Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 mu m(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them. (c) 2005 Elsevier B.V. All rights reserved.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive. (c) 2005 Elsevier B.V. All rights reserved.

Layer-by-layer assembly of viral capsid for cell adhesion

Cowpea mosaic virus (CPMV)-based thin films are biologically active for cell culture. Using layer-by-layer assembly of CPMV and poly(diallyldimethylammonium chloride), quantitatively scalable biomolecular surfaces were constructed, which were well characterized using quartz crystal microbalance, UV-vis and atomic force microscopy. The surface coverage of CPMV nanoparticles depended on the adsorption time and pH of the virus solution, with a greater amount of CPMV adsorption occurring near its isoelectric point. It was found that the adhesion and proliferation of NIH-3T3 fibroblasts can be controlled by the coverage of viral particles using this multilayer technique.

Protective immunity induced by CpG ODNs against white spot syndrome virus (WSSV) via intermediation of virus replication indirectly in Litopenaeus vannamei

The worldwide shrimp culture is beset with diseases mainly caused by white spot syndrome virus (WSSV) and suffered huge economic losses, which bring out an urgent need to develop the novel strategies to better protect shrimps against WSSV. In the present study, CpG-rich plasmid pUC57-CpG, plasmid pUC57 and PBS were employed to pretreat shrimps comparatively to evaluate the protective effects of CpG ODNs on shrimps against WSSV. The survival rates, WSSV copy numbers, and antiviral associated factors (Dicer, Argonaute, STAT and ROS) were detected in Litopenaeus vannamei. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in pUC57-CpG-pretreatment shrimps than those in pUC57- and PBS-pretreatment shrimps after WSSV infection. The Argonaute mRNA expression in pUC57-CpG-, pUC57- and PBS-pretreatment shrimps after WSSV infection was significantly higher than that of shrimps post PBS stimulation on the first day. The ROS levels in pUC57-CpG-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection on the first day. These results together demonstrated that pUC57-CpG induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei. (C) 2009 Elsevier Ltd. All rights reserved.

Four viral proteins of white spot syndrome virus (WSSV) that attach to shrimp cell membranes

White spot syndrome virus (WSSV) is a major shrimp pathogen that has a widespread negative affect on shrimp production in Asia and the Americas. It is known that WSSV infects shrimp cells through viral attachment proteins (VAP) that bind with shrimp cell receptors. However, the identity of both WSSV VAP and shrimp cell receptors remains unclear. We used digoxigenin (DIG)labeled shrimp hemocyte and gill cell membranes to bind to WSSV proteins immobilized on nitrocellulose membranes, and 4 putative WSSV VAP (37 kDa, 39 kDa and 2 above 97 kDa) were identified. Mass spectrometric analysis identified the 37 kDa putative VAP as the product of WSSV gene VP281.

Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection

The Zhikong Scallop, Chlamys farreri, is one of the most Important bivalve mollusks cultured in northern China However, mass mortality of the cultured C farreri has posed a serious threat to the maricultural Industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators Scallops challenged by AVNV at 25 C developed typical disease signs 2 days after virus injection Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery The plasma SOD activity, however, augmented consistently following virus injection Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops (C) 2010 Elsevier Ltd All rights reserved