Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Microstructure, deformation and failure of polymer bonded explosives

Polymer bonded explosives (PBXs) are highly particle filled composite materials comprised of explosive crystals and a polymeric binder (ca. 5-10% by weight). The microstructure and mechanical properties of two pressed PBXs with different binder systems were studied in this paper. The initial microstructure of the pressed PBXs and its evolution under different mechanical aggressions were studied, including quasi-static tension and compression, ultrasonic wave stressing and long-pulse low-velocity impact. Real-time microscopic observation of the PBXs under tension was conducted by using a scanning electron microscope equipped with a loading stage. The mechanical properties under tensile creep, quasi-static tension and compression were studied. The Brazilian test, or diametrical compression, was used to study the tensile properties. The influences of pressing pressures and temperatures, and strain rates on the mechanical properties of PBXs were analyzed. The mesoscale damage modes in initial pressed samples and the samples insulted by different mechanical aggressions, and the corresponding failure mechanisms of the PBXs under different loading conditions were analyzed.

Embryo-damage induced nucleation of microcracks in an aluminum-alloy under impact loading

The nucleation of microdamage under dynamic loading was investigated through planar impact experiments accomplished with a light gas gun. The microscopic observation of recovered and sectioned specimens showed that microcracks were nucleated only by cracking of brittle particles inside material. However, for comparison the in situ static tensile tests on the same material conducted with a scanning electron microscope showed that the microcracks were nucleated by many forms those were fracture of ductile matrix, debonding particles from matrix and cracking of brittle particles. The quantitative metallographic observations of the specimens subjected to impact loading showed that most of the cracked particles were situated on grain boundaries of the aluminium matrix. These facts suggested the concept of critical size and incubation time of submicroscopic cavities in the dynamic case and the mechanism of embryo-damage induced nucleation by fracture of brittle particles in the aluminium alloy under impact loading was proposed.

Influence Of Gravity On Structure Of Colloidal Crystal Using Simulated Microgravity

Liquid mixtures of water and deuterium oxide as the liquid phase, were used to match the density of charged colloidal particles. Kossel diffraction method was used to detect the crystal structures. The experiments under the density-matched (g=0) and unmatched (g=1) conditions are compared to examine the influence of gravity on the crystal structures formed by self-assembly of 110 nm (in diameter) polystyrene microspheres. The result shows that die gravity tends to make the lattice constants of colloidal crystals smaller at lower positions, which indicates that the effect of gravity should be taken into account in the study of the colloidal crystals.

Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better Preservation of plasma and acrosome integrity than TEST Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Characterization of the Rana grylio virus 3 beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

The 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3 beta-HSD gene homolog,was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3 beta-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3 beta-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3 beta-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3 beta-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3 beta-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3 beta-HSD might be a protein involved in host-virus interaction. @ 2006 Elsevier Inc. All rights reserved.

Rickettsia-like organism infection in a freshwater cultured fish Ophiocephalus argus C. in China

From 2001 to 2002, a new and emergent infectious disease of Ophiocephalus argus occurred in a fishery in Hubei Province, China, with an incidence of 60% similar to 70% and a mortality as high as 100 %. The diseased fish showed an enlarged abdomen, the millet-like nodules in internal organs, and the swollen kidney which was composed of 5 similar to 10 sarcoma-like bodies in cream or gray-white colour or ulcerated into beandregs-like substance. Light microscopic observation revealed the basophilic or acidphilic inclusions in cytoplasm of the cells and the granulomas, a diffusive chronic inflammation in internal organs. Further analysis under an electron microscope indicated that the intracytoplasmic inclusions were rickettsia-like organisms (RLOs) that are either spherical or coccoid, with variable size, ranging from 0.5 similar to 1.5 mum in diameter, and enclosed within membrane-bound cytoplasmic vacuoles. RLO had a central nucleoid region with some fine filamentous structures and an electron-dense granule. Its cytoplasm contained abundant ribosomal bodies. Occasionally, RLO appeared to be divided by binary fission. RLOs were also observed in the homogenized tissue of infected fish. The results suggested that the death of cultured O. argus was caused by RLO infection.

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells. (C) 2004 Published by Elsevier B.V.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone I-Il as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

Influence of high-temperature AIN buffer thickness on the properties of GaN grown on Si(111)

The influences of AlN buffer thickness on the optical and the crystalline properties of metalorganic chemical vapor deposition wurtzite GaN layers on Si(I 11) substrate have been investigated. High-resolution X-ray diffraction and photoluminescence measurement reveal that the thickness of AlN buffer exerts a strong influence on the distribution of dislocation and stress in GaN epilayer. The evidence is further reinforced by atomic force microscopic observation of AlN nucleation process. The optimum thickness of AlN buffer to effectively suppress Si diffusion has been determined by secondary-ion mass spectroscopy to be in the range of 13-20 nm. In addition, it is found that appropriate Si diffusion in AlN buffer helps to compensate the tensile strain in GaN, which subsequently improves the optical quality of GaN on Si(I 1, 1), and reduces the cracks over the GaN surface. (C) 2003 Elsevier B.V. All rights reserved.

The morphology and thermal properties of multi-walled carbon nanotube and poly(hydroxybutyrate-co-hydroxyvalerate) composite

Nanocomposites based on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and multi-walled carbon nanotubes (MWNTs) were prepared by solution processing. Ultrasonic energy was used to uniformly disperse MWNTs in solutions and to incorporate them into composites. Microscopic observation reveals that polymer-coated MWNTs dispersed homogenously in the PHBV matrix. The thermal properties and the crystallization behavior of the composites were characterized by thermogravimetric analysis, differential scanning calorimetry and wide-angle X-ray diffraction, the nucleant effect of MWNTs on the crystallization of PHBV was confirmed, and carbon nanotubes were found to enhanced the thermal stability of PHBV in nitrogen.

STUDIES ON MORPHOLOGY OF SOLUTION-CAST FILMS OF IPP/APP BLENDS

Morphologies of solution-cast films of iPP/aPP blends have been studied by means of electron microscopy and X-ray scattering techniques. Microscopic observation showed that solution-cast film of iPP consists of two kinds of structural regions, cross-hatched and lath-liked structures. The addition of small amount of aPP (less than or equal to 30%) into iPP did not change iPP's characteristic crystallization behavior. It is noticed that when the content of aPP in its blend was over 80%, iPP formed a very loosely woven-like network composed of very long lamellae with wide-angle lamellar branchings. The X-ray data showed that aPP did not cocrystallize with iPP.

Effect of temperature and irradiance on the growth and reproduction of Enteromorpha prolifera J. Ag. (Chlorophycophyta, Chlorophyceae)

Effect of temperature and irradiance on growth and reproduction of Enteromorpha prolifera that bloomed offshore along the Qingdao coast in summer 2008, was studied. It was showed that E. prolifera propagated mainly asexually with specific growth rate (SGR) of 10.47 at 25A degrees C/40 mu mol m(-2)s(-1). Under this condition, gametes with two flagellate formed and released in 5 days. At the beginning of the development, the unicell gamete divided into two cells with heteropolarity, and then the apical cell developed into thalli primordial cells, whereas the basal cell developed into rhizoid primordial cells. In 8-day culture, the monoplast gamete developed into juvenile germling of 240 mu m in length. Unreleased gametes can develop directly within the alga body. E. prolifera could either reproduce through lateral branching or fragmenting except apomixis revealed by Microscopic observation. On aged tissue of E. prolifera, although the degraded pigments partially remained in faded algal filaments, numerous vegetative cells could still divide actively in the algal tissues.

Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment

Objective To study the transfer of paralytic shellfish toxins (PST) using four simulated marine food chains: dinoflagellate Alexandrium tamarense -> Arterriia Artemia salina -> Mysid shrimp Neomysis awatschensis; A. tamarense-N. awatschensis: A. taniarense A. salina -> Perch Lateolabrax japonicus; and A. tamarense -> L. japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels iii the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly ibrough the vector of A. salina was then studied. The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense (2 000 cells(.)mL(-1)) for 70 minutes, the content of ChLa in A. salina and N. awatschensis reached 0.87 and 0.024 mu g-mg(-1), respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU(.)g(-1), respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in arternia sample collected on the 1st day was estimated to be 1.65x10(-5) pg STX equa Vindividual. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly froin the vector of A. salina was also studied. The mice injected with extracts from L. japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. taniarense directly or indirectly via the food chains.

Improvement and observation of immunoelectron microscopic method for the localization of frog Rana grylio virus (RGV) in infected fish cells

In this paper, to understand the roles of amorphous structures which were observed within the viromatrix of Rana grylio virus (RGV), an improved immunoelectron microscopy (IEM) method was developed to detect the localization of RGV in carp Epithelipma papulosum cyprinid (EPC) cells. Infected EPC cells were fixed with 4% paraformaldehyde-0.25% glutaraldehyde mixture, dehydrated completely, and embedded in LR White resin. This method allowed good ultrastructural preservation and specific labeling with anti-RGV antibodies. The results of IEM showed that colloidal gold mainly bound to the capsids of viral particles at the stage of viral assembly, while during the viral maturation colloidal gold bound to the envelop of virions. In addition, within the viromatrix, the amorphous structures, including dense floccules, membranous materials and tubules, also had strong colloidal gold signals, revealing that those amorphous structures were participated in RGV assembly. In contrast, no significant gold labeling signals were obtained in negative controls. The present study not only provided further evidence that amorphous structures within the viromatrix were involved in the process of RGV assembly, but also developed an improved IEM method for studying the interaction between iridovirus and host cells. (C) 2006 Elsevier Ltd. All rights reserved.