Autophagy Benefits the Replication of Porcine Epidemic Diarrhea Virus

The new porcine epidemic diarrhea (PED) outbreak has been documented in China since late 2010 and now with global distribution, resulting in enormous economic losses to swine industry. Autophagy is a highly conserved intracellular degradation process and be manipulated by some viruses for their benefits. Our previous proteomic data indicated that autophagy might participate in PEDV infection. However, the concrete role of autophagy is unknown. In the present study, we first detected the conversion of LC3-I to LC3-II, measured autophagic flux by monitoring p62/SQSTM1 degradation. Transmission electron microscope (TEM) was also used to observe autophagy induction. In addition, we demonstrated that whether the alteration of cellular autophagy by autophagy regulators and RNA interference affected PEDV replication.

The Vero cells were cultured in DMEM supplemented with 10% FBS to 80% confluence. Subsequently, cells were infected with PEDV (Accession no. KF761675) and incubated with serum-free DMEM containing 8 μg/mL trypsin. For pharmacological experiments, Vero cells were pretreated with optimal concentrations of drugs for 4 h prior to viral infection, and then infected with PEDV. For RNA interference, Beclin 1 was knocked down by transfected with Beclin 1 and scrambled siRNA with Lipofectamine 2000. Immunoblotting was performed to determine the conversion of endogenous LC3-I to LC3-II and p62/SQSTM1 degradation. The virus titers were determined by Reed–Muench method.

To determine whether PEDV infection can induce autophagy, the conversion from LC3-I to LC3-II was monitored at 6 h, 18 h and 30 h post PEDV infection. It demonstrated that the conversion from LC3-I to LC3-II was significantly enhanced after PEDV infection. Meanwhile, the p62 degradation indicated that PEDV infection can increase the level of autophagic flux in infected Vero cells. TEM observation showed that autophagosome-like vesicles were significantly increased in Vero cells post-infection compared with mock-infected. When treatment with autophagy inducer rapamycin, the virus yield was increased, while treatment with the inhibitor 3-MA, the virus yield was reduced. Knockdown of the essential endogenous Beclin 1 also reduced PEDV infection.

In the present study, we first demonstrated that PEDV infection can induce autophagy, and then assessed the impact of autophagy on viral replication. We postulate that autophagy might be a potential mechanism that PEDV manipulated to benefit replication.

This work was supported by grants from the China Agricultural Research System (CARS-36).

Disclosure of Interest: None Declared.