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Spodoptera frugiperda (Lepidoptera: Noctuidae) is a globally distributed lepidopteran crop pest that has developed resistance to most insecticides. The G-quadruplex (G4) is a secondary structure in the genome enriched in the promoters for regulating gene expression. However, little is known about G4 in S. frugiperda, especially whether G4 is involved in insecticide resistance and pest control. In this study, 387,875 G4 motifs in the whole genome of S. frugiperda were identified by bioinformatics prediction. We found that 66.90 % of theseG4 structures were located in genic regions and highly enriched in the upstream regions of start codons. Functional and pathway analyses showed that the genes with G4 enriched in promoter regions participate in several metabolic processes. Further analyses showed that G4 structures occurred more frequently in the promoters of P450 and CarE gene families. It was also investigated that G4 ligand N-methyl mesoporphyrin IX (NMM) decreased P450 protein activity in larval midgut tissue. Cytotoxicity and bioassay results revealed that NMM and pesticides had synergistic effects on toxicity. In conclusion, our findings suggest that G4 motif could be a new potential target for pest control.

Alzheimer’s disease (AD) is a neurodegenerative disorder that primarily affects elderly individuals, and is characterized by hallmark neuronal pathologies including extracellular amyloid-β (Aβ) plaque deposition, intracellular tau tangles, and neuronal death. However, recapitulating these age-associated neuronal pathologies in patient-derived neurons has remained a significant challenge, especially for late-onset AD (LOAD), the most common form of the disorder. Here, we applied the high efficiency microRNA-mediated direct neuronal reprogramming of fibroblasts from AD patients to generate cortical neurons in three-dimensional (3D) Matrigel and self-assembled neuronal spheroids. Our findings indicate that neurons and spheroids reprogrammed from both autosomal dominant AD (ADAD) and LOAD patients exhibited AD-like phenotypes linked to neurons, including extracellular Aβ deposition, dystrophic neurites with hyperphosphorylated, K63-ubiquitin-positive, seed-competent tau, and spontaneous neuronal death in culture. Moreover, treatment with β- or γ-secretase inhibitors in LOAD patient-derived neurons and spheroids before Aβ deposit formation significantly lowered Aβ deposition, as well as tauopathy and neurodegeneration. However, the same treatment after the cells already formed Aβ deposits only had a mild effect. Additionally, inhibiting the synthesis of age-associated retrotransposable elements (RTEs) by treating LOAD neurons and spheroids with the reverse transcriptase inhibitor, lamivudine, alleviated AD neuropathology. Overall, our results demonstrate that direct neuronal reprogramming of AD patient fibroblasts in a 3D environment can capture age-related neuropathology and reflect the interplay between Aβ accumulation, tau dysregulation, and neuronal death. Moreover, miRNA-based 3D neuronal conversion provides a human-relevant AD model that can be used to identify compounds that can potentially ameliorate AD-associated pathologies and neurodegeneration.

Chlorophyll is the major light absorbing pigment for plant photosynthesis. While evolution has selected for higher chlorophyll content in leaves, previous work suggests that domesticated crops grown in modern agricultural environments overinvest in chlorophyll production thereby lowering light use and nitrogen use efficiency. To investigate the potential benefits of reducing chlorophyll level, we created ethanol inducible RNAi tobacco mutants that suppress Mg-chelatase subunit I (CHLI) with small RNA (sRNA) within 3 hours of induction and reduce chlorophyll within 5 days in field conditions. We initiated chlorophyll reduction later in plant development to avoid the highly sensitive seedling stage, and to allow young plants to have full green leaves to maximize light interception before canopy formation. This study demonstrated that >60% reduction of leaf chlorophyll could be tolerated without penalty on above-ground biomass or canopy photosynthesis in field conditions. Leaf chlorophyll reduction during seed filling stages increased tobacco seed nitrogen concentration as much as 17%, while biomass and seed yields were maintained. These results indicate that time-specific reduction of chlorophyll could be a niche strategy that decouples the inverse relationship between yield and seed nitrogen by utilizing saved nitrogen from the reduction of chlorophyll while maintaining full carbon assimilation capacity.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown etiology; however, apoptosis of lung alveolar epithelial cells plays a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis remain elusive. Here, we report that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. Our results suggest that bacteria shedding corisin are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of staphylococci in pulmonary fibrosis., Idiopathic pulmonary fibrosis is associated with increased abundance of Staphylococcus and Streptococcus in the lungs. Here, the authors identify a Staphylococcus nepalensis-derived peptide, named corisin, to induce apoptosis of lung epithelial cells and exacerbation of pulmonary fibrosis in mice.

Avian infectious bronchitis virus (IBV), a coronavirus, causes infectious bronchitis leading to enormous economic loss in the poultry industry worldwide. Hypericin (HY) is an excellent compound that has been investigated in antiviral, antineoplastic, and antidepressant. To investigate the inhibition effect of HY on IBV infection in chicken embryo kidney (CEK) cells, 3 different experimental designs: pre-treatment of cells prior to IBV infection, direct treatment of IBV-infected cells, and pre-treatment of IBV prior to cell infection were used. Quantitative real-time PCR (qRT-PCR), immunofluorescence assay (IFA), flow cytometry, and fluorescence microscopy were performed and virus titer was determined by TCID50. The results revealed that HY had a good anti-IBV effect when HY directly treated the IBV-infected cells, and virus infectivity decreased in a dose-dependent manner. Furthermore, HY inhibited IBV-induced apoptosis in CEK cells, and significantly reduced the mRNA expression levels of Fas, FasL, JNK, Bax, Caspase 3, and Caspase 8, and significantly increased Bcl-2 mRNA expression level in CEK cells. In addition, HY treatment could decrease IBV-induced reactive oxygen species (ROS) generation in CEK cells. These results suggested that HY showed potential antiviral activities against IBV infection involving the inhibition of apoptosis and ROS generation in CEK cells.

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

In recent years, porcine epidemic diarrhea (PED) has become widespread and caused huge economic losses for the global pig industry. There is growing evidence that frequent outbreaks of diarrhea are caused by the variants of porcine epidemic diarrhea virus (PEDV) with high pathogenicity. Herein, an epidemic strain of PEDV HLJ strain was isolated and characterized from Heilongjiang Province of China, and the whole genomic expression profile of intestinal porcine epithelial cells (IPEC-J2) infected with HLJ strain was investigated in comparison with classical CV777 strain. A total of 26,851 genes were identified, of these, 25,880 were known genes and 971 were novel genes. There were 258 differentially expressed genes (DEGs) identified between PEDV HLJ-infected and uninfected cells at 24 h post infection (hpi), and 201 DEGs between PEDV HLJ and CV777 infection. A comparative analysis revealed that 258 DEGs were enriched in 468 gene ontology (GO) terms and mapped to 179 KEGG pathways, and 201 DEGs in 1120 GO terms and mapped to 115 KEGG pathways for HLJ-infected cells in contrast to the uninfected and CV777-infected cells, respectively. Specifically, PEDV HLJ strain could activate anti-viral innate immune response and inflammation more intensively than CV777, in which mRNA levels of interferon (IFN-β), chemokines (CCL5 and CXCL10) and pro-inflammatory cytokines (IL-8 and TNF-α) were induced earlier and more strongly. Subsequently, 20 DEGs and 5 proteins were selected and validated by real-time fluorescence quantitative PCR (RT-qPCR) and western blot, and the results were consistent with the transcriptomic analysis. Overall, this study may be helpful for understanding the pathogenesis mechanism of PEDV variants, and contribute to the effective prevention and control of PEDV infection.

Infectious bronchitis virus (IBV) is a pathogenic coronavirus with high morbidity and mortality in chicken breeding. Macrophages with normal biofunctions are essential for host immune responses. In this study, the HD11 chicken macrophage cell line and chicken peripheral blood mononuclear cell-derived macrophages (PBMCs-Mφ) were infected with IBV at multiplicity of infection (MOI) of 10. The dynamic changes of their biofunctions, including cell viability, pathogen elimination function, phagocytic ability, and gene expressions of related proteins/mediators in innate and acquired immunity, inflammation, autophagy and apoptosis were analyzed. Results showed that IBV infection decreased chicken macrophage viability and phagocytic ability, and increased pathogen elimination function. Moreover, IBV augmented the gene expressions of most related proteins in macrophages involved in multiple host bioprocesses, and the dynamic changes of gene expressions had a close relationship with virus replication. Among them, MHCII, Fc receptor, TLR3, IFN-α, CCL4, MIF, IL-1β, IL-6, and iNOS showed significantly higher expressions in IBV-infected cells. However, TLR7, MyD88, MDA5, IFN-γ, MHCII, Fc receptor, MARCO, CD36, MIF, XCL1, CXCL12, TNF-α, iNOS, and IL-10 showed early decreased expressions. Overall, chicken macrophages play an important role in host innate and acquired immune responses to resist IBV infection, despite early damage or suppression. Moreover, the IBV-induced autophagy and apoptosis might participate in the virus-host cell interaction which is attributed to the biological process.

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Background Encephalomyocarditis virus (EMCV) is a positive single-stranded small RNA virus without envelope, which can infect a variety of mammals. Swine are the most susceptible animals, which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows. Diseases caused by EMCV have a wide range of effects on the global swine industry. Methods The tissue grinding fluid of aborted fetus was inoculated on baby hamster kidney 21 (BHK-21) cells to obtain HLJ isolate by cell passage. The virus was identified by reverse transcriptase polymerase chain reaction (RT-PCR), electron microscopic observation and indirect immunofluorescence assay (IFA). HLJ strain was inoculated into mice for animal regression experiment. The whole genome of HLJ strain was amplified and sequenced. The molecular and phylogenetic analysis of its sequence was generated. Results HLJ strain caused the specific cytopathic effect (CPE) on BHK-21 cells and severe pathological changes in mice. Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5–99.7%. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage I, II, III, IV, and V based on the nucleotide sequences of the entire open reading frame (ORF) and VP1 gene. HLJ isolate was grouped into lineage I. Conclusions In this study, a strain of EMCV was isolated from an aborted fetus in Northeast China. The isolation of HLJ strain enriches the epidemiological database of EMCV.

Infectious bronchitis virus (IBV) is a coronavirus, causes infectious bronchitis (IB) with high morbidity and mortality, and gives rise to huge economic losses for the poultry industry. Aminopeptidase N (APN) may be one of the IBV functional receptors. In this study, Gallus gallus APN (gAPN) protein was screened by phage-displayed 12-mer peptide library. Two high-affinity peptides H (HDYLYYTFTGNP) and T (TKFSPPSFWYLH) to gAPN protein were selected for in depth characterization of their anti-IBV effects. In vitro, indirect ELISA showed that these two high-affinity ligands could bind IBV S1 antibodies. Quantitative real-time PCR (qRT-PCR) assay, virus yield reduction assay and indirect immunofluorescence assay results revealed 3.125–50 μg/ml of peptide H and 6.25–50 μg/ml of peptide T reduced IBV proliferation in chicken embryo kidney cells (CEKs). In vivo, high-affinity phage-vaccinated chickens were able to induce specific IBV S1 antibodies and IBV neutralizing antibodies. QRT-PCR results confirmed that high-affinity phages reduced virus proliferation in chicken tracheas, lungs and kidneys, and alleviated IBV-induced lesions. By multiple sequence alignment, motif ‘YxYY’ and ‘FxPPxxWxLH’ of high-affinity peptides were identified in IBV S1-NTD, while another motif ‘YxFxGN’ located in S2. These results indicated that high affinity peptides of gAPN could present an alternative approach to IB prevention or treatment.

The modeling of crack initiation and propagation is of vital importance in the design and safety evaluation of structural components and engineering structures. Great advances have been achieved in the crack simulation during the past decades. Although the phase-field theory and the newly proposed nonlocal macro-meso-scale consistent damage (NMMD) model can predict the crack initiation, propagation and load-deformation curves for quasi-brittle materials correctly, the energetic degradation function, which bridges the energy-based damage and the topologic damage and plays an important role in these models, is still more or less empirically determined. In the present paper, a physical interpretation and quantitative modeling of the energetic degradation function via the embedded meso-scale mechanism of damage in the NMMD model is proposed. For this purpose, the NMMD model is firstly outlined. In this model, the meso-scale damage is firstly defined based on the degradation of bonds of material point pairs according to the irreversible elongation. The macro-scale topologic damage is then evaluated by averaging the meso-scale damage in the influence domain. It is then inserted into the thermodynamically consistent framework of continuum damage mechanics via the energetic degradation function bridging the topologic damage and the energy-based damage. To physically determine the energetic degradation function, the Helmholtz free energy of the damaged and undamaged materials is evaluated from the meso-scale mechanism of damage. In addition, it is theoretically proved that such determined energetic degradation function is invariant against the meso-scale parameters, including the radius of influence domain and the critical elongation quantity, and is weakly related to the strain state. Four numerical examples involving mode-I and mix-mode failures of isotropic quasi-brittle materials under static loading are studied to verify the NMMD model with the physically determined energetic degradation function. It is demonstrated that by the proposed approach there is no need to prescribe the initial crack and potential propagation path, the crack propagation can be captured automatically and the mesh size sensitivity is circumvented. Problems to be further studied are also discussed.

In this paper, a solution of the Dirichlet problem on the upper half space for a fast growing continuous boundary function is constructed by the generalized Dirichlet integral with this boundary function.

Porcine parvovirus (PPV) can cause reproductive failure in swine, resulting in economic losses to the industry. Antiviral effects of diammonium glycyrrhizinate (DG) have been reported on several animal viruses; however, to date it has yet to be tested on PPV. In this study, the antiviral activity of DG on swine testis (ST) cell infection by PPV was investigated using an empirically determined, non-toxic concentration of DG and three different experimental designs: (1) pre-treatment of virus prior to infection; (2) pre-treatment of cells prior to infection; and (3) direct treatment of virus-infected cells. The results showed that DG possesses potent inhibitory effects on PPV when the virus was treated before incubation with ST cells and that virus infectivity decreased in a dose-dependent manner. Results were confirmed by indirect immunofluorescence assays and real-time quantitative PCR. In addition, deoxycholate was used as a control to exclude the possibility that DG acted as a detergent to inhibit PPV infectivity. The study clearly indicates that DG has a direct anti-PPV effect in vitro.

Background Transmission of SARS-associated coronavirus (SARS-CoV) is now well controlled, nevertheless, it is important to develop effective methods to identify this virus from other pathogens. Objectives The purpose of this study was to identify potential ligands and develop a novel diagnostic test to SARS-CoV using phage display technology. Study design The SARS-CoV spike 1 (S1) protein containing the receptor binding region (RBD) was used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After four rounds of biopanning, 10 monoclonal phages with specific binding activity to the S1-RBD protein were obtained and subjected to binding and diagnostic assays. Results DNA sequencing showed that two phage displayed peptides HHKTWHPPVMHL (phage-H) and SQWHPRSASYPM (phage-S) that were specific ligands to the S1 protein. Moreover, the selected phage-H and phage-S were capable of differentiating SARS-CoV from other coronaviruses in indirect enzyme-linked immunosorbent assays. Conclusion The peptides identified in this study are useful reagents for detection of SARS-CoV.

The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin–esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection. Electronic supplementary material The online version of this article (doi:10.1007/s11262-013-0926-y) contains supplementary material, which is available to authorized users.

Each solution of infinite order of the stationary Schrodinger equation defined in a smooth cone and continuous in the closure can be represented in terms of the modified Poisson integral and an infinite series vanishing continuously on the boundary.

The FPV140 gene encodes an envelope protein of fowlpox virus (FPV). In this study, the FPV140 gene of FPV Chinese isolate HH2008 was cloned and the comparison of its sequence with other FPV isolates showed it to be highly conserved across all FPV isolates. A recombinant plasmid pET-FPV140 carrying FPV140 gene was constructed and transformed into Escherichia coli. The optimal expression condition for the FPV140 gene was developed and purified FPV140 recombinant protein was used to produce rabbit polyclonal antibody. An indirect ELISA using this anti-FPV140 polyclonal antibody was capable of distinguishing avian FPV isolates from other common avian pathogens such as mycoplasma gallisepticum, infectious laryngotracheitis virus, avian influenza virus, infectious bursal disease virus, and avian infectious bronchitis virus. This ELISA will serve as a useful diagnostic tool for the detection of FPV in clinical samples.

The objective of this study was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequence alignment, four primers were designed amplifying the M gene of type II PRRSV and were subsequently utilized in an RT-LAMP assay. The RT-LAMP product had a ladder-like pattern of bands and the optimal reaction condition for this assay was determined to be 40 min at 63°C. Comparative analysis indicated that the RT-LAMP method was more sensitive than a conventional RT-PCR assay and comparable to a real-time PCR assay. In addition, the RT-LAMP assay was capable of detecting type II PRRSV in field samples and differentiating type II PRRSV from seven other porcine viruses which are all associated frequently with similar clinical symptoms.

Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and responsible for eliciting neutralizing antibodies in immunized animals. In this study, the gene encoding VP2 of PPV was amplified by PCR. The VP2 gene was then cloned into the prokaryotic expression vector, pET-32a followed by expression in Escherichia coli Rosetta. The VP2 protein expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit polyclonal antiserum was generated using the recombinant VP2 protein. The optimal titer of the anti-VP2 antibody was determined by ELISA. The anti-VP2 antibody was able to distinguish PPV from other viruses in ELISA.

Highlights ► Generation of three distinct DNA constructs encoding S protein of PEDV, the N-terminal fragment (S1) of PEDV and porcine interleukin-18 (pIL-18). ► Immune responses of above-mentioned DNA constructs were evaluated systematically using mouse model. ► PEDV-S and the combination of PEDV-S1 with pIL-18 induced the strongest responses but that pIL-18 had no adjuvant effects when given in combination with PEDV-S1., Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA constructs in the eukaryotic expression plasmid pVAX1; one encoding the S protein [pVAX1-(PEDV-S)], the second encoding the N-terminal fragment (S1) [pVAX1-(PEDV-S1)] containing potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18) [pVAX1-(IL-18)]. Immunofluorescence assays in BHK-21 cells demonstrated successful protein expression from all 3 constructs. Kunming mice were injected separately with each of these constructs or with a pVAX1-(PEDV-S1)/pVAX1-(IL-18) combination, an attenuated PEDV vaccine, or vector only control. Animals were examined for T lymphocyte proliferation, anti-PEDV antibodies, IFN-γ and IL-4 protein levels, and cytotoxic T cell function in mouse peripheral blood and spleen. In all cases, results showed that pVAX1-(PEDV-S) and the combination of pVAX1-(PEDV-S1) with pVAX1-(IL-18) induced the strongest responses; however, pIL-18 had no adjuvant effects when given in combination with pVAX1-(PEDV-S1).

Based on the bioinformatics analysis of the gene encoding glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) isolate HH08, two gene fragments were amplified by polymerase chain reaction (PCR), deleting the signal peptide and transmembrane sequences in GP5 gene. Both gene fragments were designated GP5a and GP5b, respectively. They were ligated with a linker and cloned into prokaryotic expression vector, pET-30a. Expression of the protein of interest was induced by isopropyl beta-d-1-thiogalactopyranoside. The purified protein was used as an immunogen to elicit antibody in rabbit. The immunoreactivity of the protein was determined using ELISA and Western blot. Biologically active GP5 and anti-GP5 antibody inhibited cell infection by PRRSV. Moreover, the antibody produced in this study was capable of detecting the cell infection by PRRSV and distinguishing this virus from other viruses.

Background The lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (PEDV) pathogenesis. Vero cell, an African green monkey kidney cell line, was often used to isolate and propagate PEDV. Nonetheless, the target cells of PEDV in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. The immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. Type I interferon (IFN) plays an important role in antiviral immune response. Limited reports showed that PEDV could inhibit type I interferon production. In this study, porcine small intestinal epithelial cells (IECs), the target cells of PEDV, were used as the infection model in vitro to identify the possible molecular mechanisms of PEDV-inhibition IFN-β production. Results PEDV not only failed to induce IFN-β expression, but also inhibited dsRNA-mediated IFN-β production in IECs. As the key IFN-β transcription factors, we found that dsRNA-induced activation of IFN regulatory factor 3 (IRF-3) was inhibited after PEDV infection, but not nuclear factor-kappaB (NF-κB). To identify the mechanism of PEDV intervention with dsRNA-mediated IFN-β expression more accurately, the role of individual molecules of RIG-I signaling pathway were investigated. In the upstream of IRF-3, TANK-binding kinase 1 (TBK1)-or inhibitor of κB kinase-ε (IKKε)-mediated IFN-β production was not blocked by PEDV, while RIG-I-and its adapter molecule IFN-β promoter stimulator 1 (IPS-1)-mediated IFN-β production were completely inhibited after PEDV infection. Conclusion Taken together, our data demonstrated for the first time that PEDV infection of its target cell line, IECs, inhibited dsRNA-mediated IFN-β production by blocking the activation of IPS-1 in RIG-I-mediated pathway. Our studies offered new visions in understanding of the interaction between PEDV and host innate immune system.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that induces persistent diarrhoea in swine, resulting in severe economic losses in swine-producing countries. Insights into the interplay between PEDV infection and the innate immune system are necessary for understanding the associated mechanism of pathogenesis. The transcription factor NF-κB plays an important role in regulating host immune responses. Here, we elucidated for the first time to our knowledge the potential mechanism of PEDV-mediated NF-κB activation in porcine small intestinal epithelial cells (IECs). During PEDV infection, NF-κB p65 was found to translocate from the cytoplasm to the nucleus, and PEDV-dependent NF-κB activity was associated with viral dose and active replication. Using small interfering RNAs to screen different mRNA components of the Toll-like receptor (TLR) or RIG-I-like receptor signalling pathways, we demonstrated that TLR2, TLR3 and TLR9 contribute to NF-κB activation in response to PEDV infection, but not RIG-I. By screening PEDV structural proteins for their ability to induce NF-κB activities, we found that PEDV nucleocapsid protein (N) could activate NF-κB and that the central region of N was essential for NF-κB activation. Furthermore, TLR2 was involved in PEDV N-induced NF-κB activation in IECs. Collectively, these findings provide new avenues of investigation into the molecular mechanisms of NF-κB activation induced by PEDV infection.

The spike (S) protein of porcine transmissible gastroenteritis virus (TGEV) is located within the viral envelope and is the only structural protein that possesses epitopes capable of inducing virus-neutralizing antibodies. Among the four N-terminal antigenic sites A, B, C, and D, site A and to a lesser extent site D (S-AD) induce key neutralizing antibodies. Recently, we expressed S-AD (rS-AD) in recombinant form. In the current study, we used the rS-AD as an immobilized target to identify peptides from a phage-display library with application for diagnosis. Among the 9 phages selected that specifically bound to rS-AD, the phage bearing the peptide TLNMHLFPFHTG bound with the highest affinity and was subsequently used to develop a phage-based ELISA for TGEV. When compared with conventional antibody-based ELISA, phage-mediated ELISA was more sensitive; however, it did not perform better than semi-quantitative RT-PCR, though phage-mediated ELISA was quicker and easier to set up.

The full-length genome DNA of pseudorabies virus (PrV) was transfected into swine testis (ST) cells using calcium phosphate method to generate the infectious viruses. The resulting viruses were identified by indirect immunofluorescence and were used to infect African green monkey kidney (Vero) cells. Our results showed that the produced viruses had similar plaque morphology and growth properties with PrV vaccine strain, Bartha-K61. This method may produce PrV at an inexpensive cost.Key words: Pseudorabies virus, transfection, virus plaque, cytopathic effect.

Porcine epidemic diarrhoea virus (PEDV) is one of the important pathogens that may cause severe diarrhoea in piglets. In this study, the nucleocapsid (N) gene of a Chinese PEDV isolate designated HLJBY was cloned. The phylogeny of PEDV strains was investigated by constructing a phylogenetic tree based on the N protein sequences. The results indicate that there are two major groups of Chinese PEDVs, a Japanese PEDV group and a Korean PEDV group. High-level expression of the N protein was achieved in Escherichia coli. The immunoreactivity between PEDV particles or the bacterially expressed N protein and rabbit anti-PEDV serum was confirmed by immunofluorescence assays and Western blot. Both PEDV N protein and the polyclonal antibody generated in this study are valuable diagnostic reagents for PEDV surveillance.

Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that can cause piglet diarrhea with high mortality rates. TGEV membrane (M) protein not only plays a vital role in the process of virus assembly and budding, but also induces the production of interferon-α during infection. In this study, a monoclonal antibody (MAb) designated 7G7, against the TGEV M protein was generated by inoculating BALB/c mice with TGEV followed by hybridoma technique. Immunofluorescence assays indicated that MAb 7G7 was capable of detecting cell infection by TGEV. Virus-based ELISA demonstrated that MAb 7G7 can be used as a highly specific diagnostic reagent for TGEV.

Background The context and purpose of the study included 1) bacterial expression of viral protein 6 (VP6) of porcine rotavirus (PRV) and generation of rabbit polyclonal antiserum to the VP6 protein; 3) establishment of a discrimination ELISA to distinguish PRV from a panel of other porcine viruses. Results The VP6 gene of PRV isolate DN30209 amplified by reverse transcription-PCR was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. Sequence comparison and phylogenetic analysis indicated that PRV DN30209 may belong to group A of rotavirus. Bacterially expressed VP6 was expressed in E.coli and anti-VP6 antibody was capable of distinguishing PRV from Porcine transmissible gastroenteritis virus, Porcine epidemic diarrhea virus, Porcine circovirus type II, Porcine reproductive and respiratory syndrome virus, Porcine pseudorabies virus and Porcine parvovirus. Conclusions PRV VP6 expressed in E. coli can be used to generate antibodies in rabbit; anti-VP6 serum antibody can be used as good diagnostic reagents for detection of PRV.