Identification of a novel B-cell epitope in the spike protein of porcine epidemic diarrhea virus.

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 (722SSTFNSTREL731) 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, 722SSTFNSTREL731) 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.

BST2 suppresses porcine epidemic diarrhea virus replication by targeting and degrading virus nucleocapsid protein with selective autophagy.

Interferon-induced BST2 (bone marrow stromal cell antigen 2) inhibits viral replication by tethering enveloped virions to the cell surface to restrict viral release and by inducing the NFKB-dependent antiviral immune response. However, the mechanism by which BST2 uses the selective autophagy pathway to inhibit viral replication is poorly understood. In this study, we showed that BST2 expression was significantly increased during porcine epidemic diarrhea virus (PEDV) infection of Vero cells by IRF1 targeting its promoter. We also showed that BST2 suppressed PEDV replication by binding and degrading the PEDV-encoded nucleocapsid (N) protein. The downregulation of N protein was blocked by macroautophagy/autophagy inhibitors but not a proteasome inhibitor, implying that the N protein was degraded via the selective autophagy pathway. Both the BST2 and N protein interacted with the E3 ubiquitin ligase MARCHF8/MARCH8 and the cargo receptor CALCOCO2/NDP52, and the ubiquitination of N protein was necessary for the degradation of N mediated by the BST2-MARCHF8 axis. The knockdown of MARCHF8 or ATG5 with small interfering RNAs blocked the selective autophagy pathway, rescued the protein abundance of PEDV N in 293T cells, and prevented the inhibition of PEDV replication by BST2 in Vero cells. Together, our data demonstrate the novel mechanism of BST2-mediated virus restriction, in which BST2 recruits MARCHF8 to catalyze the ubiquitination of the PEDV N protein. The ubiquitinated N protein is then recognized by CALCOCO2/NDP52, which delivers it to autolysosome for degradation through the selective autophagy pathway. Abbreviations: 3MA: 3-methyladenine; ATG: autophagy-related; Baf A1: bafilomycin A1; BST2: bone marrow stromal cell antigen 2; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; CT: cytoplasmic tail; DAPI: 4',6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; IRF1: interferon regulatory factor 1; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PED: porcine epidemic diarrhea; PEDV: porcine epidemic diarrhea virus; RT: room temperature; siRNA: small interfering RNA; STAT: signal transducer and activator of transcription; TCID50: 50% tissue culture infectious doses; TM: transmembrane.

Molecular characterization of new described kobuvirus in dogs with diarrhea in China.

Canine kobuvirus (CaKVs) was a newly described virus detected in dogs in the US and Italy. To learn more about CaKVs, 5 of 106 fecal samples from diarrhea dogs were positive with CaKVs in China, and the full genome of CaKVs strain CH-1 isolated from dog with diarrhea was sequenced. The genome consists of 8186 nucleotides, excluding the 3' poly (A) tail, and an open reading frame that maps between nucleotide positions 601 and 7943 which encodes a 2446 amino acid polyprotein. Based on the complete amino acid sequence of polyprotein, phylogenetic analysis showed that CH-1 was grouped along with other canine kobuvirus strains detected in the USA (US-PC0082, AN211D).

Suppression of Virulent Porcine Epidemic Diarrhea Virus Proliferation by the PI3K/Akt/GSK-3α/ß Pathway.

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/ß, 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/ß signaling pathway is activated by PEDV and functions in inhibiting PEDV replication.

Monoclonal Antibody to Bone Marrow Stromal Cell Antigen 2 Protein of Swine.

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-ß-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.