The Studies On Detection And Diagnosis Method Of Nucleic Acid For Fish Lymphocystis Disease Virus

Fish lymphocystis disease virus (LCDV) is the causative virus for lymphocystis disease that characterized by papilloma-like hypertrophy developing on the surface of the skin tissue. Lymphocystis disease epizootics, which infect of numerous salt- and freshwater fish species, occur worldwide. Since 1992, the lymphocystis disease was reported firstly, many species fish could be infected, including Pagrosomus majo, Lateolabrax japonica, Lutjanus argentimaculatus, Paralichthys olivaceus and other species. Lymphocystis disease is a chronic, benign, rarely fatal disease; infected fish usually recover spontaneously after some weeks or months. Although usually nonlethal, lymphocystis disease causes result an important economic loss because lesions associated with lymphocystis reduce the value of fish that cannot be commercialized.Virions of LCDV are isolated directly from the papilloma-like dermal lesions of caught epidermal flounders (Qingdao) and purified as follows: LCDV are isolated from 10 g specimens by mechanical homogenization and suspension in TNE buffer, and are frozen and thawed three times, then manual homogenized. After ultrasonic shatter, samples are centrifuged on sucrose gradient. By the way of density gradient centrifugation, the two bands are separated: litter virions are founded in 37%~40% band; density of virions is high in 47%~50% band. The viral particles measured 230 nm in diameter approximately and structure was intactness..After treated with proteinase K and SDS, the genomic DNA of virus is extracted according to the method of phenol/chloroform method and yield is approximately 2.30 μg/μl. Lymphocystis disease virus (LCDV, Qingdao isolate) genome DNA is digested with 7 restriction endonucleases, including Ban II, Bgl II, Sea I , EcoR I , Pst I , Hinc II, BamH I and Hpa II. After separated by electrophoreses, restriction fragments are obtained with numbers of 4, 11, 10, 10, 6, 17 and 4, respectively. Theestimated average molecular weight of the virus genome is about 37.0 X 106D (i.e. 56.0 kb), it is different from FLDV-1, FLDV-2 and LCDV-C. Like other vertebrate Iridoviride viruses, the genome of LCDV is highly methylated at cytosines in the CpG, as indicated by Hpa II and Msp I digestion analysis.The primer sets for the PCR designed from the published sequence of the major capsid protein gene of LCDV (GenBank: AF126405). The sizes of amplifications are found in each methods coincided with the predicted sizes of 348 bp and 173 bp respectively, whereas the healthy flounder DNA and control reaction (no DNA) are negative. The sensitivity of the PCR assays is investigated using 10 fold dilutions of LCDV DNA. The One-step PCR product is detected with concentrations as low as 1 X 10"4 ug of LCDV DNA by agarose electrophoresis and ethidium bromide staining. In the nested PCR amplification, the sensitivity is 105 times higher than one-step amplification, which corresponds to about 1 X 10"9 ug of viral DNA. During the optimization experiments of annealing temperature, the following conditions are tried: 47.0, 48.K 49.0> 50.2 and 51.4°C. Generally, the target DNA are amplified better in 49°C with One-step PCR and no variety is found in the nested PCR amplification. In conclusion, a specific and sensitive method is established for detection of LCDV by using one-step PCR and nested PCR assay.The DNA fragment of the One-step PCR products is used for the DNA probe. The DIG-labeled probe is generated with the PCR DIG Labeling Mixplus (Roche) that could direct labeled amplification products with digoxigenin-dUTP in the One-step PCR. Using DIG-labeled probe, Dot-Blot and in situ hybridization methods are established. The sensitivity of the Dot-Blot assays is investigated using 10 fold dilutions of LCDV DNA. This method could detect LCDV DNA with concentrations as low as 1 X 10"4 ug. In order to investigate the expression pattern of LCDV in tissues of diseased fishes infected are processed for in situ hybridization using DIG-labeled DNA probe. Positive signals characterized by blue precipitate are presented in the lymphocystis cells. These are cytorrhyctes that affirmed by hematoxylin-eosin stain. Alongside other diagnostic techniques, such as in situ hybridization, it will be useful in further studies to investigate the pathology and epidemiology of LCDV.The results of Southern hybridization using LCDV mcp gene fragment as a probe digoxigenin-labelled showed that the LCDV mcp gene is located in Ban 11 - A, Bgl 11 -C, Sea I - A, EcoR I - A, Pst I - B, Hinc II - E and BamH I - A fragments. After analysis these fragments, we presume Bgl II - C and Hinc II - E fragments may more appropriate to clone complete mcp gene.The prevalence of LCDV is investigated by developed One-step PCR, the nested PCR and in situ hybridization. The results revealed that LCDV has spread a large region and could infect many species fish, including Paralichthys olivaceus, Sebastodes fuscescens, Arothron hispidus, Scophthalmus maximus, Lepidorigla microptera and Lateolabrax japonicus. We presume LCDV may be carried by wild fishes inhabited offshore of this area and transmitted from wild fishes to cultured fishes through feeding. The PCR assays are evaluated for the ability to detect LCDV in fish tissues, including gut, gill, kidney, spleen, liver, stomach and heart. The positive reaction is always being first found in gill and spleen. We presume LCDV may be primal infect gill and accumulate in spleen, then spread through blood circulation.