Daisuke Yamane

Detection of a new bat gammaherpesvirus in the Philippines.

A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3′ part of the gB gene and the 5′ part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Cloning of the genome of equine herpesvirus 4 strain TH20p as an infectious bacterial artificial chromosome.

Equine herpesvirus 4 (EHV-4) is a major cause of respiratory tract disease in horses worldwide. The generation of recombinant viruses, which would lead to understanding of viral gene functions, has been hindered by the absence of suitable cell lines and small-animal models of the infection. In the present study, the genome of EHV-4 strain TH20p was cloned as a stable and infectious BAC without any deletions of the viral genes. Mini F plasmid sequences flanked by loxP sites were inserted into the intergenic region between genes 58 and 59. Coinfection of the recombinant virus with a recombinant adenovirus expressing Cre recombinase resulted in the excision of the BAC sequences. Importantly, the resulting recombinant EHV-4 replicated comparably to the wild-type virus in fetal horse kidney cells. The recombinant EHV-4 will facilitate EHV-4 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.

Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKβ-binding protein

Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes an economically important livestock disease worldwide. Previous studies have suggested that non-structural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role during virus infection. Extensive reports are available on HCV NS5A and its interactions with the host cellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. To identify the cellular proteins that interact with the N terminus of NS5A and could be involved in its function, we conducted a yeast two-hybrid screening. As a result, we identified a cellular protein termed bovine NIK- and IKKbeta-binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-kappaB) signalling in cells. The interaction of NS5A with NIBP was confirmed both in vitro and in vivo. Complementing our glutathione S-transferase pull-down and immunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5A colocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells. Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597-623. In addition, overexpression of NS5A inhibited NF-kappaB activation in HEK293 and LB9.K cells as determined by luciferase reporter-gene assay. We further showed that inhibition of endogenous NIBP by small interfering RNA molecules enhanced virus replication, indicating the importance of NIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and a viral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking as well as NF-kappaB signalling.

A Ku-band Dual-SPDT RF-MEMS Switch by Double-Side SOI Bulk Micromachining

This paper presents the design, fabrication method, and measurement results of a low-loss ohmic-contact radio-frequency microelectromechanical systems (MEMS) switch. A novel bidirectional electrostatic actuation mechanism has been developed for a dual single-pole double-throw switch that could be used for an X-Ku-band low-temperature cofired ceramic switched-line-type phase shifter. A high-aspect-ratio deep reactive-ion etching process and a thick gold-plating process were used to develop low-insertion-loss air-suspended MEMS waveguides and low resistive ohmic contacts. A typical insertion loss of 0.56 dB, a return loss of 19.4 dB, and an isolation of 51.4 dB were obtained at a Ku-band frequency of 12 GHz.

Functional analysis of Rousettus aegyptiacus "signal transducer and activator of transcription 1" (STAT1).

Abstract Bats are now known as the source of several diseases in humans, but few studies regarding immune responses and factors associated with bats have so far been reported. In this study, we focused on STAT1, one of the critical components in interferon (IFN)-signaling and antiviral activity, which is often targeted by viral proteins to reduce antiviral activity and increase viral replication. We found that Rousettus aegyptiacus STAT1 (bat STAT1) is phosphorylatable and translocates to the nucleus when stimulated with human IFN-α (hIFN-α). Furthermore, phosphorylation of bat STAT1 and inhibition of nuclear translocation was observed in IFN-stimulated cells infected with the HEP-Flury strain of rabies virus, in the same manner as in other mammals. Additionally, quantitative real-time RT-PCR revealed that bat STAT1 mRNA was highly expressed in the liver, while low in muscle and spleen.

Development of Multi-User Multi-Chip SOI CMOS-MEMS Processes

This paper presents a new method of integrating multiple MEMS designs with 40V class CMOS driver circuits in a multi-user-multi-chip manner. The multi-chip multi-user CMOS-MEMS process was done at 35 mm × 35 mm SOI chip. More than six different designs of SOI-bulk micromachined actuators including the pitch-tunable gratings were monolithically integrated onto the pre-fabricated high-voltage level-shifter circuits. We measured electro mechanical characteristics of the grating light valve integrated with high-voltage level-shifter and successfully demonstrated 1MHz operation.

A 12-GHz DPDT RF-MEMS Switch with Layer-Wise Waveguide/Actuator Design Technique

A novel design of double-pole double-throw (DPDT) RF-MEMS switch for 12-GHz phase shifter has been developed to minimize the electrical crosstalk between the signal waveguide and electrostatic actuator by allocating them in separate layers of an SOI wafer. Compared with the previous other reports, our design can use relatively large area on the chip to accommodate more electrostatic actuator and to have more electrical ground planes. With this newly developed method, silicon RF-MEMS devices will be able to overcome the drawback of solid-state devices in terms of performance, device size, and cost. This paper reports driving voltage of 4V with the switching speed of 12 microseconds, insertion loss of 3 dB, return loss of 12 dB, isolation of 30dB at 12 GHz.

Novel Sensor Structure and Its Evaluation for Integrated Complementary Metal Oxide Semiconductor Microelectromechanical Systems Accelerometer

This paper reports a novel sensor structure and its evaluation results for an integrated complementary metal oxide semiconductor (CMOS) microelectromechanical systems (MEMS) accelerometer with a wide detection range on a chip. The proposed sensor structure has the following features: i) a layer separation technique between the proof mass and the mechanical suspensions, ii) mechanical stoppers for the proof mass to avoid destruction, and iii) a SiO2 film underneath the proof mass to prevent stiction and electrical short. Gold was used as the MEMS structure material to reduce the proof mass size and to lower the Brownian noise to below 100 µg/√Hz. Furthermore, the micro fabrication was carried out below 310 °C for the CMOS devices to remain intact. The evaluation results indicate that the Brownian noise was 90.6 µg/√Hz. Thus, we have confirmed that the proposed MEMS structure has the potential for use in future integrated CMOS-MEMS accelerometers.

Physical interaction between bovine viral diarrhea virus nonstructural protein 4A and adenosine deaminase acting on RNA (ADAR)

Bovine viral diarrhea virus (BVDV) is a positive-sense RNA virus known to produce double-stranded RNA (dsRNA) during its replication in the cytoplasm. Extended dsRNA duplexes can be hyperedited by adenosine deaminase acting on RNA (ADAR), which catalyzes adenosine (A)-to-inosine (I) editing. A-to-I editing has been reported for various viruses. A number of cellular antiviral defense strategies are stimulated by dsRNA, and this may involve hyperediting of dsRNA by ADARs, followed by targeted cleavage by cytoplasmic endonucleases. Here, we identify ADAR as a binding partner of BVDV NS4A in vitro and in vivo and show that the N-terminal domain of NS4A is the ADAR-binding domain. We also show that ADAR has an inhibitory effect on BVDV replication when overexpressed in BVDV-infected bovine cells. Our findings suggest a role of NS4A in the interaction of BVDV with ADAR that favors virus replication.

Stabilization of Mode-Locked Fiber Lasers Using Bismuth-Oxide-Based Highly Nonlinear Fiber

We propose and demonstrate a stabilization method of actively mode-locked fiber lasers using a short length of Bismuth-oxide-based highly nonlinear fiber. The supermode noise could be successfully suppressed in a mode-locked fiber laser at 10GHz.