Morphogenesis And Ultrastructural Studies In DPV Artificially Infected Ducks And The Discovering And Preliminary Studying Of DPV DUTPase Gene

Duck plague virus (DPV) can cause an acute, septiaemic and contagious disease in the anseriformes including ducks, geese and swans. In duck-producing areas of the world where the disease has been reported, it has produced significant economic losses in domestic ducks and wild waterfowls. Electron microscopy is an important method in studying the viral structures, identifying new viruses, clarifying viral morphogenesis and pathology of host cells. DPV blongs to the large family Herpesviridae which comprises a large sum of enveloped, double stranded DNA virus. Compared with other alphaherpesviruses, the total level of DPV study is much lower. Ducks was artificially infected with DPV CHv strain and studies were carried on the viral morphogenesis and ultrastructural pathology. The apoptosis induced by DPV was also investigated. The dUTPase is a very important enzyme in nucleotide metabolism. The viral dUTPase may play a role in minimizing miscorporation of uracil into the viral DNA and ensure the fidelity of genome replication and enhance vial replication in nondivding host tissues. Viral dUTPase also has potential roles in virulence and viral mutation rate. A 1344bp ORF was found in the DPV gene library constructed in our laboratory and identified as DPV dUTPase gene by bioinformatics analysis. A set of primer was designed based on this fragment and used for PCR amplifying to DPV DNA. Southern hybridization method was used to identify the localization of dUTPase on the DPV genome. The contents was summarized as follows:1. The ultrastruetural changes of artificially infected ducks The ultrastructural changes of ducks being infected experimentally by duck enteritis virus (DPV) CHv strain were observed and analysed using electron microscopic techniques. The investigation showed that the ultrastructural changes appeared in liver and kidney at first. But the most serious changes were in immunological organs and digestive organs. Most of the cells were necrotic, including swollen mitochondria, dilated endoplasmic reticulum, clumped or lysed chromatin. However, most of the lymphocytes were contracted when ducks died, nucleus condensed like a black hole, with homologous and electron dense cytoplasm.2. The morphogenesis of DPV in artificially infected ducks The morphogenesis and distribution of duck enteritis virus (DPV) were observed in the tissues of ducks infected experimentally with DPV virulent strain by electron microscopy. The investigation showed that a few typical herpesvirus virions and nucleocapsids(NC) were observed in the spleen and bursa of fabricus 12 hours post inoculation, and then in the liver, small intestine, spleen, thymus, bursa of Fabricius 24 hours post inoculation. NC were divided into four types according to their structures. The nucleocapsids obtain their tegument in the nucleus and envelope from the inner nuclear membrane, or enter the cytoplasm through the nuclear membrane, obtain their tegument in the cytoplasm, envelope from the plasma membrane, finally virions were released by necrosis, exocytosis or other ways. With the replication, assembly and maturation of DPV, inclusion bodies, compact particles, microtubes, hollow tubes and coated electron dense bodies were observed in infected cells.3. The apoptosis induced by DPV virlent strain The ultrastructural investigation of tissues of ducks being infected experimentally by DPV CHv strain was carried. It shows that on the process of DP, some lymphocytes in spleen, thymus, bursa of fabricius and lamina propria of small intestine were apoptotic. Lymphocytic apoptosis was also identified by in situ terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) and agarose gel electrophoresis method. The results showed that the lymphocytes in thymus, bursa of Fabricius and spleen underwent apoptosis and necrosis at the same time. DEV induced morphological changes characteristic of apoptotic and necrotic cell death since 24 hr aider infection. The apoptosis was also demonstrated by DNA fragmentation of a 200bp ladder and TUNEL positive cells in lymphoid tissues. It was concluded that the apoptosis and necrosis of lymphocytes induced by DEV infection resulted in the depletion of lymphocytes, and the apoptosis of lymphocytes may play an important role in the pathogenesis of duck plague.4. The molecular character of DPV dUTPase gene A combinant plasmid from DPV gene library was sent for sequencing. A complete ORF was discovered and considered as a dUTPase gene by BLASTN and ORF Finder. The amino acid sequence deduced from this ORF was clusted with avian herpesvirus dUTPase from Genbank. By bioinformatics analysis, the DNA fragment was primarily identified as the dUTPase gene of DPV. The full gene was 1344bp in length, and it coded a 447 amino acid multipeptide. Compared with the amino acid sequences with other herpesvirus dUTPase, their phylogenetic tree was drawn. The results indicated that this dUTPase had the high identity withα-herpesvirus dUTPase. Furthermore, the specialized features of the encoded protein, including signal peptide cleavage site, N-glycosylation sites, codon bias, secondary stricture and hydrophobicity were predicted and analyzed.5. DPV dUTPase gene cloning and localization in genome A set of primer was designed based on DPV dUTPase gene and used for amplifying DPV DNA. The amplified fragments were cloned and sequenced. The gene and its protein sequence were accepted by GenBank with accession number DQ486149. DPV genome was extracted and cleaved with restriction enzymes and then transferred to NC Membrane, biotin-labbeled dUTPase gene probe was used to determine the location of dUTPase gene in the DPV genome. The results showed that DPV dUTPase gene located on theⅠfragment of HindⅢand M fragment of SacⅠ.6. The differential PCR based on DPV dUTPase gene The primers DU1 and DU2 were used as detect primers to amplify several other duck pathogens including DPV Cha strain and tissue samples from DPV artificially infected ducks. The results showed that the specific PCR fragment (about 1340bp) was amplified only from DNA of DPV CHv strain, but not form either DPV Cha strain and other avian pathogens. The specific PCR product was also amplified from 15 DNA samples extracted from different tissues of DPV CHv strain artificially infected ducks. The results showed that this PCR method based on DPV dUTPase gene can be applied in the diagnosis and detection of DPV, and it could also be used in differential diagnosis of DPV virulent strain and attenuated strain.