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West Nile virus (WNV) is a pathogen to cause West Nile encephalitis when the infection occurs in the brain. Previous studies in mice identified the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene as a determining factor for resistance to WNV infection. In addition, it has been suggested that human OAS1 and OASL are associated with the resistance to the WNV infection. WNV is maintained in nature through a complex life cycle involving wildbirds and mosquitoes. Birds are not only susceptible to the WNV, but also act as reservoir hosts, thus participating in the spread of the disease. It has previously been reported that chicken OASL possesses the oligoadenylate synthetase activity. However, until now the antiviral activity of chicken OASL has not been determined. In this study, we investigated the putative antiviral activity of chicken OASL by ectopic expression of this enzyme in mammalian cells and then infecting these cells with WNV replicon. We demonstrate that chicken OASL has an antiviral activity against the WNV. This is the first report to show that chicken OASL is associated with the resistance to the WNV infection.

High viral load carriage of EBV is one of the risks for PTLD in transplant recipients. We reviewed retrospectively in pediatric renal transplant recipients with EBV seronegative. EBV loads in peripheral blood and EBV-CTLs were measured every 1-3 months in 13 patients after grafting. Immunosuppressants were reduced when the patients were considered to have persistent high EBV loads (>1000 copies/μgDNA for over six months). All showed primary EBV infection: six with asymptomatic persistent high EBV loads (group A) and seven with neither EBV-associated symptoms nor persistent high EBV loads (group B). No patient developed PTLD in either group. Chronic rejection occurred in one patient in group A after immunosuppressants' reduction. There was no difference in renal dysfunction rates between the two groups. The maximum and increase rates in EBV loads were significantly higher in group A. The CTLs' percentage was significantly lower in group A when EBV loads first rose above 100 copies/μg DNA. This study suggests the possibility that EBV loads and CTLs' monitoring may be useful for avoidance of PTLD, as patients with asymptomatic persistent high EBV loads had higher EBV loads and lower percentages of CTLs., (© 2011 John Wiley & Sons A/S.)

Background: The diagnosis of infectious mononucleosis (IM) is usually on serologic tests. The responses of anti-Epstein-Barr virus (anti-EBV) antibodies are weak in infants. The authors encountered some IM infants in whom anti-EBV antibodies were undetectable during early stage, although EBV genome was found in their blood. The aim of the present study was therefore to clarify the frequency of anti-EBV-antibody negative IM cases., Methods: The EBV serostatus of 104 IM children diagnosed on Sumaya criteria was retrospectively studied. The EBV genome in peripheral blood mononuclear cells was measured., Results: The anti-viral capsid antigen-IgM (anti-VCA-IgM)-positive rate in the acute phase was only 25% in infants but 80% in patients ≥ 4 years of age. Twenty percent of the infants were negative for all anti-EBV antibodies and required repeated serologic tests. For infants, the significant rise in anti-VCA-IgG was the most sensitive marker. Three seronegative infants with IM symptoms, with circulating EBV genome during acute phase, were eventually considered as having IM on anti-VCA-IgG seroconversion thereafter., Conclusions: To diagnose IM in infants the serologic test alone in the acute phase is not sensitive enough. It is proposed that the EBV genome be evaluated in peripheral blood mononuclear cells when infants presenting with IM symptoms are negative for anti-EBV antibodies during the acute phase., (© 2010 Japan Pediatric Society.)

While angiotensin II, which is produced by the renin-angiotensin-aldosterone system, is considered to be the major regulator molecule that controls both the blood pressure and fluid system, there is an increasing body of evidence that this bioactive peptide and its receptor might also contribute to the immune system. However, there are few details known about the direct effect that angiotensin type I receptors (AT1R) have on the cytotoxic T cell (CTL). To clarify the relationship between angiotensin II and its CTL receptor, we used murine splenic and antigen-specific CTLs. Murine CTLs constantly expressed AT1R, with the activation of the AT1R expression strengthened by both anti-CD3 Ab and the use of an antigen-specific methodology. Moreover, the production of IFN-gamma and TNF-alpha through CTL stimulation can be inhibited by the selective AT1R inhibitor, Losartan. In particular, the TNF-alpha production from activated CTL that had been magnified by angiotensin II, was nullified by the AT1R inhibitor. However, a cytotoxic assay indicated it did not have any effect on the cognate interaction of the CTLs. In addition, the antigen-specific CTL induction by immunization with the CTL antigenic peptide was reduced by angiotensin II type 1 receptor blocker (ARB) in vivo. These findings suggest that ARBs might have the ability to suppress excessive antigen-specific activation and induction of CTLs promoted by angiotensin II.

Osteonecrosis of the femoral head often occurs in patients under the age of 50 years. In this study, the authors evaluated the effectiveness of high-degree posterior rotation in terms of regaining the spherical contour of severely collapsed necrotic femoral head that was moved medially. They also investigated whether or not subchondral fracture disappeared on the medial femoral head on postoperative anteroposterior radiographs as a result of remodeling after this procedure.

Polymyositis is an uncommon manifestation as a complication of chronic graft-versus-host disease (GVHD). We report a case of a 55 years' old woman diagnosed as polymyositis 2 years after bone marrow transplantation against T-cell lymphoma. Muscle weakness and the elevation of CPK value were compatible with pathognomonic findings of polymyositis. However, the muscle weakness was distributed particularly into distal lower extremities and neck. It is different from that of the typical findings in autoimmune polymyositis. Histological findings showed atrophy and anisocytosis of muscles without invasion of mononuclear cells. This might be a case of GVHD-induced polymyositis occurring symptomatically after substantially progressing under the treatment with immunosuppressive agents to control chronic GVHD after bone marrow transplantation. The treatment with prednisone (1 mg/kg) brought the rapid improvement of muscle weakness and CPK value as well as mouth dryness and cholestatic liver dysfunction like in primary biliary cirrhosis. Moreover, dose up of cyclosporine and addition of mizolibine allowed for the use of lower dose of prednisone. This case suggested that the mononuclear cells invasion into muscles in a chronic GVHD patient could not always be a definitive finding of chronic GVHD-associated polymyositis because of prior use of immunosuppressive agents.

Retrobulbar granuloma is one of the serious complications in Wegener's granulomatosis and often shows resistance to conventional therapy during long-term treatment. The outcome of this complication includes visual loss, orbital and facial deformity, fistula formation, as well as infection. There has been increasing evidence that shows the efficacy of rituximab, a chimeric anti-B cell mAb, for the treatment of autoimmune diseases including Wegener's granulomatosis. We present a 22-year-old Japanese woman who was diagnosed with Wegener's granulomatosis complicated by refractory retrobulbar granuloma. She was admitted to our hospital with pain of the right eye and right proptosis during treatment with monthly IVCY for Wegener's granulomatosis. We diagnosed refractory retrobulbar granuloma by computed tomography (CT) scan and biopsy. She showed a refractory growth of retrobulbar granuloma in spite of negative ANCA. She was also complicated with pulmonary granulomatous lesions in bilateral apices. After approval by an institutional ethical committee and informed consent of this patient, rituximab 375 mg/m2 was intravenously administered weekly four times. Concomitant prednisolone 0.5 mg/kg was also administered for 2 weeks and gradually tapered. Treatment of rituximab resulted in prompt relief of symptoms in this case and the reduction of the granuloma. BVAS score also improved from 6 to 0 at 3 months and was kept in remission for 12 months. Circulating CD19-positive cells were kept less than 0.1% during the follow-up. There were no serious adverse events. This case suggests that rituximab is effective for refractory retrobulbar granuloma complicated in Wegener's granulomatosis even when ANCA titers are negative.

SARS-CoV has four major structural proteins: the N, S, M, and E proteins. To investigate the mechanism of SARS-CoV assembly, we cloned the genes encoding these four proteins into the eukaryotic expression vector pCAGGS and transfected them into 293T cells. When all four expression vectors were co-transfected VLP formed, as confirmed using electron microscopy. Using a rabbit polyclonal antibody specific to the N protein, N-protein-containing particles similar in size to the VLP were also observed by immunoelectron microscopy, indicating that the VLP contained the N protein. Co-immunoprecipitation analyses demonstrated an interaction between the N and M proteins, suggesting that N protein binds directly to M protein to be incorporated into VLP.

Background: Bronchial asthma-like symptoms such as wheezing are commonly associated with respiratory tract infection including respiratory syncytial virus (RSV) infection in infants. No study on the association of wheezing with cytomegalovirus (CMV) infection in infancy has been reported, although CMV infection has been observed to play some role in prolonged and intractable wheezing in limited cases., Methods: The present study investigated 40 hospitalized infants who presented with first-episode wheezing between October 2003 and September 2004. Nasopharyngeal aspirates were tested for RSV, and serum antibodies against CMV were measured. As controls, age-matched infants with no wheezing were examined for CMV serostatus., Results: RSV-antigen was detected in 21 subjects (53%), and seven (18%) were considered primary CMV infection serologically. Primary CMV infection was found more often in the wheezers than in the controls although the difference was not statistically significant (P = 0.06). The incidence of splenomegaly was significantly higher in wheezers with CMV infection (86%) than in those with RSV infection or without either infection. The duration of wheezing, fever, and radiographic and laboratory findings during hospitalization were not significantly different., Conclusions: CMV infection based on serologic diagnosis should be considered in infants with first wheezing episode and particularly those with splenomegaly.

Background: Epstein-Barr virus (EBV) infection can lead to life-threatening post-transplant lymphoproliferative disorder (PTLD). The aim of the present study was to establish EBV monitoring methods to prevent PTLD., Methods: EBV-DNA load was investigated, using real-time polymerase chain reaction (PCR) and anti-EBV antibody titers, in peripheral blood mononuclear cells of 21 renal transplant patients (seven recipients who were EBV-seronegative, R[-]; 14 who were EBV-seropositive, R[+]) before grafting. The mean age at entry and the mean follow-up period was 7.8 years of age (range, 3.3-12.0 years) and 1.8 years (range, 0.4-4.0 years), respectively, in the R(-) group, and 12.5 years of age (range, 3.9-17.7 years) and 3.8 years (range, 0.8-8.2 years) in the R(+) group, respectively., Results: The mean maximum load of the EBV genome was 1071 copies/microg DNA (range, 106-20700 copies/microg DNA) in the R(-) group, and 61 copies/microg DNA (range, <50-552 copies/microg DNA) in the R(+) group. During follow up no patient in the R(+) group had any noticeable symptoms that could be related to EBV, but three recipients in the R(-) group developed EBV-related symptoms including adenoid hypertrophy, cervical lymphadenopathy, and PTLD (B cell lymphoma), in one patient each. In the R(-) group the first leukocyte-associated viremia was detected at 30-180 days, and seroconversion at 43-266 days after transplantation., Conclusions: Viral DNA detection using PCR is a useful tool for EBV surveillance, but the maximum EBV load was not markedly elevated (2474 copies/microg DNA) in a patient with PTLD. Therefore, EBV surveillance using only monitoring of EBV load in peripheral leukocyte may be insufficient. Histology may therefore be necessary to accurately diagnose PTLD.

A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon.

Atrial fibrillation is an uncommon feature of hyperthyroidism in childhood. We report a 14-year-old male who was referred to our hospital with hyperthyroidism and atrial fibrillation. He had a family history of atrial fibrillation. Spontaneous conversion of atrial fibrillation to sinus rhythm occurred 20 weeks after achieving euthyroid state by an antithyroid agent and a beta-blocker. Atrial fibrillation reoccurred after reduction of antithyroid medication and persisted for 19 weeks. Successful electrical cardioversion was performed resulting in conversion of heart rhythm to sinus. Usually, hyperthyroidism associated atrial fibrillation spontaneously reverts to sinus rhythm several weeks after achieving a euthyroid state. Control of thyroid function and heart rate is the goal of therapy for this type of atrial fibrillation.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed for the diagnosis of Crimean-Congo hemorrhagic fever (CCHF) using a novel monoclonal antibody, 1B7, to the recombinant nucleoprotein (rNP) of CCHF virus (CCHFV) Chinese strain 8402. This ELISA detected at least 2 ng/100 microl of CCHFV rNP of 8402 and of the Nigeria strain Ibr 10200, and also detected authentic nucleoproteins (NP) of Chinese strains. Although the sensitivity of the ELISA was lower than that of nested reverse transcription polymerase chain reaction (RT-PCR), it was able to detect nucleoproteins in acute sera of CCHF patients. The presence of anti-CCHFV IgG decreased the sensitivity of the ELISA, possibly due to competition with 1B7, and this would tend to limit application of the ELISA. However, the method may be useful for the diagnosis of CCHF patients in the acute stage of illness, especially in laboratories not equipped with RT-PCR testing capabilities., ((c) 2005 Wiley-Liss, Inc.)

After the first documented outbreak of Marburg hemorrhagic fever identified in Europe in 1967, several sporadic cases and an outbreak of Marburg hemorrhagic fever have been reported in Africa. In order to establish a diagnostic system for Marburg hemorrhagic fever by the detection of Marburg virus nucleoprotein, monoclonal antibodies to the recombinant nucleoprotein were produced. Two clones of monoclonal antibodies, MAb2A7 and MAb2H6, were efficacious in the antigen-capture enzyme-linked immunosorbent assay (ELISA). At least 40 ng/ml of the recombinant nucleoprotein of Marburg virus was detected by the antigen-capture ELISA format. The epitope of the monoclonal antibody (MAb2A7) was located in the carboxy-terminus of nucleoprotein from amino acid position 634 to 647, while that of the MAb2H6 was located on the extreme region of the carboxy-terminus of the Marburg virus nucleoprotein (amino acid position 643-695). These monoclonal antibodies strongly interacted with the conformational epitopes on the carboxy-terminus of the nucleoprotein. Furthermore, these two monoclonal antibodies were reacted with the authentic Marburg virus antigens by indirect immunofluorescence assay. These data suggest that the Marburg virus nucleoprotein-capture ELISA system using the monoclonal antibodies is a promising technique for rapid diagnosis of Marburg hemorrhagic fever., (Copyright 2005 Wiley-Liss, Inc.)

An enzyme-linked immunosorbent assay (ELISA) using recombinant nucleoprotein (rNP) was reported for the detection of immunoglobulin G (IgG) antibodies to Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV). The immunoglobulin M (IgM)-capture ELISA was developed for the diagnosis of CCHFV infections, using CCHFV rNP as an antigen. These newly developed assays were applied to a study of a CCHF-outbreak and evaluated with sera collected from patients diagnosed as having CCHF by positive reverse transcription-polymerase chain reaction (RT-PCR) and by detection of IgG response. IgM antibodies to CCHFV were detected in 10 of the 13 patients. IgM antibodies to the rNP of CCHFV were detected by the CCHFV rNP-based IgM-capture ELISA in all 6 patients in whom IgG responses were demonstrated, while it was not detected in the 10 patients in whom IgG responses were not demonstrated. Furthermore, the IgM antibodies were detected in 6 of the 61 residents living a CCHF endemic area during the endemic season, while it was not detected in any of the 48 Japanese residents that had never visited the CCHF endemic area. It is concluded that this newly developed CCHFV rNP-based IgM-capture ELISA is a useful method for the diagnosis of CCHFV infections.

The case of a child with Crimean-Congo hemorrhagic fever (CCHF) presumably infected with CCHF virus from her 27-year-old mother is described. The mother with CCHF was treated with ribavirin and did not present with any symptoms of obvious hemorrhage. The child developed fever on the 5th day after the mother's onset. The partial virus genome was amplified by RT-PCR, and nested PCR from the child and the genome sequence were identical to that from the mother, indicating possible transmission of the virus from mother to child. This case indicates the importance of preventive measures for in-house outbreaks of CCHF.

We treated a male patient with Crimean-Congo hemorrhagic fever (CCHF). The diagnosis of CCHF was confirmed by reverse transcription-PCR and recombinant nucleoprotein (rNP)-based immunoglobulin G (IgG) and IgM capture enzyme-linked immunosorbent assays of serially collected serum samples. The patient was treated with intravenous ribavirin and recovered with no consequences. The study indicates that rNP-based CCHF virus antibody detection systems are useful for confirming CCHF virus infections. This case also suggests that intravenous ribavirin therapy may be promising for the treatment of CCHF patients.

Chronic active Epstein-Barr virus infection (CAEBV) is a high-mortality and high-morbidity disease. To clarify the prognostic factors, a national survey was performed in Japan, and data for 82 patients who met the criteria for CAEBV were analyzed. Of these 82 patients, 47 were alive and 35 had already died. Multivariate analysis revealed that thromobocytopenia and age at disease onset were correlated with mortality. The probability of 5-year survival was 0.45 for older patients (onset age, > or = 8 years), 0.94 for younger patients (P<.001), 0.38 for patients with thrombocytopenia (platelet count < 12 x 10(4) platelets/microL at diagnosis), and 0.76 for patients without thrombocytopenia (P=.01). Furthermore, patients with T cell infection by EBV had shorter survival times than patients with natural killer cell infection (probability of 5-year survival, 0.59 vs. 0.87; P<.009). Patients with CAEBV with late onset of disease, thrombocytopenia, and T cell infection had significantly poorer outcomes.

Small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9) conjugates SUMO-1 to target proteins and modulates cellular processes such as signal transduction, transcription regulation, and cell growth regulation. We demonstrated here that the nucleocapsid protein (NP) of Hantaan virus (HTNV) was associated with Ubc9 and SUMO-1 in vivo. Analysis of the interaction between the truncated NPs and Ubc9 revealed that the amino acid residues at the positions between 101 and 238 in the NP were responsible for the interaction. Furthermore, a consensus binding motif of Ubc9 and SUMO-1, MKAE, within this region, especially the second amino acid of the motif, K residue, was crucial for the interaction, and the interaction was essential for the NP to be localized in the perinuclear region. These results indicate that the assembly of the HTNV-NP is regulated by the interaction between the NP and Ubc9. This is the first report to demonstrate the interaction of Ubc9 with a structural protein of negative-strand RNA viruses.

We previously reported the identification of a novel gene, Bdm1/NDRG4, that was expressed predominantly in the postnatal rat brain and might possibly play a role in this process. We describe here the characterization of a NDRG4 protein in a developing and maturing rat brain. Antibody raised against glutathione S-transferase (GST)-NDRG4 fusion protein recognized four protein species of 38, 39, 41, and 45 kDa on Western blotting of proteins from differently staged rat brains. The 38-kDa form was revealed after birth, and the amount of this species peaked on postnatal day 15. The 39-kDa form became detectable after postnatal week 6. The 41-kDa form appeared late in embryogenesis, increased by postnatal day 15, and disappeared at postnatal week 6. The 45-kDa form was abundant during the late embryonic period and slightly decreased after birth. Subcellular fractionation of cerebra indicated that the NDRG4 protein was distributed mainly in the mitochondria and endoplasmic reticulum (ER). Detergent solubility assays and protease susceptibility demonstrated that in the ER NDRG4 protein is membrane-associated and luminally oriented. The 45-kDa isoform was induced during NGF-mediated neuronal differentiation of PC12 cells, but not by tunicamycin which causes ER stress. Differential expressions of NDRG4 protein isoforms may be a mechanism for modifying the NDRG4 function and for the formation of a functioning nervous system.

NDRG4, a member of the new NDRG gene family, was originally cloned as a gene that was expressed predominantly in the early postnatal rat brain. To determine whether the NDRG4 protein contributes to differentiation of neural cells, the effect of lowering the cellular NDRG4 protein level on the nerve growth factor (NGF)-induced neurite formations and transcription factor activations in PC12 cells was examined. An antisense construct of rat NDRG4 cDNA was made and transfected to PC12 cells, which constitutively express a basal level of the NDRG4 protein. Of the stably transfected antisense cell clones that expressed exogenous NDRG4 antisense RNA, six clones showed reduced levels of the NDRG4 protein, but unexpectedly two clones showed quite higher levels of NDRG4 protein than the control cells. The clones having decreased levels of the NDRG4 protein extended shorter neurites than control cells in response to NGF or dibutyryl cAMP. In contrast, the NDRG4 protein-highly expressing clones did not show suppressed neurite outgrowth induced by NGF. NGF-mediated activation of the transcription factor AP-1 was found to be suppressed in the NDRG4 protein-diminished clone and enhanced in the NDRG4 protein-upregulated clone as compared with those in the control cells. These results suggest that NDRG4 plays a role in neurite outgrowth and has an influence on an NGF-stimulated AP-1 activation by an undefined mechanism in PC12 cells.