Background: Porcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied.Methods: To accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies.Results: Five antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666-789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 ( 722 SSTFNSTREL 731 ) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure.Conclusions: A mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, 722 SSTFNSTREL 731 ) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV.
Porcine epidemic diarrhea virus (PEDV) has recently caused high mortality in suckling piglets with subsequent large economic losses to the swine industry. Many intracellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, are activated by viral infection. The PI3K/Akt pathway is an important cellular pathway that has been shown to be required for virus replication. In the present study, we found that the PEDV JS-2013 strain activated Akt in Vero cells at early (5–15 min) and late stages (8–10 h) of infection. Inhibiting PI3K, an upstream activator of Akt, enhanced PEDV replication. Inhibiting GSK-3α/β, one of the downstream effectors of PI3K/Akt pathway and regulated by Akt during PEDV infected Vero cells, also enhanced PEDV replication. Collectively, our data suggest that PI3K/Akt/GSK-3α/β signaling pathway is activated by PEDV and functions in inhibiting PEDV replication.
The bone marrow stromal cell antigen 2 (BST-2) protein was identified as a novel virus restriction factor that potently restricts the replication and egress of enveloped viruses. In this study, we generated monoclonal antibodies (MAbs) against porcine BST-2 encoding 34-112 aa of porcine BST-2, which was cloned and inserted into the prokaryotic expression vector pCold-I to construct a recombinant plasmid pCold-pBST-2. The recombinant porcine BST-2 protein (rpBST-2 protein) was induced by isopropyl-β-D-thiogalactoside in Escherichia coli BL21 (DE3). Then, BALB/c mice were immunized with the purified rpBST-2 protein to prepare MAbs of BST-2. After subcloning, one strain of hybridoma cells named 1B2 secreting porcine BST-2 protein monoclonal antibody (MAb) was obtained. Indirect immunofluorescence assay and western blot analysis showed that the MAb was specifically reacted with the overexpressed porcine BST-2 protein in Vero cells. The specific MAb of porcine BST-2 provides a valuable tool for further studies of BST-2 to restrict virus infection.
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