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Pseudorabies virus (PRV) is an economically important viral agent affecting the swine industry in China. Accurate, rapid and simple detection is critical to PRV control and eradication. In the present study, a visible and low equipment-dependent recombinase-aid amplification assay integrated with lateral flow assay (RAA-LFA) was successfully developed to detect the PRV against the gE gene. The RAA-LFA did not react with the other swine pathogens, indicating the method has a good specificity. The limit of detection (LOD) for this RAA-LFA method was 21 copies per reaction against standard plasmids containing gE gene. Notably, the RAA-LFA can detect as low as 6.0 × 100 50 % tissue culture infective dose (TCID 50) viral titer per reaction under nucleic-acid-extraction free condition. Clinical detection showed that the results detected by RAA-LFA were completely consistent with that of the qPCR assay. Taken together, the developed PRV RAA-LFA method provides approachability, comparable accuracy and sensitivity tool for PRV point-of-care testing (POCT), which is valuable to PRV control in areas where equipment and personnel resources are scarce. • A RAA-LFA detection method was successfully developed against PRV gE gene. • The RAA-LFA method was tolerance to crude samples without extraction of viral DNA. • The RAA-LFA method is suitable for the application of POCT. [ABSTRACT FROM AUTHOR]

Gram-positive enhancer matrix (GEM) nanoparticles are often used in mucosal immunity, preparation of subunit vaccines or as an immune adjuvant due to its good immunological activities in recent years. Here, we designed and screened out a high affinity peptide ligand PL23, which could specifically target the non-epitope region of Classic Swine Fever Virus (CSFV) E2 protein, by virtual screening technology, enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) test. The OD value of PL23 at 450 nm was reached 1.982, and the K D value of it was 90.12 nM. Its binding capacity to protein was verified by SDS-PAGE as well. PL23 was subsequently conjugated to GEM nanoparticles by dehydration synthesis generating GEM-PL23 particles, and the GEM-PL-E2 particles were assembled after incubated with CSFV E2 protein. The cytotoxic test indicated that PL23, CSFV E2 protein, GEM nanoparticles, GEM-PL23 particles and GEM-PL-E2 particles were not toxic to cells and GEM nanoparticles could significantly promote the growth of APCs at high concentration for 1 h, p<0.001. In addition, GEM nanoparticles could promote the uptake of antigen by APCs. The cytokines tests suggested that GEM-PL-E2 particles could promote innate immune responses, regulate adaptive immune responses generated by T cells and APCs, and promote the differentiation and maturation of dendritic cells without producing inflammasomes. The results of immunological activity identification showed GEM-PL-E2 particles induced higher levels of both neutralizing antibodies and anti-CSFV antibodies than CSFV E2 protein in mice. This strategy provided a new, simpler, faster and cheaper method for assembling GEM nanoparticles, using an affinity peptide ligand replaced the protein anchor (PA), and provided a better application prospect for the application of GEM particles. [ABSTRACT FROM AUTHOR]

Vesicular stomatitis (VS), characterized by vesicular lesions, produces significant economic losses in livestock industry. Infection by its causative agent, VS virus (VSV), has been previously shown to be mediated by the glycoprotein (G) during attachment, endocytosis and membrane fusion. In the current study, we revealed a novel role of VSV G protein in negative regulation of host cell pro-inflammatory responses. We determined that VSV G protein inhibited lipopolysaccharide (LPS)-induced pro-inflammatory responses as naïve VSV virions in murine peritoneal macrophage-like cell line RAW 264.7. Furthermore, we identified that VSV G protein suppressed nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK)-mediated pro-inflammatory pathways in a dose-dependent manner. Moreover, we demonstrated that α2-3-linked sialic acids on VSV G protein were involved in antagonizing NF-κB- and MAPK-mediated pro-inflammatory responses. All these results expand the knowledge of VSV pathogenesis and strengthen the importance of VSV G protein in host innate immunity, which support implications for the development of VSV-based vaccination and oncolysis. [ABSTRACT FROM AUTHOR]

Egg drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production, and in the quality of the eggs when it infects chickens. Until now, EDSV virus-cell interactions are poorly understood, and the cellular receptor is still unknown. In the present study, we determined the atomic structure of the fiber head of EDSV (residues 377–644) at 2.74 Å resolution. Structure comparison with the (chick embryo lethal orphan) CELO long fiber head and human adenovirus fiber heads reveals that the avian adenovirus may interact with the same attachment factor in a unique fashion. Based on the previous studies of CELO virus, we assumed that the chicken coxsackievirus and adenovirus receptor (CAR) may be the attachment factor. We then demonstrate that the chicken CAR serves as a cellular attachment factor for EDSV based on three lines of evidences. Taken together, the results presented here are helpful for further exploring the pathogenesis related to the interaction between EDSV and host cells, and may be used for vaccine development and intervention strategies against EDSV infection. [ABSTRACT FROM AUTHOR]

• Generation a double deletion mutant Marek's disease virus absence of both meq and lorf9 genes. • Double deletion mutant Marek's disease virus has similar growth characteristics with parental virus in vitro. • Double deletion mutant Marek's disease virus significantly reduced replication in vivo. • Double deletion of meq and lorf9 genes overcome lymphoid organ atrophy induced by meq-deletion Marek's disease virus. • Double deletion mutant Marek's disease virus is fully attenuated. Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes rapid onset of T cell lymphomas in chickens. MDV continues to break through vaccinal immunity due to the emergence of highly virulent field strains. Earlier studies revealed that deletion of the meq gene from MDV results in attenuated vaccines that protect against disease when chickens are infected with highly virulent strains. However, meq -deleted viruses still retain the ability to induce lymphoid organ atrophy, which raises safety concerns. In an earlier study, we found that deletion of lorf9 counteracts this lymphoid organ atrophy. Here, we describe the generation of a double deletion mutant virus lacking virus-encoded meq and lorf9. In vitro studies revealed that during replication, the mutant virus had kinetic characteristics similar to the parental virus; however, in vivo the replication capability was significantly reduced. Results of animal studies revealed no obvious MDV-specific symptoms and lesions. Importantly, the double deletion mutant virus lost the capacity to induce lymphoid organ atrophy, which has been the main obstacle during development of a good vaccine candidate. [ABSTRACT FROM AUTHOR]

The nucleocapsid (N) protein is a suitable candidate for early diagnosis of porcine epidemic diarrhea virus (PEDV). Here, we identified the linear B-cell epitopes of the PEDV N-protein by integrating a computational-experimental framework and constructed three-dimensional (3D) structure model of the N protein using the ColabFold program in Google Colaboratory. Furthermore, we prepared the monoclonal antibodies against the predicted epitopes and recombinant N protein, respectively, and selected pairing mAbs (named 9C4 and 3C5) to develop a double-antibody sandwich immunochromatographic test strip using CdSe/ZnS quantum dots (QDs)-labelled 9C4 and 3C5 as capture and detection antibodies, respectively. This strip can specifically detect PEDV within 10 min with a detection limit of less than 6.25 × 103 TCID 50 /mL. In comparison with RT-PCR for testing 90 clinical samples, the relative sensitivity and specificity of the strip were found to be 98.0% and 100%, respectively, with a concordance rate of 98.9% and a kappa value of 0.978, indicating that QDs-ICTS is a reliable method for the application of PEDV detection in clinical samples. • Discovery of novel B-cell liner epitopes in the N protein of PEDV in silico. • Preparation of mAbs against predicted epitopes and recombination N protein of PEDV. • A quantum dot-based immunochromatographic test strip was developed to detect PEDV. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel electrochemical immunosensor was developed using nanohybrid materials and anti-monensin monoclonal antibody (Ab). • Zn/Ni-ZIF-8 800@graphene/AuNPs nanocomposites were used to modify glassy carbon electrode (GCE) for the first time. • The synergistic effect of Zn/Ni-ZIF-8 800, graphene and AuNPs had enhanced the conductivity and sensitivity of the biosensor. • The nanocomposites exhibited high analytical performance for detecting monensin in milk sample. Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important disease in swine-producing area, and interferon beta (IFN-β) is the first responder against the animal virus infection. However, whether PRRSV could induce the production of IFN-β is controversial. In this paper, we first time found that PRRSV could phosphorylate IFN-regulatory factor 3 (IRF-3) and weakly activate the IFN-β promoter in MARC-145 cells in early infection, but the activations of IRF-3 and IFN-β promoter were rapidly inhibited in the following infection. Furthermore, which components or products of the invading PRRSV cause PRRSV to inhibit IFN-β promoter activity attracted our attentions. The obtained results showed that PRRSV nsp1 could inhibit Poly(I:C)-induced IFN-β promoter activity in MARC-145 cells by down-regulating the protein level of IRF-3 and inhibiting the phosphorylation of IRF-3. In conclusion, our results suggested that PRRSV could be sensed by professional IFN-β-producing system and had mechanisms to inhibit this action in MARC-145 cells. [Copyright &y& Elsevier]

Abstract: An immunochromatographic strip was developed for the serological detection of type O foot-and-mouth disease (FMD) in swine. In the strip, the expressed protein of VP1, the main protective antigen of FMDV, labeled with colloidal gold was used as the detector, the staphylococcal protein A (SPA) and swine anti-foot-and-mouth disease virus (FMDV) antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. 296 swine serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial liquid-phage blocking ELISA (LPB ELISA) kit and peptide ELISA kit. The strip was shown to be of high specificity and sensitivity. Furthermore, the dipstick assay based on the strip is rapid (5min) and easy to perform with no requirement of professional skills, reagents nor equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis. [Copyright &y& Elsevier]

• PRRSV infection induces vimentin rearrangement during post-entry stage. • Vimentin enwraps PRRSV replication complex at the perinuclear site. • Vimentin rearrangement is beneficial for PRRSV replication. • Vimentin (Ser38) phosphorylation by CaMKIIγ is responsible for rearrangement. Vimentin, a member of intermediate filaments, has been documented to be involved in viral infections. Despite several studies focusing on its involvement in porcine reproductive and respiratory syndrome virus (PRRSV) infection, the detailed mechanisms by which vimentin takes effect remain to be fully elucidated. In the present study, we identified a previously unrecognized role of vimentin rearrangement in PRRSV replication. We monitored that PRRSV infection induced vimentin reorganization during post-entry stage, which was beneficial for viral replication. In detail, the serine residue of vimentin was phosphorylated at position 38 (Ser38) by calcium calmodulin-dependent protein kinase II gamma (CaMKIIγ), and vimentin filaments reorganized into cage-like structures enwrapping PRRSV replication complex (RC) at the perinuclear location. Taken together, these results expand the knowledge of PRRSV replication, and provide novel targets for prevention and control of PRRSV. [ABSTRACT FROM AUTHOR]

• Elastase is highly expressed in HP-PRRSV-infected lung tissues. • Elastase facilitates HP-PRRSV entry independent of low pH and CME. • Elastase mediates HP-PRRSV entry via membrane fusion at the cell surface. • HP-PRRSV GP5 is cleaved by elastase. Infection by enveloped viruses includes endocytosis and/or membrane fusion at the plasma membrane, where host cell proteases play an essential role. Among them, elastase-mediated infection has been documented for several enveloped viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically critical factor in global swine industry, is previously reported to infect host cells via low pH-dependent clathrin-mediated endocytosis (CME) and undergo membrane fusion in recycling endosomes. In the current study, we identified that elastase was significantly elevated in the lung tissues of highly pathogenic PRRSV (HP-PRRSV)-infected pigs compared to the mock-infected ones. We subsequently demonstrated that elastase contributed to HP-PRRSV infection in both MARC-145 cells and porcine alveolar macrophages (PAMs). Mechanistically, HP-PRRSV entered host cells at the cell surface via elastase-mediated membrane fusion, independent of low pH and CME, and its glycoprotein 5 (GP5) was cleaved by the protease during this process. All these findings deepen our understanding of HP-PRRSV infection, and are beneficial for prevention and control of the disease. [ABSTRACT FROM AUTHOR]

• A highly sensitive monoclonal antibody (mAb) against bacitracin was produced. • An immunoassay strip was developed for monitoring BAC in milk. • This strip was accurate and reliable based on the results of recovery test. In this study, a murine monoclonal antibody (mAb) of 6D2-G10 against bacitracin zinc (BAC) was produced and applied to an immunochromatographic strip (ICS) for the initial detection of BAC in milk. The ICS with a cut-off value of 25 ng/mL could be perceived by the naked eye within 10 min. With the assist of the strip reader, the limit of detection (LOD) was measured as 0.82 ng/mL, the half-maximal inhibitory concentration (IC 50) was recorded as 3.16 ng/mL, and the linear detection range was from 0.97 to 10.30 ng/mL. The recoveries ranged from 87.7% to 96.0% with the highest coefficient of variation (CV) of 9.1% in the intra-assay and from 84.3% to 90.2% with the highest CV of 10.7% in the inter-assay. In short, the established ICS provided a serviceable analytical tool for qualitatively and quantitatively monitoring BAC in milk. [ABSTRACT FROM AUTHOR]

• This assay can determine zeranol and its five family analogues in milk. • The cutoff values for zeranol and its analogues were all 10 ng/mL coincidentally. • The lower detection limits for zeranol and its analogues were all below 0.3 ng/mL. • The detection time of this assay is less than 15 min. 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 IC 50 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. [ABSTRACT FROM AUTHOR]

• The recombinant fiber protein of EDSV is expressed in Escherichia coli with highly solubility. • The recombinant protein retains the native trimeric structure, with high HA-titer. • A single inoculation with the structure-stabilized fiber protein could protect chickens from virus challenge. Egg-drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production and in the quality of the eggs when it infects chickens. In this report, we engineered truncated fiber protein containing the entire knob domain and part of the shaft region as a vaccine candidate. The protein was obtained in the soluble fraction in Escherichia coli (E. coli), and expression level after nickel-affinity purification was 126 mg/L. By means of multiple characterization methods, it is demonstrated that the recombinant protein retains the native trimeric structure. A single inoculation with the structure-stabilized recombinant protein, even at the lowest dose of 2 μg, stimulated hemagglutination inhibition (HI) antibody responses in chickens, for at least 16 weeks. Neutralizing titers in sera from the protein immunized groups was similar to that of inactivated vaccine immunized group. The lymphocyte proliferation response and cytokine secretion were also induced in immunized SPF chickens. In addition, immunization with the fiber protein also significantly reduced the viral load in the liver. Taken together, these results suggest the truncated fiber protein as an effective single dose, long lasting and rapidly effective vaccine to protect against EDSV. [ABSTRACT FROM AUTHOR]

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 NH 2 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 (Ab 2 -HRP) and horseradish peroxidase (HRP). The constructed hemin@MOFs/AuPt-Ab 2 -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. • An ultrasensitive biosensor for MD detection was constructed by using hemin@MOFs/AuPt-Ab 2 -HRP/HRP as bioprobes. • A linear calibration curve of MD ranged from 0.1 ng mL−1 to 50 ng mL−1 was obtained with LOD as low as 0.045 ng mL−1. • A multiple signal amplification strategy was triggered by the co-synergistic effect of hemin@MOFs, AuPt and HRP. • The proposed immunosensor shows a great potential for sensitive detection of small molecule drug residues. [ABSTRACT FROM AUTHOR]