| Lymphocystis disease virus, which is iridoviridae lymphocystivirus and spreads all over on the world, can lead to lymphocystis disease in more than 100 different seawater and freshwater fish species. The infection ratio is about 70% after exposure. Lymphocystis disease virus can lead to pathological changes in cells of the seawater and freshwater fish and cyst on surface of fish. Such change will eventually result in a much lower market value and related loss in aquaculture. The infection will also lead to fish death.Since 1962, when Walker isolated lymphocystis disease virus firstly, a prodigious progress have been achieved in LCDV study. The shape, the configuration, chemical component and characteristics of LCDV have been studied. The study of many years indicated that there was prodigious difference in different isolated LCDV. in 1997, Tidona obtained the genome sequences of LCDV-1 and revealed that the genome had 102653 bp which coding 195 open reading frames(ORF). In China, lymphocystis disease badly broke out in Weihai for the first time. lymphocystis disease virus China(LCDV-cn) was isolated from paralichthys olivaceus by Prof. Sun Xiuqin in 1998. LCDV-cn was then studied systemically and the part sequence of mcp gene was revealed in 2000. The homologous relationship of mcp gene in LCDV-1 compared with that of in LCDV-cn was 78%. Zhang qiya acquired the genome sequences of LCDV-C in 2004. Whereas, the genome of LCDV-C has 186250 bp and 240 ORF.In order to reveal phylogenetic station of LCDV-cn, phylogenetic relationships of LCDV-rc ,LCDV-cn, LCDV-jf, LCDV-sb, LCDV-rf, LCDV-1 and other iridovirus were analysed on the molecular level. Taking mcp gene as the molecular marker, partial nucleotide sequences(1356 bp) containing 178 infromative sites of mcp gene were acquired by PCR and cloning sequencing. Phylogenetic trees were reconstructed using maximum parsimony, maximum likelihood, neighbor joining and bayesian methods based on mcp gene sequences. Supporting values were also calculated. The results indicates that the mcp gene of LCDV-cn was same as the mcp gene of LCDV-C. One cluster belongs to genotypeâ…£contains LCDV-rc isolated from China cobio and LCDV-sb isolated from Korea weever. Another cluster belongs to genotypeâ…¡contains LCDV-cn isolated from China flounder and LCDV-jf isolated from Japan flounder. The third cluster belong to genotypeâ…¢contains LCDV-rf isolated from Japan rockfish. LCDV-1 is the last cluster and belong to genotypeâ… .By PCR, cloning and sequencing, sequences of eleven genes had been acquired and identified as same genes of LCDV-C. So, the genome of LCDV-C as the central reference sequence was been analysed. The result is that there are 119 ORF without homological sequences in iridovirus; there are 52 ORF without homological sequences in LCDV.Zinc-finger proteins play various roles in a lot of organisms, i.e. adjusting the cellular differentiation, the embryogenesis. Further more, zinc-finger proteins are related to diseases. Two ORF (lcn61 and lcn140) of lymphocystis disease virus China (LCDV-cn) are predicted to encode putative zinc finger proteins. They were amplified and inserted into pET24a(+) vector, then expressed in E. coli BL21 (DE3). His-tag fusion proteins with the high yield were obtained. It was found that fusion proteins existed mainly as inclusion body in E. coli. Analysis of LCN61 and LCN140 protein by bioinformatic software indicated that LCN61 was C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs and LCN140 is C3H1 type zinc-finger protein containing four C3H1 zinc-finger motifs. The expression of lcn61 and lcn140 genes and analysis of LCN61 and LCN140 protein set a satisfactory foundation for their functional researches.Viral bacterins have gained distinct achievements, but there are some problems in the scopes: the inhibition of viral proteins may affect the function of proteins in the host; the antigenic excursion induced by the gene mutation can make viral bacterins study frustrate continuously. RNA interference(RNAi) is a phenomenon of specific silencing of genes in diverse organisms including Drodophia, Caenorhabditis elegans, epiphyte, mammalian and etc. RNAi also exists in aquicolous organisms. RNAi is an effective method to study the gene function of aquicolous organisms as was validated zebra fish, ascidian and so on. Controlling viral infection in aquiculture is a technological challenge worldwide, and RNAi could be a way of viral infection of cultured animals. siRNAs were designed based on LCDV-cn own genes and LCDV own genes. siRNAs were injected into China flounder in order to study the effect of LCDV-cn copy and infection. The results indicated that the expression quantity of mcp gene in LCDV-cn was reduced in three days after infection of mcp-siRNA1 and mcp-siRNA3. This show that RNAi can restrain the copy of LCDV-cn. |
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