Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with-full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron Microscopy, Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV. (c) 2008 Published by Elsevier Inc.

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays. (c) 2007 Elsevier B.V. All rights reserved.

A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129 bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments. (c) 2007 Elsevier B.V. All rights reserved.

Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp, Penaeus monodon with reference to haematological, biochemical and immunological changes

Immunostimulants are the substances, which enhance the non-specific defence mechanism and provide resistance against the invading pathogenic micro-organism. In order to increase the immunity of shrimps against the WSSV, the methanolic extracts of five different herbal medicinal plants like Cyanodon dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and Eclipta alba were selected and mixed thoroughly in equal proportion. The mixed extract was supplemented with various concentrations viz. 100 (A), 200 (B), 400 (C), and 800 (D) mg kg(-1) through artificial diets individually. The prepared diets (A-D) were fed individually to WSSV free healthy shrimp Penaeus monodon with an average weight of 8.0 +/- 0.5 g for 25 days. Control diet (E), devoid of herbal extract was also fed to shrimps simultaneously. After 25 days of feeding experiment, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps succumbed to death within 7 days when fed on no herbal immunostimulant diet (E). Among the different concentrations of herbal immunostimulant supplemented diets, the shrimps fed on diet D (800 mg kg(-1)) significantly (P < 0.0001) had more survival (74%) and reduction in the viral load. Also the better performance of haematological, biochemical and immunological parameters was found in the immunostimulant incorporated diets fed shrimps. The present work revealed that the application of herbal immunostimulants will be effective against shrimp viral pathogenesis and they can be recommended for shrimp culture. (c) 2006 Published by Elsevier Ltd.

Twin-spot launch for enhancement of multimode-fiber communication links

A novel twin-spot launch is proposed for multimode-fiber (MMF) links. Experimental and theoretical investigation of the launch indicates a penalty reduction of ≈50% of the 10 Gigabit Ethernet allocation for EDC-enabled links over worst-case MMF. © 2007 Optical Society of America.

Construction and characterization of a novel recombinant single-chain variable fragment antibody against White Spot Syndrome Virus from shrimp

An antibody phage display library against White Spot Syndrome Virus (WSSV) was constructed. After four rounds of panning against WSSV, 192 out of 480 clones displayed WSSV binding activity. One of the positive clones, designated A1, had relatively higher activity specifically binding to WSSV A1-soluble, single-chain fragment variable (scFv) antibody has an affinity constant (K-aff) of 2.02 +/- 0.42 x 10(9) M-1. Dot blot assays showed that A1-soluble scFv could detect WSSV directly from shrimp hemolymph after 24-h feeding infection by WSSV. A1 scFv has potential for the development of a cheap, simple and sensitive diagnostic kit for WSSV in the field. (C) 2003 Elsevier Science B.V. All rights reserved.

Purification and characterization of White Spot Syndrome Virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii

Penaeid shrimp is the natural host of White Spot Syndrome Virus (WSSV) that can cause high mortality in the infected hosts. Attempts to obtain sufficient amounts of purified intact WSSV for characterization have been unsuccessful. Using crayfish, Cambarus clarkii as a proliferation system, a large amount of infectious WSSV was reproduced and intact WSSV viral particles were purified with a new isolation medium by ultra-centrifugation. Purified WSSV particles were very sensitive to organic solvents and the detergent, Triton X-100. The size of the rod-shape, somewhat elliptical, intact WSSV was 110-130 x 260-350 mm with a long, tail-like envelope extension. The naked viral nucleocapsid was about 80 x 350 nm, and it possessed 15 spiral and cylindrical helices composed of 14 globular capsomers along its long axis, and a 'ring' structure at one terminus. Distinct WSSV genome DNA patterns were obtained when the purified genomic dsDNA of WSSV was digested with five different restriction enzymes (HindIII, XhoI, B(BamHI, SalI, and SacI). In addition, at least 13 major and distinct protein bands could be observed when purified intact WSSV viruses were separated by SDS-PAGE followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 190, 84, 75, 69, 68, 58, 52, 44, 28, 27.5, 23, 19, and 16 kD, respectively. Both the 44 and 190 kD proteins were easily removed if the hemolymph from the: WSSV infected crayfish was transiently treated with 1%, Triton X-100 before it was subjected to gradient centrifugation, indicating that both of them are located on the surface of the viral envelope. These characteristics are consistent with WSSV isolated from the penaeid shrimp. (C) 2001 Elsevier Science B.V. All rights reserved.

Fabrication and optical optimization of spot-size converters with strong cladding layers

A silicon-on-insulator (SOI) optical fiber-to-waveguide spot-size converter (SSC) overlaid with specially treated silica is investigated for integrated optical circuits. Unlike the conventional process of simply depositing the hot silica on silicon waveguides, two successive layers of silicon dioxide were grown on etched SSC structures by PECVD (plasma-enhanced chemical vapor deposition). The two layers have 0.8% index contrast and supply stronger cladding for an incident light beam. Additionally, this process is able to reduce the effective refractive index of the input mode to less than 1.47 (extremely close to that of the fiber), substantially weakening the unwanted back reflection. Exploiting this technology, it was demonstrated that the SSC showed a theoretical low mode mismatch loss of 1.23 dB for a TE-like mode and has an experimental coupling efficiency of 66%.

1.55-mu m ridge DFB laser integrated with a buried-ridge-stripe dual-core spot-size converter by quantum-well intermixing

A ridge distributed feedback laser monolithically integrated with a buried-ridge-stripe spot-size converter operating at 1.55 mu m was successfully fabricated by means of low-energy ion implantation quantum-well intermixing and dual-core technologies. The passive waveguide was optically combined with a laterally exponentially tapered active core to control the mode size. The devices emit in a single transverse and single longitudinal mode with a sidemode suppression ratio of 38.0 dB. The threshold current was 25 mA. The beam divergence angles in the horizontal and vertical directions were as small as 8.0 degrees x 12.6 degrees, respectively, resulting in 3.0-dB coupling loss with a cleaved single-mode optical fiber.

Laser diode monolithically integrated with an electroabsorption modulator and dual-waveguide spot-size converter

A 1.60-mu m laser diode and electroabsorption modulator monolithically integrated with a dual-waveguide spot-size converter output for low-loss coupling to cleaved single-mode optical fiber is demonstrated. The devices emit in a single transverse and quasi-single longitudinal mode with a side mode suppression ratio of 25.6 dB. These devices exhibit a 3-dB modulation bandwidth of 16.0 GHz, and modulator extinction ratios of 16.2 dB dc. The beam divergence angle is about 7.3x10.6 deg, resulting in 3.0-dB coupling loss with cleaved single-mode optical fiber. (c) 2005 Society of Photo-optical Instrumentation Engineers.

Monolithically integrated semiconductor optical amplifier and electroabsorption modulator with dual-waveguide spot-size converter input and output

We have demonstrated an electroabsorption modulator and semiconductor optical amplifier monolithically integrated with novel dual-waveguide spot-size converters (SSC) at the input and output ports for low-loss coupling to a planar light-guide circuit silica waveguide or cleaved single-mode optical fibre. The device was fabricated by means of selective-area MOVPE growth, quantum well intermixing and asymmetric twin waveguide technologies with only a three-step low-pressure MOVPE growth. For the device structure, in the SOA/EAM section, a double ridge structure was employed to reduce the EAM capacitances and enable high bit-rate operation. In the SSC sections, buried ridge structure (BRS) was incorporated. Such a combination of ridge, ATG and BRS structure is reported for the first time in which it can take advantage of easy processing of the ridge structure and the excellent mode characteristic of BRS. At the wavelength range of 1550-1600 nm, lossless operation with extinction ratios of 25 dB dc and more than 10 GHz 3 dB bandwidth is successfully achieved, The beam divergence angles of the input and output ports of the device are as small as 8.0 degrees x 12.6 degrees, resulting in 3.0 dB coupling loss with a cleaved single-mode optical fibre.

Monolithically integrated laser diode and electroabsorption modulator with dual-waveguide spot-size converter input and output

We have demonstrated a 1.60 mu m ridge-structure laser diode and electroabsorption modulator monolithically integrated with buried-ridge-structure dual-waveguide spot-size converters at the input and output ports for low-loss coupling to a cleaved single-mode optical fibre by means of selective area growth and asymmetric twin waveguide technologies. The devices emit in single transverse and quasi-single longitudinal modes with a side mode suppression ratio of 25.6 dB. These devices exhibit 3 dB modulation bandwidth of 15.0 GHz and modulator extinction ratios of 14.0 dB dc. The output beam divergence angles of the spot-size converter in the horizontal and vertical directions are as small as 7.3 degrees x 10.6 degrees, respectively, resulting in 3.0 dB coupling loss with a cleaved single-mode optical fibre.

Lossless electroabsorption modulator monolithically rntegrated with a semiconductor optical amplifier and dual-wavegulde spot-size converters

Semiconductor optical amplifier and electroabsorption modulator monolithically integrated with dual-waveguide spot-size converters at the input and output ports is demonstrated by means of selective area growth, quantum-well intermixing, and asymmetric twin waveguide technologies. At the wavelength range of 1550 similar to 1600 nm, lossless operation with extinction ratios of 25-dB dc and 11.8-dB radio frequency and more than 10-GHz 3-dB modulation bandwidth is successfully achieved. The output beam divergence angles of the device in the horizontal and vertical directions are as small as 7.3 degrees x 10.6 degrees, respectively, resulting in 3.0-dB coupling loss with cleaved single-mode optical fiber.

Laser diode integrated with a dual-waveguide spot-size converter by low-energy ion implantation quantum well intermixing

A ridge laser diode monolithically integrated with a buried-ridge-structure dual-waveguide spot-size converter operating at 1.58 mu m is successfully fabricated by means of low-energy ion implantation quantum well intermixing and asymmetric twin waveguide technology. The passive waveguide is optically combined with a laterally tapered active core to control the mode size. The devices emit in a single transverse and quasi single longitudinal mode with a side mode suppression ratio of 40.0dB although no grating is fabricated in the LD region. The threshold current is 50 mA. The beam divergence angles in the horizontal and vertical directions are as small as 7.3 degrees x 18.0 degrees, respectively, resulting in 3.0dB coupling loss With a cleaved single-mode optical fibre.