| This scientific dissertation concentrates on studies on some biological characteristics of Duck Enteritis Virus CH virulent isolate and is summarized as follows:1. Studies on adaptation of Duck Enteritis Virus CH virulent strain (DEV-CHv) to duck embryo fibroblast (DEF) for growth. Ten-day-old duck embryos were decapitated and cut into pieces and then trypsinized for 3 minutes at 37℃. The cells were resuspended, dispensed and cultivated in minimum essential medium containing 10% calf bovine serum. DEF monolayer usually was ready for use 36 hours after incubation. DEV-CHv was adapted to growth on DEF monolayer by the following methods: Liver suspension from artificially infected ducks showing mortality was serially passaged in 10-day-old embryonating duck eggs for 2 times through the route of chorioallantoic cavity and the chorioallantoic fluid collected was inoculated onto confluent DEF cell sheet. Cytopathic effects of DEV-CHv initially adapted to grow on DEF cell cultures were characterized by DEF becoming brilliant and round in appearance. And the cytopathic effects of DEV-CHv fully adapted to grow on DEF sheets by six serial passages were characterized by DEF cell sloughing from DEF monolayer and forming characteristic round-shaped plaques on cell sheet. DEF sloughing usually was observed 30 hourspostinfection and distinct plaques were detected 36 hours postinfection and over 80% of the DEF cells in monolayers showed cytopathic effect by 48 hours postinfection. Intranuclear inclusion bodies could be observed in DEF infected with DEV-CHv and polykaryocytes could be found among DEF infected by DEV-CHv.2. Ultrathin sectioning and transmission electron microscopy of infected duck embryo fibroblasts were employed to investigate the morphology of DEV-CHv in DEF. The nucleic acid of DEV-CHv was round in shape with diameter of 35 nm-45 nm and was often in a cluster in the nucleus of DEF. The nucleocapsid of DEV-CHv was round in shape with diameter of 90 nm-100 nm and could be observed both in nucleus and cytoplasm of DEF. Based on their mophological characteristics, DEV-CHv nucleocapsids could be divided into 4 distinct groups which were hollow nucleocapsid, nucleocapsid with several nucleic acid particles closely attached to its inner wall, concentri circle nucleocapsid and infarctate nucleocapsid, of which nucleocapsid with several nucleic acid particles closely attached to its inner wall could be subdivided into nucleocapsids with nucleoids of triangle, quadrangle, pentacle, hexagon respectively. The mature DEV-CHv which had the structures of envelop and tegument was spherical in shape with diameter of 150 nm-300 nm and was usually observed in cytoplasmic vacuoles. Intracytoplasmic inclusion bodies and intranuclear inclusion bodies could be observed respectively in the cytoplasm and nucleus of infected DEF. With the appearance of progeny DEV-CHv in DEF, some densely electron-stained virus-related structures which were rod-shaped, U-shaped, or of circle, semicircle or concentric circle in appearance could be observed in the cytoplasm of DEF. Attached DEV-CHv penetrated the DEF cell by direct fusion between the viral envelop and the plasma membrane. Progeny nucleocapsids assembled in the nucleus and exited from this compartment into the cytoplasm by budding through the inner nuclear leaflet into the interconnected perinuclear cisterna and cisternae of endoplasmic reticulum. DEV tegumentation occurred in cisternae of endoplasmic reticulum and the tegumented progeny nucleocapsids obtained their final envelop by budding into cytoplasmic vesicles. Intravesicular mature DEV-CHv particles were discharged from the cell through exocytosis of the cytoplasmic vesicles by DEF, or through rupture of thevirus-containing vesicles.3. The methods of light microscopy of DEF monolayers processed by hematoxylin-eosin staining and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) respectively, DNA fragmentation analysis, and electron microscopy were employed to evaluate the function of DEV-CHv inducing apoptosis. And the results showed that DEV-CHv-induced cell death exhibited features consistent with a typical form of apoptosis.4. Real-time PCR method was developed for DEV-CHv detection. Real-time PCR primers and TaqMan-MGB probe were designed according to the DEV-CHv nucleotide sequence obtained by sequencing the 498-bp conventional PCR product which was generated by amplifying DEV-CHv DNA template with primers designed according to nucleotide sequence of certain other DEV strain archived in Genbank. The assay protocol was optimized with the reaction volume of 25 ul to include 1 *PCR Buffer, 5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 0.5 umol/L each primers, 0.2 (xmol/L TaqMan-MGB probe, 1 U/25ul Taq, 2 ul DNA template. The real-time PCR cycling consisted of 60 s at 95°C, 50 cycles of 30 s at 94°C and 30 s at 60°C. Standard curve generated by utilizing the 10-fold serial dilution of 498-bp purified PCR product was employed for quantification.5. DEV-CHv growth pattern in DEF was studied by the established real-time PCR method. Studies on DEV-CHv amount in DEF at specific interval indicated that progeny DEV-CHv began to be released into supernatant of MEM cultivation 22 hours postinfection and could attain a 103-fold increase at 50-hour postinfection. Increase of DEV-CHv amount in DEF was prominent at 14-hour, 22-hour, 26-hour and 30-hour postinfection respectively and DEV-CHv amount at 50-hour postinfection was 103-fold higher compared with at 6-hour postinfection; Compared with the MEM culture supernatant, DEF was the main part containing DEV-CHv and the latter was 102-103 higher than the former; Studies concerning effect of DEV-CHv inoculum concentration on DEV-CHv yields indicated that DEV-CHv content in supernatant and DEF cell were in positive relation with inoculum concentration at 14-hour and 26-hour postinfection but not at 38-hour postinfection. Adsorption time of 60 min, 120 min,180 min andinoculum casting or not imposed no effect on DEV-CHv amount in supernant and DEF.6. Characteristics concerning DEV-CHv propagation in tissues of artificially infected ducks were studied by the established real-time PCR method. The results indicated that DEV-CHv did not perform noticeable propagation until 2 days after challenge. Marked high DEV-CHv content observed 2-day and 3-day postinfection in bursa of Fabricius indicated that this organ was an important target in which DEV-CHv replicates. Bursa of Fabricius, esophagus, liver, ileum-cecum conjunction part, spleen, duodenum and lung were specimen examined with high DEV-CHv content and in these specimen DEV-CHv could yield an increase of 105 fold, whereas specimen with low DEV-CHv content such as cerebrum and kidney could yield an increase of 103 fold. A rapid propagation of DEV-CHv could be observed preceding mortality. High content in all the specimen examined indicated that DEV-CHv is of less specific tissue tropism.7. DEV-CHv purification was perfonned and the result indicated that after processing of the cell lysate with the ultrafiltration membrane with nominal pore size of 0.22 um and nominal cut-off molecular weight of 1000 K Daltons respectively, the concentrated DEV fluid was processed by Sepharose 4B chromatography and sucrose density gradient ultracentrifugation with sucrose concentration of 30%, 40%, 50%, 60% (W/W) respectively, highly purified DEV-CHv virions could be obtained.8. The purified DEV-CHv virion was analyzed by the method of SDS-PAGE and the result indicated that 28 proteins with molecular weights of 263, 203, 150, 104, 97, 88, 72, 56, 53, 50, 44, 42, 39, 38, 34, 32, 29, 28, 25, 24, 23, 20, 19, 17, 16, 15, 13, 12 KD respectively were detected, of which 72, 32, 17, 16 and 15 KD proteins were the main ones. Through Western Blot analysis, 4 bands corresponding to proteins with molecular weight of 72, 56, 44 and 34 KD respectively were detected. |
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