Duck Enteritis Virus Genome Replication Screening Of Non-essential Regions

Duck viral enteritis (DVE), also known as duck plague, is an acute, contagious and lethal disease of ducks and geese, caused by duck enteritis virus (DEV) and characterized by vascular damage,tissue hemorrhages, digestive mucosal eruptions, lesions of lymphoid organs,and degenerative changes in parenchymatous organs. DEV is currently classified to the family Herpesviridae, belonging to the Alpha herpesvirus subfamily,but has not been grouped into any genus yet.Although there has been about 56 genes registed in the GenBank,the content and organization of the DEV genome are still unknown. In this dissertation,the genomic DNA of DEV was extracted and analyzed by restriction enzyme; three replicative unessential regions in duck enteritis virus genome were also screened.1.DEV vaccine strain VAC and isolate strain SD-01 were propagated in chicken embryo fibroblast (CEF) cells respectively. SD-01 could be adapted to CEF cells after 2 generation of passage. Virus titers rose gradually with the increasing of passage in CEF cells.2.The genomic DNA of DEV was extracted by two methods. One was to purify virion first and then obtain genomic DNA by phenol/chloroform extraction. The other was combination of infected cells permeabilization by a Triton X-100 lysis buffer, digestion of cellular DNA by micrococcal nuclease, and subsequent extraction of viral DNA which is protected from nuclease by virion capsids. The genomic DNA samples were digested by EcoRⅤ;the results showed that the nuclease treatment resulted in significant reduction of cellular DNA. The PCR test indicated the samples were exactly DEV genomic DNA. The genomic DNA of vaccine strain and isolate SD-01 were digested by 8 restriction enzymes. The restriction enzyme maps of the two strain produced by XboⅠand EcoRⅤare nearly identical.Maps produced by MluⅠ,HindⅢ,and KpnⅠdiffers by only few fragments. The mobility in the two maps produced by EcoRⅠ,BglⅡand ApaLⅠis much different.3.The TK gene of DEV was amplified by PCR and cloned into pMD18-T vector. The expression cassette of EGFP from plasmid pEGFP-C1, including EGFP gene controlled by CMV promoter and enhancer was inserted into the EcoRⅤsite of TK gene to give rise to the transferring vector pTK-EGFP.A 1.9 kb BamHⅠfragment containing US1-US10 gene was used to construct transferring vector. The fragment was cut with MluⅠand NruⅠto delete 583 bp from 3'terminal of US1 ORF to 5'terminal of US10.The EGFP expression cassette was inserted into the MluⅠand NruⅠsite,giving rise to the transferring vector pUS1-EGFP-US10. The US2 gene of DEV was amplified by PCR and cloned into pGM-T vector.The recombinant plasmid was digested by BamHⅠand EcoRⅤ.The expression cassette of EGFP from plasmid pEGFP-C1, including EGFP gene controlled by CMV promoter and enhancer was inserted into the digested recombinant plasmid to give rise to the transferring vector pUS2-EGFP.4.The three transferring vectors with combination of Lipofectamine 2000 were transfected into CEF cells infected DEV VAC strain respectively.All of the transferring vectors could generate recombinant viruses expressing the green fluorescence protein after 3~4 times selection and purification in CEF cells. The results suggest that TK,US1-US10 and US2 genes belong to unessential regions of DEV replicating on CEF cells.