JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Development of an Immunochromatographic Strip Test for the Rapid Detection of Zearalenone in Corn.
J. Agric. Food Chem.
PUBLISHED: 11-10-2014
Show Abstract
Hide Abstract
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 ?g/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.
Related JoVE Video
Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs.
J. Gen. Virol.
PUBLISHED: 09-10-2014
Show Abstract
Hide Abstract
The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.
Related JoVE Video
Development of a blocking ELISA for detection of antibodies against avian hepatitis E virus.
J. Virol. Methods
PUBLISHED: 03-27-2014
Show Abstract
Hide Abstract
A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (HEV). In the bELISA, the coating antigen was a truncated protein containing C-terminal 268-amino acid region of ORF2 from an avian HEV strain isolated in China (CaHEV) and blocking antibody was a monoclonal antibody (mAb) 1H5 recognizing the epitope within amino acids 384-414 in the C-terminal 268-amino acid region. The concentration of blocking mAb 1H5 was determined as that yielded an OD450nm value of 1.0 for binding to the coating antigen and the antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 20.7% at the mean percent inhibition plus 3 standard deviations was determined by testing 265 negative sera. The bELISA had a sensitivity of 98.3% by testing 116 positive sera from chickens infected experimentally with CaHEV and had no cross-reaction with other anti-avian virus antibodies. The compliance rates of the bELISA with indirect ELISA and Western blot were 83.7% and 93.3%, respectively, by testing 300 field chicken sera. These results suggested that the bELISA developed in this study can be used for detection of antibodies against avian HEV and showed high reproducibility compared with indirect ELISA and Western blot methods.
Related JoVE Video
Anti-idiotypic antibodies reduce efficacy of the attenuated vaccine against highly pathogenic PRRSV challenge.
BMC Vet. Res.
PUBLISHED: 01-29-2014
Show Abstract
Hide Abstract
The inability of current vaccines to provide effective protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection is not fully understood. One of the reasons might be the presence of anti-idiotypic antibodies (Ab2s) to the envelope glycoprotein GP5 induced by PRRSV infection since our previous studies demonstrated the presence of auto-Ab2s (aAb2s) in pigs infected with PRRSV. To test this hypothesis, PRRSV negative piglets were injected with a monoclonal Ab2 (Mab2-5G2) and aAb2s that are specific for anti-GP5 antibody, vaccinated with the attenuated PRRSV vaccine CH-1R and then challenged with the highly pathogenic PRRSV HuN4 strain. The animals were evaluated for clinical signs, pathological changes of the thymus and lungs, viremia, levels of serum antibodies and cytokines.
Related JoVE Video
Heme oxygenase-1 acts as an antiviral factor for porcine reproductive and respiratory syndrome virus infection and over-expression inhibits virus replication in vitro.
Antiviral Res.
PUBLISHED: 01-25-2014
Show Abstract
Hide Abstract
Virus replication depends upon host-cell processes in infected cells, and this is true for porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS that is a worldwide threat to the swine industry. Heme oxygenase-1 (HO-1) is a ubiquitously expressed inducible isoform of the first and rate-limiting enzyme for heme degradation. Our previous research suggested that HO-1 may play an important role in PRRSV infection. However, the function of HO-1 in PRRSV infection is unclear. In the present study, Marc-145, PK-15(CD163) cell lines and porcine alveolar macrophages (PAMs) were used to evaluate the effects of HO-1 induction and over-expression on the replication of two different PRRSV strains. Induction of HO-1 markedly decreased the replication of PRRSV strains in the different cells. Similarly, adenoviral-mediated over-expression of HO-1 also greatly decreased the replication of PRRSV. In contrast, ablation of HO-1 using small interfering RNA concomitantly increased PRRSV replication. Therefore, the data were consistent with HO-1 acting as an antiviral factor and these findings suggested that over-expression or induction of HO-1 may provide a potential therapeutic strategy against PRRSV infection.
Related JoVE Video
Phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in central China based on the ORF3 gene and the main neutralization epitopes.
Arch. Virol.
PUBLISHED: 09-06-2013
Show Abstract
Hide Abstract
Since 2010, porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central China. To investigate the epidemic characteristics of PEDV, the complete ORF3 genes of 14 PEDV field strains from central China during 2012 to 2013 were cloned, sequenced and compared with reference strains. Phylogenetic analysis based on the complete ORF3 gene showed that the PEDVs in central China and the reference strains could be divided into three groups: G1, G2, and G3. The 14 PEDV isolates were classified as G1 and showed a close relationship to some Chinese strains isolated previously in central China and differed genetically from recent isolates from southern China, Korean strains (SM98 and DB1865, 2012), the Chinese LZC strain (2007), and the vaccine strain (CV777) being used in China. Our findings suggested that the PEDVs circulating between 2012 and 2013 in central China might have evolved from earlier strains in the local region. To determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the S gene. The results showed that the neutralizing epitopes at aa 245-252 were highly conserved, but most of the amino acid changes occurred in the epitope regions aa 7-146 and 271-278. We speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of PEDV and consequently result in vaccination failure. Together, these findings may be useful for understanding the epidemiology of PEDV and may be relevant for designing of new and more efficacious vaccines.
Related JoVE Video
Antibody-dependent enhancement of PRRSV infection down-modulates TNF-? and IFN-? transcription in macrophages.
Vet. Immunol. Immunopathol.
PUBLISHED: 08-08-2013
Show Abstract
Hide Abstract
Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Previous studies have shown that Fc? receptor (Fc?R)-mediated entry of infectious PRRSV immune complexes into macrophages plays a pivotal role in the pathogenesis of the disease. This study demonstrates that PRRSV was able to suppress the transcription of key antiviral genes tumor necrosis factor-? (TNF-?) and interferon-? (IFN-?), when infection was via the ADE pathway. Investigation of this infection pathway found that PRRSV suppresses the antiviral genes by disrupting the transcription of the genes coding for the associated transcription factors interferon regulatory factor-1 (IRF-1), interferon regulatory factor-3 (IRF-3) and nuclear factor kappa B (NF-?B). The ADE pathway of infection allows PRRSV to specifically target antiviral genes and alters the innate intracellular immune responses in macrophages. The ADE mechanism described in this study furthers our understanding of pathogenesis following PRRSV infection and is of general relevance to virally induced disease and in relation to antiviral vaccination strategies.
Related JoVE Video
Rapid and sensitive detection of ?-agonists using a portable fluorescence biosensor based on fluorescent nanosilica and a lateral flow test strip.
Biosens Bioelectron
PUBLISHED: 05-13-2013
Show Abstract
Hide Abstract
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.
Related JoVE Video
The zinc-finger domain was essential for porcine reproductive and respiratory syndrome virus nonstructural protein-1? to inhibit the production of interferon-?.
J. Interferon Cytokine Res.
PUBLISHED: 02-21-2013
Show Abstract
Hide Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) has caused one of the most economically devastating and pandemic diseases of swine. Previous studies have documented that PRRSV nonstructural protein-1? (nsp1?) was an interferon antagonist, but the mechanism by which nsp1? inhibited the interferon (IFN)-? production was unclear. Here, by site-directed mutagenesis of the predicted zinc-coordinating residues of the zinc-finger (ZF) domain of nsp1? or by deletion of the ZF domain of nsp1?, we explored whether the ZF domain was required for nsp1? to disrupt the IFN-? production. The results showed that both mutagenesis of the predicted zinc-coordinating residues of the ZF domain and deletion of the ZF domain made nsp1? lose its interferon antagonism activity. In conclusion, our present work indicated that the ZF domain of nsp1? was necessary for nsp1? to inhibit the IFN-? induction.
Related JoVE Video
Impairment of the antibody-dependent phagocytic function of PMNs through regulation of the Fc?Rs expression after porcine reproductive and respiratory syndrome virus infection.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Porcine reproductive and respiratory syndrome (PRRS) is identified as one of the most important etiological agents in multifactorial respiratory disease of swine and can predispose pigs to secondary infections by other pathogens, usually bacteria. To understand the mechanism for an increased susceptibility to secondary bacterial infections, we investigated the antibody-dependent phagocytosis behaviour and killing ability of PMNs after infection by PRRSV strains BJ-4 or HN07-1. PMNs antibody-dependent phagocytosis and their ability to kill E.coli were both noticeably decreased following PRRSV infection, in particular with the highly pathogenic strain HN07-1. As the change in this function of the PMNs may reflect a variation in the expression of Fc?Rs, the expression profiles of the activating and the inhibitory Fc?Rs were examined. We found that RNA expression of the inhibitory receptor Fc?RIIB was up-regulated post-infection, and this was greater after infection with the more virulent PRRSV strain HN07-1. The activating receptor Fc?RIIIA RNA expression was on the other hand inhibited to the same extent by both PRRSV strains. Neutralizing antibody titers post-infection by PRRSV strains BJ-4 or HN07-1 were also detected. All of the pigs in infection groups showed viraemia by the end of the study (56 DPI). These observations may help to understand the mechanism of increased susceptibility to secondary bacterial infections following PRRSV infection.
Related JoVE Video
Rapid detection of Listeria monocytogenes in raw milk with loop-mediated isothermal amplification and chemosensor.
J. Food Sci.
PUBLISHED: 10-20-2011
Show Abstract
Hide Abstract
Loop-mediated isothermal amplification (LAMP) allows a rapid amplification of nucleic acids under isothermal conditions. It can be combined with a rhodamine-based dual chemosensor for much more efficient, field-friendly detection of Listeria monocytogenes. In this report, LAMP was performed at 63 °C for 10 min, followed by a rapid reaction of DNA amplification and the byproduct, pyrophosphate ion, with a rhodamine-based dual chemosensor and Cu(2+) is visualized as a disappearance of red color. The detection limit of L. monocytogenes by the LAMP-chemosensor was 8 to 10 cells per reaction tube, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 10560 reference method. The results showed that the LAMP-chemosensor method has the advantages of better sensitivity and speed and less dependence on equipment than the standard Polymerase Chain Reaction for specifically detecting low levels of L. monocytogenes DNA, and this can be useful in the field as a routine diagnostic tool.
Related JoVE Video
Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of olaquindox residues.
J. Agric. Food Chem.
PUBLISHED: 08-23-2011
Show Abstract
Hide Abstract
A rapid immunochromatographic lateral flow test strip of competitive format has been developed for the specific determination of olaquindox (OLA) residues in pig urine and muscle tissues. The sensitivity of the test strip was found to be 1.58 ± 0.27 ?g/kg and 1.70 ± 0.26 ?g/kg of OLA in pig urine and muscle tissues, and the lower detection limit was 0.27 ± 0.08 ?g/kg and 0.31 ± 0.07 ?g/kg respectively. For negative pig urine and muscle samples spiked with 4, 12, and 36 ?g/kg, the recovery range was 83.0-94.0% and 78.8-87.4% and the coefficient of variation scope [CV (%)] was 3.17-7.41% and 4.66-7.64% respectively. Parallel analysis of OLA samples from pig urine and muscle tissue showed comparable results from the test strip and HPLC. Each test requires 5-8 min, and the test strip can provide a useful screening method for quantitative, semiquantitative, or qualitative detection of OLA residues.
Related JoVE Video
Porcine Fc?RIIb mediates enhancement of porcine reproductive and respiratory syndrome virus (PRRSV) infection.
PLoS ONE
PUBLISHED: 08-11-2011
Show Abstract
Hide Abstract
Antibody-dependent enhancement (ADE) of virus infection caused by the uptake of virus-antibody complexes by Fc?Rs is a significant obstacle to the development of effective vaccines to control certain human and animal viral diseases. The activation Fc?Rs, including Fc?RI and Fc?RIIa have been shown to mediate ADE infection of virus. In the present paper, we showed that pocine Fc?RIIb, an inhibitory Fc?R, mediates ADE of PRRSV infection. Stable Marc-145 cell lines expressing poFc?RIIb (Marc-poFc?RII) were established. The relative yield of progeny virus was significantly increased in the presence of sub-neutralization anti-PRRSV antibody. The Fab fragment and normal porcine sera had no effect. Anti-poFc?RII antibody inhibited the enhancement of infection when cells were infected in the presence of anti-PRRSV antibody, but not when cells were infected in the absence of antibody. These results indicate that enhancement of infection in these cells by anti-PRRSV virus antibody is Fc?RII-mediated. Identification of the inhibitory Fc?R mediating ADE infection should expand our understanding of the mechanisms of pathogenesis for a broad range of infectious diseases and may open many approaches for improvements to the treatment and prevention of such diseases.
Related JoVE Video
Development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus.
J. Virol. Methods
PUBLISHED: 07-13-2011
Show Abstract
Hide Abstract
The genes encoding the Erns and E2 antigen epitopes of classical swine fever virus (CSFV) were expressed as a chimeric protein in Escherichia coli BL21 by pET expression system. The antigenicity of the expressed protein CnC2 was identified by indirect enzyme-linked immunoabsorbant assay (ELISA) and immunoblot with anti-CSFV antibodies. Based on the CnC2 protein, an immunochromatographic strip was developed to evaluate the antibody titer of serum samples from swine vaccinated with CSFV vaccine rapidly. The chimeric protein used as a detector was labeled with colloidal gold. Staphylococcal protein A (SPA) and anti-CnC2 monoclonal antibodies (mAbs) were blotted onto the nitrocellulose membrane as the test and control lines, respectively. The strip assay could be performed within 5min, which did not require any special equipment or skills. Through testing sera against various strains of CSFV, the sensitivity of the strip was determined to be 97.0% (65/67) and the specificity was 100% (98/98). The strip results were consistent with those of the existing commercial ELISA kit, and their correlation coefficient was 0.935. In conclusion, the immunochromatographic strip was an acceptable method for surveying CSFV-antibody titers in pigs.
Related JoVE Video
The nonstructural protein 1 papain-like cysteine protease was necessary for porcine reproductive and respiratory syndrome virus nonstructural protein 1 to inhibit interferon-? induction.
DNA Cell Biol.
PUBLISHED: 03-27-2011
Show Abstract
Hide Abstract
Porcine reproductive and respiratory syndrome virus nonstructural protein 1 (nsp1) could be auto-cleaved into nsp1? and nsp1?, 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-? 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-? production. The results showed that mutations that inactivated papain-like cysteine protease activity of nsp1? made nsp1 lose its IFN antagonism activity, whereas mutations that inactivated papain-like cysteine protease activity of nsp1? did not influence the IFN antagonism activity of nsp1. In conclusion, our present work indicated that the papain-like cysteine protease activity of nsp1? was necessary for nsp1 to inhibit IFN-? induction.
Related JoVE Video
Identification of host cell binding peptide from an overlapping peptide library for inhibition of classical swine fever virus infection.
Virus Genes
PUBLISHED: 03-03-2011
Show Abstract
Hide Abstract
The envelope proteins of classical swine fever virus (CSFV) mediate the binding of CSFV to cell surface molecules and allow CSFV subsequent to enter host cells. However, the proteins binding to host cells and their binding sequences are uncertain. The results showed that the protein E1, E2, and Erns were displayed on the surfaces of T7 phages. The E2 and Erns phage clones showed high binding affinity to host cells, in which the E2 phage clone interacted more specifically with host cells than with other cells, while the Erns phage clone interacted with all tested cells. A 30-mer phage displaying peptide library was constructed and screened against immobilized host cells, in which each peptide was overlapped 10aa to another peptide and spanned all amino acid sequences of Erns and E2. Fifty-eight clones with specific binding to host cells were isolated. Amino acid sequence analyses for two phage clones (P2 and P6) demonstrated the strongest binding positions were at 101-130 (S2) in Erns, and 141-170 (S6) in E2, respectively. The synthetic peptides (S2 and S6) could inhibit the binding of phage clones (P2 and P6) and CSFV to cell. About 86.74 and 74.24% inhibition rates of CSFV infection were achieved at 55 ?M of the synthetic peptides S2 and S6. The results also indicated that the S2 (LAEGPPVKECAVTCRYDKDADINVVTQARN) and S6 (AVSPTTLRTEVVKTFRRDKPFPHRMDCVTT) from CSFV were host cell binding peptides, and both of them had potential for research of CSFV entering host cells.
Related JoVE Video
Endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit IFN-? induction.
Mol. Immunol.
PUBLISHED: 01-10-2011
Show Abstract
Hide Abstract
Previous studies have shown that PRRSV nsp11, which was an endoribonuclease, was an interferon antagonist, however, the mechanism that nsp11 inhibited IFN-? production was unclear. To explore whether the endoribonuclease was required for nsp11 to disrupt the IFN-? 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-? promoter activity. In conclusion, our present work indicated that the endoribonuclease activity of nsp11 was essential for nsp11 to inhibit the IFN-? induction.
Related JoVE Video
Cloning and characterization of ovine immunoglobulin G Fc receptor III (Fc?RIII).
Vet. Immunol. Immunopathol.
PUBLISHED: 08-12-2010
Show Abstract
Hide Abstract
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 (Fc?RIII). The newly cloned ovine Fc?RIII 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 Fc?RIII 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 Fc?RIII 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 Fc?RIII on the surface. COS-7 cells expressing Fc?RIII were able to bind chicken erythrocytes sensitized with ovine IgG1, but not IgG2. Identification of ovine Fc?RIII will further our understanding of the ovine immune system.
Related JoVE Video
Porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages.
Vet. Microbiol.
PUBLISHED: 07-21-2010
Show Abstract
Hide Abstract
In 2006 China experienced outbreaks of a severe form of porcine reproductive and respiratory syndrome (PRRS) characterized by high fever, morbidity and mortality in swine irrespective of age. It is thought that secondary bacterial infections may contribute to the generation of this severe form of the disease. To determine the mechanisms by which a highly pathogenic PRRSV strain causes high fever we used an in vitro model to investigate the production of the pro-inflammatory cytokines IL-1? and TNF-? by macrophages in response to inoculation with PRRSV with or without LPS. Firstly we demonstrated, through an animal inoculation trial, that the isolate HN07-1 was a highly pathogenic strain and sequencing showed that the virus had the same genomic characteristics as previously described isolates. Porcine alveolar macrophage (PAM) cultures infected with PRRSV strains showed increased cytokine secretion and this was greater in the more virulent strain. Addition of LPS further increased cytokine secretion and again the effect was greater with the more virulent strain. Incubation of PAMs with PRRSV strain HN07-1 resulted in a significant increase in surface CD14 expression. This may explain the synergistic action between PRRSV and LPS in the induction of inflammatory cytokine secretion seen in the PAMs and so offer an explanation for the high fever that is characteristic of infections by the highly pathogenic PRRSV.
Related JoVE Video
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.
Virus Res.
PUBLISHED: 05-27-2010
Show Abstract
Hide Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important disease in swine-producing area, and interferon beta (IFN-beta) is the first responder against the animal virus infection. However, whether PRRSV could induce the production of IFN-beta is controversial. In this paper, we first time found that PRRSV could phosphorylate IFN-regulatory factor 3 (IRF-3) and weakly activate the IFN-beta promoter in MARC-145 cells in early infection, but the activations of IRF-3 and IFN-beta promoter were rapidly inhibited in the following infection. Furthermore, which components or products of the invading PRRSV cause PRRSV to inhibit IFN-beta promoter activity attracted our attentions. The obtained results showed that PRRSV nsp1 could inhibit Poly(I:C)-induced IFN-beta 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-beta-producing system and had mechanisms to inhibit this action in MARC-145 cells.
Related JoVE Video
Development of a peptide-based immunochromatographic strip for differentiation of serotype O Foot-and-mouth disease virus-infected pigs from vaccinated pigs.
J. Vet. Diagn. Invest.
PUBLISHED: 05-11-2010
Show Abstract
Hide Abstract
An immunochromatographic strip for discriminating Foot-and-mouth disease virus (FMDV) infected from vaccinated pigs was developed based on synthetic peptide. Five peptides designed from the amino acid sequences of nonstructural proteins (NSP) of FMDV were synthesized, and pep5 located in NSP 3B reacted strongly with serum from FMDV-infected pigs but did not react with serum samples from healthy vaccinated pigs. An immunochromatographic strip was developed by using colloidal gold labeled with pep5 as the detector. Staphylococcal protein A and rabbit against peptide-conjugated ovalbumin antibody immunoglobulin G were blotted on the nitrocellulose membrane for the test and control lines. In comparison with 2 commercial NSP enzyme-linked immunosorbent assays, the peptide-based strip showed good specificity and sensitivity. The apparent agreements of this new assay with Ceditest(R) ELISA and UBI(R) ELISA were 98.59% and 96.63%, respectively. These results indicate that the strip can be adequately used to discriminate FMDV-infected animals from vaccinated animals.
Related JoVE Video
Mareks disease virus-encoded microRNAs: genomics, expression and function.
Sci China Life Sci
PUBLISHED: 03-24-2010
Show Abstract
Hide Abstract
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 Mareks 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. Mareks 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.
Related JoVE Video
Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O.
J. Virol. Methods
PUBLISHED: 01-18-2010
Show Abstract
Hide 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 (5 min) 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.
Related JoVE Video
Genetic characterization and ligand specificity of the ovine Fc gamma receptor I (ovFc gamma RI).
Vet. Immunol. Immunopathol.
PUBLISHED: 01-05-2010
Show Abstract
Hide Abstract
Receptors for the Fc region of IgG (Fc gamma Rs) play a critical role in the immune system and host protection against infection. In this study, we describe the cloning, mRNA expression and IgG subclass specificity of ovine Fc gamma receptor I (ovFc gamma RI). The ovFc gamma RI cDNA contains a 1047bp open-reading frame, and is predicted to encode a 349 amino acid trans-membrane glycoprotein composed of three immunoglobulin-like domains, a trans-membrane region and a short cytoplasmic tail. The overall identity of the ovine Fc gamma RI to its cattle, human and mouse counterparts at the level of the amino acid sequence was 92%, 61% and 54%, respectively. Rosetting analysis shows that COS-7 cells were transfected with an expressional vector carrying the cDNA open-reading frame of ovFc gamma RI and expressed this receptor on the surface. Identification of ovine Fc gamma RI will aid in the understanding of molecular basis of the ovine immune system and further studies of the receptor function.
Related JoVE Video
Development of immunoassays for the detection of sulfamethazine in swine urine.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess
PUBLISHED: 08-15-2009
Show Abstract
Hide Abstract
The use of sulfonamides, such as sulfamethazine (SM2), in pig production is recognized as a public health risk as it inevitably results in sulfamethazine residues in pork. This study is aimed at establishing rapid, simple, reliable methods, with both sensitivity and specificity, for detecting sulfamethazine residues. For this purpose, monoclonal antibodies against sulfamethazine were prepared and characterized. No cross-reaction of the monoclonal antibodies was identified with other sulfonamides or analytes. Based on the competitive immunoassay principle, an indirect competitive ELISA kit (SM2 kit) and a rapid detection strip for detecting sulfamethazine residues were developed using monoclonal antibodies and the colloidal gold technique. The indirect competitive ELISA kit and the strip assay could be performed within 2 h and 5-10 min, respectively. The results showed that the detection limits were 1 ng ml(-1) for the indirect competitive ELISA kit and 8 ng ml(-1) with the unaided eye and 1 ng ml(-1) with the strip reader for the rapid strip assay. Comparing the HPLC method with the SM2-kit or the strip in pig urine spiked with standards of SM2, the difference was <4.6% for SM2-kit and 4.3% for the strip. The two methods are suitable for the rapid screening of sulfamethazine residues in swine urine. Experimental data revealed that the two methods, especially the strip, proved to be sensitive, specific, rapid and easy to use for the quantitative, semi-quantitative or qualitative detection of SM2 residues in swine urine.
Related JoVE Video
Cloning and characterization of ovine immunoglobulin G Fc receptor II (FcgammaRII).
Vet. Immunol. Immunopathol.
PUBLISHED: 07-01-2009
Show Abstract
Hide Abstract
Immunoglobulin G (IgG) Fc receptors (FcgammaRs) bind to immune complexes through interactions with the Fc region of IgG to initiate or inhibit the defense mechanism of the leukocytes on which they are expressed. In this study, we describe the cloning, sequencing and characterization of ovine FcgammaRII. By screening a translated expression sequence tag (EST) database with the protein sequence of bovine IgG Fc receptor II, we identified a putative ovine homologue. Using rapid amplification of cDNA ends (RACE), we isolated the cDNA encoding ovine FcgammaRII from peripheral blood leucocyte RNA. The ovine FcgammaRII cDNA contains an 894bp open-reading frame, encoding a 297 amino acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory motif (ITIM). The glycoprotein encoded by the cloned cDNA was then expressed on the surface of COS-7 cells and immunoglobulin-binding assays show that it binds ovine IgG1, but not IgG2. Identification of the ovine FcgammaRII will aid in the understanding of the molecular basis of IgG-FcgammaR interaction.
Related JoVE Video
Characterization and ligand specificity of sheep IgG2 receptor.
Immunogenetics
PUBLISHED: 03-06-2009
Show Abstract
Hide Abstract
Neutrophils and macrophages in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2, termed boFcgamma2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass. Although related to other mammalian FcgammaRs, boFcgamma2R belongs to a novel gene family that includes the human killer Ig-like receptor and FcalphaRI (CD89) proteins. In this study, we describe the presence and characterization of this novel class of FcgammaR in sheep. The comparative analysis of this novel FcgammaR has allowed us to begin an exploration of some immunological characteristic of ruminants.
Related JoVE Video
Expression, purification and characterization of a functional extracellular domain of porcine FcgammaRII.
Protein Expr. Purif.
PUBLISHED: 02-19-2009
Show Abstract
Hide Abstract
FcgammaRs are involved in regulating a multitude of innate and adaptive immune responses, which makes them attractive targets for the development of novel immunotherapeutic approaches. In this report, we describe a simple method for the production of a large quantity of recombinant porcine FcgammaRII. The extracellular domain of the porcine FcgammaRII (poFcgammaRII) gene was constructed and cloned into the Escherichiacoli expression vector pET-28a. The recombinant protein was expressed at high level in E. coil BL21 (DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6M guanidine hydrochloride and purified by Ni-chelation, and refolded by rapid dilution. After purification and renaturation, the recombinant soluble protein (rsFcgammaRII) coated on high-binding ELISA plates, showed concentration dependent binding of porcine IgG and the binding of porcine IgG to the surface bound rsFcgammaRII was inhibited in a dose-dependent manner by soluble rsFcgammaRII itself. Then by the inhibition assay we evaluated the effectiveness of the rsFcgammaRII in inhibiting the IgG binding to the whole molecule of poFcgammaRII expressed on the Marc-145 cell surface, the rsFcgammaRII inhibited the binding of porcine IgG to the transfected Marc-145 cells surface, with an IC(50) value of 0.87 microM, demonstrating that rsFcgammaRII manifests the similar specificity as native poFcgammaRII. The method for highly efficient production of biologically active poFcgammaRII may be employed for both basic research and potential clinical applications.
Related JoVE Video
Immunochromatographic lateral flow strip tests.
Methods Mol. Biol.
PUBLISHED: 01-23-2009
Show Abstract
Hide Abstract
The immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid identification of various analytes at the point of care. We have developed lateral flow strip tests for the specific qualitative or semiquantitative detection of antigens, antibodies, and haptens, such as drug residues. Here, we describe in detail the preparation of three examples of the strip tests for detection of (a) the infectious bursal disease virus; (b) Trichinella specific antibodies, and (c) Clenbuterol residues in urine samples.
Related JoVE Video
Amino acid at position 176 was essential for porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 1? (nsp1?) as an inhibitor to the induction of IFN-?.
Cell. Immunol.
Show Abstract
Hide Abstract
Previous studies have shown that porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1? (nsp1?) was the interferon (IFN) antagonist. However, the mechanism was unclear. In the present study, deletion of the carboxyl-terminal extension (CTE) (167-180 amino acid (aa)) made nsp1? lose its inhibitory ability to the induction of IFN-?. And a series of C-terminal truncated mutants for nsp1? showed that 1-176 aa of nsp1? was able to inhibit the induction of IFN-? and deleting or mutating the amino acid F176 made nsp1? not inhibit the induction of IFN-?. In conclusion, the CTE and the amino acid F176 were critical for nsp1? as the IFN antagonist and the region representing 167-176 was the minimal subunit of the CTE for nsp1? to retain its suppressive activity to the induction of IFN-?.
Related JoVE Video
Development of an enzyme linked immunosorbent assay and an immunochromatographic assay for detection of organophosphorus pesticides in different agricultural products.
PLoS ONE
Show Abstract
Hide Abstract
Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment.
Related JoVE Video
Development of an immunochromatographic strip for the detection of antibodies against Porcine circovirus-2.
J. Vet. Diagn. Invest.
Show Abstract
Hide Abstract
A rapid (<5 min) immunochromatographic strip using a colloidal gold-labeled antigen probe was successfully developed and applied for the detection of Porcine circovirus-2 (PCV-2) antibodies in swine. Recombinant Cap protein truncated nuclear localization signal of PCV-2, was expressed and labeled with colloidal gold. This conjugate was dispensed on a conjugate pad as the detector. Staphylococcal protein A and purified porcine anti-PCV-2 antibodies were blotted on a nitrocellulose membrane for the test and control lines, respectively. Sensitivity and specificity of this strip test was evaluated using PCV-2 antisera as well as other sera from pigs infected with a variety of swine viruses. For the validation of this strip test, 500 clinical swine serum samples were assessed both by the strip and a commercial enzyme-linked immunosorbent assay (ELISA) kit. The agreement between the immunochromatographic strip and ELISA kit was 94.00%. This strip possesses high sensitivity and specificity and may be useful as a candidate for rapid diagnosis of PCV-2 antibodies in the field.
Related JoVE Video
Immunomodulatory roles and functional analysis of pre-B lymphocyte DT40 cells with the bursal-derived BSP-II treatment.
Peptides
Show Abstract
Hide Abstract
The bursa of Fabricius, the acknowledged central humoral immune organ, is vital to B cell differentiation. However, the regulatory function of the bursal-derived peptide on avian B cell proliferation has not been reported. BSP-II is a recently reported bursal-derived bioactive peptide. In this paper, 75 days-old chicks were twice subcutaneously immunized with BSP-II and inactivated avian influenza virus (AIV, H(9)N(2) strain). It was proved that BSP-II induced a strongly AIV-specific HI antibody production in the immunized chicks. Also, BSP-II could enhance avian pre-B lymphocyte DT40 cell viability. To investigate the global patterns of gene expression in DT40 cells after BSP-II treatment, gene microarray was carried out. It was identified that the differentially expressed genes were involved in various pathways, of which six pathways were associated with signaling transductions, including ErbB signaling, MAPK signaling, Toll-like receptor signaling, Notch signaling, mTOR signaling, and Wnt signaling. Finally, RT-qPCR was used to confirm the microarray expression data. These results indicated the molecular basis of pre-B lymphocyte viability with BSP-II treatment, which provided a potential mechanism of the bursa of Fabricius on pre-B lymphocyte viability, differentiation, and development. These results are valid for the mechanism of the bursa of Fabricius on B lymphocytes development.
Related JoVE Video
Construction of a human scFv antibody library with VH regions randomized and its application.
Biotechnol. Lett.
Show Abstract
Hide Abstract
A non-immunized human single chain variable fragment (scFv) library containing 2.5 × 10(7) individual clones was constructed from antibody variable region genes of 200 non-immunized donors. ScFv gene repertories were generated by randomly combining rearranged variable regions of heavy chain (VH) and natural occurring light chain (VL) using overlapping extension PCR (OE-PCR). Five recombinant protein antigens from different species were successfully used to select specific binders. Phage ELISA showed that the recombinant phage particle could specifically bind to non-structural protein 1 of Avian influenza virus. This method can therefore efficiently generate a phage antibody library.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.