Isolation and charaterization of a rifiabdovirus from diseased flounder Paralichthys olivaceus

The family Rhabdoviridae contains the genera Vseiculovirus, Lyssavirus, Ephemerovirus, Novirhabdovirus I Cytohabdovirus and Nucleorhabdovirus I belong to the order Mononegavirales. More than 175 rhabdoviruses have been reported from many different species of fish and other life forms. Rhabdoviruses constitute one of the largest groups of viruses isolated front teleost fish. The viruses are mostly associated with epizootics and heavy losses in piscine aquaculture. A rhabdovirus pathogenic virus isolated from diseased fish flounder Paralichthys olivaceus (PoRV). The tissues extracted from the diseased flounder, filtered through a filter membrane to get rid of bacteria, and 12 fish cell lines I Ctenopharngodon idellus ovaries (GCO), Epilhelioma papulosum cuprini (EPC), Ctenopharyngodon idellus fins (GCF), Pimephales promelas (FHM), Cyprinus carpio leucocyte cell (CLC), Brown Bullhead ( BB), Flounder embryo (FE), Ctenopharngodon idellus kidney (CIK), Cobiocypris rarus ovary (GRO), Hypophthalmichthys molitrix blastula (HMB), Grouper proboscis (GP) and Goldfish fin (CAR), etc. were challenged with the extracts. Observation of the cytopathogenic effect appeared in GCO, EPC, FHM, GCF, CLC, FE and BB 7 of these cell lines. It is supported that PoRV was the viral pathogen of the diseased flounder. The plaques of PoRV virus were produced in GCO cells plate with 10(-3), 10(-4), 10(-5), 10(-6), 10(-7) series dilutions, and after plaque assays and isolation, PoRV titers of about 10(6.5) TCID50/mL were obtained by infecting the grass carp ovary ( GCO) cells. The growth curve of PoRV was determined, a growth curve for the virus obtained from persistently infected GCO cell layers. The growth curves of the virus showed an initial exponential rise and reached a maximal constant value front 0 to 36 hours. The viruses were purified by differential centrifugation and sucrose gradient centrifugation from GCO cells infected. Electron microscopy observation showed that the viral particles of ultrathin sections from host cells and negatively stained front purified virus had typical bullet-shape and was about 60nm x 200nm in size. The physical and chemical properties were detected. Monolayers of GCO cell cultures were infected with 10 - fold dilutions of the tissues isolated after incubated 30 to 60min at 50 and 56 degrees C. PoRV isolate was mixed with 1/4 volume of chloroform I their the mixtures were shaken and centrifuged at 2000r/min for 10min to separate the chloroform from the aqueous phase of the treated sample. Stability at selected pH levels, from pH1 to 12, was tested by incubating PoRV isolate in medium adjusted to each pH by the 1 mol/L HCl or I mol/L NaOH, after I h inculation at 25 degrees C, and the samples were adjusted to pH 7. PoRV isolation was treated with 1-beta-d-arabinofuranosylcytosine (Ara-c). Then all the treated samples of PoRV infectivity were titrated as compared to the control (no treatment, or treatment other viruses). The results were shown that PoRV was temperature and lipid solvent sensitivity, but insensitivity to pH and Ara-c. SDS - PAGE analysis of the purified PoRV particles indicated that the structural proteins of PoRV were mainly composed of L (Polymerase), G ( Glycoprotein). N ( Nucleoprotein), P ( Phosphoprotein) and M (Matrix protein) 5 potypeptide with the molecular weights about 250, 67, 44, 30 and 23 W, respectively.The data shows that PoRV is a kind of fish rhabdovirus and very closely resembled Scophthalmus maximus rhabdovirus (SMRV) and Spring viraemiaof carpvirus (SVCV)