Wir sind dankbar, Junko Wada für ihre hervorragende technische Unterstützung. Wir danken Prof. Akio Nomoto für die freundliche Bereitstellung eines Baculovirus-Expressionsvektors für PVR-IgG2a. Diese Studie wurde teilweise unterstützt von Grants-in-Aid für die Förderung der Ausrottung der Kinderlähmung und Forschung für neu auftretende und neu auftretender Infektionskrankheiten vom Ministerium für Gesundheit, Arbeit und Soziales.

1. Vorbereitung der Wartung Medium (MM) und Wachstumsmedium (GM) Für die Herstellung von MM und GM finden Sie auf einer Polio-Labor Handbuch des WHO 1. Dulbecco modifiziertem Eagle-Medium (DMEM) mit 2% fetalem Kälberserum (FCS) und 10% FCS als Ersatz von MM und GM verwendet werden könnten, bzw. ergänzt. 2. Herstellung von anti-PV-Antikörper <ol><li> Rekonstituieren anti-PV-Antikörper (RIVM PV Eingabe Antiserum) durch Zugabe von Wasser (0,5 ml pro Flasche) zu jeder Flasche des Anti-PV-Antikörper (Typ 1, 2 oder 3). Fügen Sie diese rekonstituierte Antikörper-Lösungen (0,5 ml) auf 4,5 mL MM in separaten beschrifteten Röhrchen. Hinweis: Einige viele anti-PV-Antikörper werden als gebrauchsfertige Form (0,5 ml pro Flasche) zur Verfügung gestellt. Für eine solche Menge von anti-PV-Antikörper, ist der Zusatz von Wasser für die Zubereitung nicht erforderlich. </li><li> Mix gleichen Volumen der rekonstituierten Anti-PV-Antikörper. Für anti-PV1 +2, 2 +3 oder 3 +1: Mix 1,8 ml jedes rekonstituierte Antikörper (1,8 ml + 1,8 ml = 3,6 ml für jede Mischung). Für anti-PV1 +2 +3: Mix 1,2 ml jedes rekonstituierte Antikörper (1,2 ml + 1,2 ml + 1,2 ml = 3,6 mL). Hinweis: PV-Proben konnte ein Gemisch aus verschiedenen Serotypen von PV enthalten. Um einen Serotyp der PV in Proben mit einer Mischung aus PVs zu identifizieren, ist Pools von anti-PV-Antikörper (dh 1 +2, 2 +3, 3 +1, 1 +2 +3) notwendig. </li><li> Add 1 / 5 Volumen von FCS (0,72 ml) in jede anti-PV (1 +2, 2 +3, 3 +1 oder 1 +2 +3) Antikörper (3,6 mL) (final 18,2% FCS). Hinweis: Der Zusatz von FCS ist zur Reduzierung von unspezifischen Agglutination von Gelatine Partikel durch Antikörper verursacht. </li><li> Halten Sie die Antikörper-Pools bei -20 ° C bis zur Verwendung in PA Assay. </li></ol> 3. Rekonstitution der sensibilisierten Gelatine Teilchen <ol><li> Fügen Sie 1,5 ml Rekonstitutionspuffer im Kit mit einem Fläschchen sensibilisiert Gelatine-Teilchen (sofern als gefriergetrocknetes Pulver) enthalten. </li><li> Gut mischen durch leichtes Klopfen </li><li> Halten Sie die rekonstituierte sensibilisiert Partikel bei 4 ° C. Rekonstituierte Gelatine Teilchens sein könnte aktiven mindestens 2 Wochen bei 4 ° C aufbewahrt, sollte aber kurz vor dem Test vorbereitet werden. </li></ol> Wichtiger Hinweis: Mischen Sie keine unterschiedlichen viel sensibilisierter Gelatinepartikel, weil die Nicht-Agglutination Bild sensibilisiert Gelatinepartikel anders sein könnte, indem die Menge der Teilchen. Für jedes Experiment, soll eine einzige Menge sensibilisiert Gelatine Partikel verwendet werden. 4. PA-Assay für PV Identifikation <ol><li> Bereiten Sie eine Mikrotiterplatte. Zur Identifizierung eines Virus-Probe, wir brauchen 5 Brunnen + mindestens eine negative Kontrolle (sensibilisiert Teilchen ohne Virus und Antikörper, sowie Nr. 6 in unten. See No Virus und Antikörper-Steuerung im Beispiel). Anzahl der Bohrungen beträgt 5 x Proben + mindestens eine Kontrolle in dem Test. Wichtiger Hinweis: Bitte nehmen Sie sich mindestens eine No-Virus und Antikörper-Kontrolle in den Test. Eine Durchstechflasche sensibilisiert Teilchen ist etwa für die Identifizierung von 10 Virusproben. </li><li> Add 12 ul / well anti-PV-Antikörper oder GM, die Mikrotiterplatte. Die Anordnung der Platte ist wie folgt (siehe auch Beispiel unten). In Proben ohne Antikörper (auch Nr. 1 und 6), fügen Sie 12 ul GM anstelle von anti-PV-Antikörper. Wichtiger Hinweis: Wenn viele PV-Proben in diesem Test geprüft werden sollte, würde Mehrkanal-Pipetten-oder Dispenser hilfreich sein für diesen Schritt. <table border="1"><tbody><tr><td> Nun # 1 </td><td> Nun # 2 </td><td> Nun # 3 </td><td> Nun # 4 </td><td> Nun # 5 </td><td> Nun # 6 </td></tr><tr><td> GM </td><td> Anti-P1 +2 +3 </td><td> Anti-P1 +2 </td><td> Anti-P2 3 </td><td> ani-T1 +3 </td><td> GM </td></tr></tbody></table></li><li> Add 12 ul / well des Virus Probe. In einer Probe ohne Virus (auch # 6), fügen Sie 12 ul GM anstelle von Virus-Lösung. Hinweis: Virus Probe Kulturüberstand der infizierten Zellen, in der Regel L20B Zellen oder RD-Zellen. Wichtiger Hinweis: Wenn viele PV-Proben in diesem Test geprüft werden sollte, würde Mehrkanalpipette hilfreich sein für diesen Schritt. <table border="1"><tbody><tr><td> Nun # 1 </td><td> Nun # 2 </td><td> Nun # 3 </td><td> Nun # 4 </td><td> Nun # 5 </td><td> Nun # 6 </td></tr><tr><td> Virenprobe </td><td> Virenprobe </td><td> Virenprobe </td><td> Virenprobe </td><td> Virenprobe </td><td> GM </td></tr></tbody></table></li><li> Nehmen Sie rekonstituierte sensibilisiert Partikel-Lösung von 4 ° C bis Raumtemperatur, und klopfen Sie leicht vollständig aussetzen Partikel. Wichtiger Hinweis: Nach der vollständigen Suspension der Partikel durch leichtes Klopfen, sollten die Teilchen sofort zu den Platten gegeben werden, bevor die suspendierten Teilchen Niederte und bilden nicht-homogene Lösung. </li><li> Fügen Sie 25 ul / Vertiefung der sensibilisierten Partikel in jede Vertiefung. Wichtiger Hinweis: Nach Zugabe von sensibilisierten Partikel, sollte die Platte sofort auf Platte Mischen unterzogen werden, um eine einheitliche Reaktion der Proben beginnen. Deshalb sollte neben der sensibilisierten Partikel durch Spender zu einer zeitlichen Verzögerung zwischen den Proben in der Platte zu minimieren durchgeführt werden. </li><li> Legen Sie die Mikrotiterplatte auf einen Teller Mixer und mischen die Platte für 30 Sekunden. </li><li> Legen Sie die Mikrotiterplatte auf weißem Papier (zur Erleichterung der visuellen Beobachtung) auf einem Tisch bei Raumtemperatur (15 ~ 30 ° C) für 2 h. Wichtiger Hinweis: Bewegen Sie sich nicht die Platte, während und nach der Reaktion für die Beobachtung der Ergebnisse und Fotos zu machen. Die gebildeten Agglutination ist nicht stabil und einfach durch Verschieben der Platten gestört. </li><li> Machen Sie Fotos von der Platte. Wichtiger Hinweis: Teilansicht des Fotos konnte nicht genügend Daten für eine spätere Untersuchung und Überprüfung der Ergebnisse für die Interpretation. Mehrere Fotos zu ergreifen, um all die Vertiefungen der Platten mit Schwerpunkt auf die Agglutination Bild der Brunnen decken. </li></ol> 5. Die Interpretation der Ergebnisse <ol><li> Identifikation von positiven und negativen Brunnen. Vergleichen Sie die Agglutination in den Proben mit der No-Virus und Antikörper-Kontrolle (Fällung von Gelatine Partikeln, nicht-Agglutination) (Abbildung 1 und 2). Bilder der Agglutination, die eindeutig aus kein Virus und keine Antikörper-Steuerung (non-Agglutination, Negativ-Kontrolle) unterschieden werden sollte als positiv beurteilt werden, und Bilder von Agglutination, die ähnlich wie die negative Kontrolle sind als negativ beurteilt werden. Proben, die klar, aber nur teilweise Agglutination zeigen könnte als positive, aber die Aufnahme als &quot;positiv (Teil-Agglutination)&quot; beurteilt werden. </li><li> Identifikation von PV. Interpretieren Sie die Ergebnisse auf positive und negative Brunnen mit anti-PV-Antikörper gegen PV-Serotypen identifizieren. Prinzip der Interpretation des Ergebnisses ist ähnlich dem der Neutralisationstest in Zellkultur-System (zB positiv für anti-P1 +2 Antikörper zeigt, dass die Probe vom Typ 3 PV enthält). </li></ol> 6. Repräsentative Ergebnisse Repräsentatives Ergebnis von PA Assay PV Identifikation ist in Abbildung 1 dargestellt. Alle PV-Stämme gebildet Agglutination der sensibilisierten Gelatine-Partikel (keine Antikörper-Proben). In Anwesenheit von entsprechenden anti-PV-Antikörper, Fällung von Gelatine-Partikel (non-Agglutination) ähnlich kein Virus und keine Antikörper-Steuerung (negative Kontrolle) beobachtet wurde (z. B. PV1 (Sabin) in Gegenwart von anti-P1 +2 oder anti-P3 1-Antikörper). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Identifikation von PV durch PA Assay. Virus-Lösungen von PV1 (Sabin), PV2 (Sabin) und PV3 (Sabin) (Virustiter von 1,2 x 10 8 CCID 50 / 12 ul, 8,2 x 10 7 CCID 50 / 12 ul, und 3,5 x 10 7 CCID 50 / 12 ul,), wurden für die Identifizierung von PA-Assay verwendet. Kein Virus und keine Antikörper-Kontrolle zeigte Fällung von Gelatine-Partikel (non-Agglutination, Negativ-Kontrolle). <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Die Interpretation der Positive and Negative Brunnen. Virus-Lösungen von PV1 (Sabin), PV2 (Sabin) und PV3 (Sabin) (Virustiter von 2,4 x 10 8 CCID 50 / 25 ul, 1,7 x 10 8 CCID 50 / 25 ul, und 7,3 x 10 7 CCID 50 / 25 ul bezeichnet) wurden verdünnt 1 / 20 bis 1 / 320 und dann 25 ul der verdünnten Proben wurden durch PA-Assay ohne Anti-PV-Antikörper untersucht.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

Souji Masujima ist ein Mitarbeiter der New Product Design Department, Fujirebio Inc.

Die einzigartige Punkt dieser PA-Assay ist die Nutzung von PVR-Molekül statt Anti-PV-Antikörper für den Nachweis von PV 2. Dies ermöglicht eine spezifische Wechselwirkung der sensibilisierten Gelatinepartikel mit PV mit einer einheitlichen Affinität zu allen drei Serotypen des PV 3-6. Wir benutzten einen Immunoadhäsin Form von PVR (PVR-IgG2a) für die Sensibilisierung der Gelatine Partikeln, weil monomeren Formen der PVR nur moderate Affinitäten zu den einzelnen Bindungsstelle am Virion (Dissoziationsk...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Sensibilisiert-Gelatine-Partikel </td><td> Fujirebio Inc. </td><td></td><td> Test-Kit </td></tr><tr><td> Rekonstitutionspuffer </td><td> Fujirebio Inc. </td><td></td><td> Test-Kit </td></tr><tr><td> Mikrotiterplatte </td><td> Fujirebio Inc. </td><td></td><td> Test-Kit </td></tr><tr><td> Anti-PV-Antikörper </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

In der Global Polio Eradication Initiative spielt Labordiagnostik eine entscheidende Rolle durch die Isolierung und Identifizierung von PV aus der Stuhlproben von akuten schlaffen Lähmungen (AFP) Fällen. In der World Health Organization (WHO) Global Polio Laboratory Network, PV Isolierung und Identifizierung werden derzeit durch Zellkultur-System und real-time RT-PCR bzw. durchgeführt. In der post-Eradikation Ära der PV, wäre eine einfache und schnelle Identifizierung Verfahren hilfreich sein für die schnelle Bestätigung der Polio-Fälle auf der nationalen Laboratorien. In der vorliegenden Studie zeigen wir das Verfahren von neuen PA-Assay für PV Identifikation entwickelt. Das PA-Assay nutzt Zusammenspiel von PV-Rezeptor-(PVR)-Molekül und Virion, dass bestimmte und einheitliche Affinität zu allen Serotypen von PV ist. Das Verfahren ist einfach (eine Schritt-Verfahren in Reaktion Platten) und schnelle (Ergebnisse können innerhalb von 2 h Reaktionszeit erhalten werden), und das Ergebnis wird visuell beobachtet (Beobachtung der Agglutination von Gelatine-Partikel).

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

Eine kürzlich entwickelte neuartige Partikelagglutinations (PA)-Assay unter Verwendung von Virus-Rezeptor-Molekül erlaubt eine schnelle und einfache Identifizierung des Poliovirus (PV). In diesem Artikel werden wir das Verfahren für die PA-Assay für PV ausweisen.

Partikelagglutinations Methode für Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

particle-agglutination-method-for-poliovirus-identification

Research

JoVE Journal

Immunology and Infection

15.2K Aufrufe.  National Institute of Infectious Diseases. Eine kürzlich entwickelte neuartige Partikelagglutinations (PA)-Assay unter Verwendung von Virus-Rezeptor-Molekül erlaubt eine schnelle und einfache Identifizierung des Poliovirus (PV). In diesem Artikel werden wir das Verfahren für die PA-Assay für PV ausweisen.

Serie: Particle Agglutination Method for Poliovirus Identification

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Ein 1,5 Stunden-Verfahren für die Identifizierung von Enterococcus Spezies direkt aus Blutkulturen

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Forschung

Immunologie und Infektion

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Peptid-basierte Identifizierung von Functional Motive und ihre Bindungspartner

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Tests für die Identifizierung neuer Antivirale gegen Blauzungenvirus

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS-vermittelte Stürmer Genetics Screening zur Identifizierung von Genen in Nichtwirtresistenz Beteiligte


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Schnelle Identifizierung von Gram-negativen Bakterien aus Blutkulturbrühe unter Verwendung von MALDI-TOF-Massenspektrometrie

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Mittelung der Virushülle Glykoprotein-Spikes von Electron Cryotomography Rekonstruktionen mit Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Kapsel Serotypisierung<em&gt; Streptococcus pneumoniae</em&gt; Durch Latexagglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Frühe das Eindringen des Virus-Assays für die Ermittlung und Bewertung von antiviralen Verbindungen

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Durchflusszytometrische Analyse von partikelgebundenen Bet v 1 Allergens in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...