| Duck hepatitis virus1is worldwide distributed picornavirus that causes acute, rapidly spreading and highly fatal disease in young ducklings, characterized primarily by hepatitis. The disease can cause mortality up to95%in7-day old ducklings and economic lost to all duck raising farms because of the high mortality. At present, researeh of duck hepatitis mainly focus on genome sequeneing and diagnosis and controling of DHV-1. There have been, however, comparatively few research on the pathogenic mechanism of duck hepatitis and the host response mechanism has not been reported.In order to investigate the role of apoptosis in duck viral hepatitis pathogenesis,4-day(4D)and21-day old(21D)ducklings were inoculated with DHV-1, and sacrificed postinfection (p.i.). Expression profiles of apoptosis related genes including caspase-3,8,9and Bcl-2in liver, kidney, spleen and Bursa of Fabricius were examined and the quantity of virus in blood was analyzed by using real-time PCR. In addition, terminal transferase end labeling (TUNEL) was performed to detect the apoptotic cell. There are three parts in the thesis.(1) Based on the analysis of quantitative PCR, the amount of virus was tested in blood of the4D and21D ducklings group. The amount of virus increased and reached the peak at24h p.i. followed with the change of apoptotic hepatocyte’percentage in4D ducklings. The maximum amount of the virus in the blood was1063viral genome equivalents at24h p.i.. After infection, virus in blood of21D ducklings increased from2to48h p.i.(2) Expression profiles of apoptosis related genes including Caspase-3,8,9and Bcl-2in different tissues of the4D ducklings were examined by using real-time PCR. In spleen of4D ducklings group, the transcription of Bcl-2was increased firstly, then decreased from12h p.i. and expression of Caspase-8and Caspase-3was increased. In Bursa of Fabricius (BF), the transcription of Caspase-9was almost unchanged at all the time points after infection. The expression of Caspase-3and Caspase-8were drown-regulate, while Bcl-2was induced and reached a peak at12h p.i.. In kidney Caspase-9,3,8and Bcl-2were almost suppressed at all the time points except for a slight increase at48h p.i. when compared to the controls. As for the transcription of Caspase-3,8,9, there were almost no significant increases in liver infected compared to the controls, while transcription of Bcl-2decreased.(3) Expression profiles of apoptosis related genes in different tissues of the21D ducklings were examined with real-time PCR. In spleen and Bursa of Fabricius of infected group, the expression of Caspase-3, Caspase-8and Bcl-2was increased significantly and reached a peak at12h p.i., and then decreased to the normal value at48h p.i.. The transcription of Bcl-2and Caspase-3was induced at6h p.i. and the expression of Caspase-8and Caspase-9was not changed in liver. In kidney the expression of Bcl-2was increased and reached a highest value at24h p.i. and expression of Caspase-3decreased to the normal level. As for the transcription of Caspase-8and Caspase-9, there was on significantly changes.(4) In4D ducklings group, TUNEL analysis demonstrated that apoptosis rate in liver increased significantly compared with controls from2h p.i., except for a puniness rebound at first6h and12h p.i. In spleen, apoptosis rate of infected group was higher compared to control group from6h p.i., significant at6h p.i. and12h p.i. In21D ducks group, there was a decreased trend in liver from2h p.i., and no significant difference was observed between infected and control group except for2and12h p.i.. In spleen, the percentage of apoptotic cells was higher from6to48h p.i., and significantly at6h and24h p.i. compared to the controls, but significantly lower at12h p.i. At other time points, there were no obvious differences between the treatments and the controls.In summary, our study showed that duck hepatitis virus induced apoptosis of liver, spleen and BF in4D ducklings, and promoted the spread of the virus, while apoptosis was not significant in21D ducklings. |
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