Avian Reovirus Infection Induces Autophagy In SPF Chickens And Its Relationship With Viral Replication

Avian reovirus (ARV) is one of the important pathogens, and its infection causes viral arthritis, tenosynovitis, and induce immunosuppression in chicken and turkey, resulting in significant economic losses for poultry industry. Our previous study has confirmed that ARV infection can induce autophagy which is conductive to viral replication in infected cells, and associated with the pathogenicity of ARV, but whether ARV infection is capable of inducing autophagy which facilitates viral replication in vivo, still need further validation.Microtubule associated protein 1 light chain 3 (microtubule-associated protein 1 light chain 3, LC3/Atg8) is autophagy marker proteins on the membranes of the autophagosome and its expression level in some extent reflects the number of autophagosome. First of all,1 d SPF chickens were infected with ARV strain GX/2010/1, and set up a blank and positive control group, then collected organs for the detection of LC3 to value that whether ARV infection could induce autophagy in vivo. The study revealed that the conversion of LC3-Ⅰ to LC3-Ⅱ occurred in heart, liver, spleen, kidney, caecum, bursa, thymus of ARV-infected chickens and in heart, caecum and bursa was apparent. To further validate these results, we observed heart, caecum and bursa in ARV-infected chicken by transmission electron microscope and found that many typical structures of autophagy could be seen in ARV-infected chicken tissues such as degenerative mitochondria and two limiting membranes of the autophagosome and so on. Altogether, it indicated that ARV infection could induce autophagy in SPF chickens.Then, chickens were treated with PBS, starvation, ARV (GX/2010/1) for 48h,72h,96h, and 120h, respectively. Hearts, caecum and bursas were collected to detect the LC3 conversion. The results shown that the ratio of LC3-Ⅱ/β-actin in heart, caecum and bursa of ARV-infected chickens grown incrementally over time and markedly increased at 72h or 96h, but decreased after 96h or 120h, respectively. Then, we used the tissues for detecting viral titer by TCID50 assay at the same time. It showed that, regardless of the heart, caecum or bursa, the virus yields were significantly increased at 72h in ARV-infected chickens and then decreased. This suggested that a certain time dependence between viral replication and autophagy induced by ARV infection. It was not only capable of inducing autophagy, and could utilize autophagy to promote transient but obvious virus production.Last, 1d SPF chickens were pretreated with CQ or vehicle and then inoculated with ARV or sham-infected. Hearts, caecum and bursa were collected from chicken of all groups at 96 h, and the extracted proteins from these organs were analyzed for the accumulation of LC3-II by western blot and viral titer by TCID50 assay. We examined the marked LC3-II protein accumulation in heart, and little accumulation of LC3-Ⅱ in caecum and bursa of the ARV-infected chickens compared with sham-infected chickens, indicating that CQ could inhibit autolysosome formation as evidenced by the increased LC3II accumulation in tissues of ARV-infected chickens. Further detected the viral titer and the results shown that CQ treatment decreased virus production of heart and caecum in ARV-infected chickens compared to treatment with virus alone, but this phenomenon was not obvious in bursa, suggesting that CQ could inhibit viral replication by inhibiting autophagy in ARV-infected chickens.In conclusion, we present evidence that ARV infection induces autophagy in SPF chicken, but this kind of autophagy sustains short time, and it utilizes autophagy to promote transient but obvious virus production. ARV-induced autophagy in chicken can be attenuated by pretreatment with CQ, resulting in the accumulation of LC3-Ⅱ and inhibition of viral replication.