The major capsid protein of lymphocystis disease virus isolated from Rachycentron canadum (LCDV-rc) was amplified and analysed. The 457bp DNA core fragment was amplified with the degenerate primers designed according to the conserved sequences of MCP gene of iridoviruses, then the flaking sequences adjacent to the core region were amplified by inverse PCR, and the complete sequence was obtained by combining all of them. The open reading frame of the gene is 1380bp in length, encoding a putative protein of 459 aa with molecular weight 51.12 kD and pI 6.87. Constructing the phylogenetic tree for comparing the MCP amino acid of iridoviruses, the results indicated that LCDV-rc is most homologous to the other Lymphocystis viruses and all of them constitute a branch. Accordingly LCDV-rc is identified as Lymphocystivirus.
Animals ; Base Sequence ; Capsid Proteins ; genetics ; Iridoviridae ; classification ; genetics ; Molecular Sequence Data ; Perciformes ; microbiology ; Phylogeny ; Polymerase Chain Reaction ; methods

Virus infection consists of entry, synthesis of macromolecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus (SGIV) assembly in the medaka haploid embryonic stem (ES) cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluorescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site (VAS). Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell. Taken together, VP088 is an excellent marker for visualizing the SGIV infection process. Our results provide new insight into macromolecular component recruitment and SGIV assembly.
Animals ; Cell Line ; Embryonic Stem Cells ; metabolism ; pathology ; virology ; Fish Diseases ; genetics ; metabolism ; virology ; Humans ; Iridoviridae ; physiology ; Oryzias ; Viral Proteins ; genetics ; metabolism ; Virus Assembly ; physiology

A virus was isolated from cultured sick giant salmander (Andrias davidianus ) in a farm, Shanxi Province, China. Skin ulceration and necrosis of the distal limbs are main clinical symptoms. Virus propagated and caused CPE at 10 degrees C to 30 degrees C in BF-2, CO, CHSE, FHM cells. The optimum condition of replication was in BF-2 cells at 25 degrees C. The virus was proved to be senstive to chloroform, heat, pH3 and pH10 treatment. Viral replication was inhibited by 5-Fluoro-2-deoxyuridine (FUDR). These results indicated that the virus possessed an envelope and DNA as the genome. Electron-microscopic observation of thin-section showed numerous hexagonal viral particles measuring 130 nm to 150 nm in diameter orderly arranged in a lattice form in cytoplasm of BF-2 cells. The particles showed typical iridovirus morphology. A 413 bp fragment was amplified from the viral main capsid protein gene by PCR. The fragments was sequenced and analysed. The results showed the isolate shared more than 96% nucleotide identity with some Ranaviruses. We suggested that this virus was named as Andrias davidianus iridovirus (ADIV) tentatively.
Animals ; Base Sequence ; Iridovirus ; genetics ; isolation & purification ; Molecular Sequence Data ; Urodela ; virology

During the summer of 2009, mass mortality was observed in cage-cultured Rock Bream (Oplegnathus fasciatus; Temminck and Schlegel) in the Liaoning Province. Histopathogic studies of the affected fish showed enlarged basophilic cells in the kidney and spleen. These necrotic cells were stained purple using haematoxylin and eosin (HE). GF cell cultures showed advanced cytopathic effects after infection with virus supernatants from diseased fish homogenate. Transmission electron microscopy revealed hexagonal outlines virions in the cytoplasm of the spleen, kidney, liver, intestine cells. The viral particles consisted of a central nucleocapsid (100-110 nm) and envelope, and were 150-180 nm in diameter. These results suggested that the virus belonged to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570-bp fragments were amplified from the viral DNA in spleen, kidney, gill, intestine, heart and brain of diseased fish with the primers derived from red sea bream Iridovirus (RSIV). In addition, a specific fragment of 1 400 bp of the major capsid protein (MCP) gene of the Iridovirus was amplified by PCR. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Megalocytivirus. The tree demonstrated that RSIV-LN09 virus existed in the same branch as the RSIV-U1 et al. Our present results indicated that RSIV was the causative agent.
Animals ; DNA, Viral ; genetics ; Iridovirus ; classification ; genetics ; isolation & purification ; physiology ; Microscopy, Electron ; Perciformes ; virology ; Phylogeny ; Polymerase Chain Reaction

The Rana grylio virus (RGV) is a member of the genus Ranavirus. It belongs to the family Iridoviridae, and contains the gene 67R encoding dUTPase. In order to investigate the function of 67R in the replication and infection of RGV, we constructed Δ67R-RGV, a recombinant virus with deletion of 67R. First, we constructed the plasmid pGL3-67RL-p50-EGFP-67RR which carried an enhanced green fluorescence gene (EGFP) as a selectable marker. After homologous recombination between pGL3-67RL-p50-EG- FP-67RR and the RGV genome, Epithelioma papulosum cyprini (EPC) cells were infected with the resulting mixture. Through ten successive rounds of plaque isolation via EGFP selection, all plaques emitted green fluorescence, and finally Δ67R-RGV was generated. Total DNA of Δ67R-RGV infected cells was extracted for PCR analyses. Simulateously, mock infected and wild-type RGV (wt-RGV) infected cells were used as a comparison. Results showed that 67R could be detected in wt-RGV infected cells, but that only the EGFP gene was detected in Δ67R-RGV infected cells. Furthermore, one-step growth curves of wt-RGV and Δ67R-RGV were similar. Therefore, 67R and its encoding product dUTPase might not be essential for the growth of RGV. These results suggest that, homologous recombination and recombinant rana- virus could be used to study the gene function of viruses in aquatic animals.
Genes, Viral ; physiology ; Genome, Viral ; Polymerase Chain Reaction ; Pyrophosphatases ; genetics ; Ranavirus ; genetics ; Recombination, Genetic