吉川 哲史

Background: Cytokines and chemokines induced by human herpesvirus 6 (HHV-6) infection may play an important role in the observed HHV-6-associated clinical complications. However, basic data for cytokine and chemokine synthesis in primary HHV-6 infected patient without complication is lacking. Objective: Aim of this study was to elucidate basic kinetic data for expressions of cytokines and chemokines in patients with primary HHV-6 infection without complication. Study design: Twenty-six patients suffering from fever were enrolled in this study. Fourteen biomarkers were measured in 74 serially collected sera samples from 26 patients. Additionally, serum samples obtained from 14 healthy children were used for control. Results: Twenty of the 26 patients were diagnosed with primary HHV-6 infection based on viral isolation and serological analysis. The mean age (P = 0.1289) and proportion of males to females (P = 0.9999) between the patients with and without primary HHV-6 infection were not statistically different. At the acute phase of the disease, three cytokines (IFN-gamma; P = 0.0046, IL-2; P = 0.0366, and IL-4; P = 0.0255) and one chemokine (MCP-1; P = 0.0019) were significantly higher in patients with primary HHV-6 infection compared to those without infection. Interleukin-5 levels during the convalescent period were significantly higher in patients with HHV-6 infection (P = 0.0205). By 1 month post-infection, cytokine and chemokine expression had returned to almost basal levels. Conclusion: As suggested by the previous in vitro studies, present in vivo analysis also suggests that HHV-6 has potency for induction of cytokines and chemokines. (C) 2010 Elsevier B.V. All rights reserved.

Two genetic diagnosis systems using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology were evaluated: one for detecting the HA gene of the pandemic influenza A/H1N1 2009 virus (H1pdm RT-LAMP) and the other for detecting the matrix gene of the influenza A virus (TypeA RT-LAMP). The competence of these two RT-LAMP assay kits for the diagnosis of the pandemic influenza A/H1N1 2009 virus was compared using real-time RT-PCR assays developed recently on viruses isolated and clinical specimens collected from patients with suspected infection. TypeA RT-LAMP and H1pdm RT-LAMP showed almost the same sensitivity as real-time RT-PCR for viruses isolated. The sensitivity and specificity of TypeA RT-LAMP and H1pdm AT-LAMP were 96.3% and 88.9%, respectively, for clinical specimens. Considering that the ability of the two RT-LAMP assay kits for detection of the pandemic influenza A/H1N1 2009 virus was comparable to that of the real-time RT-PCR assays, and that the assays were completed within 1 hr and did not require any expensive equipment, these two RT-LAMP assays are promising rapid diagnostic tests for the pandemic influenza A/H1N1 2009 virus at the hospital bedside. J. Med. Virol. 83:10-15, 2011. (C) 2010 Wiley-Liss, Inc.

Background: As congenital cytomegalovirus (CMV) infection causes significant clinical consequences not only at birth but also later as neurological sequelae, it is critical to establish a strategy for screening congenitally infected newborns. Previous studies have identified an insufficient sensitivity in screening methods based on the use of dried blood spots (DBSs). Objectives: To evaluate the feasibility of the authors' recently developed method for large-scale screening for congenital CMV infection and to identify risk factors for congenital infection. Methods: More than 21 000 newborns were enrolled at 25 sites in six geographically separate areas of Japan. Urine was collected onto filter cards placed in the diapers, which were then analysed by quantitative PCR using the filter disc directly as a template. Clinical and physical findings of the newborns were extracted from their medical records. CMV strains from the cases and their siblings were genetically compared. Viral loads in DBSs obtained from some of the cases were compared with those in the urine filters. Results: Congenital CMV infection was identified in 0.31% (95% CI 0.24% to 0.39%) of the newborns, and 30% of the cases (20/66) had typical clinical manifestations and/or showed abnormalities in brain images at birth. Although the positive predictive value of our screening was 94%, the lack of any comparison with a gold standard assay prevented calculation of the negative predictive value. Almost two-thirds of the cases had siblings, a significantly higher frequency than for uninfected newborns. Most of the cases (21/25) excreted CMV strains identical to those of their siblings. CMV DNA was undetectable in three out of 12 retrievable DBS specimens. Conclusions: Implementation of an effective large-scale screening programme for congenital CMV infection is feasible. Siblings are the major risk factor for congenital CMV infection, which emphasises the need for education of mothers-to-be as well as vaccine development.

In order to determine whether six other human herpesviruses, aside from herpes simplex virus, are associated with non-herpetic acute limbic encephalitis in immunocompetent individuals, real-time PCR was used to detect the DNA of herpesviruses in CSF collected from 61 patients with this form of encephalitis. Five of the human herpesviruses tested were not detected in any of the 61 CSF samples. EBV DNA was detected in one CSF sample. The EBV DNA-positive patient was a 36-year-old woman who presented with fever, headache, mild somnolence, and the typical neuroimaging findings.

Background: Human herpes virus 6 (HHV-6) is frequently reactivated and detected in plasma of patients in the early phase of hematopoietic stem cell transplantation (HSCT). HHV-6 reactivation has been implicated to be associated with significant morbidity, including encephalitis, bone marrow suppression or acute graft-versus-host disease (aGVHD). However the clinical manifestation and management of HHV-6 reactivation in patients undergoing HSCT are not subject to a clear consensus. We investigated the incidence of HHV-6 reactivation in relation to mortality and morbidity in children. Patients and methods: Between April 2005 and October 2009, 62 consecutive pediatric recipients of allogeneic HSCT were weekly monitored for HHV-6 loads measured by quantitative polymerase chain reaction. Hematological malignancies were diagnosed in 57 patients and non-malignant disorders in five patients. Twenty patients received related bone marrow, 24 patients unrelated bone marrow, 15 patients unrelated cord blood, and three patients related peripheral blood stem cell. Serological HLA disparities were 0 (n = 44), 1 (n = 15), and 2 (n = 3). HHV-6 reactivation was treated in the presence of clinical symptoms. Results: HHV-6 reactivation was observed in 36 of 62 patients (58%) by day 100 after allogeneic HSCT. HHV-6 loads more than 500 copies/mcg DNA were associated with higher nonrelapse mortality (P = .070 using a generalized Wilcoxon test). Twenty-two patients developed aGVHD and the incidence of HHV-6 reactivation was 14 of 22 (64%) and 22 of 40 (55%) with or without aGVHD. The incidence of aGVHD was 3 out of 10 (30%) and 11 out of 26 (42%), with or without administration of gancyclovir or cidofovir, respectively. Administration of antiviral agents was related to the reduced incidence of aGVHD using a multivariate logistic regression analysis (P = .086). Conclusion: HHV-6 reactivation is common after HSCT in children and is associated with signifi cant morbidity such as aGVHD. Prompt administration of antiviral agents suppresses symptoms caused by the reactivation of HHV-6 and thereby may reduce the development of aGVHD.

We tried to reveal the strain specificity of neutralizing mAbs against H3N2 influenza viruses in individuals. A large number of B lymphocytes of a pediatrician were collected by apheresis and two Ab libraries were constructed at 2004 and 2007 by using the phage-display technology. The libraries were screened against 12 different H3 strains Of flu isolated between 1968 and 2004. Large numbers of clones that bound to the Ags were isolated and mAbs that specifically bound to H3 strain viruses were selected. Their binding activity to the 12 strains and neutralizing activity were studied by ELISA and focus reduction test, respectively. Furthermore, the binding activity to hemagglutinin (HA) was examined by Western blot. The majority of clones showing the neutralizing activity turned out to be anti-HA mAbs and could be divided into three major groups showing distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003. (C) 2009 Elsevier Inc. All rights reserved.

We sought to clarify clinical features of exanthem subitum associated-encephalitis/encephalopathy, generally caused by primary human herpesvirus-6 infection in Japan. A two-part questionnaire was sent to hospitals between January 2003-December 2004. Of 3357 questionnaires, 2357 (70.2%) were returned, and 2293 (68.3 %) were eligible for analysis. Eighty-six cases of exanthem subitum-associated encephalitis/encephalopathy were reported. Seventy-seven (89.5%) of 86 patients were diagnosed with human herpesvirus-6 infection by virologic examination. Although 41 (50.6%) of 81 patients had no sequelae, 38 (46.9%) had neurologic sequelae. Moreover, two fatal cases (2.5%) were reported. Pleocytosis was evident in only 4 (7.5%) of 53 patients, and cerebrospinal fluid protein levels were within normal range (23.4 +/- 14.6 mg/dl, S.D.) in all patients. Human herpesvirus-6 DNA was detected in 21 (53.8%) of 39 patients. Abnormal computed tomography findings were a predictor of neurologic sequelae (P = 0.0097). As a consequence of this survey, we estimate that 61.9 cases of exanthem subitum-associated encephalitis occur every year. The disease prognosis was unexpectedly poor. (C) 2009 by Elsevier Inc. All rights reserved.

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used it combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV In a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region Seven representative clones front these 14 groups had a strong gB654 binding affinity by Surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity In vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate tumoral immunity to the selected pathogen. (C) 2009 Elsevier Masson SAS. All rights reserved.