Abstract
BackgroundMuscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old. It shares common physicochemical properties with avian reovirus (ARV) and differs in coding assignment and pathogenicity. The ARV p17 protein has been shown to trigger autophagy via activation multiple signaling pathways, which benefits virus replication. Since MDRV lacks the p17 protein, whether and how MDRV induces autophagy remains unknown. The aim of this study was to explore whether MDRV induces autophagy and which viral proteins are involved in MDRV-induced autophagy.MethodsThe autophagosome-like structures in MDRV-infected cells was observed under transmission electron microscopy. MDRV-induced autophagy was examined by analyzing the LC3-II level and phosphorylated form of mammalian target of rapamycin (mTOR) by Western blot assays. The effects of 3-methyladenine, rapamycin, chloroquine on viral yields were measured with quantitative(q) real-time reverse transcription (RT)-polymerase chain reaction (PCR) and 50% tissue culture infective dose (TCID50) assays, respectively. Additionally, to determine which viral protein is responsible for MDRV-induced autophagy, both p10.8- and σNS-encoding genes of MDRV were cloned into the pCI-neo-flag vector and transfected into DF-1 cells for detection of LC3-II.ResultsThe typical double-membrane vesicles containing cytoplasmic inclusions were visible in MDRV-infected immortalized chicken embryo fibroblast (DF-1) cells under transmission electron microscopy. Both primary Muscovy duck embryo fibroblasts (MDEF) and DF-1 cells infected with MDRV exhibited a significant increased levels of LC3-II accompanied with downregulation of phosphorylated form of mTOR, further confirming that MDRV is capable of inducing autophagy. Autophagy could be suppressed by 3-methylademine and induced by rapamycin and chloroquine. Furthermore, we found that σNS induces an increased levels of LC3-II, suggesting that the MDRV σNS protein is one of viral proteins involved in induction of autophagy. Both qRT-PCR and TCID50 assays showed that virus yield was increased in rapamycin treated DF-1 cells following MDRV infection. Conversely, when infected cells were pretreated with chloroquine, virus yield was decreased.ConclusionsThe MDRV σNS nonstructural protein is responsible for MDRV-induced autophagy and benefits virus replication.
Highlights
Muscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old
Autophagy is regulated by several cellular signaling pathways, one of which involves the class I phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin is well-studied, which negatively regulate autophagy, while the class III PI3K/Beclin-1 pathway positively regulates autophagy [12, 13]. Many other factors such as p53, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and adenosine monophosphate-activated protein kinase (AMPK), and Protein kinase R (PKR)-Eukaryotic initiation factor 2α (eIF2α) have been demonstrated to regulate the formation of autophagosome [14]
To determine whether MDRV is capable of MDRV induces autophagy in Immortalized chicken embryo fibroblast (DF-1) and Muscovy duck embryo fibroblasts (MDEF) cells via suppression of mammalian target of rapamycin (mTOR) phosphorylation To further confirm that autophagy is induced in MDRVinfected DF-1 cells, light chain 3 (LC3) type II (LC3-II), an important hallmark of autophagy induction, was detected by immunoblot assays
Summary
Muscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old. It shares common physicochemical properties with avian reovirus (ARV) and differs in coding assignment and pathogenicity. Muscovy duck reovirus (MDRV) infects 4 to 45-day-old Muscovy ducklings and causes "liver white spot disease" with a high morbidity and mortality. This acute infectious disease is characterized clinically by soft foot, diarrhea, stunted growth and white necrotic foci in liver and spleen [1,2,3], resulting in huge economic losses in Muscovy duck production. It is of great importance to elucidate the pathogenesis and to propose efficient ways for prevention and control of MDRV infection
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