Autophagy Inhibitors Reduce Avian Reovirus-mediated Apoptosis In Cultured Cells And In Chicken Embryos

Avian reovirus infection cause arthritis and tenosynovitis in chicken and turkey. Meanwhile, ARV infection can also cause immune suppression, resulting in heavy losses for the poultry industry. Some researches have demonstrated that ARV can induce autophagy and apoptosis in infected cells. Furthermore, apoptosis has a role in boosting the incidence of chickens. However, methods for effectively reducing ARV-triggered apoptosis remain to be explored. This study was designed to explore the effection of autophagy inhibitor CQ and E64d +pepstatinA on ARV-induced apoptosis.First of all, we detected the effect of two autophagy inhibitors on ARV-induced cell growth inhibition by MTT assay. The result showed that ARV induced growth inhibition of DF1 cells was significantly reduced by the two autophagy inhibitors. Then morphological observation showed that ARV infection led to obvious cytopathic effect in DF1 cells, both CQand E64d plus pepstatin A dramatically reduced the ARV-induced cytopathic effectFinally, we performed FACS analysis using an annexin-V/PI double staining assay to determine ARV induced apoptosis in DF1 cells in the presence or absence of these compounds. The result showed that ARV infection-induced apoptosis was robustly inhibited in the presence of either CQ or E64d plus pepstatin A.The cleaved caspase-3 usually be identified as a hallmark of apoptosis. We detected caspase-3 and cleaved caspase-3 of ARV infected DF1 cells in the presence or absence of the two autophagy inhibitors by western blot. The level of cleaved caspase-3 significantly reduced when pretreated with CQ or E64d plus pepstatin A in ARV infected DF1 cells. The expression of ARV capsid protein σC was also detected by western blot and the viral titer detected by TCID50 assay. The level of viral replication significantly reduced in the presence of these compounds.Apoptosis is a crucial strategy utilized by which ARV exerts its pathological effects in infected birds. Given that the inhibition of autophagy can decrease ARV-induced apoptosis in cultured cells, we next examined whether the ARV-induced death of chicken embryos would be affected by autophagy inhibitors.11-day old SPF chicken embryos were pretreated with either CQ or E64d plus pepstatin A or vehicle. These chicken embryos were then inoculated with ARV (107TCID50/100μl) or sham-infected. Determination of the virus yield from the fluids of the chicken embryos revealed that both CQ or E64d plus pepstatin A treatment decreased virus production in ARV-infected embryos compared to treatment with virus alone.Attenuated cleavage of caspase-3 were detected in the bursas, hearts and intestines of the ARV-infected chicken embryos that were pretreated with CQ or E64d plus pepstatin A compared to virus infection alone. Survival curves showed that chick embryos mortality statistics significantly reduced after pretreated with CQ or E64d plus pepstatin A.This research demonstrates that based on the inhibition of autophagy, CQ or E64d plus pepstatin A can significantly inhibit the ARV(GX/2010/1 strain) induced apoptosis and reduced the level of viral replication. For chick embryo, the mortality statistics reduced by ARV significantly reduced when pretreated with the two autophagy inhibitors.