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.
•The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French «S-InDel» PEDV strain (PEDV/FR/001/2014) and in case of positive ...semen to determine the infectivity of that semen.•Both infected boars had diarrhea after inoculation and shed virus in feces.•In addition, PEDV genome was detected by RT-qPCR in the sperm-rich fraction of semen from the two boars infected with the «S-InDel» PEDV strain.•The PEDV positive semen («S-non-InDel» and «S-InDel») sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer’s patches by RT-qPCR except for one pig.
PEDV is mainly transmitted by the oro-fecal route although PEDV shedding in semen has already been shown for an S-non-InDel PEDV strain infection. The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French S-InDel PEDV strain (PEDV/FR/001/2014) and in case of positive semen to determine the infectivity of that semen. Both infected boars had diarrhea after inoculation and shed virus in feces. PEDV genome was also detected by RT-qPCR in the sperm-rich fraction of semen (6.94 × 103 and 4.73 × 103 genomic copies/mL) from the two boars infected with the S-InDel PEDV strain but only once at 7DPI. In addition, PEDV RNA in Peyer’s patches and in mesenteric lymph nodes was also present for the two inoculated boars. The PEDV positive semen (S-non-InDel and S-InDel) sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. The inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 DPI. But, PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer’s patches by RT-qPCR except for one pig. Even if PEDV genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected PEDV.
Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhoea, has recently caused significant economic damage worldwide. Porcine aminopeptidase N (pAPN) has been reported to be the ...receptor for PEDV, although robust evidence is lacking. In the present study, we explored whether pAPN functions as a receptor for PEDV. Human HeLa cells expressing pAPN and pAPN-positive porcine CPK cells failed to support PEDV infection, but were susceptible to infection by transmissible gastroenteritis virus (TGEV), which utilizes pAPN as a functional receptor. In contrast to TGEV, PEDV did not bind soluble porcine aminopeptidases (pAPs) and infection was not inhibited by the soluble form of pAPs. However, overexpression of pAPN in porcine CPK cells (CPK-pAPN cells) slightly increased the production of PEDV, and the increased replication in CPK-pAPN cells was inhibited by bestatin, an inhibitor of the protease activity of aminopeptidase N. These results suggest that pAPN is not a functional receptor for PEDV, but promotes the infection of PEDV through its protease activity.
MicroRNAs (miRNAs) are a class of noncoding RNAs involved in posttranscriptional regulation of gene expression and many critical roles in numerous biological processes. Porcine epidemic diarrhea ...virus (PEDV), the etiological agent of porcine epidemic diarrhea, causes substantial economic loss in the swine industry worldwide. Previous studies reported miRNA involvement in viral infection; however, their role in regulating PEDV infection remains unknown. In this study, we investigated the regulatory relationship between miRNA-221-5p and PEDV infection, finding that miR-221-5p overexpression inhibited PEDV replication in a dose-dependent manner, and that silencing endogenous miR-221-5p enhanced viral replication. Our results showed that miR-221-5p directly targets the 3' untranslated region (UTR) of PEDV genomic RNA to inhibit PEDV replication, and that miR-221-5p overexpression activates nuclear factor (NF)-κB signaling via p65 nuclear translocation, thereby upregulating interferon (IFN)-β, IFN-stimulated gene 15, and MX1 expression during CH/HBTS/2017 infection. We subsequently identified NF-κB-inhibitor α and suppressor of cytokine signaling 1, negative regulators of the NF-κB pathway, as miR-221-5p targets. These results demonstrated the ability of miR-221-5p to inhibit PEDV replication by targeting the 3' UTR of the viral genome and activating the NF-κB-signaling pathway. Our findings will aid the development of preventive and therapeutic strategies for PEDV infection.
Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United ...States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5'-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV.
The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.
Coronaviruses are important pathogens of humans and animals, and vaccine developments against them are imperative. Due to the ability to induce broad and prolonged protective immunity and the ...convenient administration routes, live attenuated vaccines (LAVs) are promising arms for controlling the deadly coronavirus infections. However, potential recombination events between vaccine and field strains raise a safety concern for LAVs. The porcine epidemic diarrhea virus (PEDV) remodeled TRS (RMT) mutant generated in this study replicated efficiently in both cell culture and in pigs and retained protective immunogenicity against PEDV challenge in pigs. Furthermore, the RMT PEDV was resistant to recombination and genetically stable. Therefore, RMT PEDV can be further optimized as a backbone for the development of safe LAVs.
Porcine epidemic diarrhea (PED) which is caused by porcine epidemic diarrhea virus (PEDV), is an intestinal communicable disease. In recent years, though pigs have been immunized with the vaccines in ...pig farms, PED still broke out and caused severe economic losses to the swine industry in the northeast China. In this study, the sample was positive for PEDV variant strains via the nano-nest PCR. The strain was successfully isolated from positive samples and was serially passaged in Vero-E6 cells. In addition, the strain was identified via electron microscopy observation, indirect immunofluorescence assay and infection experiment in newborn piglets and named PEDV CH/JLDH/2016 strain (Accession No. MF346935). Phylogenetic analysis of the S gene showed that the CH/JLDH/2016 strain was clustered into G2b subgroup. Comparing with the CV777 vaccine strain, amino acid sequence analysis of CH/JLDH/2016 strain showed that 15 nucleotides were inserted and 9 were absent in S gene, whose amino acid sequence it educed insertions of 5 amino acids(58NQGX61 and 145N) and absences of 3 amino acids(164RD165 and 1204Y). Our strain, in the SS2 epitope have no amino acid, variant while in SS6 epitope, Y changed into S in 776th amino acid. The results indicated that PEDV G2b variant strains have been emerged in Jilin province. The identification of new types of PEDV variant strains would stimulate the development of effective vaccines for the prevention and control of PED. The novel vaccines that based on these newly identified PEDV variant strains may contribute to the control of PED outbreaks in China.
•The isolated variant PEDV from Dehui, China, was distributed into the G2b subgroup.•The differences of strains help to explain why PEDV vaccines have no protection in most piglets in China.•Our variant PEDV with the absence of Y in the 776th aa was from G2b subgroup while others were from G1a subgroup.
•A newly isolated PEDV SH strain contains a unique continuous 12-aa deletion (aa 399--410) in the NEP-1C9 epitope of the N protein.•PEDV SH is highly pathogenic to newborn piglets.•Mice inoculated ...with inactivated SH produce antibodies against the N protein, but not against the NEP-1C9 peptide (aa 398--406).•The deletion in N protein does not influence the pathogenicity or immunogenicity of the SH strain.
Since late 2010, highly virulent PEDV G2-genotype strains have emerged globally extracting heavy losses on the pork industries of numerous countries. We investigated the characteristics of a field strain of PEDV (PEDV strain SH) isolated from a piglet with severe diarrhea on a farm in Shanghai China. Whole genome sequencing and analysis revealed that the SH strain belonged to subtype G2b and has a unique 12-aa deletion (aa 399–410) including the antigenic epitope NEP-1C9 (aa 398–406) of the N protein. PEDV SH strain is highly pathogenic to challenged newborn piglets, resulting in 100 % morbidity and mortality. Pathological examination revealed significant villus atrophy in the jejuna of infected piglets. Mice inoculated with inactivated PEDV SH produced antibodies against the N protein, but no antibodies against the deletions. These results illustrated that deletion of the NEP-1C9 epitope had no effect on the immunogenicity or pathogenicity of PEDV, providing evidence of the necessity to monitor the genetic diversity of the virus. Our study also contributes to development of candidate for vaccines and diagnostics that could differentiate pigs seropositive due to vaccination by conventional strains from wild virus infection.
Type III interferons (IFNs) play a vital role in maintaining the antiviral state of the mucosal epithelial surface in the gut, and in turn, enteric viruses may have evolved to evade the type III IFN ...responses during infection. To study the possible immune evasion of the type III IFN response by porcine epidemic diarrhea virus (PEDV), a line of porcine intestinal epithelial cells was developed as a cell model for PEDV replication. IFN-λ1 and IFN-λ3 inhibited PEDV replication, indicating the anti-PEDV activity of type III IFNs. Of the 21 PEDV proteins, nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, open reading frame 3 (ORF3), E, M, and N were found to suppress type III IFN activities, and IRF1 (interferon regulatory factor 1) signaling mediated the suppression. PEDV specifically inhibited IRF1 nuclear translocation. The peroxisome is the innate antiviral signaling platform for the activation of IRF1-mediated IFN-λ production, and the numbers of peroxisomes were found to be decreased in PEDV-infected cells. PEDV nsp1 blocked the nuclear translocation of IRF1 and reduced the number of peroxisomes to suppress IRF1-mediated type III IFNs. Mutational studies showed that the conserved residues of nsp1 were crucial for IRF1-mediated IFN-λ suppression. Our study for the first time provides evidence that the porcine enteric virus PEDV downregulates and evades IRF1-mediated type III IFN responses by reducing the number of peroxisomes.
Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that emerged in swine in the United States and has caused severe economic losses. PEDV targets intestinal epithelial cells in the gut, and intestinal epithelial cells selectively induce and respond to the production of type III interferons (IFNs). However, little is known about the modulation of the type III IFN response by PEDV in intestinal epithelial cells. In this study, we established a porcine intestinal epithelial cell model for PEDV replication. We found that PEDV inhibited IRF1-mediated type III IFN production by decreasing the number of peroxisomes in porcine intestinal epithelial cells. We also demonstrated that the conserved residues in the PEDV nsp1 protein were crucial for IFN suppression. This study for the first time shows PEDV evasion of the type III IFN response in intestinal epithelial cells, and it provides valuable information on host cell-virus interactions not only for PEDV but also for other enteric viral infections in swine.
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