Dynamic Relationship Between Inflammation-related Factors And Splenic Lesions In Duckling Infected Duck Reovirus

Duck reovirusis is a disease caused by Duck Reovirus(DRV). The disease is characterized with yellowish-white focal necrosis on the surface of spleen and bursal atrophy. The virus often induce dsecondary infection and cause enormous economic losses in breeding industry. Further research about the mechanism of mammalian splenic injury had been done which indicated that PKR takes an important role in different injuries. During the process of splenic injury, the function of PKR was not recorded and whether PKR participated in splenic injury after DRV infection was also unclea in duck. So it is essential for exploring the function of PKR in splenic injury. In this study, we established an artificial infection model, and researched the fuction of PKR-mediated inflammatory signaling pathways in spleen injury after DRV infection in ducklings which was included the two points. 1.Preparation of DuTNF-α polyclonal antibodyIn this study,we used duckling spleen RNA as the templates, the DuTNF-α gene was amplificated by RT-PCR. DuTNF-α cDNA was cloned into pET-28 a. Recombinant plasmid was harvested after enzyme cleavage by BamHⅠand HindⅢand selected the corrected sequence. Thereafter,recombinant plasmid was transfected into E. coil and induced under the conditions 1mmol/L of IPTG for 4h, 37℃. The DuTNF-α gene was highly expressed. Interest protein purificated by Ni column, and used to immunize rabbit as antigen 3 times. The rabbit-derived antiserum was collected and purified. The results of immunohistochemistry staining and Elisa showed that polyclonal antibodies had better reactogenicity. 2.Establishment of 7-day-old Cherry Valley DRV infection modelThe ducklings were sacrificed and the spleen were collected in 1d, 3d, 5d, 7d, and 14 d post infection. The degree of spleen injury was determined by histopathology. By histopathological and immunohistochemistry staining, the inflammation was detected. The mRNA expression level dynamic changes were detected by technology of RT-qPCR in infected spleen included PKR, MKK6, AP-1, IPS-1, NF-κB, TNF-α and IL-6. Spleen paraffin sections of 5 time points in experimental and control group were selected for immunohistochemistry, 3 repetitions in each group. Using digital slice scanner for slice scan and the results was standed by a basis of the positive rate per unit area.In gross pathology, noticeable splenomegaly and dark red to tan in some areas were observed during 1th and 3th day post infected. But the spleen returned to normal colour, small to large yellow-white lesions on the surface were seen in 5th day post infection. In histopathology, severe splenic and white pulp lymphocytopenia in 3 days post infectied, focal necrosis well demarcated in 5th day post infected were obvious. The granuloma structures were seen in spleen 7th day post infection. Base on the Opriessning T standard, the result of spleen histopathologic evaluation was that severe hemorrhage in 3d, necrosis in 5d, the formation of granulomas structure in 7d post infection. Immunohistochemistry with specific inflammatory cells antibodies and anti-DRV antibodies indicated that spleen developed severe inflammation. The expression of related factors in PKR mediated inflammatory pathways were detected by fluorescent quantitation. In 1th, 3th and 5th day post infection, PKR was up-regulation, reached peak in 5th day post infection. Compared to the control group, significant change(P<0.05) occurred in 7th, 14 th and 5th day post infection. It was positively correlated with the expression of DRV. The correlation in PKR and DRV showed that PKR might be participatde in the ds RNA identification which activated downstream factor result in splenic lesions. In all the infective stage, MKK6, AP-1 and IPS-1 were not up-regulation, but the NF-κB mRNA was up-regulation all the time. The phenomenon indicated that PKR participated distinguish the virus and activated NF-κB transcription into nuclear launch related factor. Downstream inflammatory cytokines TNF-α expression is consistent with the trend of NF-κB. In 3d post infection, IL-6 was up-regulated and to peak in 7d which indicated TNF-α and IL-6 participate spleen inflammation. Immunohistochemistry statistical showed that TNF-α protein was up-regulated and to peak in 5 day post infection.It was consistented with fluorescence quantitative results.Conclusion: When DRV infected, the structure and function of the spleen were affected after ducklings DRV infection. The spleen PKR inflammation signaling pathway was activated and the activation state was associated with degree of splenic injury. Inflammatory cytokines TNF-α, IL-6 were significantly up-regulated which indicated PKR inflammatory signaling pathways and TNF-α, IL-6 expression were participation in splenic injury post DRV infection.