Purification And Analysis Of Genomes And Structural Proteins Of Muscovry Duck Reovirus (DRV) Isolated In China

OBJECTIVEO: Recently,a new disease of Muscovry duck broke out and was prevalent in southemeast of china .At necropsy, in most cases,liver showed small white necrotic foci,so the disease named as "white dot liver"disease of Muscovry duck.Because of high mortality and significant economic losses in infected ducklings,many researchers have conducted on investigations of aetiology and epidemiology ,but these conclusions exist great difference.Wu bao cheng et al first proved that the phathogency of this prevalence is a Muscovry duck reovirus by sires of experiments .A isolate of Muscovry duck reovirus in china is not consistent with those reported isolates of avian reovirus in the fields of phathogenicity and tissue tropism .In order to study the molecular basis of avian reoviruses,we chose a reference strain S1133 and a virulent (field) strain DRV-YH as models,and have compared the viral genomes and proteins of the two strains from in infected cells by immunoprecipitation tests.METHODS: DRV-YH plaque-purified third with solid culture medium in Muscovry duck embryo fibroblast (DEF) was grown with 199 sultion in semiconfluent monolayers of chicken embryo fibroblast (CEF) .The infected cells was disrupted by three freeze-thaw cycles,and then differential centrifugation, ultracentrifugation and sucrose discontinious gradient ultracentrifugation were adopted to concentrate and purify the virus. Electron microscopy(EM),TCID₅₀ test and spectrometer were used for detection of the purified virus.The extracted RNA of the virus was analysised with agarose electrophorisis , and analysis for the viral polypeptides by SDS-PAGE.RESULTS: DRV-YH plaque-purified third named as DRV-YJL. TCID₅₀ of DRV-YJL and S1133 were 10^6.5/0.02ml and 10^8.0/0.02ml respectively.The two purifieds of DRV-YJL and S1133 lie in the layers of 20-35% and 35-50% sucrose .Electron microscopic evaluation revealed that a great number of virions in the purifieds of 35-50% sucrose were 60—80nm in diameter,and that morphology was consistent with a number of the family Reoviridae. TCID50 of the two purifieds in 35-50% sucrose were 10^8.8/0.02ml and 10^10.3 /0.02ml respectively,and the contents of viral proteins were 5.2mg/ml and 5.7mg/ml respectively. The extracted RNA of the virus was analysised with agarose electrophorisis ,the result comfirmed that the genomes of the virus consists of 10 RNA fregments ,and the rates of RNA migration in agarose gels had no differeneces except for M3 gene. Analysis for the viral polypeptides by SDS-PAGE showed that 12 polypeptides'molecular weights occurred in gels were 149.0, 129.0, 111.0, 79.0, 75.0, 50.0,42.0,39.0,37.0,35.9,32.0,29.0 ku ,and 5 of them were the same as those of avian reovirus S1133 referred as control in this test.CONCLUSION: the above results demonstrated that DRV-YH has a common electrophoretic mobilities of avian reovirus.