三浦 浩樹

Human herpesviruses exhibit diverse pathogenic outcomes and the molecular reasons are not fully understood. Human herpesvirus 6B (HHV-6B) causes exanthema subitum and encephalitis, whereas the closely related HHV-6A is typically asymptomatic. Here, we show that cellular APOBEC3 enzymes restrict HHV-6A replication but not HHV-6B. HHV-6B expresses higher levels of the U28 protein, which binds multiple APOBEC3 proteins and promotes their relocalization and degradation. In contrast, HHV-6A fails to counteract APOBEC3 activity and accumulates extensive mutations in both cell- and patient-derived viral genomes. Individual APOBEC3 gene ablation enhances HHV-6A replication and reduces the viral mutation burden. Together, our studies suggest that differential susceptibility to APOBEC3 restriction may help to shape the evolvability and clinical manifestations of HHV-6A and HHV-6B.

ABSTRACT 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.

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.

Human herpesviruses (HHVs) cause central nervous system (CNS) infections; however, the role of neural autoantibodies remains unclear. We aimed to assess the presence of anti-N-methyl-D-aspartate receptor (anti-NMDAR) and anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in HHV-associated CNS infections. Seventeen adults with HHV DNA in the cerebrospinal fluid were tested using flow cytometry-based assays. None of the patients tested positive for anti-NMDAR antibodies. Anti-MOG antibodies were detected in two patients with VZV-associated CNS infection, one appearing after deterioration and the other at onset. Both patients recovered without sequelae. Anti-MOG antibodies may arise in VZV-associated CNS infections, warranting the consideration of autoimmune mechanisms.

Varicella-zoster virus (VZV) causes varicella in children, establishes lifelong latency and reactivates to cause herpes zoster later in life. Implementation of routine varicella vaccination in Japan since 2014 has reduced varicella cases, however, breakthrough varicella still occurs. This study aimed to clarify the current distribution of VZV clade among pediatric varicella patients and adults with VZV-associated central nervous system (CNS) infections in Japan. Skin swabs were collected from varicella patients (< 15 years) in Aichi Prefecture (September 2015-August 2017). Cerebrospinal fluid (CSF) samples were obtained from adult patients (> 15 years) with VZV-associated CNS infections (November 2014-June 2023). VZV DNA was detected by PCR, and its clade was determined by sequencing open reading frame (ORF) 22 and ORF37 regions. Wild-type and Oka vaccine strains were distinguished by loop-mediated isothermal amplification (LAMP) method. Of 124 pediatric swab samples and 62 adult CSF samples 94.4% belonged to clade 2 and 4.8% clade 1. No clade 1 samples were detected in CSF samples. No vaccine strain was detected. Clinical characteristics did not differ significantly among clades. Clade 2 VZV predominates in both pediatric varicella and adult VZV-related CNS infections in Japan with sporadic clade 1 varicella cases.

Background: Recent studies have reported the possible link between adeno-associated virus 2 (AAV2) and severe pediatric acute hepatitis. It has been suggested that aberrant AAV2 replication initiated by coinfection with “helper viruses” such as human adenovirus and human herpesvirus-6B (HHV-6B) may induce abnormal immune responses. Encephalitis/encephalopathy is a severe complication of primary HHV-6B infection, but the underlying mechanisms remain unclear. This study analyzed the association between AAV2 coinfection and neurologic complications of primary HHV-6B infection in children. Methods: Preserved serum samples obtained from 36 patients with HHV-6B-associated encephalitis/encephalopathy, 39 with febrile seizure, and 83 without neurologic complications were retrospectively analyzed. Primary HHV-6B infection was confirmed if HHV-6B DNA was detected and the HHV-6B antibody was negative in serum. AAV2 and HHV-6 DNA loads were quantitated using real-time PCR. Results: AAV2 was detected in 4 (11%) and 3 (8%) patients in the encephalitis/encephalopathy and febrile seizure groups, respectively. In contrast, AAV2 was undetectable in 83 patients without neurologic complications. AAV2 detection frequency was significantly higher in the encephalitis/encephalopathy and febrile seizure groups compared with the no neurologic complications group (P = 0.01 and P = 0.03, respectively). Among 4 patients with HHV-6B-associated encephalitis/encephalopathy, AAV2 DNA was detected in the cerebrospinal fluid of 2 patients. Serum HHV-6B DNA load was not significantly different among patients who were AAV2 positive or AAV negative and with or without neurologic complications. Conclusions: These findings suggest that coinfection of AAV2 and HHV-6B is associated with neurologic complications such as encephalitis/encephalopathy and febrile seizure in children.

OBJECTIVE: To elucidate the trends and clinical features of virologically diagnosed breakthrough varicella (BV) 9 years after implementation of the universal vaccination program in Japan. PATIENTS AND METHODS: Study participants were patients with suspected varicella less than 15 years of age who visited 1 of 15 pediatric clinics in the Nagoya VZV Study Group between September 2015 and August 2023. Practitioners collected patient samples and information such as background characteristics, clinical symptoms, and immunization status. All patients had varicella confirmed by real-time polymerase chain reaction assays. RESULTS: Of 719 patients with suspected varicella, 512 had laboratory-diagnosed varicella and available information on vaccination status. They were divided into 3 groups: 167 with natural varicella, 250 with BV and 1 dose of vaccine, and 95 with BV and 2 doses. The monthly number of patients with varicella decreased gradually during the observation period. Typical seasonal peaks were observed until the 2019-2020 season. The proportion of patients with BV, particularly BV after 2 doses of vaccine, gradually increased. Patients with BV and 2 doses had a significantly lower median age (5 years) than those with 1 dose (6 years) (p < 0.001). The transmission route for BV was unknown in approximately 30-50 % of patients. Duration of fever was significantly longer (p = 0.0138) and the number of skin eruptions was also significantly higher (p = 0.0013) in the 1-dose group than in the 2-dose group. CONCLUSIONS: Although the number of pediatric patients with varicella declined after implementation of national immunization with 2 doses of varicella vaccine, the proportion of patients with BV, especially those who received 2 doses, gradually increased. Clinical symptoms were significantly milder in patients with BV and 2 doses. Laboratory diagnosis of varicella is becoming increasingly important due to an increase in the proportion of patients with BV who have mild symptoms.

ABSTRACT The recent clinical features of Epstein‐Barr virus (EBV) and cytomegalovirus (CMV) infections in young children in developed countries remain unclear. This study investigated the clinical features of EBV and CMV infections and the latest seroepidemiology in Japan. Seroprevalence was analyzed 303 stored serum samples using commercial Enzyme Immunosorbent Assay kits, and viral infections were investigated in a cohort of febrile children under 5 years of age. After maternal antibody levels declined, the seroprevalences of EBV and CMV gradually increased by adolescence to 42.9% and 57.1%, respectively. Among 2,732 febrile children, serum EBV and CMV DNAs were detected in 1.76% and 1.24%, respectively. Of 25 primary EBV–infected patients, 15 (60.0%) had infectious mononucleosis (IM) with significantly higher IM frequency, WBC, atypical lymphocyte ratios, AST, ALT, LDH, and EBV DNA load compared to EBV–reactivated patients. No CMV DNA–positive patients had IM. Among primary EBV–infected patients, those with IM were older and had more atypical lymphocytes and higher EBV DNA load than those without IM. The age of primary EBV infection appears to have decreased compared to reports from Western countries in the 1990s. Even among children under 5 years of age, 60.0% of those with primary EBV infection developed IM.

This report describes the case of a previously healthy nine-year-old girl who developed a left temporal lobe brain abscess following spontaneous exfoliation of a healthy deciduous tooth. She presented in convulsive status epilepticus without fever or focal neurological symptoms, and the initial MRI revealed a ring-enhancing lesion with adjacent dural inflammation. Surgical drainage and intravenous antibiotic therapy led to a full recovery with no neurological sequelae after six weeks of treatment. Pus culture revealed Streptococcus intermedius and Aggregatibacter aphrophilus, both of which are part of the normal oral flora. The route of infection in this case is suggested to be hematogenous spread from the site of tooth exfoliation, as evidenced by the ipsilateral location of the abscess. This highlights the potential for serious intracranial infections to originate from the exfoliation of a deciduous tooth in healthy children, particularly those with orthodontic appliances.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

To elucidate the seroprevalence and rate of asymptomatic infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Japanese children, serological analysis was performed using serum samples collected from March 2020 to February 2023. A total of 1493 serum samples were collected during the first study period (March 2020 to February 2021). None of the serum samples was positive for SARS-CoV-2 antibody. In the second period (March 2021 to February 2022), seven of the 1055 patients (0.7%) experienced SARS-CoV-2 infection. The third period (March 2022 to February 2023) was divided into three terms: from March to June 30, 2022; from July to October 2022; and from November 2022 to February 2023. The seroprevalence gradually increased throughout this period, with rates of 6.0%, 18.6%, and 30.4% in the three terms, respectively. Pediatric cases of asymptomatic SARS-CoV-2 infection occurred after the surge of Omicron variants. Since none of the SARS-CoV-2 antibody-positive patients had a previous history of coronavirus disease 2019, the seroprevalence rate in this study may represent the rate of asymptomatic infection.

We encountered a previously healthy 3-year-old girl with interstitial pneumonitis that initially developed due to human adenovirus type 2 infection and exacerbated by primary human herpesvirus 7 infection. A comprehensive serum biomarker analysis showed patterns that differed by viral infection, suggesting that respiratory and lymphotropic viral infections might have different pathophysiology in interstitial pneumonitis.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and greater risks of other complications, including malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol(SH)-linked alkylation for the metabolic (SLAM) sequencing and Cleavage Under Target & Release Using Nuclease analysis (CUT&RUN), we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNA polymerase II with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-κB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitors, OTX015 and MZ1, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals.

OBJECTIVES: Intestinal rotavirus (RV) vaccine replication and host immune response are suggested to be affected by several factors, including maternal antibodies, breastfeeding history, and gut microbiome, which are thought to be similar in pairs of twins. The aim of this study was to determine whether viral shedding from the fecal RV vaccine strain Rotarix® (RV1) and IgG and IgA responses to RV show similarity in pairs of twins. METHODS: Quantitative reverse transcription polymerase chain reaction specific to RV vaccine strain RV1 was used to monitor fecal RV1 viral shedding. RV IgG and IgA titers were measured using an in-house enzyme-linked immunosorbent assay. Fecal RV1 viral shedding and immune responses were compared between twins and singletons with mixed effects and fixed effects models. RESULTS: A total of 347 stool and 54 blood samples were collected from four pairs of twins and twelve singletons during the observation period. Although the kinetics of fecal RV1 viral shedding and immune responses differed among vaccinated individuals, they appeared to be similar within twin pairs. RV shedding after the first dose (P=0.049) and RV IgG titers during the entire observation period (P=0.015) had a significantly better fit in the fixed effect model that assumed that twins have the same response versus the model that assumed that twins have a different response. CONCLUSIONS: The similarity of RV vaccine viral replication in intestine and host immune responses in twin pairs was demonstrated using statistical analysis.

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and possesses greater risks of having other complications, which include malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol-Linked Alkylation for the Metabolic Sequencing and Cleavage Under Target & Release Using Nuclease analysis, we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNAPII with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-κB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitor, OTX015, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals.

Abstract Nonpharmaceutical interventions (NPIs) to control COVID‐19 have decreased the incidence of many pediatric infectious diseases. The epidemiology of β‐ and γ‐herpesvirus infections might have been affected by NPIs. The aim of this study was to elucidate changes in trends in β‐ and γ‐herpesvirus infections and complex febrile seizures (cFS) of viral etiology before and during the COVID‐19 pandemic. Between April 2017 and March 2021, febrile children aged ≤5 years were enrolled. Detection of EBV, CMV, HHV‐6B, and HHV‐7 DNA in serum was performed using real‐time PCR. The epidemiology of viral infections and cFS were compared between the prepandemic and pandemic periods. During the observation period, 1432 serum samples were collected. The mean number of febrile children decreased during the pandemic period, but the number of patients with HHV‐6B infection increased from 35 (9.3% of all febrile children) per year before the pandemic to 43 (15.5%) during the pandemic. The change in the proportion of patients with primary HHV‐6B infection was 6.50% (95% confidence interval [CI], 2.05%–11.3%; p = 0.0047). The mean number of patients with cFS decreased during the pandemic period, but the number of patients with HHV‐6B–associated cFS was stable throughout the observation period. Therefore, the change in proportion of patients with cFS caused by primary HHV‐6B infection was 49.5% (95% CI, 12.2%–60.5%; p = 0.0048). The disease burden of primary HHV‐6B infection among patients in the emergency room remained unchanged, with a significant increase in the relative proportion after the COVID‐19 pandemic began.

Central nervous system (CNS) involvement in anaplastic large cell lymphoma (ALCL) at diagnosis is rare and leads to poor prognosis with the use of the standard ALCL99 protocol alone. CNS-directed intensive chemotherapy, such as an increased dose of intravenous MTX, increased dose of dexamethasone, intensified intrathecal therapy, and high-dose cytarabine, followed by cranial irradiation, has been shown to improve survival in this population. In this paper, the authors describe a 14-year-old male with an intracranial ALCL mass at onset who received CNS-directed chemotherapy followed by 23.4 Gy of whole-brain irradiation. After the first systemic relapse, the CNS-penetrating ALK inhibitor, alectinib, was applied; it has successfully maintained remission for 18 months without any adverse events. CNS-penetrating ALK inhibitor therapy might prevent CNS relapse in pediatric ALK-positive ALCL. Next-generation ALK inhibitors could be introduced as a promising treatment option, even for primary ALCL with CNS involvement, which could lead to the omission of cranial irradiation and avoid radiation-induced sequalae. Further evidence of CNS-penetrating ALK inhibitor combined therapy for primary ALK-positive ALCL is warranted to reduce radiation-induced sequalae in future treatments.