S1D (residues 636-789) is a neutralizing epitope region on the spike protein (S) of porcine epidemic diarrhea virus (PEDV). To accurately identify epitopes on S1D, the S1-phage library containing the gene encoding the S1D region of PEDV S protein was micropanned by six specific monoclonal antibodies (McAbs) against the S1D region. These micropanned epitope regions (MER) were focused on 696-779 amino acids of the S protein. To further map epitopes of the MER, seven overlapping mini-fragments covering MER nucleotides were separately synthesized and expressed in Escherichia coli BL21 with a GST tag. These mini-GST fusion proteins were scanned by ELISA and Western blotting with the six McAbs, and the result showed that S1D5 (residues 744-759) and S1D6 (residues 756-771) are two linear epitopes of the PEDV S protein. The antisera of the epitopes S1D5 and S1D6 could react with the native S protein of PEDV. Furthermore, Pepscan of the two linear epitopes demonstrated that SS2 ((748)YSNIGVCK(755)) and SS6 ((764)LQDGQVKI(771)) are two core epitopes on S1D5 and S1D6, respectively, located on the S protein of PEDV.

Porcine epidemic diarrhea virus (PEDV) is the cause of an economically important swine disease. Previous studies suggested that PEDV does not elicit a robust IFN response, but the mechanism(s) used to evade or block this innate immune response was not known. In this study, we found that PEDV infection blocked synthetic dsRNA-induced IFN-b production by interfering with the activation of interferon regulatory factor 3 (IRF3). We identified PEDV replicase encoded papain-like protease 2 (PLP2) as an IFN antagonist that depends on catalytic activity for its function. We show that levels of ubiquitinated proteins are reduced during PEDV infection and that PEDV PLP2 has deubiquitinase (DUB) activity that recognizes and processes both K-48 and K-63 linked polyubiquitin chains. Furthermore, we found that PEDV PLP2 strongly inhibits RIG-Iand STING-activated IFN expression and that PEDV PLP2 can be co-immunoprecipitated with and deubiquitinates RIG-I and STING, the key components of the signalling pathway for IFN expression. These results show that PEDV infection suppresses production of IFN-b and provides evidence indicating that the PEDV papain-like protease 2 acts as a viral DUB to interfere with the RIG-I-and STING-mediated signalling pathway.

a b s t r a c tSeveral studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.

Since early 2006, porcine epidemic diarrhea virus (PEDV) has been reemerging in immunized swine herds. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. The entire ORF3 genes of 12 PEDV field strains and one vaccine strain were sequenced. The ORF3 genes of Chinese PEDV field strains (excluding CH/GSJIII/07) contain a single 672- or 675-nucleotide (nt) ORF, which encodes a 223- or 224-aa-long peptide. However, the CV777 vaccine strain and CH/GSJIII/07 contain a 276-nt ORF because of a 49-nt deletion at nt 245-293. The Chinese PEDV field strains and PEDV reference strains are divided into three groups based on the phylogenetic relationship of their ORF3 genes. Chinese PEDV field strains (excluding CH/GSJIII/07) have a close phylogenetic relationship to Korean strains and are genetically different from the PEDV vaccine strains. However, CH/GSJIII/07 has a close phylogenetic relationship to two vaccine strains, suggesting that it might have evolved from a live vaccine strain. Chinese PEDV field strains (excluding CH/GSJIII/07) can be differentiated from PEDV vaccine strains by a nested RT-PCR method.

In contrast to type I interferons that target various types of cells and organs, interferon lambda (IFN-L) primarily acts on mucosal epithelial cells and exhibits robust antiviral activity within the mucosal surface. Porcine epidemic diarrhea virus (PEDV), which causes high morbidity and mortality in piglets, is an enteropathogenic coronavirus with economic importance. Here, we demonstrated that both recombinant porcine IFN-L1 (rpIFN-L1) and rpIFN-L3 have powerful antiviral activity against PEDV infection of both Vero E6 cells and the intestinal porcine epithelial cell line J2 (IPEC-J2). Both forms of rpIFN-L inhibited two genotypes of PEDV (strain CV777 of genotype 1 and strain LNCT2 of genotype 2). rpIFN-L1 primarily controlled viral infection in the early stage and had less antiviral activity in IPEC-J2 than in rpIFN-L3 cells infected with PEDV. In addition, rpIFN-L1 exhibited greater antiviral activity against PEDV infection of IPEC-J2 cells than that of porcine IFN-alpha. Consistent with this finding, rpIFN-L1 triggered higher levels of certain antiviral IFN-stimulated genes (ISGs) (ISG15, OASL, and MxA) in IPEC-J2 cells than porcine IFN-alpha. Although IPEC-J2 cells responded to both IFN-alpha and lambda, transcriptional profiling of ISGs (specifically ISG15, OASL, MxA, and IFITMs) differed when induced by either IFN-alpha or rpIFN-L. Therefore, our data provide the experimental evidence that porcine IFN-L suppresses PEDV infection of IPEC-J2 cells, which may offer a promising therapeutic for combating PED in piglets.

Since late 2010, porcine epidemic diarrhea virus (PEDV) has rapidly disseminated all over the China and caused considerable morbidity and high mortality (up to 100%) in neonatal piglets. 79.66% (141 of 177) pig farms in 29 provinces (excluding Tibet and Hainan, China) and 72.27% (417 of 577) samples were positive for PEDV confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The full-length S genes of representative field strains were sequenced. 33 field strains share 93.5%–99.9% homologies with each other at the nucleotide sequence level and 92.3%–99.8% homologies with each other at the amino acids sequence level. Most field strains have nucleotide deletion and insertion regions, and show lower homologies (93.5%–94.2%) with Chinese classical strain CH/S, however higher homologies (97.1%–99.3%) with recent strain CHGD-1. The phylogenetic analysis showed there are classical strains and variants prevailing in pig herd in China. PEDV has a high detection rate in pig herds in China. Sequence analysis indicated the S genes of recent field strains have heterogeneity and the variants are predominant.

Induced pluripotent stem cells (iPSCs) have fewer and immature mitochondria than somatic cells and mainly rely on glycolysis for energy source. During somatic cell reprogramming, somatic mitochondria and other organelles get remodeled. However, events of organelle remodeling and interaction during somatic cell reprogramming have not been extensively explored. We show that both SKP/SKO (Sox2, Klf4, Pou5f1/Oct4) and SKPM/SKOM (SKP/SKO plus Myc/c-Myc) reprogramming lead to decreased mitochondrial mass but with different kinetics and by divergent pathways. Rapid, MYC/c-MYC-induced cell proliferation may function as the main driver of mitochondrial decrease in SKPM/SKOM reprogramming. In SKP/SKO reprogramming, however, mitochondrial mass initially increases and subsequently decreases via mitophagy. This mitophagy is dependent on the mitochondrial outer membrane receptor BNIP3L/NIX but not on mitochondrial membrane potential (ΔΨ) dissipation, and this SKP/SKO-induced mitophagy functions in an important role during the reprogramming process. Furthermore, endosome-related RAB5 is involved in mitophagosome formation in SKP/SKO reprogramming. These results reveal a novel role of mitophagy in reprogramming that entails the interaction between mitochondria, macroautophagy/autophagy and endosomes.

In 2011, outbreaks of viral diarrhea were observed on most swine-breeding farms in most of the provinces of China. The disease is characterized by vomiting, severe diarrhea, and a high mortality rate (82.3%) in newborn piglets. The clinical appearance was similar to that of porcine epidemic diarrhea virus (PEDV) infection. PEDVs were detected in samples (feces or small intestines) from most farms. In order to investigate whether there is a PEDV variant circulating in China, we sequenced and analyzed the complete genome of the recently identified field strain, CH/FJND-3/2011. The sequence data indicate that this PEDV variant prevails in China.