Molecular Characteristics And Biological Functions Of Some Genes In Duck Enteritis Virus

Duck viral enteritis (DVE), also known as duck plague, is an acute, heat, haemorrhagic and lethal disease. The disease is caused by duck enteritis virus (DEV), which was epidemic in many countries and regions and caused economic losses. DEV is a member of of 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. The genomic structure of DEV was still unknown due to the molecular biology of the virus lagging behind other herpesvirus, which limited the research on pathogenesis of the virus and control of the disease.According to the genomic sequences of DEV clone-03 submitted in GenBank, we selected the conserved genes of glycoprotein UL27, major capsid protein UL19 and the conserved gene cluster of UL22-TK-UL24 as targeted genes for cloning. Homologues of alphaherpesviruses were used as reference strains to analyze the conserved motifs of five proteins. Multiple alignment showed that UL19 and UL27 were more conserved, however, UL22 showed week level homology. The TK possessed the signature motifs of thymidine kinase: one is ATP binding mogif and the other is nucleoside binding motif. Similar to HSV-1, DEV clone-03 UL24 also contained 5 conserved sites. Protein UL22 and UL27 were typical transmembran glycoprotein, seven N-linked glycosylation sites and six transmemebrane regions were predicted in UL22 protein, and nine N-linke glycosylation sites and three transmemebrane regions existed in UL27 protein. In addition, UL22 protein contained signal peptide sequence, which located at the 1～20 amino acids. Phylogenetic analysis on the five proteins showed that DEV clone-03 had closer relationship with genus Mardivirus but showing a far relationship with Iltovirus. From the tree view of the five proteins, although all five proteins showed closely relationship with genus Mardivrus, each protein formed separate branch. We suggest that DEV should be classified into family Herpesviridae, subfamily Alphaherpesvirinae.To identify the structural genes of DEV clone-03, the UL19, UL27 and UL6 were cloned and expressed in prokaryotic system and the proteins were used to immunize the rabbit to acquire antiserum. Western blot and indirect immunity fluorenscence methods were used to detect the reaction of antiserum to DEV. The result showed that the antiserum of UL19-N, UL19-M and UL19-C could react with DEV and recognized a specific band with molecular weight of approximately 150kDa. The size of specific protein band was consistent with the prediction. Together with the information that DEV UL19 possessed conserved region with the homologues of alphaherpesviruses, we considered UL19 as a structural protein of DEV clone-03. UL19 may function as capsid protein. The UL27 protein could not be detected by Western blot but could be detected by indirected immuno fluorenscence. The result showed that antiserum of UL27-M could react with DEV. We predicted that UL27 was composition of DEV clone-03. We presumed that UL27 may be glycoprotein of DEV because UL27 contained conserved region that existed in other alphaherpesviruses and had the proterties of transmembrane glycoprotein. The antiserum of UL6 could react with DEV clone-03, and a specific protein band with a molecular weight of 88kDa was detected, which was consistent with our prediction. We thought UL6 as a structure protein of DEV clone-03.The antigenic epitopes of DEV UL19 were predicted from the aspect of hydrophilicity, antigenic and surface probability plot by using DNAStar software. The result showed that the amino fragment, middle fragment and carboxyl fragment possessed antigenetic epitopes, so we randomly select the UL19-C fragment to identify antigenic epitopes of DEV clone-03 UL19. The UL19-C protein was taken as antigen to prepare monoclonal antibody. After three times clone, we acquired 4 strains (1B5, 3A1, 4F9, 6F6) stable hybridoma cells. DEV UL19-C was truncated into two overlapped fragment to express in prokaryotic system, we named the amino terminal as UL19-C1 and the carboxyl terminal as UL19-C2. Western blot detection showed that 4 monoclonal antibody strains could not recognize UL19-C2 but UL19-C1. The 4 monoclonal antibody strains were specific for epitopes of UL19-C1. There were antigenic epitopes in 921～1162 amino acid of DEV clone-03 UL19 carboxyl terminal fragment, which could be recognized specifically by 4 monoclonal antibody strains.