Development of an antigen ELISA to detect sapovirus in clinical stool specimens.

Human sapovirus (SaV) strains are etiological agents of mild and/or acute gastroenteritis in children and adults. In this study, we describe the development of a novel antigen enzyme-linked immunosorbent assay (ELISA) detection system that was based on hyperimmune rabbit and guinea pig antisera raised against SaV genogroup I (GI) virus-like particles. The ELISA had 100% specificity, and sensitivities of 60% and 25% when compared to single-round PCR and nested PCR, respectively. Our results have shown the ELISA was useful in detecting SaV GI antigens in clinical stool specimens collected two days after the onset of illness.

Deletion analysis of the sapovirus VP1 gene for the assembly of virus-like particles.

Human sapovirus (SaV) strains are agents of gastroenteritis. They cannot be grown in cell culture. In this study, constructs containing SaV N- and C-terminal-deleted recombinant capsid proteins (rVP1) were expressed in a baculovirus expression system to allow us to better understand the sequence requirements for the formation of virus-like particles (VLPs). Only proteins derived from N-terminal-deleted rVP1 constructs that began 49 nucleotides downstream assembled into VLPs, which included both small and native-size VLPs. Our results were similar to those reported in a rabbit hemorrhagic disease virus (RHDV) N- and C-terminal-deleted rVP1 expression study but were distinct from those reported in a norovirus N- and C-terminal-deleted rVP1 expression study, suggesting that SaV and RHDV may have similar expression requirements.

Characterization of polyclonal antibodies raised against sapovirus genogroup five virus-like particles.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains were recently divided into five genogroups (GI to GV). We characterized novel polyclonal antibodies raised against SaV GV virus-like particles (VLPs) by Western blot analysis, and both antibody and antigen enzyme-linked immunosorbent assays (ELISAs). Our results have indicated SaV GI and GV VLPs were antigenically distinct by Western blotting and ELISAs. These reagents may be useful for genogroup specific detection of SaV.

Cross-reactivity among sapovirus recombinant capsid proteins.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains are divided into five genogroups (GI-GV) based on their capsid (VP1) sequences. Human SaV strains are noncultivable, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV. In this study, rVP1 constructs of SaV GI, GII, and GV strains were expressed in a baculovirus expression system. The structures of the GI, GII, and GV VLPs, with diameters of 41-48 nm, were morphologically similar to those of native SaV. However a fraction of GV VLPs were smaller, with diameters of 26-31 nm and spikes on the outline. This is the first report of GII and GV VLP formation and the first identification of small VLPs. To examine the cross-reactivities among GI, GII, and GV rVP1, hyperimmune rabbit antisera were raised against Escherichia coli-expressed GI, GII, and GV N- and C-terminal VP1. Western blotting showed the GI antisera cross-reacted with GV rVP1 but not GII rVP1; GII antisera cross-reacted weakly with GI rVP1 but did not cross-react with GV rVP1; and GV antisera reacted only with GV rVP1. Also, hyperimmune rabbit and guinea pig antisera raised against purified GI VLPs were used to examine the cross-reactivities among GI, GII, and GV VLPs by an antigen enzyme-linked immunosorbent assay (ELISA). The ELISA showed that the GI VLPs were antigenically distinct from GII and GV VLPs.

Detection of norovirus and sapovirus infection among children with gastroenteritis in Ho Chi Minh City, Vietnam.

This report describes norovirus (NoV) and sapovirus (SaV) infections in hospitalized children with acute sporadic gastroenteritis in Ho Chi Minh City, Vietnam. Stool specimens collected between December 1999 and November 2000 were examined for NoV and SaV using reverse transcription-PCR and phylogenetic analysis. NoVs were detected in 72 of 448 rotavirus-negative specimens, counted as part of an overall annual detection rate of 5.4% (72 of 1,339 children). This included four NoV genogroup I (GI) strains and 68 NoV GII strains. Only one SaV GI strain was detected in the rotavirus-negative specimens. Over 73% of the NoV sequences belonged to GII/4 (Lordsdale cluster) and were detected in all months except March. We also detected GII/3 strains (Saitama U201 cluster), a naturally occurring recombinant NoV, between January 2000 and March 2000 but not after this period. Other NoV strains belonging to GI/4, GI/8, GII/1, and GII/7 were also detected but were infrequent. In addition, two almost identical NoV GII strains (strains 026 and 0703) collected six months apart were classified into a new genotype that includes the Mc37 strain, which was previously shown to be a recombinant NoV. During this one-year study, the NoV prevailed at the end of the rainy season and the beginning of the dry season. Further epidemiological studies may be necessary to determine whether the GII/4 strains continue to dominant in this region.

Inhibition of attachment of virions of Norwalk virus to mammalian cells by soluble histone molecules.

Viral infection is usually initiated by the binding of virus particles to specific receptor molecule(s) on the host cell surface. Blocking of this step prevents the following step, penetration into the cell. In the present study, we investigated the virus-cell interactions of virions of Norwalk virus (NV), a major etiological agent for viral diarrhea. We found that histone was an extremely strong NV-binding protein. Histone H1, a heterologous histone molecule, appeared to be able to interact not only with NV particles, but also with the cell surface. Histone H1 appeared capable of effectively preventing the attachment of NV to intestinal cells, but not of other viruses. No cytotoxic effects of histone were observed under the assay conditions. These results indicate that nonsecretory histone molecules may inhibit the attachment of viruses to cells like lactoferrins. Our results suggest that by searching virus-binding molecules, we might find antiviral agents, even for new viruses.

Loci on murine chromosomes 7 and 13 that modify the phenotype of the NOA mouse, an animal model of atopic dermatitis.

The NOA (Naruto Research Institute Otsuka Atrichia) mouse is an animal model of allergic or atopic dermatitis, a condition characterized by ulcerative skin lesions with accumulation of mast cells and increased serum IgE. We reported earlier that a major gene responsible for dermatitis in the NOA mouse lay in the middle of chromosome 14, and that the incidence of disease clearly differed according to parental strain; the mode of inheritance was autosomal recessive with incomplete penetrance. In the study reported here, we searched for genes that might modify the NOA phenotype, and we identified two candidate loci that appeared to contain genes capable of modifying atopic or allergic dermatitis, one in the middle of chromosome 7 (chi2 = 14.66; P = 0.00013 for D7Mit62) and the other in the telomeric region of chromosome 13 (chi2 = 15.352; P = 0.000089 for D13Mit147). These loci correspond to regions of synteny in human chromosomes where linkages to asthma, atopy, or related phenotypes, such as serum IgE levels, have been documented.

Interaction of recombinant norwalk virus particles with the 105-kilodalton cellular binding protein, a candidate receptor molecule for virus attachment.

Norwalk virus (NV), responsible for outbreaks of acute gastroenteritis, comprises the species of the genus Norwalk-like viruses in the family Caliciviridae. Although the study of the molecular biology of NV has been hampered by a lack of culture systems or small experimental animal models, virus-like particles (VLPs) generated with recombinant baculoviruses harboring the capsid protein gene of NV provide a useful tool for investigating NV-cell interactions. In this study, the attachment of the recombinant VLPs derived from the Ueno virus (UEV), a strain belonging to the genogroup II NVs, to mammalian and insect cells was examined. Kinetic analyses of the binding of the recombinant VLPs of the UEV (rUEVs) to Caco-2 cells demonstrated that the binding was specific and occurred in a dose-dependent manner. Approximately 7.5% of the prebound rUEVs were internalized into the Caco-2 cells. Enzymatic and chemical modification of Caco-2 cell surface molecules suggested that the binding was directly mediated by a protein-protein interaction. A virus overlay protein-binding assay (VOPBA) indicated that rUEVs appeared to bind to a 105-kDa molecule, designated as the NV attachment (NORVA) protein. Furthermore, the assay indicated that its native conformational structure was indispensable for the binding activity. In Caco-2 cells, the NORVA protein was detected when VOPBA was carried out with the VLPs from Seto and Funabashi viruses, which are serologically different NVs from UEV, used as probes. The binding of rUEVs to NORVA protein was also observed in six mammalian cell lines other than Caco-2. These data suggest that the attachment of NV to mammalian cells is mediated by NORVA protein, which is ubiquitously expressed in the mammalian cells. The present study is the first report on the role of the cellular molecule in the binding of recombinant VLPs of NV.

Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen.

The second open reading frame (ORF2) gene of the Chitta virus (CHV) was cloned to construct a recombinant baculovirus. The CHV ORF2 is predicted to encode a capsid protein of 535 amino acids (aa). CHV showed a high aa identity in the capsid region with genogroup II Norwalk virus (NV) (65-85%), but a low aa identity with genogroup I NV (44-46%). Phylogenetic analysis of the ORF2 gene demonstrated that CHV is genetically closely related to the Hawaii virus included in genogroup II NV. The recombinant capsid protein of CHV (rCHV) self-assembled to form empty virus-like particles (VLPs) when expressed in insect cells with the recombinant baculovirus. An enzyme-linked immunosorbent assay (ELISA) based on antisera to rCHV was developed to detect CHV antigen in stools. The antigen ELISA appeared to be highly specific to both rCHV and CHV-like strains. In addition, combined use of antigen ELISAs using antibodies against two antigenically distinct recombinant VLPs, the recombinant Chiba virus (rCV) and recombinant Seto virus (rSEV), enabled us to determine the genetic as well as antigenic relationship among these three viruses.

Serotype-specific antigen ELISA for detection of Chiba virus in stools.

Chiba virus (CV), a Norwalk-like virus (NLV), was first identified as a cause of oyster-associated outbreak of gastroenteritis that occurred in Chiba prefecture, Japan, in 1987. An enzyme-linked immunosorbent assay (ELISA), based on hyperimmune antisera to recombinant baculovirus-expressed capsid proteins of CV (rCV), was developed to detect CV antigen in stools. No cross-reactions were observed with other enteric viruses including enteroviruses, rotaviruses, astroviruses, or enteric adenoviruses. The ELISA was used to screen 101 stools collected from 16 oyster-associated outbreaks of acute gastroenteritis. Twelve stools (11.9%) from seven outbreaks were positive for CV antigen. Ten rCV ELISA-positive strains were confirmed by RT-PCR and nucleotide sequencing. ELISA-positive strains showed 96-100% nucleotide sequence identity to each other, though they were obtained nine years apart. Phylogenetic analysis demonstrated that all ten strains clustered with the prototype CV in genogroup I viruses. We concluded that the antigen ELISA described in this study is highly type-specific, and that this method should be useful for epidemiological surveys of Chiba virus infections.

Molecular cloning, expression, and antigenicity of Seto virus belonging to genogroup I Norwalk-like viruses.

The viral capsid protein of the Seto virus (SeV), a Japanese strain of genogroup I Norwalk-like viruses (NLVs), was expressed as virus-like particles using a baculovirus expression system. An antigen detection enzyme-linked immunosorbent assay based on hyperimmune antisera to recombinant SeV was highly specific to homologous SeV-like strains but not heterologous strains in stools, allowing us type-specific detection of NLVs.

Mapping of a gene responsible for dermatitis in NOA (Naruto Research Institute Otsuka Atrichia) mice, an animal model of allergic dermatitis.

The NOA (Naruto Research Institute Otsuka Atrichia) mouse is an animal model of allergic or atopic dermatitis, a condition characterized by ulcerative skin lesions with accumulation of mast cells and increased serum IgE. These features of the murine disease closely resemble human atopy and atopic disorders. We performed linkage analysis in NOA back-cross progeny, as a step toward identifying and isolating a gene responsible for the NOA phenotype. We crossed NOA mice with five other murine strains (C57BL/6J, IQI, C3H/HeJ, DBA/2J, and BALB/cByJ) and then bred back-cross animals. Using microsatellite markers, we scanned the entire genomes of 559 N2 offspring from the five parental strains. Linkage analysis revealed a significant association between ulcerative skin lesions and markers on murine chromosome 14. Statistical analysis indicated that the critical region was assigned to the vicinity of D14Mit236 and D14Mit160.

Significantly elevated expression of PF4 (platelet factor 4) and eotaxin in the NOA mouse, a model for atopic dermatitis.

The NOA (Naruto Research Institute Otsuka Atrichia) mouse, an animal model of allergic or atopic dermatitis, exhibits ulcerative skin lesions associated with accumulation of mast cells and eosinophils, a significantly increased level of serum IgE, and scratching behavior. To investigate genetic contributors to the pathological process of dermatitis in this murine model, we looked for genes that were expressed differently in spleens of NOA mice compared with controls, by means of a differential display method. We cloned and characterized one gene that revealed a significantly higher expression in the NOA mouse than in control strains. Its cDNA consisted of 570 nucleotides, including 315 nucleotides of open reading frame encoding 105 amino acids. The deduced amino acid sequence identified this gene as the murine homologue of rat and human platelet factor (PF) 4s (89% identity and 64% identity in 105 amino acids, respectively). PF4 is a heparin-binding protein that is released from alpha-granules of activated platelets and belongs to the family of chemokine molecules that contain a CXC motif. Our results suggested that increased expression of PF4 may play an important role in the etiology of allergic dermatitis.

[Sudden death during Holter electrocardiographic monitoring in a patient with hypertrophic cardiomyopathy].

A 56-year-old man visited a doctor because of arrhythmia and a diagnosis of hypertrophic cardiomyopathy was made in 1990. On January 9th, 1997, when aged 63 years old, he visited another doctor because of paroxysmal atrial fibrillation tachycardia. Since his clinical symptoms did not ameliorate, he visited our hospital on April 14th, and Holter electrocardiographic monitoring was started on April 16th. While walking on the street, his heart rate increased up to 107 beat/min when premature ventricular contraction with a long coupling interval of 400 msec occurred, followed by polymorphous ventricular tachycardia, ventricular fibrillation and death. He had no risk factors for sudden death such as early onset of symptoms, history of syncope, family history of sudden death, documented ventricular tachycardia, and congestive heart failure. Although many cases of hypertrophic cardiomyopathy end in sudden death, no electrocardiographic recordings at the time of sudden death have been available. This is the first case of electrocardiographic recording at the time of sudden death in a patient with hypertrophic cardiomyopathy.

Nipradilol inhibits DNA synthesis by regulating nitric oxide synthesis in cultured rat mesangial cells.

To elucidate whether nipradilol modulates mesangial cell function, we assessed the effects of nipradilol on DNA synthesis in the presence and absence of N-nitro-L-arginine methyl ester (L-NAME) in cultured rat mesangial cells stimulated with 1% fetal bovine serum. Nipradilol inhibited [3H]thymidine incorporation into mesangial cells in a dose- and time-dependent manner. In addition, the anti-mitogenic effect of 100 microM nipradilol was significantly inhibited in the presence of 10 microM L-NAME. Moreover, nipradilol increased intracellular cyclic guanosine monophosphate (cGMP). These results suggest that nipradilol exerts its efficacy in the treatment of several types of glomerulonephritis with mesangial cell proliferation by increasing in intracellular cGMP.

Anti-mitogenic effects of sarpogrelate in cultured rat mesangial cells.

We investigated the effects of the new 5-HT2A receptor antagonist, sarpogrelate, on DNA synthesis in renal mesangial cells stimulated with 5-HT in the presence and absence of platelet-derived growth factor (PDGF)-BB. Both 5-HT and PDGF-BB demonstrated a mitogenic effect on these cells. When mesangial cells were incubated in the absence of PDGF-BB, sarpogrelate inhibited DNA synthesis in these cells in a dose-dependent manner. In the presence of PDGF-BB, sarpogrelate had a weaker anti-mitogenic effect in mesangial cells stimulated with 5-HT. Sarpogrelate was cytotoxic at concentrations over 10(-5) M according to the results of LDH release assays, and it reduced the S1 phase in mesangial cells stimulated with 5-HT by a flow cytometry. These findings suggest that sarpogrelate may be effective in the treatment of some glomerulonephritis associated with mesangial cell proliferation.

[Effects of recombinant human erythropoietin (rHuEpo) on the interactions between glomerular endothelial cells and mesangial cells: regulation of cell proliferation via endothelin (ET)-1].

To elucidate the effects of recombinant human erythropoietin (rHuEpo) on the interactions between glomerular endothelial cells (GENs) and mesangial cells (GMCs), we investigated whether or not cultured bovine GENs alter endothelin (ET)-1 secretion from bovine MCs and MC proliferate under basal or rHuEpo-stimulated conditions. Incubation for 24 hours with synthetic ET-1 stimulated MC in a dose-dependent manner. In addition, 100 pg/ml of ET-1 significantly stimulated MC proliferation after more than 12 hours incubation. Moreover, rHuEpo stimulated ET-1 secretion from GENs in a dose-dependent manner and showed less stimulation of ET-1 secretion from GMCs than GENs. DNA synthesis in both GENs and GMCs was significantly stimulated with more than 5 U/ml of rHuEpo. ET-1 secretion from GENs co-cultured with GMCs was higher than that from cultured GENs only. Conditioned medium, obtained from co-culture of GENs and GMCs, stimulated the proliferation of GMCs that were significantly inhibited with 10(-6) M approximately 10(-5) M BQ-123, a ETA receptor antagonist. These results suggest that rHuEpo directly stimulates the proliferation of GENs and GMCs, and this stimulatory effect is in part due to ET-1 secreted from these cells, especially GENs.

Crystallization and preliminary X-ray diffraction studies of an endoglucanase from Aspergillus aculeatus.

Most fungal cellulases are found in multiple forms varying in size and substrate specificity. Aspergillus aculeatus is known to produce nine cellulolytic enzymes including an endoglucanase (FI CM-cellulase, M(r) = 24,002) as the major component. Single crystals of FI CM-cellulase from Aspergillus aculeatus have been prepared by sitting-drop vapour diffusion using ammonium sulphate as a precipitant. The cellulase crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 52.79(2) A, b = 106.40(4) A and c = 33.15(1) A. The crystals contain one enzyme molecule per asymmetric unit. They diffract to at least 2.0 A resolution and are very stable against X-ray irradiation.