Cloning And Analysis Of Partial Genomic Sequence Of Duck Enteritis Virus

Duck viral enteritis (DVE), or duck plague (DP), was caused by duck enteritis virus (DEV). DVE was an acute , heat and haemorrhagic contagious viral disease that naturally affecting birds of the order Anseriformes (ducks, geese and swans). DEV is one of the members of the family Herpesviridae. Although some research on biologic character of DEV has been done, it was still limited in molecular biology field. The DEV genomic features and physical map have not been elucidated, so the molecular study of DEV has fallen behind those of other herpesviruses. Until now, we only knew the entire open reading frame (ORF) sequences of TK, UL24 and gH gene of DEV, and the partial nucleotide sequences of UL6, UL7 and UL30 gene of the virus. About 90 percent genomic sequence was still unknown. And the basic research of DEV, such as the function of viral proteins encoded by viral genes, virus replication mechanism in host cell and the infection course, and the mechanism of the host against virus infection, was not totally clear. At the same time, we should show emphasis on the prevention and control measure, and on the research of genetically engineered vaccine based on the DEV genome sequence.We took the genomic unknown sequence of DEV Clone-03, purified by plague assay, as the main subject, and took the molecular biology technique as the main method to provide the molecular characteristics of genes of the virus for further research. First, plague assay purification, propagation and PCR identification of DEV; Second, amplification of the unknown gene of DEV by the method of targeted gene walking PCR; Third, the prediction of ORF and sequence analysis of DEV genes; At last, the prokaryotic expression of UL6 gene and the preparation of high immune serum of rabbits against the fusion proteins.We purified a DEV Chinese commercial vaccine strain by plaque test on a monolayer of CEF and the plaque-purified virus was designated DEV clone-03 vaccine (DEV Clone-03)strain, then it was passaged once in 10-day-old SPF chicken embryos by inoculating it onto chorioallantoic membrance. Based on the published partial nucleotide sequence of UL6 gene of DEV LA (Lake Andes) strain on GenBank (Accession Number: AF043730), two primers P1 and P2 were designed. Based on the published partial nucleotide sequence of UL30 gene region of DEV vaccine (DP-VAC) strain on GenBank (Accession Number: AF064639), two primers P11 and P12 were designed. The two pairs of primers were used to detected DEV Clone-03 DNA by using PCR assay. Then the primers P1 and P2 were used to determine the distribution of DEV Clone-03 in chicken embryos. The result revealed that the DEV Clone-03 DNA was found in chorioallantoic membrance, heart, liver, spleen, lung, kidney and muscle skin from the dead chicken embryos infected with DEV Clone-03. The sensitive test of the PCR assay showed that the 10-3 diluted DNAs from 100μl...