Identification Of The Types Of Thymocyte Death Induced By HP-PRRSV And Activation Of JAK/STAT Pathway

Porcine reproductive and respiratory syndrome(PRRS)is an immunosuppressive infectious disease caused by PRRS virus(PRRSV).It is characterized by severe reproductive failure in sows as well as pneumonia.In addition,it has been considered as an increased risk of secondary bacterial infections in piglets and growing pigs.Since the first outbreak of highly pathogenic PRRS in China in 2006,HP-PRRS has caused huge economic losses in swine industry.It is generally believed that PRRSV induces lesions in the immune organs of piglets leading to severe host immunosuppression.Compared with classical PRRS,HP-PRRS is characterized by high morbidity,high mortality,high body temperature,and mixed or secondary infection.In 2010,we reported that the infection of weaned piglets by HP-PRRSV(HuN4 strain)showed signidicant atrophy in thymus and the degree of atrophy was as high as 90%.Furthermore,the mechanisms of thymus lesions induced by HP-PRRSV HuN4 were analyzed.We confirmed that the thymic atrophy was associated with cell apoptosis and,interestingly,most of the apoptotic cells were identified as non-virus infected cells.In this study,we will further investigate the mechanisms of thymocytes reduction induced by HP-PRRSV infection,and a primary thymus cells culture system(PTCS)in vitro will be used to identify the type of cell death and the mechanisms of activation of JAK/STAT signaling pathway.Our results will provide a theoretical basis for further elucidation the mechanisms of HP-PRRSV-induced immune suppression and the immune response against HP-PRRSV.The thymus of 21-day-old SPF pigletwas selected for preparing the PTCS that was applied to identify the types of thymocyte death induced by HP-PRRSV and mechanisms of activation of JAK/STAT pathway.The morphology of PTCS was observed by electron microscopyat different time point post infection.The results showed that the type of cell death was consistent with the characterization of cell apoptosis and a large number of apoptotic cells were detected at 40 hpi,which means that apoptosis is responsible for the death of thymocytes induced by HP-PRRSV in PTCS.It has been confirmed that HP-PRRSV infection can cause the amounts of STAT-1 protein,that exists in the JAK/STAT classical pathway,changes in the HP-PRRSV infected thymus significantly.Therefore,the effect of HP-PRRSV infection on JAK/STAT signaling pathway in PTCS were analyzed in this study.Firstly,we found out that the infection of HP-PRRSV was inhibited in PTCS,when the cells were pretreated with IFN-?.The changes of STAT-1 protein after HP-PRRSV infection was detected by wetern blot,and the results showed that the expression of STAT-1 protein was up-regulated by HP-PRRSV infection by activating the JAK/STAT pathway.It has been reported that the JAK/STAT can be activated through two pathways:one is through the ubiquitinated proteasome pathway and the other is through the autophagosome lysosome pathway.As known,MG132 is an inhibitor of ubiquitination and 3-MA has the effect of inhibiting autophagy.Therefore,in this study,thesetwo drugs were applied to analyze the JAK/STAT pathway in PTCS infected by HP-PRRSV and the amounts of related protein changes were detected by Western blot.The results showed that the amounts of STAT-1 protein in cells treated with MG132 was significantly increased when compared with control group,but no significantly difference can be detectedin the case of 3-MA treatment group.These results indicated that HP-PRRSV infection activated the JAK/STAT signaling pathway through the ubiquitination pathway.In conclusion,these results demonstrated that HP-PRRSV infection can induce apoptosis that was activated the JAK/STAT signaling pathway via ubiquitination pathway and was responsible for the death of thymocytes.