Signal Transduction Pathways Of Apoptosis Induced By Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus In Thymi Of Infected Piglet

Porcine productive and respiratory syndrome(PRRS), caused by PRRS virus(PRRSV), which is an important immunosuppressive disease. PRRS can cause respiratory disorders in piglet and reproductive failure in pregnant sows. In 2006, the highly pathogenic PRRS(HP-PRRS) outbreak in China, which resulted in a large number of pig deaths and caused great economical loss to porcine industry. Compared to the classical PRRSV, HP-PRRSV can lead to a higher mortality rate and more severe inflammatory responses. Our previous studies have demonstrated that HP-PRRSV(HuN4 strain) can cause severe thymus atrophy of piglets, and the atrophy was related to the thymocytes apoptosis. In this study, we further investigated the signal transduction pathways of apoptosis induced by HP-PRRSV in thymi of infected piglet.In order to clarify whether the Caspase-mediated pathways related to the thymocytes apoptosis induced by HP-PRRSV, the changes in mitochondrial membrane potential and thymocytes apoptosis were detected by flow cytometry and immunofluorescence method, respectively. The results showed that the mitochondrial membrane potential depolarized and apoptosis increased significantly on 7 and 10 days after HP-PRRSV infection. Besides, Caspase-3 localization was also detected by immunohistochemical method and Caspase-3 protein increased significantly. These results demonstrated that the thymocytes apoptosis induced by HP-PRRSV is related to the Caspase-mediated pathways.In order to clarify signal transduction mechanisms of apoptosis caused by HP-PRRSV in the infected pigs, the changes of apoptosis-related factors were detected by Real-time qPCR, Western blot and flow cytometry. The results showed that: HP-PRRSV infection caused significant increases in the numbers of cells that can express the two proteins and mRNA of apoptosis-related factor(Fas, FasL) on 7 and 10 d after virus infection, and the other extrinsic-mediated protein(TNF, TNFR and Caspase-8 protein) also increased, which indicated the extrinsic signaling pathway of apoptosis was activated. Besides, the endogenous signal transduction of apoptosis was also investigated. The results showed that Mitochondrial membrane potential changed, mRNA expression of endogenous apoptosis-related gene Bax increased, accompanied by Bcl-2 gene decreased, pro-apoptotic Bax protein increased, anti-apoptotic proteins(Bcl-2) decreased and effector protein Caspase-9 activated. These results demonstrated that endogenous signaling pathway of apoptosis is activated. Finally, the increased and activation of Caspase-3 protein indicated that HP-PRRSV can induce apoptosis via mitochondrial and death receptor pathway in the thymi of infected piglets.