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Foot and mouth disease virus (FMDV) is a highly contagious pathogen propagating among cloven-hoofed animals. As a major immunogenic protein, VP1 plays a pivotal role in the induction of neutralizing antibodies, which therefore is an ideal target for developing subunit vaccines. In current study, four prokaryotic expression clones (rV4C, rC4V, rV5F and rF5V) were constructed by fusing truncated calreticulin (CRT) (120–250 aa or 120–308 aa) at the N/C terminal of vp1 gene, and co-expressed with chaperone trigger factor 16 (Tf16) in E.coli, respectively. The soluble recombinant CRT-fused VP1 proteins could form into homogeneous reactive polymers with average hydrodynamic diameters around 100 nm according to the dynamic light scattering (DLS) data. Immunization of guinea pigs with 10 μg purified CRT-fused VP1 proteins induced high levels of antibodies against naked-VP1 through indirect ELISA. Sandwich ELISA showed that only rC4V could elicit the same level of antibody against FMD virus as commercial inactivated vaccine after booster. The lymphocyte cytokines secretion of immunized rC4V was higher than the other CRT-fused VP1 proteins in guinea pigs. These results showed that the soluble CRT-fused VP1 proteins, especially rC4V, expressed with Tf16 in E. coli might have potential to be used as subunit vaccine candidate against FMDV.

Glycoprotein (GP) 2a was a minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV) and was one of crucial proteins for PRRSV to bind cell receptor, which indicated that there were neutralizing epitopes on GP2a. In the present work, we used mouse anti-GP2a41-208aa serum and one GP2a41-208aa specific monoclonal antibody (McAb) to identify B-cell epitopes of GP2a by peptide-based ELISA. A liner B-cell epitope F194PTPGSRPKLHDFQQ208 was identified. However, the results of virus neutralization experiment showed that the McAb could not reduce the titers of PRRSV, which indicated that the identified epitope was not the neutralizing epitope of PRRSV. While the amino acid sequence of this epitope was conserved in North American (type 2) PRRSV, which suggested that this epitope might be diagnostic potential for type 2 PRRSV strains. In conclusion, our present work identified a new epitope on GP2a and this epitope might be diagnostic potential for type 2 PRRSV strains.

The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499-638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV.

A facile, efficient and sensitive electrochemical immunosensor based on Au nanoparticles/Zn/Ni-ZIF-8-800@graphene composites was developed to detect the monensin in milk. Zn/Ni-ZIF-8-800 with high stability was obtained via pyrolysis of the Zn/Ni-bimetallic metal-organic frameworks (MOFs) precursor Zn/Ni-ZIF-8. Benefitting from the synergistic effects including the high porosity of the Zn/Ni-ZIF-8-800 and the excellent electrical conductivity of graphene, the electrochemical signal got greatly enhanced. The AuNPs were electrodeposited on the Zn/Ni-ZIF-8-800@graphene surface for antibody-antigen capture. Then the constructed hybrid materials not only possess high specific surface area, but also exhibit excellent biocompatibility and electron transfer ability. Morphology and elemental analyse of these composites were conducted via scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). In addition, the crystal structure was analyzed by X-ray diffraction (XRD). Afterwards, the preparation process of the biosensor was investigated by cyclic voltammetry (CV), and the detection procedure was evaluated by differential pulse voltammetry (DPV). Under the optimal conditions, the achieved linear range was 0.25–100 ng mL−1, and the calculated detection limit was 0.11 ng mL−1. The as-prepared biosensor in this work offers an effective approach to measure monensin and may be a promising candidate for detecting other veterinary drugs in food products.

Two different genotypes of porcine epidemic diarrhea virus (PEDV), the classical and variant strains, are classified by multiple insertions and deletions in their S genes. It is critical to detect and differentiate two genotypes in the pork industry to prevent PEDV outbreaks. In the present study, a novel duplex TaqMan RT-PCR was developed for detecting and differentiating PEDV strains in China. There was no cross-amplification between the two probes when using standard recombinant plasmids, and the specificity was further confirmed by using other seven non-PEDV swine pathogens. The minimum copies required for the detection of both classical and variant PEDV were 4.8 × 102 DNA copies/reaction. The repeatability of TaqMan RT-PCR was evaluated using standard recombinant plasmids and gave coefficients of variation 0.19–4.93. In recent 5 years, 79 clinical samples were collected from piglets with severe diarrhea in the Central China. Among these clinical samples, 51 were confirmed as PEDV positive by conventional RT-PCR, whereas 63 variant PEDV, 3 co-infections and 1 classical PEDV were confirmed by this duplex TaqMan RT-PCR, with viral loads of 102–108, 102–103, and 104 copies/reaction, respectively. Therefore, the duplex TaqMan RT-PCR could be a useful method for detecting and differentiating variant and classical PEDV strains. The results showed that variant PEDV was prevalent in clinical samples in central China. Moreover, in this study, co-infection by PEDV strains was detected for the first time and might help explain the emergence of the novel recombinant PEDV in recent years.

Based on colloidal gold and broad-spectrum monoclonal antibody that binds to zeranol and its five analogues with high sensitivity, a lateral flow immunochromatographic assay (LFIA) in a competitive format was developed to specifically determine residues of zeranol, an illegal growth promoter in livestock. In this study, the assay had high sensitivity and was broad-spectrum only for zeranol and its five analogues, and the results were obtained within 10 min without needing sophisticated procedures. The cutoff values for zeranol and its five analogues were 10 ng/mL, and the IC50 values for zeranol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol and zearalenone were 1.250, 1.800, 1.775, 1.225, 1.709 and 1.319 ng/mL, respectively. The recovery rates were ranged from 85.6 to 93.9%, with the coefficient of variations less than 12.4%. The results demonstrated that the LFIA could be used for rapid, simultaneous, semi-quantitative and quantitative detection of residues of zeranol and its five analogous in milk.

Marek׳s disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-β binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-β1, with suppression of TGF-β signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.

Maduramicin (MD) is a type of monoglycoside polyether ionophore antibiotic that can effectively treat coccidiosis and facilitate animal growth. However, its extensive and excessive use brings potential risk to human health. Herein, an electrochemical immunosensor based on indirect competitive format was fabricated for analysis of MD residue in eggs by a multiple signal amplification system. Initially, Au nanoparticles were deposited onto glassy carbon electrode surface to load the coating antigen MD-BSA and to improve conductivity. Then the signal amplification platform was constructed by encapsulating hemin into Fe-MIL-88 NH2 metal-organic frameworks (hemin@MOFs), and then the obtained composites were decorated with AuPt nanoparticles. The synthesized hemin@MOFs/AuPt was not only used as a signal amplification mediator, but also utilized as a carrier for immobilization of horseradish peroxidase-conjugated affinipure goat anti-mouse antibody (Ab2-HRP) and horseradish peroxidase (HRP). The constructed hemin@MOFs/AuPt-Ab2-HRP bioconjugates could effectively amplify the current signal since hemin@MOFs, AuPt and HRP all exhibited high catalytic activity towards the hydrogen peroxide. Moreover, the established immunosensor showed high sensitivity and stability during the detection procedure. With the synergistic catalytic effect of hemin@MOFs, AuPt and HRP, a wide detection range of 0.1–50 ng mL−1 and a low detection limit of 0.045 ng mL−1 were achieved (S/N = 3), respectively. Ultimately, the developed method displayed excellent performance in practical applications, providing a promising probability to detect other veterinary drug residues to guarantee food safety.

A portable fluorescence biosensor with rapid and ultrasensitive response for Clenbuterol (CL) has been built up with fluorescent nanosilica and a lateral flow test strip. Quantitative detection of CL was realized by recording the fluorescence intensity of fluorescent nanosilica captured on the test line. The sensing results indicated that the sensitivity of the fluorescent nanosilica-based strip was better than that of conventional colloidal gold-based strips. The visual limit of detection of the strip for qualitative detection was 0.1 ng/mL while the LOD for quantitative detection could down to 0.037 ng/mL by using fluorescence biosensor. The recoveries of test samples were from 89.3% to 97.7%. The assay time for CL detection was less than 8 min, suitable for rapid testing on-site.

Previous studies have shown that PRRSV nsp11, which was an endoribonuclease, was an interferon antagonist, however, the mechanism that nsp11 inhibited IFN-β production was unclear. To explore whether the endoribonuclease was required for nsp11 to disrupt the IFN-β production, substitutions of the presumed catalytic histidine and lysine residues of nsp11 were introduced into plasmid pcDNA 3.1-FLAG. The results showed that mutation that inactivated endoribonuclease made nsp11 lose its ability to inhibit Poly(I:C)-induced IFN-β promoter activity. In conclusion, our present work indicated that the endoribonuclease activity of nsp11 was essential for nsp11 to inhibit the IFN-β induction.

Receptors for the Fc regions of immunoglobin G (IgG) play a critical role in immunoregulation and immune defenses against pathogens. In this study, we describe the cloning, eukaryotic expression and IgG subclass specificity of ovine Fc gamma receptor III (FcγRIII). The newly cloned ovine FcγRIII cDNA contains a 940 bp open-reading frame (ORF), and is predicted to encode a 250 amino acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail. The overall identity of the ovine FcγRIII amino acid sequence to its cattle, pig and human counterparts was 83.2%, 62.0%, 60.7%, respectively. Overlapping PCR was performed with the extracellular domain of ovine FcγRIII and the transmembrane and intracellular region of ovine Fc gamma chain to construct a chimeric receptor. Rosetting analysis showed that transfected COS-7 cells required Fc receptor gamma chain for the expression of FcγRIII on the surface. COS-7 cells expressing FcγRIII were able to bind chicken erythrocytes sensitized with ovine IgG1, but not IgG2. Identification of ovine FcγRIII will further our understanding of the ovine immune system.

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL −1 . The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.

This article aims to establish an efficient assay for screening monoclonal antibodies (McAbs) against the membrane proteins of chicken embryo fibroblast (CEF) for further studies of the cellular receptors of infectious bursal disease virus (IBDV). McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-biotin postinfection. Then, the cells were reacted with streptavidin-horseradish peroxidase (HRP) and finally stained by 3-amino-9-ethylcarbazole (AEC). The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them (1A5, 1H11, 2B12, 3G1, 4D10, and 4B8) have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs, which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.