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Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles--now considered "quasi-enveloped particles"--are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/ PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses. IMPORTANCE Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles present in feces and bile are nonenveloped, while those in circulating blood and culture supernatants are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help to elucidate the entry mechanisms and receptors for HEV infection in the future. This is the first report to characterize the detailed morphological features of membrane- associated HEV particles. [ABSTRACT FROM AUTHOR]

Abstract Micro/Nano imprinting or hot embossing is currently a target of interest for industrial production of micro and Nano devices for the low cost aspect. In Fluidic MEMS (Micro Electromechanical Systems) applications, polymer materials have been widely employed for their low cost to fabricate the economical products (Becker and Heim in Sens Acuators A 83:120?135,2000; Becker and Gaertner in Mol Biotechnol 82:89?99,2001). However glasses are much more suitable for the higher temperature applications or under the stronger chemical environments. Moreover UV absorption of glass materials is much less than that of polymers, which is the advantage for bio-analysis. In Optical MEMS as well, glasses are good candidate materials for the better optical properties, such as high refractive index, low UV absorption and others. Although wet etching of glasses is widely employed for fabrication of fluidic MEMS devices, the wet etching is not satisfactory for the low machining resolution, the isotropic etched profile and poor roughness of the fabricated structures. Dry etching of glasses is then an alternative for Micro/Nano structuring, but the etching rate is extremely low (order of 0.1??m/min) and the cost is too high because of the expensive RIE (Reactive Ion Etching) facility. Above mentioned is the reason why we are interested in hot embossing or imprinting of glasses of Micro/Nano scale. In our previous study, Micro/Nano imprinting was developed for Pyrex glasses using GC (Glassy Carbon) mold prepared by FIB machining (Takahashi et al. in Symposium on DTIP 2004 pp 441?446,2004). The disadvantage of FIB machining is limited area of etching. The typical area of FIB is less than several hundreds micrometer square. This is the reason why we tried the large area of embossing using GC mold fabricated by dicing machine. Micro hot embossed test structures were successfully demonstrated with good fidelity. Fabricated micro structures can be applied for fabrication of microchamber array for PCR (Akagi et al. in Sci Techol Adv Mater 5:343?349,2004; Nagai et al. in Anal Chem 73:1043?1047,2001). [ABSTRACT FROM AUTHOR]

Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/△ORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/△ORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE: The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for in vitro assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEVinfected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. [ABSTRACT FROM AUTHOR]

Abstract  In our previous works we had shown that the use of ultrasonic vibration in micro hot embossing processes proved to be effective in improving the molding accuracy. We then decided to extend this technology of ultrasonic vibration to nanoimprint lithography also, and investigate its effect on nanoimprints experimentally. This task also required the use of a heater capable of sustaining three kinds of stresses, namely loading force, thermal stress, and ultrasonic vibration in the molding process. This work led to the development of an ultrasonic nanoimprint system with a built-in pyrolytic graphite/pyrolytic boron nitride all-in-one heater. The material chosen for nanoimprinting was polycarbonate with its glass transition temperature being 150°C. The research in the area showed that the timing point at which an impression of ultrasonic vibration begins is an important factor. When an ultrasonic vibration was impressed at an early stage in a molding process the height of the imprinted pattern seemed to increase where high amplitudes of the acoustical vibration were involved. Moreover, when the molding accuracies of line/space pattern with line widths of 500, 750 nm, and 1 μm were compared among themselves, the effect of assistance from ultrasonic vibration became quite noticeable in the case of small lines patterns; this was the case even where the amplitudes of the ultrasonic vibration were small. As for the application of ultrasonic vibration on nanoimprinting is concerned, it was found to greatly improve the molding accuracy of the process. [ABSTRACT FROM AUTHOR]

The surface quality of a mold is one of the major factors in imprint lithography as it determines the final surface quality and the minimum size of the replicated patterns. This paper describes a process for O2-based reactive ion etching (RIE) of glassy carbon (GC) without encountering any micro masking effect. Glassy carbon, because of its attractive properties such as its surface inertness, thermal stability and extraordinary hardness, has drawn much interest as a mold material for high-temperature imprinting on glasses and metals. Etch profiles with highly smooth surfaces free of micro masking effects were achieved by adding SF6 to the etching gas. The fraction of SF6 in the gas mixture (ranging from 0.1 to 0.6) showed little change in the quality of the etched surface, but it did lead to a proportional decrease in the etch rate of GC. A reasonable GC etch rate (115-120 nm min[?]1) and a smooth etch surface were obtained using SF6 at a fraction 0.2 or below. Using electron beam lithography (EBL), and processing under the established SF6/O2 RIE conditions, GC molds were fabricated and successfully applied to thermal imprinting onto glass and metals. [ABSTRACT FROM AUTHOR]

Abstract  We succeeded in making a Si stencil mask for deep X-ray lithography (DXL) by using MEMS fabrication technologies. In order to make this mask, a 200 μm thick Si wafer was etched through its entire thickness where the remaining silicon served as the absorber for the mask. The minimum line width on the mask was 20 μm. DXL experiments were executed by using this Si stencil mask on the beamline BL2 at the NewSUBARU SR facility of the Laboratory of Advanced Science and Technology for Industry, University of Hyogo. In our experiment we succeeded in the X-ray exposure of PMMA sheets through their entire thicknesses of 0.5 and 1 mm. This means a success in line/space patterning with 20 μm line width that can lead to the fabrication of PMMA structure of maximum aspect ratio of 50. Moreover, the sticking was prevented by substituting water with hydrofluoroether employed for after-develop wash operation This Si stencil mask enabled a transcript of more precise pattern using the beamline BL2 at the NewSUBARU SR facility as compared with results from a stainless stencil mask and an Au/polyimide mask. [ABSTRACT FROM AUTHOR]

We demonstrated fabricating a needle array of polycarbonate (PC) and polymethyl methacrylate (PMMA) by using a 3-D LIGA (lithografie, galvanoformung, abformung) process. The diameter of the bottom of the needle was about 50 m, and the height was 135 m. Although the LIGA process is commonly applied for making structures with vertical sidewalls, the use of an x-ray grayscale mask in the LIGA process has made it possible to fabricate needle-shaped structures. The x-ray grayscale mask was composed of a Si x-ray absorber and an SU-8 membrane. The sidewall of the x-ray absorber was diagonally processed by Si tapered-trench-etching technology such that the transmission intensity of x rays could be changed locally. The x-ray lithography experiment was executed by using this x-ray grayscale mask on a beamline BL-4 in the TERAS synchrotron radiation facility at National Institute of Advanced Industrial Science and Technology (AIST). By using this facility, a PMMA resist master with three-dimensional (3-D) structures was made. A Pt layer was then sputter-deposited as a seed layer on the PMMA resist master, and a Ni mold was fabricated by electroforming technology. In addition, a needle array of PC and PMMA was produced by hot embossing technology. Self-assembled monolayers (SAMs) of a release agent were required on the surface of the mold pattern to achieve a complete molding. Thus, we succeeded in extending the LIGA process to three dimensions by the use of an x-ray grayscale mask. [ABSTRACT FROM AUTHOR]

We propose a new fabrication method of an x-ray grayscale mask using micro-electro-mechanical-systems (MEMS) technologies, and also report on successful fabrication of three-dimensional (3D) microstructures on a polymethylmethacrylate (PMMA)sheet by using only a single x-ray exposure. We showed that silicon can be diagonally etched by optimizing the etching condition in a reactive-ion-etching (RIE) process. It is well known that the absorbers of an x-ray mask can be made into 3-D shapes. Here, we describe how this process can be extended to fabricate an x-ray grayscale mask by using a tapered-trench-etching technique. With such a mask, we carried out experiments on x-ray lithography (XRL) using a beam line BL-4 in the synchrotron radiation facility TERAS of National Institute of Advanced Industrial Science and Technology (AIST). The dose energy used for the exposure was 150 mA⋅h, and the subsequent resist development was done by a GG developer at room temperature for 16 h. The sidewalls in the upper part of the PMMA resist structure were inclined and rounded. In particular, the shape of the PMMA resist structure of the lines with 20-μm width (also referred as 20-μm lines) could be processed to achieve a halberd-like shape. Thus, the effectiveness of the grayscale mask in adjusting to the varying thicknesses of absorber was confirmed by XRL experiments. Moreover, we showed that the final shape of PMMA resist structures after XRL was predictable by calculations. [ABSTRACT FROM AUTHOR]