Despite autophagy’s pivotal role in the replication of viruses such as duck Tembusu virus (DTMUV), which has caused massive economic losses to the poultry industry in the world, the specific relationships between DTMUV and cellular autophagy remain largely unknown. In response, we investigated the interactions between autophagy and DTMUV, the effects of the structural and non-structural proteins of DTMUV on autophagy, and the autophagy-related signaling pathways induced by DTMUV. Among the results, DTMUV increased the autophagy flux in duck embryo fibroblasts (DEF) and BHK-21 cells, while autophagy facilitated viral replication. After we pharmacologically induced autophagy with rapamycin (RAPA), the replication of DTMUV increased by 15.23-fold compared with the control group of DEF cells. To identify which DTMUV protein primarily induced autophagy, all three structural proteins and seven non-structural proteins of DTMUV were transfected into cells, and the results showed that non-structural protein 3 (NS3) induced significant autophagy in DEF cells. By means of Western blot, immunofluorescence, and transmission electron microscopy, we confirmed that NS3 protein could significantly induce autophagy and autophagy flux. Furthermore, we showed that NS3 induced autophagy in DEF cells through extracellular signal-regulated kinase 2 (ERK2) and phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways using specific inhibitors and RNA interference assays. Finally, autophagy induced by NS3 promoted DTMUV replication. These results provide novel insight into the relationship between DTMUV and autophagy, broadening the current understanding of the molecular pathogenesis of DTMUV.

Background: Avian hepatitis E virus (HEV) is the pathogenic agent of big liver and spleen disease (BLS) and hepatitis-splenomegaly syndrome (HS) in chickens, which has caused economic losses to the poultry industry in China. Eighteen samples of BLS chickens were collected in this study to understand the molecular epidemiology characteristics of avian HEV in Shandong province, China.Results: Gross and microscopic lesions of clinical samples were observed, then virology detection and genetic analysis of avian HEV were performed. The results showed that there were significant swelling and rupture in the liver, and spleen was enlarged. Microscopic lesions demonstrated that obvious hemorrhage in the liver, with infiltration of heterophilic granulocytes, lymphocytes, and macrophages, the reduction of lymphocytes in the spleen. Eleven out of the 18 samples were positive for HEV, with a positive rate of 61.11%. More importantly, all HEV positive samples were mixed infections. Among them, the mixed infections of avian HEV and chicken infectious anaemia virus (CIAV) and fowl adenovirus (FAdV) were the most common. In addition, the genetic evolution analysis showed that all obtained HEV isolates did not belong to the reported 4 genotypes, and they constituted a novel genotype. Conclusions: These results of this study further enriched the epidemiological data of avian HEV in Shandong province and proved the genetic diversity of HEV in China, but also uncovered the complicated mixed infections of avian HEV in clinical.