential markers of SARS-CoV-2-associated severe AE, although further studies are needed to validate their clinical utility. All Authors: No reported disclosures