ISOLATION AND CHARATERIZATION OF A RHABDOVIRUS FROM DISEASED FLOUNDER PARALICHTHYS OLIVACEUS

The family Rhabdoviridae contains the genera Vseiculovirus, Lyssavirus, Ephemerovirus, Novirhabdovirus, Cytohabdovirus and Nucleorhabdovirus, belong to the order Mononegavirales1More than 175 rhabdoviruses have been reported from many different species of fish and other life forms1 Rhabdoviruses constitute one of the largest groups of viruses isolated from teleost fish1 The viruses are mostly associated with epizootics and heavy losses in piscine aquaculture1 A rhabdovirus pathogenic virus isolated from diseased fish flounder Paralichthys olivaceus (PoRV) 1 The tissues extracted from the diseased flounder, filtered through a filter membrane to get rid of bacteria, and 12 fish cell lines, Ctenopharngodon idellus ovaries (GCO), Epithelioma 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), Gobiocypris rarus ovary (GRO), Hypophthalmichthys molitrix blastula (HMB), Grouper proboscis (GP) and Goldfish fin (CAR), etc1 were challenged with the extracts1 Observation of the cytopathogenic effect appeared in GCO, EPC, FHM, GCF, CLC, FE and BB 7 of these cell lines1 It is supported that PoRV was the viral pathogen of the diseased flounder1 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 10615 TCID50/mL were obtained by infecting the grass carp ovary (GCO) cells1 The growth curve of PoRV was determined, a growth curve for the virus obtained from persistently infected GCO cell layers1 The growth curves of the virus showed an initial exponential rise and reached a maximal constant value from 0 to 36 hours1 The viruses were purified by differential centrifugat ion and sucrose gradient centrifugation from GCO cells infected1 Electron microscopy observation showed that the viral particles of u-l trathin sections from host cells and negatively stained from purified virus had typical bulle-t shape and was about 60nm @ 200nm in size1 The physical and chemical properties were detected1 Monolayers of GCO cell cultures were infected with 10- fold dilutions of the tissues isolated after incubated 30 to 60min at 50 and 56e 1 PoRV isolate was mixed with 1/4 volume of chloroform, then the mixtures were shaken and centrifuged at 2000r/min for 10min to separate the chloroform from the aqueous phase of the treated sample1 Stability at selected pH levels, from pH1 to 12, was tested by incubating PoRV isolate in medium adjusted to each pH by the 1mol/L HCl or 1mol/L NaOH, after 1h inculation at 25 e, and the samples were adjusted to pH 71 PoRV isolation was treated with 1-B-d-arabinofuranosylcytosine (Ara- c)1 Then all the treated samples of PoRV infectivity were titrated as compared to the control (no treatment, or treatment other viruses)1 The results were shown that PoRV was temperature and lipid solvent sens-i tivity, but insensitivity to pH and Ara- c1 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)