Wir danken David Wang, Anatoly Urisman, Amy Kistler, Kael Fischer, Patrick Tang, und Alexander Greninger für die initiale Entwicklung und Optimierung der Virochip Protokoll. Diese Arbeit wird durch ein NIH K08 zu gewähren und Abbott Discovery Award (CC) und dem Howard Hughes Medical Institute (bis JLD) unterstützt.

Die Schritte des Virochip Assay umfassen (1) Nukleinsäure-Extraktion aus klinischen Proben, (2) reverse Transkription der RNA extrahiert und 2 nd-cDNA-Synthese, (3) PCR-Amplifikation von cDNA zufällig grundiert, (3) Cy3 Fluoreszenzfarbstoff Einbau (4), die Hybridisierung von markierten Materials auf die Virochip Microarray, und (5) Scannen und Analyse (Abbildung 1). Das Protokoll unten wird die Nutzung des Virochip Assay auf einem Nasenabstrich Probe aus einem Kind zeigen, mit einem Influenza-ähnliche Erkrankung. Primersequenzen Primer A 5&#39;-GTTTCCCAGTCACGATA-(N 9) -3 &#39; Primer B-5&#39;-GTTTCCCAGTCACGATA-3 &#39; 1. Nukleinsäure-Extraktion <ol><li> Nukleinsäure-Extraktion aus klinischen Proben sollten unter Biosafety Level 2 (BSL-2) oder höher Bedingungen durchgeführt werden, da diese Proben potenziell infektiös sind. </li><li> Der Nasenabstrich Probe wird in virale Holding Medium transportiert. Aliquot 200 ul der Probe in ein 1,5 ml Eppendorf Reaktionsgefäß. </li><li> Optional: pass Probe durch ein 0,22 um-Filter (für virale Partikel zu reinigen) </li><li> Optional: Vorbehandlung Probe mit DNAse (zum Host genomische DNA abbauen und reinigen für geschützte enkapsidiert viralen Nukleinsäure) mit dem Turbo DNAse Kit (Ambion) gemäß den Anweisungen des Herstellers </li><li> Entpacken Sie die Probe mit dem Zymo ZR Viral RNA Extraction Kit oder Qiagen UltraSens Virus Kit gemäß den Anweisungen des Herstellers. </li></ol> 2. Reverse Transkription und 2 nd-cDNA-Synthese (&quot;Round A&quot;) <ol><li> Add 1 UL 40 pmol / ul Primer A bis 4 uL extrahierte RNA. Hitze bis 65 ° C für 5 min in einem Thermocycler (zB GeneAmp PCR System 9700) und dann abkühlen lassen bei Raumtemp x 5 min. </li><li> Add 5 ul einer Master-Mix bestehend aus 2 ul 5X RT-Puffer, 1 ul 12,5 mM dNTP, 1 ul Wasser, 0,5 ul 0,1 M DTT und 0,5 ul SSIII RT (Invitrogen). Inkubation bei 42 ° C x 60 Minuten. </li><li> In einem programmierbaren Thermocycler, Hitze bis 94 ° C x 2 min (zum Abbruch der Reverse-Transkriptase-Reaktion), kühl bis 10 ° C, Puls Zentrifuge, und fügen Sie dann 5 ul Sequenase Mix, bestehend aus 1 ul 5X Sequenase Puffer, 3,8 ul Wasser, und 0,15 ul Sequenase (USB Corporation). Für 2 nd-Strang-Synthese, Rampe von 10 ° C bis 37 ° C über 8 min, 8 min halten, dann Hitze auf 94 ° C x 2 min (zu Sequenase inaktivieren) und kühlen bis 10 ° C (halten). </li><li> Optional: Rd Ein Beispiel cDNA kann an dieser Stelle bei -20 ° C gelagert werden </li></ol> 3. PCR-Amplifikation zufällig Primed cDNA (&quot;Round B&quot;) <ol><li> Add 5 ul Rd Eine Stichprobe von 45 ul einer Master-Mix bestehend aus 5 ul 10x PCR-Puffer, 1 ul 12,5 mM dNTP, 1 ul 100 pmol / ul Primer B, 1 ul KlenTaq LA (Sigma) und 37 ul Wasser. </li><li> Run PCR-Protokoll wie folgt: 94 ° C, 2 min → (25 Zyklen von 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( halten) </li><li> Überprüfen Sie die Rd B-Probe auf einem 1,5% Agarose Elektrophorese-Gel. Ein Abstrich aus ca. 200 - 1000 bp sollte visualisiert werden (Abb. 2A) </li></ol> 4. Cy3 Fluoreszenzfarbstoff Incorporation <ol><li> Optional: ein weiteres Beispiel kann mit dem Cy5 Fluoreszenzfarbstoff markiert werden und hybridisiert die gleichen Microarray. </li><li> Zur Einarbeitung aa-dNTP, mit 5 ul Rd B-Probe zu 45 uL einer Master-Mix bestehend aus 5 uL 10x PCR-Puffer, 1 uL 12,5 mM aa-dNTP (Invitrogen), 1 uL 100 pmol / ul Primer B, 1, 1 UL KlenTaq LA (Sigma) und 37 uL Wasser. Run PCR-Protokoll wie folgt: 94 ° C, 2 min → (15 Zyklen von 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( halten) </li><li> Reinigen Sie die Rd C Probe mit den DNA Clean and Concentrator-5 Kit (Zymo Research) gemäß den Anweisungen des Herstellers. Eluieren in 10 ul. </li><li> Add 1 ul 1M Bicarbonat (Sigma) und 1 uL Cy5 (GE Healthcare), die Rd C Probe. </li><li> Inkubieren x 1 Stunde im Dunkeln. </li><li> Reinigen Sie die Cy3-markierten Probe mit den DNA-Clean and Concentrator-5 Kit (Zymo Research) gemäß den Anweisungen des Herstellers. Eluieren in 12 ul. </li></ol> 5. Hybridisierung zu Virochip Microarray <ol><li> Optional: Verwendung 1,5 ul der cDNA-Konzentration und der Menge an Farbstoff auf einem Nanodrop Spektrophotometer aufgenommen zu überprüfen (zur Normalisierung der Proben) </li><li> In einer PCR-Streifen Rohr add 1 ul 25X Fragmentierung Puffer, 5 ul 5X Blockierungspuffer, 9,5 ul (oder die Menge zu normalisieren) von Cy3-markierten Probe, und Wasser zu einem Gesamtvolumen von 25 ul (Agilent) </li><li> Add 25 ul 2X GEx Hybridisierungspuffer (Agilent). Spin down kurz, um Luftblasen zu entfernen. </li><li> Legen Sie 40 ul der Probe auf Dichtung schieben. </li><li> Ort Agilent-gedruckt Virochip array (University of California, San Francisco / Agilent) (Seite mit der Aufschrift &quot;Agilent&quot; down) auf der Oberseite der Dichtung schieben und befestigen Sie Schraube fest. </li><li> Hybridisierung über Nacht in 65 ° C Backofen, Drehzahleinstellung bei 10 Umdrehungen pro Minute. </li><li> Um Arrays, Vorwärmen Puffer 2 (Agilent) auf 37 ° C waschen Nehmen Sie Abstand Dichtung schieben / array Sandwich Unterwasserwelt in Puffer 1 (Agilent). Wash in Puffer 1 x 1 min. Wash in vorgewärmten Puffer 2 x 1 min. Langsam entfernen Folie aus Puffer 2, wobei darauf zu Blasen zu vermeiden (Verwendung Kimwipe weg Docht zusätzliche Feuchtigkeit wird sorgfältig vermieden direkt berühren aktive Seite des Arrays) </li><li> Legen Sie gleiten in Diahalter und insert into Array-Scanner. </li></ol> 6. Virochip Scanning und Analyse <ol><li> Scan Cy3-markierten Array mit 5 &amp;mgr; m oder 2 um je nach Instrument der Standard-Scan-Protokoll (Abbildung 2B). </li><li> Analysieren Virochip Microarrays durch visuelle Inspektion, Cluster-Analyse oder die automatisierte Virochip Analyse-Programm, E-Predict 11. Mit jeder dieser drei Methoden ist Influenzaviren leicht in der Nasenabstrich Probe (von einem Kind mit Grippe-ähnliche Erkrankung) (Abbildung 2C) erkannt </li></ol> 7. Repräsentative Ergebnisse: Wenn das Protokoll korrekt erledigt ist, sollte ein Abstrich auf Agarosegelelektrophorese nach dem Rd B Schritt (Abbildung 2A) gesehen werden. Die Virochip Microarray auf der Agilent-Plattform wird schwierig sein, visuell zu analysieren (Abbildung 2B), aber wenn ein Virus vorhanden ist, sollte leicht durch Sichtkontrolle, Cluster-Analysen und / oder E-Predict (Abbildung 1C) identifiziert werden. Als positive Kontrolle für den Test dienen, können Proben mit gemessenen Mengen von Nukleinsäure aus einem bekannten Virus (MS2 Bakteriophage zB Tabak-Mosaik-Virus) versetzt werden. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Schematische Darstellung der Virochip Microarray-Verarbeitung und Analyse. Extrahierte Nukleinsäure aus klinischen Proben werden nach dem Zufallsprinzip amplifiziert, markiert mit Fluoreszenzfarbstoffen und hybridisiert die Virochip Microarray. Microarrays sind bei 2 mm Auflösung gescannt und analysiert mit einer Vielzahl von rechnerischen Werkzeugen, einschließlich E-vorherzusagen. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Schritte in die Virochip Assay Nach der Verstärkung durch zufällige PCR, ein Abstrich von 200 -. 1000 bp sichtbar gemacht werden durch Gelelektrophorese (A) (B) Drei Virochip Microarrays aus dem 8 Arrays / Objektträger aus Glas dargestellt, mit einer kleinen Region. eines Microarray aufgeblasene im Einschub auf der rechten unteren Ecke. (C) Automated Microarray-Analyse mit viralen E-Predict enthüllt die Anwesenheit von Influenza A-Virus in der klinischen Probe. Die hohe Ähnlichkeit Score (s = 0,55) entspricht einem p-Wert, der nahe bei Null liegt, wodurch dieser eine äußerst signifikante Vorhersage.

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

Die Virochip Protokoll, wie hier beschrieben ist komplex und erfordert eine sorgfältige und qualifizierte Forschungs-Techniker. Richtig Reagenz-Konzentrationen und Bedingungen für die PCR, Kennzeichnung und Hybridisierung sind kritisch. Die Virochip Protokoll kann leicht modifiziert werden, um die Analyse der erkrankten Geweben durch die Verwendung eines Gewebes Extraktionsmethode wie TRIzol (Invitrogen) zur Nukleinsäure-Extraktion unterzubringen. Probes können hinzugefügt oder gelöscht werden, wie notwendig, um das gewünschte Spektrum und Breite der Abdeckung zu erzielen, z. B. Sonden zum Nachweis von viralen Ziele wie Bakterien und Pilze können entwickelt und hinzugefügt werden &quot;on-the-fly&quot;. Einige Anwendungen des Virochip Assay derzeit in Entwicklung sind Breitspektrum-virale Diagnostik im klinischen Labor, Ausbruch Untersuchung, Reinheit Screening von Medikamenten und Impfstoffen sowie neuartige virale Erreger entdeckt.

<table border="1"><tbody><tr><th> Name des Reagenz / Material </th><th> Name des Reagenz / Material </th></tr><tr><td> Primera </td><td> 5&#39;-GTTTCCCAGTCACGATA-(N9) -3 &#39; </td></tr><tr><td> Primer B </td><td> 5&#39;-GTTTCCCAGTCACGATA-3 &#39; </td></tr><tr><td rowspan="5"> RT Master Mix (5 ul) </td><td> 2 ul 5X RT-Puffer </td></tr><tr><td> 1 ul 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 ul Wasser </td></tr><tr><td> 0,5 ul 0,1 M DTT </td></tr><tr><td> 0,5 ul SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 ul) </td><td> 1 ul 5X Sequenase Puffer </td></tr><tr><td> 3,8 ul Wasser </td></tr><tr><td> 0,15 ul Sequenase (USB Corporation) </td></tr><tr><td rowspan="5"> Rd B PCR Master Mix (45 ul) </td><td> 5 ul 10x PCR-Puffer </td></tr><tr><td> 1 ul 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 ul 100 pmol / ul Primer B </td></tr><tr><td> 1 ul KlenTaq LA (Sigma) </td></tr><tr><td> 37 ul Wasser </td></tr><tr><td rowspan="5"> Rd C PCR Master Mix (45 ul) </td><td> 5 ul 10x PCR-Puffer </td></tr><tr><td> 1 ul 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 ul 100 pmol / ul Primer B </td></tr><tr><td> 1 ul KlenTaq LA (Sigma) </td></tr><tr><td> 37 ul Wasser </td></tr><tr><td rowspan="4"> Hybridisierungspuffer (25 ul) </td><td> 1 ul 25X Fragmentierung Puffer (Agilent) </td></tr><tr><td> 5 ul 5X Blocking-Puffer (Agilent) </td></tr><tr><td> 9,5 ul (oder die Menge zu normalisieren) von Cy3-markierten Probe </td></tr><tr><td> Fügen Sie Wasser bis zu einem Gesamtvolumen von 25 ul </td></tr><tr><td> Agilent Virochip Microarray </td><td> Lizenz angemeldet, verfügbar ab Chiu Labor, UCSF </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

Die Diagnose von viralen Ursachen vieler Infektionskrankheiten ist schwierig aufgrund der inhärenten Reihenfolge Vielfalt der Viren sowie die laufende Aufkommen neuer viraler Erreger wie SARS-Coronavirus und 2009 pandemische H1N1-Influenza-Virus, die nicht nachweisbar sind mit herkömmlichen Methoden. Um diese Herausforderungen anzugehen, haben wir bereits entwickelt und validiert eine pan-virale Microarray-Plattform namens Virochip mit der Fähigkeit, alle bekannten Viren sowie neue Varianten auf Basis der konservierten Sequenz Homologie 1 zu erfassen. Mit dem Virochip, haben wir das gesamte Spektrum der Viren mit Infektionen der Atemwege, einschließlich Fälle von ungeklärter critical illness bei hospitalisierten Patienten mit einer Empfindlichkeit entspricht oder überlegen gegenüber herkömmlichen klinischen Tests 2-5 assoziiert identifiziert. Die Virochip wurde auch genutzt, um neue Viren zu identifizieren, einschließlich des SARS-Coronavirus 6,7, einem neuartigen Rhinovirus Clade 5, XMRV (ein Retrovirus in Verbindung mit Prostata-Krebs) 8, Vogelgrippe Bornavirus (die Ursache eines Wasting Disease bei Papageien) 9, und eine neuartige cardiovirus bei Kindern mit Atemwegs-und Durchfallerkrankungen 10. Die aktuelle Version des Virochip hat zu einer Agilent Microarray-Plattform portiert worden und besteht aus ~ 36.000 Sonden aus über ~ 1.500 Viren in GenBank Stand Dezember 2009 abgeleitet. Hier zeigen wir die Schritte in der Bearbeitung eines Virochip Assay von Anfang bis Ende (~ 24 Stunden Bearbeitungszeit), einschließlich Probe Nukleinsäure-Extraktion, PCR-Amplifikation mit Random-Primern, Fluoreszenzfarbstoff Einarbeitung und Microarray-Hybridisierung, Scannen und Analyse.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Die Virochip ist ein pan-virale Microarray entwickelt, um gleichzeitig zu erkennen alle bekannten Viren sowie neue Viren auf der Basis von konservierten Sequenzhomologie. Hier zeigen wir, wie man ein Virochip Testlauf zu klinischen Proben auf das Vorhandensein von sowohl bekannte als auch unbekannte Viren zu analysieren.

Mit einem Pan-Viral Microarray-Assay (Virochip) zur klinischen Proben, die für virale Erreger Bildschirm

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.7K Aufrufe.  University of California, San Francisco. Die Virochip ist ein pan-virale Microarray entwickelt, um gleichzeitig zu erkennen alle bekannten Viren sowie neue Viren auf der Basis von konservierten Sequenzhomologie. Hier zeigen wir, wie man ein Virochip Testlauf zu klinischen Proben auf das Vorhandensein von sowohl bekannte als auch unbekannte Viren zu analysieren.

Serie: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Aufbau einer Liquid-bedeckten Kultur Polarized Menschliche Airway Epithelial Calu-3-Zellen, um Host-Zell-Antwort, um Atemwegspathogene Studie<em&gt; In-vitro-</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Forschung

Immunologie und Infektion

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Mit Klick-Chemie, um die Wirkung von Viral Infection auf Host-Zell-RNA Synthese Messen

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Mit fluoreszierenden Proteinen zu Glycosome Dynamics in der afrikanischen Trypanosomen auf Monitor

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Ein miniaturisiertes Glycan Microarray-Test zur Beurteilung der Avidität und Spezifität von Influenza-A-Virus Hämagglutininarten

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Messung von Influenza-Neuraminidase Inhibition Antikörpertiter durch Enzyme-Linked-Lectin-Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Verwendung von Kapseln zur Negativfärbung von Virusproben im Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic Tötung Assay Immunreaktionen gegen bakterielle Krankheitserreger zu bewerten

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Bestimmung der chemischen Inhibitor-Effizienz gegen intrazelluläre toxoplasma Gondii Wachstum mit einem Luziferase-basierten Wachstumstest

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Messung natürlich erworbener Phagozytose-induzierender Antikörper gegen Plasmodium falciparum Parasiten durch einen Flow Cytometry-based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Diagnose von bakteriellen Atemwegsinfektionen beim Menschen mittels isothermer Amplifikation

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Autor im Rampenlicht: Weiterentwicklung des schnellen Nachweises von Atemwegserregern mit mikrofluidischem Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Schneller Nachweis von bakteriellen Krankheitserregern, die Infektionen der unteren Atemwege verursachen, durch mikrofluidische Chip-basierte schleifenvermittelte isotherme Amplifikation

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...