| 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), which was epidemic in many countries and regions and caused economic losses. DEV is one of the members of the family Herpesviridae. According to the report of Eighth International Committee on Taxonomy of Virus (ICTV), DEV was assigned as member of family Herpesviridae, unclassified virus. 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, which limited the research on pathogenesis of the virus and prevention and control of the disease.We took the genomic unknown sequence of DEV Clone-03, took the molecular biology technique, basing on the published partial nucleotide sequence of UL35, UL45, UL50 and SORF3 gene of DEV Clone-03 strain on GenBank designed primers. We amplified unknown gene of DEV by the method of targeted gene walking PCR; predicted ORF and analysis the sequence of DEV using biological softwire.The sequence was determined through three times sequenced, 33 020bp sequence of DEV Clone-03 was amplified. 29 914bp was amplified in UL region, 3106bp was amplified in US region. Homology analysis using Genrunner and BLAST, eight ORFs were predicted in UL (Unique long) region and two ORFs were predicted in US (Unique short) region. They showed a high homology with UL36 protein, UL43 protein, UL44 protein, UL51 protein, UL52 protein, UL53 protein, UL54 protein , UL55 protein, US2 protein and US3 protein of Herpes simplex virus 1 (HSV-1), and the arrangement of the ORFs was similar with that of HSV-1 and Marek's disease herpesvirus 1(MDV-1). So we designated them as DEV Clone-03 UL36, UL43, UL44, UL51, UL52, UL53, UL54, UL55 US2 and US3 homolog gene of HSV-1. aliment Associate with the sequences of DEV amplified in our laboratory in the past. It is possible that DEV is similar to the majority of Marek's disease herpesvirus in the UL genome region arrangement, especially in Marek's disease herpesvirus 1(MDV-1). Multiple alignment and amino acid phylogenetic analysis of the DEV Clone-03 10 ORFs with 11αherpes virus strains with DNAMAN and DNAStar (Megalign) software. Multiple alignment showed DEV Clone-03 UL51, UL52, UL53, UL54 gene contained consensus motifs similar with Alphaherpesviruses, especially with HSV-1 and MDV-1. Amino acid phylogenetic analysis showed that DEV Clone-03 clustered in the subfamily Alphaherpesvirinae and more similar to Mardivirus.We chose DEV Clone-03 UL46 and UL49 gene according to the DEV Clone-03 sequences which have been submitted. 1 110bp in 3′of DEV Clone-03 UL46 (UL46-C) and 762bp of UL49 gene were expressed in E.coli. The expressed products were purified by metal (Ni2+) chelation affinity chromatography, the purified His-tagged UL46-C and His-tagged UL49 fusion proteins were received. Western blot detected using rabbits anti-DEV serum showed UL46-C protein had a weak reaction, but UL49 protein had a strong reaction with antiserum. The method of the targeted gene walking PCR was used to amplify 33020bp sequence of the genome of DEV Clone-03 in this study. The result showed that it was feasible to amplify the unknown sequence of DNA viral genome by the method. 10 ORFs were obtained and 7 of them were the first report gene ORFs of DEV, which provided the basis for the research of DEV genomic structure. The expression of partial gene of DEV Clone-03 UL46 (UL46-C) and UL49 gene provided a basic work for gene function research in DEV. Our results 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. At the same time, the results showed information for the pathogenicity and classification of DEV. |
PDF Full Text Request