Identification of the linear ligand epitope on classical swine fever virus that interacts with porcine kidney 15 cells.

Binding of the viral ligand to a specific receptor is the first step of virus entry into target cells. The envelope proteins Erns, E1, and E2 of classical swine fever virus (CSFV) are involved in the interaction with host cell receptors to mediate CSFV infection. The aim of this investigation was to identify epitopes that bind to porcine kidney (PK)-15 cells to prevent CSFV infection. Ten peptides representing Erns, E1, and E2 were synthesized. Immunohistochemical study showed that the SE24 peptide, which is derived from the E2 amino acid sequence, could effectively bind to PK-15 cells. Similarly, a flow cytometry assay demonstrated that SE24 binding to PK-15 cells could be blocked by CSFV. The binding of SE24 with PK-15 cells leads to decreased CSFV infection of PK-15 cells in a dose-dependent manner. These results suggest a potential new strategy for the prevention and control of CSFV infection that requires further investigation.

La liaison d'un ligand viral à un récepteur spécifique est la première étape de l'entrée du virus dans les cellules cibles. Les protéines Erns, E1, et E2 de l'enveloppe du virus de la peste porcine classique (VPPC) sont impliquées dans l'interaction avec les récepteurs de la cellule hôte afin de médier l'infection par le VPPC. L'objectif de la présente étude était d'identifier les épitopes qui se lient aux cellules de rein de porcs (PK-15) afin de prévenir l'infection par le VPPC. Dix peptides représentant Erns, E1, et E2 ont été synthétisés. Des études immunohistochimiques ont montré que le peptide SE24, qui est dérivé de la séquence des acides aminés d'E2, pouvait effectivement se lier aux cellules PK-15. De manière similaire, une épreuve par cytométrie en flux a démontré que la liaison de SE24 aux cellules PK-15 pouvait être bloquée par le VPPC. La liaison de SE24 avec PK-15 amène une diminution de l'infection des cellules PK-15 d'une manière dose dépendante. Ces résultats suggèrent une nouvelle stratégie potentielle pour la prévention et la maîtrise de l'infection par le VPPC qui nécessite de plus amples études.(Traduit par Docteur Serge Messier).

Prokaryotic expression of the extracellular domain of porcine programmed death 1 (PD-1) and its ligand PD-L1 and identification of the binding with peripheral blood mononuclear cells in vitro.

Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.

La protéine de la mort cellulaire programmée (PD-1), une molécule co-stimulatrice de la famille de CD28, a deux ligands, PD-L1 et PD-L2. Nos études antérieures ont montré que l'expression de PD-1 et PD-L1 est régulée à la hausse lors d'une infection virale chez des porcs. Des études exhaustives ont montré chez l'humain et la souris qu'un blocage de la voie PD-1/PD-L1 par des anticorps anti PD-L1 ou du PD-1 soluble permet la régénération des cellules T épuisées. Dans la présente étude les domaines extracellulaires de PD-1 et PD-L1 ont été utilisés afin d'évaluer l'attachement de PD-1 et PD-L1 avec des cellules mononucléaires du sang périphérique (CMSP). Nous avons amplifié l'ADNc codant pour les domaines extracellulaires de PD-1 et PD-L1 pour construire des plasmides d'expression recombinants et obtenir des protéines recombinants solubles, qui ont par la suite été marquées avec de l'isothiocyanate de fluorescéine (ITCF). Les protéines recombinantes His-ExPD-1 et His-ExPD-L1 étaient exprimées sous la forme de corps d'inclusion avec un poids moléculaire relatif de 33,0 et 45,0 kDa, respectivement. Nous avons par la suite préparé des anticorps polyclonaux contre ces protéines avec un antisérum titrant 1:102 400. L'attachement des protéines aux CMSP a été évalué par cytométrie en flux. Les signaux de fluorescence de His-ExPD-1-ITCF et His-ExPD-L1-ITCF étaient supérieurs à ceux pour le témoin ITCF. Ces résultats suggèrent que les protéines recombinantes solubles pourraient être utilisées afin de préparer des anticorps monoclonaux pour bloquer la voie PD-1/PD-L1.(Traduit par Docteur Serge Messier).

Homogeneous electrochemical detection of ochratoxin A in foodstuff using aptamer-graphene oxide nanosheets and DNase I-based target recycling reaction.

A simple and feasible homogeneous electrochemical sensing protocol was developed for the detection of ochratoxin A (OTA) in foodstuff on the immobilization-free aptamer-graphene oxide nanosheets coupling with DNase I-based cycling signal amplification. Thionine-labeled OTA aptamers were attached to the surface of nanosheets because of the strong noncovalent binding of graphene oxide nanosheets with nucleobases and aromatic compounds. The electronic signal was acquired via negatively charged screen-printed carbon electrode (SPCE) toward free thionine molecules. Initially, the formed thionine-aptamer/graphene nanocomposites were suspended in the detection solution and far away from the electrode, thereby resulting in a weak electronic signal. Upon addition of target OTA, the analyte reacted with the aptamer and caused the dissociation of thionine-aptamer from the graphene oxide nanosheets. The newly formed thionine-aptamer/OTA could be readily cleaved by DNase I and released target OTA, which could retrigger thionine-aptamer/graphene nanocomposites with target recycling to generate numerous free thionine molecules. Free thionine molecules were captured by negatively charged SPCE, each of which could produce an electrochemical signal within the applied potentials. Under optimal conditions, graphene-based aptasensing platform could exhibit good electrochemical responses for the detection of OTA at a concentration as low as 5.6pg/mL. The reproducibility, precision and selectivity of the system were acceptable. Importantly, the method accuracy was comparable with commercialized OTA ELISA kit when using for quantitative monitoring of contaminated wheat samples.

Hybridization-induced Ag(I) dissociation from an immobilization-free and label-free hairpin DNA: toward a novel electronic monitoring platform.

A novel silver ion (Ag(+))-assisted hairpin DNA through C-Ag(+)-C coordination chemistry was designed for immobilization-free and label-free electrochemical monitoring of human immunodeficiency virus (HIV) DNA on a negatively charged indium-tin oxide electrode, based on hybridization-induced dissociation of silver ions from the hairpin DNA.

Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine.

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.

Infectivity and propagation of attenuated infectious bursal disease virus in the chicken B-lymphocyte cell line DT40.

In this paper, the infectivity and propagation of two attenuated infectious bursal disease virus (IBDV) strains in DT40 cells were investigated. The results showed that both of the tested strains, TAD and HN(3), directly infect and proliferate in DT40 cells, requiring no adaptive passages. Unexpectedly, IBDV can be rapidly propagated and continuously harvested at high titers for a long time, accompanied by the rapid growth of host cells and showing no increase in pathogenicity. Our results provide further support to suggest that DT40 cells can be used as an ideal model for studying IBDV pathogenesis. Additionally, the DT40 cell line could also serve as a potential system for commercial IBDV vaccine preparation.

Development and evaluation of an immunochromatographic strip for trichinellosis detection.

In the present study, an immunochromatographic strip was developed for the serological detection of trichinellosis in swine. In the strip, the excretory-secretory (ES) antigens of Trichinella labelled with colloidal gold was used as the detector, and the staphylococcal protein A (SPA) and goat anti-ES antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. The evaluation of the strip was performed by comparing 60 clinical positive blood samples detected by the artificial digestion method with 46 serum samples from pigs infected with parasites other than Trichinella and 30 serum samples of parasite-free healthy pigs. The strip was shown to be of high specificity and sensitivity that were closely correlated with those of ELISA. Furthermore, the dipstick assay based on the strip is rapid (10 min) and easy to perform with no requirement of special skill, reagent or equipment. This suggests the immunochromatographic strip is an acceptable alternative to be used in clinical laboratories lacking specialized equipment as well as for field diagnosis.

Identification of neutralizing epitopes on the VP2 protein of infectious bursal disease virus by phage-displayed heptapeptide library screening and synthetic peptide mapping.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, which is one of the most important and widespread infectious diseases in commercial chickens. Conformational epitopes have been reported in the highly variable region of the VP2 protein of IBDV. In the present study, a random heptapeptide library was screened by using monoclonal antibodies (mAbs), YNW17 and YNW29, directed to the VP2 of IBDV and two peptide motifs, D-X-P-R and A-R-G, were identified. The motifs are present on the N and C terminal sequences of the highly variable region of VP2. Synthetic overlapping peptides covering the motifs on VP2 were analyzed by Dot- ELISA with the mAbs and two epitopes 197CDSSDRPRVYTIT209 and 329ARGSLAVTI337 identified. The above epitopes were also recognized by chicken anti-IBDV sera and shown to inhibit the binding of their mAbs to recombinant VP2. Both mAbs and sera from mice immunized with the conjugated epitope-peptides were able to neutralize serotype I IBDV. These results indicated that the epitopes are two neutralizing linear B-cell epitopes and would be useful for the development of peptide-based IBD vaccines.