v Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV).A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.
We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.Norwalk virus (NV) is a member of the family Caliciviridae, genus Norovirus, formerly called Norwalk-like virus and possessing a single-stranded RNA genome. NV has been a significant cause of nonbacterial infectious gastroenteritis and foodborne diseases all over the world. The virus is highly infectious and spreads through contaminated food as well as water in food-borne disease cases. In infectious gastroenteritis cases, this virus is spread from person to person within semiclosed communities such as schools, nursing homes, and hospitals. According to national food-borne disease statistics in Japan, the number of patients involved in incidents caused by NV is likely to be large. Thus, diagnosis of NV infection is extremely important for public health, since strategies for treatment of patients and control of diseases will be carried out effectively when the causative agent is diagnosed. NV has been diagnosed using electron microscopy (EM), reverse transcription-PCR (RT-PCR), and enzyme-linked immunosorbent assay (ELISA). These methods have worked efficiently in most cases. However, due to the great diversity of nucleotide sequences throughout the whole genome of NV and the capsid protein, neither ELISA nor RT-PCR detects all types of NV (3,4,5,9). In addition, the very limited numbers of particles shed in patient fecal material makes detection by EM difficult (2, 9). In cases of NV infections with homologous strains, genomic RNA detection by RT-PCR and antigen-antibody detection by ELISA can yield excellent results (1,3,4,7,8). Regardless of its variation, NV has been divided into two large genogroups, genogroup I (GI) and genogroup II (GII).We expressed a large amount of several strains of NV capsid protein in an Escherichia coli system and generated monoclonal antibodies (MAbs) against GI capsid protein (12) as well as G...
Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C 20 -prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR⅐RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.
Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin‐biotin sandwich ELISA (ABS‐ELISA) system for SEA, SEB, and SEC1 using newly‐developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78–25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantification and simplicity in detecting SEs.
Hexakis(trialkylsilyl)cyclotrisilanes 1a (trialkylsilyl = tert-butyldimethylsilyl) and 1b (trialkylsilyl = diisopropylmethylsilyl) were synthesized by the reductive coupling of the corresponding 2,2-dibromotrisilanes. Their molecular structures were confirmed by X-ray crystallography. In the absence of trapping reagents, photolysis of cyclotrisilanes 1a and 1b gave the corresponding stable tetrakis(trialkylsilyl)disilenes quantitatively. Irradiation of cyclotrisilanes 1a and 1b in (Z )-2-butene and (E )-2-butene afforded exclusively the corresponding (Z )-and (E )-2,3-dimethyl-1silacyclopropanes, respectively. Stereospecific formation of the cycloadducts indicates that the bis(trialkylsilyl)silylenes generated from cyclotrisilanes 1a and 1b would be singlets in the ground state.
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