Biological Characteristics Of New Type Gosling Viral Enteritis Virus Virulent Strain

New type gosling viral enteritis (NGVE) is an infectious disease in goslings less than 30 days of age, which caused by a DNA virus, named new type gosling viral enteritis virus (NGVEV), belonged to the adenovirus genus. In gosling-producing areas where the disease has been reported, NGVE has produced significant economic losses in the offspring of breeder geese due to high mortality. Few information was available so far for goose adenovirus, especially for pathogenicite goose adenovirus.Electron microscopy is an important method in studying the viral structures, identifying new viruses, clarifying viral morphogenesis, and pathology of host cells. The morphogenesis of NGVEV in duck embryo in vivo and duck embryo fibroblast (DEF) cells in vitro, as well as, the ultrastructural changes of host cells were observed and analysed using electron microscopic techniques (TEM). The apoptosis induced by NGVEV was also investigated.Goslings were experimentally infected with NGVEV CN virulent strain and sampled at a series of time points. Then an indirect immunoperoxidase staining and an indirect fluorescent antibody (IFA) assay were established and used to determine the histological localization of NGVEV in conventional paraformaldehyde-fixed paraffin-embedded gosling tissue post experimental infection. The apoptosis induced by NGVEV in experimentally infected goslings was further reported.Dynamic changes of the humoral and the cellular immunity response elicited by the inactivated-NGVEV vaccine with or without recombinant goose interleukin-2 (GoIL-2) adjuvant in the adult geese post vaccination (PV) were investigated by enzyme-linked immunosorbent assay (ELISA), virus neutralization antibody test (VNT) and lymphocyte proliferation assay. An assessment of the performance of the inactivated NGVEV vaccine with or without GoIL-2 djuvant following subcutaneous inoculation to breeder geese before the egg production period is reported. The contents were summarized as follows:1. The morphogenesis of NGVEV in duck embryo and the ultrastructural changes of host cells. NGVEV particles could be found in the chorioallantois, intestine, heart, liver, brain and gizzard at the different infection times. The virus particles were round in shape with diameter of 70 nm-80 nm. The virion entered the cell by attaching to cytoplasmic membrane, then replicated and matured in the nucleus. Finally, NGVEV virions were released into extracellular space through the disruption of cytoplasmic membrane. Rough endoplasmic reticulum (RER) was expanded heavily during infection. The mitochondria of chorioallantois epithelia were ultra-condensed and aggregated into compact clusters, while mitochondria in other cells were inflated and disaggregated. NGVEV could induce host cells of duck embryo undergoing apoptosis.2. The adaptation of NGVEV in duck embryo fibroblasts and the multiplication characteristic of NGVEV. NGVEV was propagated by allantoic cavity inoculation in 10-days-old duck embryonated eggs, then adapted to the duck embryo fibroblast (DEF) cells. From the fifth passages onwards, NGVEV was totally adapted to DEF, the clear and consistent cytopathic effect (CPE) were observed from 72 h post infection (PI). The TCID50 of NGVEV arrived the highest level at the fifth passage, and the maximum virus titer were reached during 96 h to 144 h PI.3. The morphogenesis of NGVEV and the characteristic ultra-structural changes of infected DEF cells. The typical NGVEV particles were round, with a diameter ranging from 75 nm to 90 nm, and they were presented both in the nucleus and the cytoplasm of the infected DEF cells. The mature virion contained nucleocapsid and nucleic acid. The virion penetrated the DEF, then replicated and matured in the nucleus, and they were finally released to the extracellular space via the budding and the disruption of the cytoplasmic membrane. With the appearance of progeny NGVEV, certain virus-related structures with densely electron stained, which were circular, U-shaped, or irregular in appearance, could be observed in the cytoplasm of infected DEF cells. Three types of intracytoplasmic inclusion bodies were observed. The mitochondria were ultracondensed and aggregated into compact clusters were showed. Apoptotic cells were detected during the NGVEV infection.4. The apoptosis of DEF cells induced by NGVEV CN virulent strain. The monolayer DEF cells were experimentally infected with NGVEV and the dynamic changes of apoptosis were detected at different infection time points by light microscopy and transmission electron microscopy (TEM). The results showed that NGVEV could induce the infected cells undergoing apoptosis and changing regularly. A series of characteristic apoptotic morphological changes including shrinkage of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies were observed. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. Annexin V-FITC/PI staining of infected cells were examined by flow cytometry analysis and fluorescence microscope. The percentage of apoptotic cells was found increase with the incubation time until reaching the maximum at 120 h PI and the positive signal of apoptotic cells were observed.5. The efficient method for purifying NGVEV, the morphology of NGVEV particles and the preparation of rabbit anti-NGVEV polyclonal serum. The conventional differential centrifugation to remove debris after cell disruption, chloroform extraction to remove the none NGVEV-protein debris, cesium chloride(CsCl) density gradient ultra-centrifugation for virus separation, and conventional differential ultracentrifugation for virus concentration were used to purify NGVEV. After that the complete and the incomplete NGVEV particles were separated. The complete NGVEV particle, was round in shape with diameter about 75 nm-90 nm, had non-enveloped capsid composed with capsomeres. Rabbit anti-NGVEV polyclonal serum was prepared, then extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography.6. Detection and localization of NGVEV in paraffin-embedded experimentally infected gosling tissues sections by indirect immunoperoxidase staining and indirect immunofluorescent assay. In order to detailed definition of the tropism of NGVEV in conventional paraformaldehyde-fixed paraffin-embedded gosling tissue sections, an SP assay and an IFA were established and optimized. NGVEV antigen could be detected in the gastrointestinal tract and lymphoid organs as early as hours 48 PI. The lymphoid organs (bursa of Fabricus, thymus, Harderian gland and spleen), the digestive organs (proventriculus, gizzard, duodenum, jejunum, ileum, cecum, rectum and liver), and kidney of infected goslings showed extensive evidence of viral antigens appearance. The lymphocytes, macrophages, mesenchymes, endothelial cells, epithelia, superficial and crypt mucosal cells, glandular cells, fibrocytes served as the principal site for NGVEV localization. The number and the intensity of positive signal increased with the infection time until 15 dPI.7. A preliminary study on apoptosis induced by NGVEV in vivo. After goslings orally inoculated with CN virulent strain of NGVEV, the apoptotic morphological changes of the internal tissues were evaluated at different infection times by performing histological analysis through light microscope observation and ultra-structural analysis through transmission electron microscope (TEM) observation, as well as, the DNA fragmentation were detected by DNA ladder analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing and formation of apoptotic bodies were observed by light microscope and TEM. Apoptotic cells were widely detected in the lymphoid organs and the digestive organs, and sporadic appeared in others organs. Many kinds of host cells undergoing apoptosis, including lymphocytes, macrophages, monocytes, endothelial cells, epithelia and intestinal cells. The number of the apoptotic cells was increased with infection times up to day 12 PI.8. The immunogenicity of inactivated-NGVEV vaccine with or without GoIL-2 adjuvant. The inactivated-NGVEV vaccine could elicit the strong humoral and cellular response in the vaccinated adult geese. The absorbance values of specific anti-NGVEV antibody, the neutralization antibody titer and the lymphocyte proliferation index (LPI) were rapidly increased and peaked at about 28 days post vaccination (PV), after that, which remained at a plateau, then had a slight decrease. The GoIL-2 adjuvant could enhance immune response, which in conjunction with inactivated-NGVEV vaccine could induce the significant higher specific anti-NGVEV antibody absorbance value, the neutralization antibody titer and the LPI than the results of none-adjuvant inactivated-NGVEV vaccine (P < 0.05). The inactivated-NGVEV vaccine (0.5 ml/per goose) with 1000 U/per goose GoIL-2 adjuvant was considered as the most effective dose.9. The protective efficacy of inactivated-NGVEV vaccine in the breeder geese. Following one application, the progeny of the inactivated-NGVEV vaccine with or without GoIL-2 adjuvant vaccinated geese showed sufficient levels of protective efficacy against NGVEV challenged during almostly six month. No clinical sign and abnormal of health status was observed in inactivated-NGVEV vaccine vaccinated breeder geese and its progeny, whereas severe NGVE lesions were observed in the dead goslings of the control group following laboratory challenge, which was affirmed by histopathological examine and IFA detection. A high correlation was observed between the NGVEV antibody titers of breeder geese serum and the maternally-derived NGVEV antibody titers of progeny yolk, which suggested that the detection of specific IgG concentration in the progeny yolk could be used as a means of antibody monitoring in commercial breeder flocks instead of serum and could be less time-consuming. These results showed that vaccination of breeder geese with an inactivated-NGVEV vaccine before the egg production period could be a safety and effective means to control of NGVE disease in the progeny goslings, moreover, the inactivated-NGVEV vaccine with GoIL-2 adjuvant was suggested more effective.