河村 吉紀

Pediatric cases of the coronavirus disease 2019 (COVID-19) are generally mild or asymptomatic, and are usually detected by virological examination following close contact with COVID-19 patients, often the children's parents. The detailed clinical features and virological data of pediatric patients with COVID-19, particularly young infants, remain unclear. Here, the clinical and virological characteristics of four children with COVID-19 including two young infants were investigated. One- and 4-month-old boys with COVID-19 were both asymptomatic, and seroconversion was demonstrated. These findings suggest that even young infants can mount an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite having weaker immune defenses than adolescents and adults. Three-year-old boy, who was SARS-CoV-2-negative, was admitted to the same room as his SARS-CoV-2-positive father due to the lack of caregivers. Although he was asymptomatic, he had seroconverted to SARS-CoV-2. Eleven-year-old boy, who was sibling of the 3-year-old boy, was also SARS-CoV-2-negative. He was isolated in his own room and did not seroconvert. If young children are SARS-CoV-2 negative, they should be isolated from their SARS-CoV-2-positive parents. This may be difficult in practice, if parents with COVID-19 are the only available caregivers. In such situations, the most appropriate measures should be taken for each patient.

Varicella-zoster virus (VZV) reactivation from the enteric nervous system can cause ileus (Ogilvie's syndrome) in adult patients. Since no pediatric cases have been described, we sought to retrospectively analyze VZV reactivation in pediatric hematology-oncology patients to determine whether VZV infection including subclinical VZV reactivation can induce gastrointestinal complications such as Ogilvie's syndrome. Thirty-five patients who received chemotherapy at our institution between September 2013 and June 2018 were included. Serum samples were collected weekly during hospitalization and every 3 months during outpatient maintenance chemotherapy. A real-time polymerase chain reaction assay was used to measure VZV DNA load in serum. The clinical features of patients with VZV infection were retrospectively analyzed. Of 1165 serum samples, 7 (0.6%) were positive for VZV DNA. VZV DNA was detected in 3 of 35 patients. In patient A, VZV DNA was detected during two episodes. The first episode involved varicella-like eruptions caused by the Oka VZV vaccine strain. The second episode involved herpes zoster (HZ) caused by the same strain. Patients B and C had a clinical course that was typical for HZ caused by wild-type VZV. No gastrointestinal symptoms were observed at the time of VZV infection in these three patients. VZV DNA was not detected in any other samples. No pediatric cases with Ogilvie's syndrome caused by VZV reactivation were demonstrated in this cohort. Additionally, no subclinical VZV reactivation was found in this cohort. Further study is needed to elucidate the precise incidence of pediatric Ogilvie's syndrome caused by VZV reactivation.

BACKGROUND: It is well known that febrile seizures are commonly occur in children with exanthem subitum. In this study, we compared the clinical features and backgrounds of patients with complex febrile seizures with and without primary human herpesvirus 6B infection. METHODS: Sixty-two patients were enrolled after experiencing their first febrile seizure. Primary human herpesvirus 6B infection was confirmed when human herpesvirus 6B DNA was detected and human herpesvirus 6B antibody was negative in serum obtained during the acute phase of infection. Patient age, gender, and features of seizures were evaluated between patients with and without human herpesvirus 6B infection. RESULTS: Thirty patients with complex febrile seizure were diagnosed with primary human herpesvirus 6B infection. Those with primary human herpesvirus 6B infection (median, 13 months; range, seven to 39 months) were significantly younger than those without primary human herpesvirus 6B infection (median, 19 months; range, 10 to 59 months) (P = 0.001), and the proportion of males was significantly higher in patients without primary human herpesvirus 6B infection (male/female, 25/7) than in those with the infection (male/female, 14/16) (P = 0.017). An interval between fever onset and seizures of more than 24 hours was significantly more common in patients with primary human herpesvirus 6B infection (15 of the 30 patients) than in those without primary HHV-6B infection (two of 32 patients) (P < 0.001). CONCLUSIONS: A younger age at onset, a different gender ratio compared with febrile seizure due to other causes, and the length of interval between fever and seizures were features of complex febrile seizure associated human herpesvirus 6B infection. These findings may suggest a mechanism of complex febrile seizure onset different from that due to other causes.

Group A rotavirus (RVA) rarely causes severe complications such as encephalitis/encephalopathy. However, the pathophysiology of this specific complication remains unclear. Next-generation sequence analysis was used to compare the entire genome sequences of RVAs detected in patients with encephalitis/encephalopathy and gastroenteritis. This study enrolled eight patients with RVA encephalitis/encephalopathy and 10 with RVA gastroenteritis who were treated between February 2013 and July 2014. Viral RNAs were extracted from patients' stool, and whole-genome sequencing analysis was carried out to identify the specific gene mutations in RVA obtained from patients with severe neurological complications. Among the eight encephalitis/encephalopathy cases, six strains were DS-1-like G1P[8] and the remaining two were Wa-like G1P[8] (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Meanwhile, eight of the 10 viruses detected in rotavirus gastroenteritis patients were DS-1-like G1P[8], and the remaining two were Wa-like G1P[8]. These strains were further characterized by conducting phylogenetic analysis. No specific clustering was demonstrated in RVAs detected from encephalitis/encephalopathy patients. Although the DS-1-like G1P[8] strain was predominant in both groups, no specific molecular characteristics were detected in RVAs from patients with severe central nervous system complications.

During local small measles outbreak in Japan, 3 adolescents with febrile skin rash suspected as having measles were diagnosed with primary human herpesvirus (HHV)-7 infection. Primary HHV-7 infection can cause exanthem subitum in not only young children but also adolescents. HHV-7 should be considered as a possible causative agent for adolescent febrile skin rash during the measles outbreak.

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.

Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1-3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1-4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.

BACKGROUND: Human herpesvirus-6B (HHV-6B) infection after allogenic hematopoietic stem cell transplantation (allo-HSCT) is known to be associated with post-transplant limbic encephalitis in adults. Meanwhile, the association between HHV-6B infection and central nervous system complications remains unclear in pediatric allo-HSCT patients. METHODS: In this study, HHV-6B infection was monitored for more than 50 days after HSCT using virus isolation and real-time PCR. Clinical information such as patient background and encephalitis status was collected retrospectively from medical records. Risk factors for HHV-6B infection were determined by the Cox proportional hazards model, and the clinical features of HHV-6B encephalitis in pediatric allo-HSCT patients were elucidated. RESULTS: Human herpesvirus-6B infection was observed in 74 (33.8%) of 219 patients at 3-47 days (median 18, interquartile range 13-20). Risk factors identified in multivariable analysis were hematological malignancy (hazards ratio [HR], 5.0; 95% confidence interval [CI], 2.3/12.5; P < .0001), solid tumor (HR, 4.8; CI, 1.5/16.3; P = .0104), unrelated donor (HR, 2.1; CI, 1.0/4.6; P = .0378), and sex-mismatched donor (HR 1.8; CI, 1.1/3.0; P = .0257). HHV-6B encephalitis occurred in only one of the 219 patients (0.46%); this patient demonstrated the typical clinical course of posterior reversible encephalopathy syndrome. CONCLUSION: Hematological malignancy, solid tumor, unrelated donor, and sex-mismatched donor were significant risk factors for HHV-6B infection after pediatric allo-HSCT. In pediatric allo-HSCT patients, the incidence of HHV-6B encephalitis was low and the clinical features differed from those in adult patients.

Immunocompetent sisters with chromosomally integrated human herpesvirus 6A (HHV-6A) transiently excreted HHV-6B genome in their saliva. They did not have past histories of exanthema subitum but had antibodies against HHV-6A and HHV-6B. This suggests that endogenous HHV-6A may modify the clinical features of HHV-6B coinfection.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

OBJECTIVE: This cohort study, based on the design of a prior study in the United States, was conducted to elucidate the clinical features of primary human herpesvirus-6B (HHV-6B) infection. METHODS: Between June 2014 and May 2016, febrile children younger than 5 years who visited the emergency room (ER) and underwent blood examination were enrolled in this study. RESULTS: Fifty-nine (12%) of the 491 patients were diagnosed with primary HHV-6B infection. The rates of both simple and complex febrile seizure were significantly higher in patients with primary HHV-6B infection than in those without (P < 0.001 and P = 0.008, respectively). The median age at primary HHV-6B infection was 15 months. Forty-seven (79.7%) of the 59 patients with primary HHV-6B infection were younger than 2-year-old. Clinical features were compared between HHV-6B-infected patients older and younger than 2 years. The frequency of apparent infection (exanthema subitum) was significantly higher in the younger patients (P = 0.01). The median leukocyte (P = 0.01) and lymphocyte (P < 0.001) counts in the patients older than 2 years were significantly lower than those in the younger patients. CONCLUSIONS: Primary HHV-6B infection accounted for 12% of ER visits. Secondary febrile seizures, in particular the complex type, were considered to be a major contributor to the disease burden of primary HHV-6B infection. The timing of primary HHV-6B infection occurred at older ages than in past reports, and the frequency of inapparent infection was higher in older patients.

BACKGROUND: Since patients with breakthrough varicella (BV) have mild symptoms, clinical diagnosis is difficult. In high vaccine coverage area, as BV occurs sporadically, point of care test is required for controlling varicella outbreak. In this study, the reliability of varicella zoster virus (VZV)-loop mediated isothermal amplification (LAMP) was evaluated for the rapid diagnosis of BV. STUDY DESIGN: A total of 328 swab samples collected from patients with suspected varicella were analyzed. For the laboratory diagnosis of varicella, VZV real-time PCR was carried out using DNA extracted from swab samples. Swab samples without DNA extraction were used for VZV-LAMP(direct-LAMP). RESULTS: VZV infection was diagnosed by real-time PCR in 285 cases, including 105 natural varicella cases and 180 BV cases. VZV DNA was detected in 250 (87.8%) of the 285 cases by direct-LAMP. The presence and duration of fever, number of skin eruptions, and VZV DNA load were significantly lower in BV than natural varicella. The sensitivity of direct-LAMP for the diagnosis of varicella and BV was 93.3% and 84.4%, respectively. CONCLUSIONS: Direct LAMP was considered to be useful for rapid diagnosis of BV as it has several advantages such as low cost, ease and rapidity, as compared to real time PCR.

INTRODUCTION: Mutations of the ATP1A3 gene are associated with a wide spectrum of neurological disorders including rapid onset dystonia-parkinsonism and alternating hemiplegia of childhood (AHC). The genotype-phenotype correlations in these cases remain unclear however. We here report a pediatric case of catastrophic early life epilepsy, respiratory failure, postnatal microcephaly, and severe developmental disability associated with a novel heterozygous ATP1A3 mutation. SUBJECT: A boy with a normal birth to nonconsanguineous parents was transferred to the NICU due to postnatal respiratory failure at 2 days. He showed extreme hypotonia, episodic oculomotor abnormality and tachycardia, and frequent epileptic seizures. Mechanical ventilation was required but his epileptic seizures were intractable to multiple antiepileptic drugs, including extremely high doses of phenobarbital. METHODS AND RESULTS: Whole exome sequencing analysis of the case and his parents identified a de novo heterozygous mutation in the ATP1A3 gene (c.2736_2738CTTdel, p.Phe913del). DISCUSSION: The Phe913 residue in the ATP1α3 protein that is deleted in our case is highly conserved among vertebrates. Notably, an amino acid deletion in the same transmembrane domain of this protein, p.Val919del, has been reported previously in typical AHC cases, suggesting that p.Phe913del is a pathogenic mutation. Several reported cases with severe symptoms and very early onset epilepsy harbor ATP1α3 mutations at structural positions in this protein that differ from that of Phe913. Further functional studies are required to clarify the relationship between the loss of Phe913 and the very distinct resulting phenotype.

Differentiation between active and latent viral infection is critical for analysis of HHV-6-associated disease. HHV-6 infection has been associated with several clinical manifestations; however, the precise role of HHV-6 in pediatric LDLT remains unclear. This retrospective cohort study included 33 pediatric patients who received LDLT. All of the recipients were monitored for HHV-6 infection using viral isolation and real-time PCR. HHV-6 infection was observed in 14 of 33 (42.4%) recipients, and HHV-6B infection occurred within 2 weeks after LDLT in 10 of 14 (71.4%) recipients. HHV-6 was isolated from 10 of 33 (30.3%) recipients. Multivariate analysis showed that independent predictors of HHV-6B infection were age (OR 0.975; 95% CI 0.943-0.999; P = .041), PELD (OR 1.091; P = .038), and biliary atresia (OR 16.48; P = .035). The occurrence of unexplained fever was significantly higher in recipients with HHV-6B infection (11/14) compared with uninfected recipients (6/19) (P = .013). Additionally, ALT levels at 8 and 9 weeks after transplantation were significantly higher in the recipients with HHV-6B infection. Younger age, high MELD/PELD score, and biliary atresia as an underlying disease were identified as risk factors for viral infection.

The objectives of the work are to elucidate the incidence and virological findings of chromosomally integrated human herpesvirus 6 (ciHHV-6) in Japanese population and to analyze an association between ciHHV-6 and the clinical manifestation of exanthema subitum (ES). Real-time polymerase chain reaction was performed to determine HHV-6 DNA loads in 2347 cord blood samples from healthy neonates (cohort A), febrile children less than 5 years old (cohort B), and hematopoietic cell transplant recipients (cohort C). CiHHV-6 was confirmed by detection of high copy numbers of viral DNA in somatic cells. The integration site was determined by fluorescent in situ hybridization analysis. In the ciHHV-6 subjects of cohorts A and B, HHV-6 antibody titers were measured, the history of ES was obtained, and the incidence of ES was compared with non-ciHHV-6 children without primary HHV-6B infection in the cohort B. CiHHV-6 was detected in 14 (0.60%) of the 2347 samples: A (6/1006, 0.60%), B (6/790, 0.76%), and C (2/551, 0.36%). The integration sites were on chromosome 22q in seven cases, Yp in two cases, and 17q and Xp in one case. No past history of ES was observed in 11 of the 12 subjects. Nine children with ciHHV-6 underwent serological analysis and were found to be positive for HHV-6 IgG antibodies. Incidence of ES was statistically higher in the control subjects than the ciHHV-6 subjects (P = 0.0039). In Japan, the frequency of ciHHV-6 was 0.60%. A high incidence of ciHHV-6A, specifically in chromosome 22, is a characteristic finding among the Japanese. CiHHV-6 may interfere with the clinical symptoms of primary HHV-6B infection.

BACKGROUND: We sought to determine whether late-phase human herpesvirus 6B (HHV-6B) infection in hematopoietic stem cell transplant (HSCT) recipients was associated with serious outcomes and mortality. METHODS: The occurrence and course of HHV-6B infection was monitored for at least 60 days after transplant using virus isolation and real-time polymerase chain reaction. Risk factors for late-phase HHV-6B infection were examined, and the propensity score was calculated with significant risk factors. The inverse probability-weighted multivariable logistic regression analysis was performed to estimate odds ratios (ORs) and the 95% confidence intervals (95% CI) for mortality. RESULTS: Late-phase HHV-6B infection was observed in 12/89 (13.5%) of the HSCT recipients. Older age (OR: 10.3, 95% CI: 2.1/72.9, P = .0027), hematologic malignancy (OR: 10.3, 95% CI: 1.8/97.1, P = .0063), unrelated donor transplantation (OR: 5.3, 95% CI: 1.1/36.0, P = .0345), and sex-mismatched donor transplantation (OR: 6.3, 95% CI: 1.4/39.5, P = .0149) were identified as risk factors for late-phase HHV-6B infection. Fifteen subjects died (17%). Inverse probability-weighted multivariable logistic model analysis revealed that late-phase HHV-6B infection was an independent risk factor for mortality (OR: 4.2, 95% CI: 1.7/11.0, P = .0012). Among 5 of the fatal cases of late-phase HHV-6B infection, viral infection might be associated with severe clinical manifestations. CONCLUSION: Late-phase HHV-6B infection in HSCT recipients was associated with worse outcomes. The full spectrum of clinical features of the infection has not been fully elucidated, and therefore, recipients with high-risk factors for late-phase HHV-6B infection should be carefully monitored.

Despite the long-standing observation that herpes simplex virus (HSV) latency-associated transcript (LAT) promoter deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 isolates with targeted mutations in the LAT promoter and LAT-associated microRNAs (miRNAs) affecting (i) the LAT TATA box; (ii) the LAT ICP4-binding site; (iii) miRNA I (miR-I) and miR-II (miR-I/II), which both target ICP34.5; and (iv) miR-III, which targets ICP0. While the LAT TATA box mutant caused milder acute infections than wild-type (WT) virus, there was no difference in the recurrence phenotype between these viruses. LAT and miRNA expression during latency was not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause an in vivo phenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant were similar to those of its rescuant, although the acute infection was slightly reduced in severity relative to that caused by the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While they do not rule out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals.IMPORTANCE While it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype.

RotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

BACKGROUND: Rotavirus can, rarely, cause severe complications such as encephalopathy/encephalitis, myocarditis, sudden death, urinary stone, and gastrointestinal (GI) bleeding; and the incidence of these severe complications remains unclear. Additionally, it has not been determined whether rotavirus (RV) vaccine could reduce cases of severe complications or not. METHODS: A two-part questionnaire was designed to determine the number and clinical features of severe complications between 1 September 2008 and 31 August 2015, including the observation periods before and after RV vaccine introduction in Aichi Prefecture. RESULTS: Twenty-four cases of encephalitis/encephalopathy, eight cases of sudden death, three cases of urinary tract stone, and three cases of GI bleeding were reported during the 2008/2009 season and the 2012/2013 seasons. Although five cases of encephalitis/encephalopathy were reported, no other cases of severe complications were reported during the 2013/2014 and 2014/2015 seasons. No age difference was noted according to type of complication. Although onset of encephalitis/encephalopathy and of sudden death was around day 2 of illness, that of urinary tract stone and GI bleeding was slightly later (day 6 and day 4). In addition to the eight sudden deaths, fatal outcome was also noted in four cases (13.8%) of encephalitis/encephalopathy, and in one case of GI bleeding. CONCLUSION: According to the questionnaire survey in Aichi Prefecture, the incidence of the four severe RV-associated complications appears to have declined as the vaccination rate has increased.

Human herpesvirus 6 (HHV-6), a member of the betaherpesvirus family, has two distinct species: HHV-6A and HHV-6B. HHV-6B real-time reverse transcription polymerase chain reaction (RT-PCR) has been used to distinguish between active and latent viral infection. In this study, we developed a real-time RT-PCR assay to detect HHV-6A-specific transcripts and evaluated its reliability for analysis of clinical samples. To develop HHV-6A-specific real-time RT-PCR assays, three different classes of gene transcripts (immediate early: U90; early: U12; and late: U100) were selected as targets. Serial d ilutions of plasmid DNAs containing target sequences and RNAs extracted from HHV-6A-infected cells were used to determine assay specificity and sensitivity. Peripheral blood mononuclear cells (PBMCs) collected from patients with either primary or reactivated HHV-6B infection, and one patient with X-linked severe combined immunodeficiency (X-SCID) with HHV-6A reactivation, were used to evaluate assay reliability. The HHV-6A-specific real-time RT-PCR assays amplified plasmids containing the target sequences at concentrations between 10 and 1 × 106 copies per reaction. The intra-assay coefficients of variation were less than 5%. The three classes of HHV-6A gene transcripts were not detected in any HHV-6B sample isolated from the patients. In the X-SCID patient, high copy numbers of HHV-6A U12 and U100 transcripts were detected in PBMC samples during viremia. Thus, we successfully established highly sensitive and reproducible real-time RT-PCR methods targeting three classes of HHV-6A gene transcripts. This method should be useful for discriminating active HHV-6A infection from either latent infection or chromosomally integrated HHV-6A (ciHHV-6A).

Integration of the complete human herpesvirus 6 (HHV-6) genome into the telomere of a chromosome has been reported in some individuals (inherited chromosomally integrated HHV-6; iciHHV-6). Since the proportion of iciHHV-6-positive individuals with integration in chromosome 22 is high in Japan, we hypothesized a founder effect. In this study, we sought to elucidate the reason for the high proportion of viral integrations into chromosome 22. We analyzed six cases of iciHHV-6A and two cases of iciHHV-6B, including one iciHHV-6A case with a matched sample from a father and one iciHHV-6B case with a matched sample from a mother. In iciHHV-6A, the same copy numbers of viral telomeric repeat sequences (TRS) and the same five microsatellite markers were detected in both the index case and paternal sample. Moreover, the same five microsatellite markers were demonstrated in four cases and the same copy numbers of viral TRS were demonstrated in two pairs of two cases. The present microsatellite analysis suggested that the viral genomes detected in some iciHHV-6A patients were derived from a common ancestral integration.

Previous studies have demonstrated the transmission of rotavirus vaccine strains from vaccinated children to nonvaccinated siblings. We sought to fully elucidate the safety of rotavirus (RV) vaccination in closed contact circumstance, such as the foster home for future assessment of the vaccine safety in an neonatal intensive care unit. Stool samples were collected from 4 RV vaccinated (160 samples) and 23 unvaccinated (766 samples) infants. RV viral RNA loads were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). RV vaccine strain RNA was persistently detected in stool samples collected from the four vaccine recipients and one unvaccinated infant, but not in the stool samples collected from the 22 other unvaccinated infants. The unvaccinated infant who tested positive for the RV vaccine strain was vaccinated prior to enrollment in this study. The quantitative real-time RT-PCR data revealed a peak viral RNA load 1 week after vaccination followed by a gradual decrease. The current study suggests that RV vaccination may be safe in a close contact environment because there was limited transmission from RV vaccinated to unvaccinated infants. J. Med. Virol. 89:79-84, 2017. © 2016 Wiley Periodicals, Inc.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hematological disorder associated with severe systemic inflammation caused by an uncontrolled and ineffective immune response resulting in cytokine storm. Epstein-Barr virus (EBV) is the most common infectious agent in patients with the viral-associated HLH. Limited numbers of cases with cardiac complication have been demonstrated in other viral-associated HLH patients. Herein, we report a pediatric case of severe EBV-associated HLH with cardiac complications. CASE PRESENTATION: A previously healthy 4-year-old Japanese female was admitted to a local hospital with a four day history of fever. Despite antibiotic treatment, her fever persisted to day 7 of the illness. Finally, the diagnosis of HLH was confirmed by fulfilling diagnostic criteria for HLH and pathological analysis of bone marrow aspiration. Real-time PCR detected a high copy number of EBV DNA in the peripheral blood mononuclear cells (PBMCs) at the time of hospital admission. During treatment according to HLH-2004 protocol, sudden cardiopulmonary arrest (CPA) occurred on day 30 of the illness and immediate resuscitation was successful. Acute myocarditis was considered the cause of the CPA. Although the treatment regimen was completed on day 88 of the illness, a remarkably high copy number of EBV DNA was still detected in her PBMCs. Based on our flow cytometric in situ hybridization analysis that revealed EBV infection of only B lymphocytes, we decided to administer rituximab to control the abnormal EBV infection. Afterwards the amount of EBV DNA decreased gradually to undetectable level on day 130 of the illness. Unfortunately, a coronary artery aneurysm was discovered at the left main coronary artery on day 180 of the illness. Finally, the patient was discharged from the hospital on day 203 of the illness without sequelae except for a coronary aneurysm. CONCLUSIONS: In this case report, EBV-HLH was complicated with cardiac symptoms such as myocarditis and coronary artery aneurysm. Although remarkably high copy number of EBV DNA was detected in PBMCs after completion of the HLH-2004 protocol, rituximab treatment resulted in a dramatic decrease of EBV DNA to undetectable levels. Rituximab treatment might have been beneficial for the patient's survival.

Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 10(6) copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 10(6) copies/tube) in the multiplex PCR assay; however, 1 × 10(6) copies/tube of HHV-6A DNA did not affect the quantification of 1 × 10(4) copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R(2)  = 0.913) and HHV-6B (R(2)  = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 10(6) copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628-1635, 2016. © 2016 Wiley Periodicals, Inc.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the pathogenesis of gastrointestinal diseases, such as rotavirus gastroenteritis (GE). Kinetics of these biomarkers were examined in paired serum samples collected from bacterial enteritis patients with Campylobacter (n = 2) and Salmonella (n = 4) and viral GE patients with rotavirus (n = 27), norovirus (n = 25), and adenovirus (n = 11). At the time of hospital admission, all viral GE patients demonstrated increased MMP-9 and decreased MMP-2 and TIMP-2 serum levels. In contrast to viral GE patients, serum MMP-9 levels were not elevated at the time of hospital admission but elevated at the time of discharge; serum MMP-2 and TIMP-2 levels were decreased both at the time of admission and discharge in bacterial enteritis patients. Interestingly, the kinetics of serum MMP-2, MMP-9, and TIMP-2 levels were similar among the viral GE patients but distinct from bacterial enteritis patients. Thus, the involvement of MMPs and TIMPs in the pathophysiology of gastrointestinal symptoms likely varies depending on the etiological agent. Further studies are required to verify whether the extent of the bacterial enteritis or age of the patients influences these serum biomarkers. J. Med. Virol. 88:1341-1346, 2016. © 2016 Wiley Periodicals, Inc.

In 1974, Japanese scientists developed a live attenuated varicella vaccine based on the Oka strain. The efficacy of the vaccine for the prevention of varicella has been primarily demonstrated in studies conducted in the United States following the adoption of universal immunization using the Oka strain varicella vaccine in 1996. Although the vaccine was developed by Japanese scientists, until recently, the vaccine has been administered on a voluntary basis in Japan resulting in a vaccine coverage rate of approximately 40%. Therefore, Japan initiated universal immunization using the Oka strain varicella vaccine in November 2014. Given the transition from voluntary to universal immunization in Japan, it will also be important to monitor the epidemiology of varicella and herpes zoster. The efficacy and safety of co-administration of the varicella vaccine and measles, mumps, and rubella vaccine have been demonstrated in many countries; however, there was no data from Japan. In order to adopt the practice of universal immunization using the Oka strain varicella vaccine in Japan, data demonstrating the efficacy and safety of co-administration of varicella vaccine and measles and rubella (MR) vaccine were required. Additionally, we needed to elucidate the appropriate time interval between the first and second administrations of the vaccine. It is also important to differentiate between wild type and Oka vaccine type strains in herpes zoster patient with past history of varicella vaccine. Thus, there are many factors to consider regarding the adoption of universal immunization in Japan to control varicella zoster virus (VZV) infections.

Some healthy individuals carry human herpesvirus-6 (HHV-6) within a host chromosome, which is called inherited chromosomally integrated human herpesvirus-6 (iciHHV-6). Because iciHHV-6 is generally considered a non-pathogenic condition, it is important to distinguish iciHHV-6 from HHV-6 reactivation in immunocompromised hosts because both conditions manifest high copy numbers of the HHV-6 in peripheral blood mononuclear cells. Although fluorescent in situ hybridization (FISH) is a reliable method for the diagnosis of iciHHV-6, HHV-6-specific FISH probes are not commercially available. In our present study, we established a simple PCR-based method for producing FISH probes that can detect the chromosomal integration site of iciHHV-6 at high sensitivity. Using these probes, we confirmed that HHV-6 signals were consistently located at the telomeric region in all of the 13 iciHHV-6 individuals examined. Interestingly, in all seven Japanese iciHHV-6A patients, signals were detected exclusively on chromosome 22q. This method provides a simple and fast approach for iciHHV-6 diagnosis in the clinical laboratory.

Rotavirus gastroenteritis causes substantial morbidity and mortality worldwide in children. We report three infants with rotavirus gastroenteritis complicated by various severity of gastrointestinal bleeding. Two patients (cases 1 and 2) recovered completely without any specific treatments. One patient (case 3) died despite extensive treatments including a red blood cell transfusion and endoscopic hemostatic therapy. Rotavirus genotypes G1P[8] and G9P[8] were detected in cases 2 and 3, respectively. Rotavirus antigenemia levels were not high at the onset of melena, suggesting that systemic rotaviral infection does not play an important role in causing melena.

OBJECTIVE: To clarify the clinical and radiological spectrum of posterior reversible encephalopathy syndrome (PRES) in children, and to identify the prognostic factors. METHODS: The records of 40 children with PRES were reviewed. Acute clinical symptoms, MRI including apparent diffusion coefficient (ADC) maps in the acute and follow-up periods and neurological sequelae, including epilepsy, were noted. RESULTS: Age at onset ranged from 2 to 16 years. Underlying disorders were hematological or neoplastic disorders (n = 20), renal diseases (n = 14) and others (n = 6). In the acute period, 31 patients had seizures, 25 had altered consciousness, 11 had visual disturbances and 10 had headache. Of 29 patients who had ADC maps in the acute period, 13 had reduced diffusivity as shown by ADC within PRES lesions. Of 26 patients with follow-up MRI, 13 had focal gliosis or cortical atrophy. No patients had motor impairment, and four patients had focal epilepsy. No clinical variables were associated with focal gliosis or cortical atrophy on follow-up MRI, but lesional ADC reduction in the acute period was prognostic for focal gliosis or cortical atrophy on follow-up MRI (p = 0.005). CONCLUSIONS: To the best of our knowledge, this is the largest cohort study to date involving PRES in children. Acute symptoms in pediatric patients are similar to those reported in adults, but altered consciousness was more frequent in children. Lesional ADC reduction in the acute period was common and was a good predictor of later, irreversible MRI lesions.

BACKGROUND: Human herpesvirus 6B (HHV-6B) is the causative agent for exanthem subitum. HHV-6B was associated with mesial temporal sclerosis (MTS), leading to mesial temporal lobe epilepsy (MTLE). In this study, we sought to elucidate the pathogenic role of HHV-6B in patients with MTLE. METHODS: Seventy-five intractable MTLE patients, including 52 MTS patients and 23 non-MTS patients, were enrolled in this study. Resected hippocampus, amygdala, and mixed samples of amygdala and uncus samples were examined by real-time polymerase chain reaction (PCR) and reverse-transcriptase PCR to detect viral DNA and messenger RNA (mRNA), respectively. Host gene expressions, including neural markers, were measured using the TaqMan Gene Expression Assay. RESULTS: Detection of HHV-6 DNA was higher in MTS patients than non-MTS patients (median/interquartile range: 19.1/0-89.2 vs 0.0/0.0-0.0 copies/µg DNA; P = .004). HHV-6B viral DNA was determined in 12/27 HHV-6 DNA-positive samples, and no HHV-6B mRNA were detected in all samples. In MTS patients, expression of monocyte chemotactic protein-1 (P = .029) and glial fibrillary acidic protein (P = .043) were significantly higher in the amygdala samples with HHV-6 DNA than those without viral DNA. CONCLUSIONS: This study suggests that HHV-6B may play an important role in the pathogenesis of MTS via modification of host gene expression.

BACKGROUND: Although myelosuppression caused by human herpesvirus 6B (HHV-6B) reactivation in transplant recipients has been extensively investigated, the pathophysiological mechanisms of severe neutropenia in primary HHV-6B infection remain unclear. PROCEDURE: Fifty-four patients with primary HHV-6B infection were evaluated. Hematological examinations and blood sampling were conducted on days 1-4 (pre) and 5-10 (post) after the onset of illness. Severe neutropenia was defined as a neutrophil count less than 500 cells/μL. Patient characteristics, clinical data, and cytokines and chemokines levels were compared between the patients with (n = 16) and without (n = 38) severe neutropenia. RESULTS: Severe neutropenia was detected in samples that were collected between days 5 and 10 after illness. Significantly lower platelet counts (pre, P = 0.048; post, P = 0.032) and regulated on activation, normal T cell expressed and secreted levels (post, P = 0.007) were detected in the patients with neutropenia. Aspartate aminotransferase levels (P = 0.008), and interferon γ-inducible protein-10 (P < 0.0001), monocyte chemoattractant protein-1 (P = 0.005), and monokine induced by interferon γ (P = 0.011) levels were significantly higher in post samples collected from the patients with neutropenia. No differences were observed in any patient characteristics and serum cytokines levels. No bacterial infections were detected during the observation period. CONCLUSIONS: Chemokines may play an important role in the pathogenesis of severe neutropenia in patients with primary HHV-6B infection.

Quenching probe PCR (QP-PCR) analysis was used to determine the frequency of ganciclovir (GCV) resistance among clinical isolates of human herpesvirus 6B (HHV-6B) obtained from patients with primary viral infection and viral reactivation. Forty-two HHV-6B clinical isolates were repeatedly recovered from 15 hematopoietic stem cell transplant (HSCT) recipients, and 20 isolates were recovered from 20 exanthem subitum (ES) patients. Of the 15 HSCT recipients, 9 received GCV during the observation period; however, none of the ES patients were treated with GCV. Two established laboratory strains, Z29 and HST, were used as standards in this study. Regions 1 and 2 of the U69 gene of all of the clinical isolates demonstrated the same melting temperature as regions 1 and 2 of the Z29 strain. For region 3, the melting temperatures of all clinical isolates fell between the melting temperature of the plasmid containing the A462D single nucleotide polymorphism (SNP) and the melting temperature of the Z29 strain, and the melting temperatures profiles of all clinical isolates were similar to the melting temperature profile of the Japanese HST strain. As expected, none of the 20 clinical isolates recovered from the ES patients and the 14 isolates recovered from the HSCT recipients who did not receive GCV treatment carried the six known SNPs associated with GCV resistance. Interestingly, these six SNPs were not detected in the 28 clinical isolates recovered from the 9 HSCT recipients who received GCV. Additional sequence analysis of the U69 gene from the 15 representative isolates from the 15 HSCT recipients identified other SNPs. These SNPs were identical to those identified in the HST strain. Therefore, the rate of emergence of GCV-resistant HHV-6B strains appears to be relatively low, even in HSCT recipients treated with GCV.

The reliability of the HHV-6B LAMP using the dry-reagent method was evaluated using serum samples obtained from febrile children. The sensitivity of the original and dry-reagent methods was 10 copies/reaction and 100 copies/reaction, respectively. The dry-reagent LAMP method was highly sensitive (94.0%) and specific (96.0%) for the detection of HHV-6B.

In order to determine whether mixed infections of human herpesvirus 6B (HHV-6B) occur in immunocompetent and immunocompromised individuals, we examined the copy numbers of telomeric repeat sequences (TRS) of clinical isolates. In clinical isolates obtained from patients with exanthem subitum caused by primary HHV-6B infection, PCR products with HHV-6B TRS ranging between 400 and 800 bp were amplified. PCR products of various sizes were amplified in four clinical isolates from drug-induced hypersensitivity syndrome (DIHS) patients and 15 isolates from hematopoietic stem cell transplant (HSCT) recipients with HHV-6B reactivation. Based on the sequence analysis of the PCR products, the copy numbers of TRS in DIHS and HSCT patients were between 42 and 82 and 22 and >90, respectively. For two of the HSCT recipients, HHV-6B TRS PCR products of different sizes were detected in several isolates from each patient, which suggests mixed HHV-6B infections. In two of the posttransplant HHV-6B encephalitis patients, the sizes of the TRS nested PCR products amplified from the reactivated virus detected in the central nervous system differed from those of the virus detected in initial isolates from peripheral blood mononuclear cells. Taken together, these results suggest that PCR analysis of TRS copy number is a reliable tool for the discrimination of HHV-6B clinical isolates. Additionally, mixed HHV-6B infections occurred in HSCT recipients, and in some cases, compartmentalization of the HHV-6B strains to the central nervous system versus the blood compartment occurred in posttransplant HHV-6B encephalitis patients.

Rapid differentiation between wild-type varicella zoster virus (VZV) and Oka-vaccine (vOka) strains is important for monitoring side reactions of varicella vaccination. To develop a high-throughput molecular diagnostic method for the differentiation of wild-type VZV and vOka strains based on cycling probe technology. The primers were designed to amplify common sequences spanning a single nucleotide polymorphism (SNP) in gene 62 of VZV. DNA-RNA chimeric probes (cycling probes) were designed to detect the SNP at nucleotide 105705. The cycling probe real-time PCR assays for VZV wild-type and vOka strains specifically amplified plasmids containing target sequences that ranged between 10 and 1×10(6) copies per reaction. The inter- and intra-assay coefficients of variation were less than 5%. After initial validation studies, the clinical reliability of this method was evaluated using 38 swab samples that were collected from patients suspected of being zoster. Compared to the loop mediated isothermal amplification method, which is defined as the gold standard, cycling probe real-time PCR was highly sensitive and specific. The cycling probe real-time PCR technology is a reliable tool for differentiating between wild-type VZV and vOka strains in clinical samples.

Epstein-Barr virus (EBV) is the most common infectious cause of non-genetic hemophagocytic lymphohistiocytosis (HLH). To investigate EBV-infected lymphocytes and immune dysfunction in EBV-associated HLH, blood samples from a 6-year-old boy were longitudinally analyzed using molecular techniques. EBV-positive lymphocytes were detected as CD5(+) , CD8(+) , and/or HLA DR(+) lymphocytes on Day 25 of the disease, mostly disappearing thereafter. CD8(+) cells specific for lytic antigen BRLF1 were detected, but cells specific for latent antigens EBNA3 and LMP2 were not. EBV genes EBNA1, LMP1, LMP2, EBER1, BARTs were detected, suggesting a latency type II gene expression pattern in this case.

Severe pneumonia and leukocytosis are characteristic, frequently observed, clinical findings in pediatric patients with pandemic A/H1N1/2009 influenza virus infection. The aim of this study was to elucidate the role of cytokines and chemokines in complicating pneumonia and leukocytosis in patients with pandemic A/H1N1/2009 influenza virus infection. Forty-seven patients with pandemic A/H1N1/2009 influenza virus infection were enrolled in this study. Expression of interleukin (IL)-10 (P = 0.027) and IL-5 (P = 0.014) was significantly greater in patients with pneumonia than in those without pneumonia. Additionally, serum concentrations of interferon-γ (P = 0.009), tumor necrosis factor-α (P = 0.01), IL-4 (P = 0.024), and IL-2 (P = 0.012) were significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without neutrophilic leukocytosis. Of the five serum chemokine concentrations assessed, only IL-8 was significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without leukocytosis (P = 0.001). These cytokines and chemokines may play important roles in the pathogenesis of childhood pneumonia associated with A/H1N1/2009 influenza virus infection.

The monitoring of active human herpesvirus 6 (HHV-6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT-PCR methods were designed to detect the three kinetic classes of HHV-6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV-6B infection) and 15 hematopoietic stem cell transplant recipients with HHV-6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1-100 ng/reaction) and C(t) value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV-6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV-6B quantitative RT-PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT-PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV-6B infection.

The aims of this study were to elucidate the kinetics of Epstein-Barr virus (EBV) DNA load in serially collected peripheral blood mononuclear cells of patients with primary EBV infection, and to determine the correlated host factors. Blood samples were collected from 24 patients with primary EBV infection. EBV DNA copy numbers were measured using real-time polymerase chain reaction. Based on the kinetics of EBV DNA load, the 24 patients were divided into two groups: rapid regression and slow regression. Eighteen of the 24 patients (75%) were included in the slow regression and 6 (25%) in the rapid regression group. No statistically significant differences were observed between the two groups in clinical features and laboratory findings. However, acute phase (3 to 10 days after the onset of the illness) serum samples from six children in the slow regression and four in the rapid regression group revealed significantly higher serum interleukin (IL)-1β (P= 0.018), IL-12 (P= 0.009), tumor necrosis factor-α (P= 0.019), interferon-inducible protein 10, and monokine induced by interferon γ concentrations in the rapid regression than the slow regression group. On the other hand, sera from six children in the slow regression and four in the rapid regression group in the convalescent phase (14 to 21 days after the onset of the illness) showed no statistically significant differences between the two groups in these biomarker concentrations. Based on this, it was concluded that the kinetics of EBV DNA load can be divided to two different patterns after primary EBV infection, and immune response might be associated with viral clearance.