Biological Characteristics Of Duck Viral Swollenhead Hemorrhagic Disease Virus Virulent Strain

Duck viral swollenhead hemorrhagic disease (DVSHD) is an acute infection disease in ducks which caused by Duck viral swollenhead hemorrhagic disease virus (DSHDV), The typical clinical symptom includes swollen head, conjunctiva hyperaemia or hemorrhage, yellow and swollen liver with hemorrhage, infected duck's body temperature being over 43℃and diarrhea with green dejecta. It has the high morbidity and mortality, all ages and varietal of ducks can be infected, DVSHD has produced significant economic losses in duckling-producing areas in China. Based on the works of epidemiologic investigation, observations of clinical signs and pathological lesions, agents isolated and identified, experimental infections, examination by transmission electron microscope, cross neutralization test, the agent was identified as Orthoreo virus. Only little information was available at present.The biological characteristics and immunogenicity of DSHDV were study in our research, the results were summarized as follows:1. The adaptation of DSHDV in duck embryo fibroblasts and the multiplication characteristic of DSHDV. After the ducklings were artificial challenged by DSHDV, the same typical clinical symptom appeared with the reports before. The rejuvenesced virus could proliferate in 10-days-old duck embryon. The virus in allantoic fluid was inoculated on the duck embryo fibroblast (DEF) cells, after 2 passages onwards, DSHDV could adapt to DEF. After 5 passages, it could be totally adapted, the clear and consistent cytopathic effect (CPE) could be observed at 72 h post infection (PI). The TCID50 of DSHDV increased with the passage times and arrived the stable level(106.55-106.91/ml) at the fifth passage. In one infection period the virus titers declined straightly during Oh-8h PI and increased rapidlly during 8 h-72 h PI, the maximum virus titers appeared during 72 h-120 h PI which was the best time for harvesting.2. The morphogenesis of DSHDV and the characteristic ultra-structural changes of infected DEF cells. The typical DSHDV particles were round or oval in shape with a pyknotic core packaged into the double shells, the average diameter ranged from 75 nm to 85 nm. The virion presented only in the cytoplasm of the infected DEF cells. The virion absorbed the membrane of DEF and penetrated, uncoated, replicated and matured in the cytoplasm, and they were finally released to the extracellular space via the disruption of the cytoplasmic membrane. The infected cells showed the nucleus deformation, chromatin gathering, the mitochondria and rough endoplasmic reticulum severity extension. The syncytia and apoptotic cells as well as viral inclusion body all appeared in DEF cells during the infection.3. The apoptosis of DEF cells induced by DSHDV virulent strain. (1)The results of light microscopy and transmission electron microscopy showed that DSHDV could induce the infected cells undergoing a series of characteristic apoptotic morphological changes after infection, such as cytoplasmic condensation, nuclear deformation, chromatin condensed and marginalization, endoplasmic reticulum and mitochondrium vacuolization, a large number of shapes of apoptotic bodies appeared. (2)The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. (3)AnnexinV FITC/PI staining of infected cells were examined by flow cytometry analysis. The percentage of apoptotic cells increased with the incubation time and reached the maximum at 96 h PI and then decreased later. (4)The positive signal of apoptotic cells were also observed under fluorescence microscope.4. The purification of DSHDV and the morphology of DSHDV particles. After the comparative study on purifying DSHDV, the pure virions can be obtained through these steps:freeze thawing repeatedly, differential centrifugation, PEG6000 precipitation,30% sucrose cushion ultra-centrifugation and cesium chloride (CsCl) discontinuous density gradient ultra-centrifugation. The results of negative staining showed that the complete DSHDV particle was globular in shape with diameter about 80 nm-85 nm, had non-enveloped capsid composed with capsomeres.5. Study on the virus structural proteins of DSHDV. The antigen including purified DSHDV virions was injected into rabbit, the rabbit anti-DSHDV polyclonal serum was prepared successfully, the rabbit anti-DSHDV IgG were extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. The results of protein denaturing gel electrophoresis showed that the complete DSHDV was composed with 10 virus structural proteins(VP), named VP1,VP2,VP3... VP10 (MW:151,129,115,90,76,66,56,52,39,35kDa). Results of immunoblotting demonstrated that virus structural proteins related to immunological activity were VP2,VP4,VP5 (MW:129,90,76kDa).