Sequencing And Analysis Of Duck Virus Enteritis New Gene 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. Plague assay purification, propagation and PCR identification of DEV, amplification of the unknown gene of DEV by the Improved method of targeted gene walking PCR; the prediction of ORF and sequence analysis of DEV genes by long ORF and Phylogenetic methods.The results showed that it was feasible to amplify the unknown sequence of DNA viral genome by the method. The sequences of two nucleotide fragments were chosen as the start point for amplification. One of the fragments is sited at UL(Unique long), with the sequences of 28 240bp , this region contained seven ORFs homogous to Herpes simplex virus 1(HSV-1)UL8 protein, UL9protein, UL10 protein, UL11 protein, UL12protein, UL13 protein, UL14 protein, UL15protein, UL16 protein, UL17protein, UL18protein, UL19 protein, UL20 protein and UL21 protein respectively. So we designated them as UL8, UL9, UL10, UL11, UL12, UL13, UL14, UL15, UL16, UL17, UL18, UL19, UL20 and UL21 homolog gene of HSV-1 of DEV Clone-03 and the sizes were 2 271 bp,2 301 bp,1 170 bp,264 bp,1 341bp,1 575 bp,1 142 bp,1 947 bp,1 089 bp,2 055 bp,912bp,4 143 bp,822 bp,1 686 bp respectively. The corresponding amino acid sizes encoded by above genes were 756,766,389,87,446,524,383,648,362,684,303,1 380,273和561 aa respectively.US (Unique short)was chosen as the second start point to amplify the unknown gene, and a 3 581 bp fragment was acquired. Seven ORFs were predicted, they showed a high homology with US1 protein, US10 protein and SORF3 protein of MDV-1, so we designated them as US1, US10 and SORF3 homolog gene of MDV-1 of DEV Clone-03. The nucleotide sizes of the seven ORFs were 990bp,507bp and 888 bp respectively, and the amino acid sizes were 329,168 and 295. All above genes except UL21 accorded with their predicted sizes, respectively.The arrangements of UL8-UL21genes are relatively conserved in herpesvirus genome especially in alphaherpesvirus genome. The arrangement of UL8-UL21 genes of DEV Clone-03 was similar with that of HSV-1 and Marek's disease herpesvirus 1(MDV-1), and the DEV Clone-03 US1-SORF3genes were also equivalent in arrangement to the Equid herpesvirus 1(EHV-1)and Marek's disease herpesvirus 2(MDV-2)homologous genes. So it is possible that DEV is similar to the majority of herpesviruses in the genome region arrangement.Sequence analysis of the 17 genes between DEV Clone-03 and reference herpesvirus strains showed that DEV Clone-03 was higher homologous to alphaherpesviruses than to betaherpesviruses or gammaherpesviruses. Amino acid phylogenetic analysis of the 17genes showed that DEV Clone-03 clustered in the subfamily Alphaherpesvirinae, and DEV Clone-03 was assigned to the subfamily Alphaherpesvirinae and was more similar to MDV. Probably, DEV will be classified as the genus Mardivirus within subfamily Alphaherpesvirinae in the future. Certainly, more molecular data about DEV is essential for further studying its classification and virulence.In conclusion, the method of the targeted gene walking PCR and fragment length amplified PCR were used to amplify 28 240 bp sequence of the genome of DEV Clone-03 in this study. It was the first report of 17 complete gene ORFs of DEV, which provided the basis for the research of DEV genomic structure and showed information for the pathogenicity and classification of DEV. The nucleotide sequences of the 17 genes were very important for the identification of the genes, the constitution of the products of the genes and the elucidation of the functions of the corresponding proteins. This study provided molecular biology proof for the establishment of diagnosis method and the research of genetically engineered vaccine in order to prevent and control DVE. All ORF sequence of the gene will be identified, the gene product of the composition and functions is important to clarify the meaning, especially UL15, UL16, UL17, UL18 and UL19 nucleocapsid Genome Research found duck plague virus nucleocapsid laid the foundation for the structure. The section of the virus genome research and application of biometrics in this section of the sequence from different angles and the same level of analysis, gene from the perspective of duck plague virus in the herpes virus genetic evolution, the possibility of duck plague virus based on the classification of duck plague virus and in-depth study to lay a solid foundation.