Ruiguang Deng

Rapid and sensitive detection of β-agonists using a portable fluorescence biosensor based on fluorescent nanosilica and a lateral flow test strip

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.

Endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit IFN-β induction.

Abstract 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.

Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

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.

Development of an Immunochromatographic Strip Test for the Rapid Detection of Zearalenone in Corn

A rapid immunochromatographic test strip has been developed for the detection of zearalenone (ZEN) residues in corn. For this purpose, a specific anti-ZEN monoclonal antibody (mAb), 4A3-F9, was obtained and identified. ZEN coupled to bovine serum albumin (BSA) via 1,4-butanediol diglycidyl ether was prepared as immunogen. The mAb showed low cross-reactivity with five ZEN analogues. Using an antibody preparation with a titer of ≥1:5.12 × 10(5), the cross-reactivity (CR) of the anti-ZEN monoclonal antibody with four of the analogues was <4%, except for zearalanone, which was 53.121%. The recovery rates of ZEN in spiked corn samples were in the range of 91.30-97.07% with coefficients of variation <5.32%. An immunochromatographic strip was developed using the specific anti-ZEN monoclonal antibody and applied to the screening of corn samples for ZEN residues. The test could be accomplished within 5-10 min. The sensitivity of the test strip in corn sample extract was confirmed to be 20 μg/kg by unaided visual assessment, and the IC50 was calculated as 3.4 ng/mL using a test strip reader. The test strip, analyzed by unaided visual assessment and strip reader, showed very good agreement with competitive indirect ELISA and high-performance liquid chromatography (HPLC) analysis for naturally contaminated corn samples.

Marek's disease virus-encoded analog of microRNA-155 activates the oncogene c-Myc by targeting LTBP1 and suppressing the TGF-β signaling pathway.

Mareks 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.

The Nonstructural Protein 1 Papain-Like Cysteine Protease Was Necessary for Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 to Inhibit Interferon-β Induction

Porcine reproductive and respiratory syndrome virus nonstructural protein 1 (nsp1) could be auto-cleaved into nsp1α and nsp1β, both of which had the papain-like cysteine protease activities. Previous studies have shown that porcine reproductive and respiratory syndrome virus nsp1 was an interferon (IFN) antagonist. However, the mechanism by which nsp1 inhibited IFN-β production was unclear. Here, we used site-directed mutagenesis that inactivated the papain-like cysteine protease activities of nsp1 to explore whether the papain-like cysteine protease activities were required for nsp1 to disrupt IFN-β production. The results showed that mutations that inactivated papain-like cysteine protease activity of nsp1α made nsp1 lose its IFN antagonism activity, whereas mutations that inactivated papain-like cysteine protease activity of nsp1β did not influence the IFN antagonism activity of nsp1. In conclusion, our present work indicated that the papain-like cysteine protease activity of nsp1α was necessary for nsp1 to inhibit IFN-β induction.

Rapid and Sensitive Detection of the Food Allergen Glycinin in Powdered Milk Using a Lateral Flow Colloidal Gold Immunoassay Strip Test

A rapid immunochromatographic lateral flow test strip in a sandwich format was developed with the colloidal gold-labeled mouse antiglycinin monoclonal antibody (mAb) and rabbit antiglycinin polyclonal antibody (pAb) to specifically identify glycinin, a soybean allergen. The test strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad, and a test membrane containing a control line and a test line. This test strip has high sensitivity, and results can be obtained within 10 min without sophisticated procedures. The limit of detection (LOD) of the test strip was calculated to be 0.69 mg/kg using an optical density scanner that measures relative optical density. The assay showed high specificity for glycinin, with no cross-reactions with other soybean proteins or other food allergens. The recoveries of the lateral flow test strip in detecting glycinin in powdered milk samples ranged between 80.5 and 89.9% with relative standard deviations of less than 5.29% (intra-assay) and 6.72% (interassay). Therefore, the test strip is useful as a quantitative, semiquantitative, or qualitative detection method for glycinin in powdered milk. In addition, the test strip can be used to detect glycinin in other processed foods and may be a valuable tool in identifying effective approaches for reducing the impact of glycinin.

Marek’s disease virus-encoded microRNAs: genomics, expression and function

MicroRNAs (miRNAs) are the recently discovered small non-coding RNA molecules that have post-transcriptional regulatory functions in many important biological processes. A large number of miRNAs have been found to be encoded by viral genomes, especially in herpesviruses. Previous research regarding miRNAs encoded by herpesviruses, including Marek’s disease virus (MDV), has demonstrated their involvement in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis. MDV is an oncogenic alphaherpesvirus, with the ability to induce tumors in natural hosts; however, formation of these tumors can be prevented by immunization with attenuated or nonpathogenic forms of the virus. Marek’s disease is considered to be a good biomedical model for investigating the biology, genetics, and immunology of tumorigenesis. In this paper, we review the discovery and identification of MDV-encoded miRNAs, along with their genomics, expression profiles, and currently known functions. We also discuss the prospects and techniques possibly applicable to the further investigation of the biological roles of MDV-encoded miRNAs.

Development of a One-Step Strip Test for the Diagnosis of Chicken Infectious Bursal Disease

Abstract A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Mareks disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 × 104 ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1–2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.

Development of a lateral-flow immunochromatographic test device for the rapid detection of difloxacin residues

Abstract In this study, a rapid competitive immunochromatographic test strip has been developed for specifically testing residues of difloxacin (DIF). 1H10-2B2, one of the three hybridoma lines that were tested and selected by enzyme linked immunosorbent assay, was used in the test strip. The monoclonal antibody has a good sensitivity with an IC50 of 2.2 ng/mL to DIF, 0.24% cross-reactivity towards danofloxacin and no cross-reactivity towards other related compounds and other drugs. The calibration curve of DIF test strip presented a typical sigmoidal curve. In practice, the lower limit of detection using a strip reader was 0.5 ng/ml while 2 ng/ml by unaided visual assessment. Samples of milk were spiked at 2–8 ng/mL, presenting recoveries between 94.5 and 107%, and the coefficient of variation (CV,%) (1.6–9%). The data above suggests that the strip meets the requirements of high sensitivity, good specificity, simplicity and speed, as well as the characteristics of reproducibility and accuracy.