Ringraziamo David Wang, Anatoly Urisman, Amy Kistler, Kael Fischer, Patrick Tang, e Alexander Greninger per lo sviluppo iniziale e l&#39;ottimizzazione del protocollo Virochip. Questo lavoro è supportato da un K08 NIH concessione e Abbott Discovery Award (per CC) e l&#39;Howard Hughes Medical Institute (a JLD).

I passi del saggio Virochip includono (1) l&#39;estrazione degli acidi nucleici da campioni clinici, (2) trascrizione inversa di RNA estratto e 2 ° fili sintesi di cDNA, (3) amplificazione di cDNA a caso innescato, (3) Cy3 incorporazione colorante fluorescente (4), l&#39;ibridazione dei materiali marcati al microarray Virochip, e (5) scansione e analisi (Figura 1). Il protocollo illustrato di seguito dimostrerà l&#39;uso del test Virochip su un campione di tampone nasale da un bambino con una malattia simil-influenzale. Primer Sequenze Primer Una 5&#39;-GTTTCCCAGTCACGATA-(N 9) -3 &#39; Primer B-GTTTCCCAGTCACGATA 5&#39;-3 &#39; 1. Estrazione acido nucleico <ol><li> Estrazione degli acidi nucleici da campioni clinici deve essere eseguita sotto livello di biosicurezza 2 (BSL-2) o più condizioni, in quanto questi campioni sono potenzialmente infetti. </li><li> Il campione tampone nasale viene trasportato in media tenendo virale. Aliquota 200 ml di campione in un tubo da microcentrifuga 1,5 ml Eppendorf. </li><li> Opzionale: passare campione attraverso un filtro a 0,22 micron (per purificare per particelle virali) </li><li> Opzionale: campione pretrattare con DNAsi (a degradare DNA genomico di accoglienza e per purificare protette, encapsidated nucleico virale) utilizzando il kit Turbo DNAsi (Ambion) secondo le istruzioni del costruttore </li><li> Estrarre l&#39;esempio utilizzando il Zymo ZR Viral RNA Extraction Kit o kit Qiagen Virus Ultrasens secondo le istruzioni del produttore. </li></ol> 2. Trascrizione inversa e 2 ° filamento sintesi CDNA (&quot;Round A&quot;) <ol><li> Aggiungere 1 uL 40 pmol / mL Primer A a 4 uL estratto RNA. Di calore a 65 ° C per 5 min in un termociclatore (ad esempio GeneAmp PCR System 9700) e poi lasciate raffreddare a temperatura ambiente x 5 min. </li><li> Aggiungere 5 ml di una master mix composto da 2 microlitri di buffer 5X RT, 1 microlitri 12,5 mm dNTP, 1 microlitri di acqua, 0,5 microlitri 0,1 M DTT, e 0,5 microlitri SSIII RT (Invitrogen). Incubare a 42 ° C x 60 minuti. </li><li> In un termociclatore programmabile, il calore a 94 ° C x 2 min (per interrompere la reazione della trascrittasi inversa), raffreddare a 10 ° C, centrifuga impulso, e poi aggiungere 5 microlitri Sequenase mix costituito da 1 tampone microlitri Sequenase 5X, 3,8 microlitri di acqua, e 0,15 Sequenase microlitri (USB Corporation). Per 2 ° filone sintesi, rampa di salita da 10 ° C a 37 ° C oltre 8 minuti, per 8 minuti, poi il calore a 94 ° C x 2 min (per inattivare Sequenase) e raffreddare a 10 ° C (in attesa). </li><li> Optional: Rd Un campione di cDNA possono essere conservati a questo punto a -20 ° C. </li></ol> 3. Amplificazione PCR di primer CDNA casuale (&quot;Rotonda B&quot;) <ol><li> Aggiungere 5 uL di Rd Un campione a 45 ml di una master mix composto da 5 microlitri di buffer 10X PCR, 1 microlitri 12,5 mm dNTP, 1 ml 100 pmol / mL B primer, 1 ml KlenTaq LA (Sigma), e 37 microlitri di acqua. </li><li> Esegui Protocollo PCR come segue: 94 ° C, 2 min → (25 cicli di 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( attesa) </li><li> Controllare il campione Rd B su un 1,5% gel di agarosio. Uno striscio da circa 200 - 1000 bp dovrebbe essere visualizzate (Figura 2A) </li></ol> 4. Cy3 incorporazione colorante fluorescente <ol><li> Opzionale: un altro campione può essere etichettato con il colorante fluorescente Cy5 e ibridizzato al microarray stesso. </li><li> Per incorporare aa-dNTP, aggiungere 5 uL di campione Rd B a 45 uL di una master mix composto da 5 uL tampone 10X PCR, 1 uL 12,5 mm aa-dNTP (Invitrogen), 1 uL 100 pmol / uL Primer B, 1, 1 uL KlenTaq LA (Sigma), e 37 uL acqua. Esegui Protocollo PCR come segue: 94 ° C, 2 min → (15 cicli di 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( attesa) </li><li> Pulire il campione Rd C con il DNA Pulire e Concentratore-5 Kit (Zymo ricerca) secondo le istruzioni del produttore. Eluire in 10 L. </li><li> Aggiungere 1 uL di bicarbonato 1M (Sigma) e 1 uL Cy5 (GE Healthcare) per il campione C Rd. </li><li> Incubare x 1 ora al buio. </li><li> Pulire il campione Cy3-etichettato con il DNA Pulire e Concentratore-5 Kit (Zymo ricerca) secondo le istruzioni del produttore. Eluire in 12 microlitri. </li></ol> 5. Ibridazione di Virochip Microarray <ol><li> Optional: usare 1,5 microlitri per verificare la concentrazione cDNA e la quantità di colorante incorporato in uno spettrofotometro NanoDrop (per la normalizzazione dei campioni) </li><li> In un tubo striscia PCR aggiungere 1 microlitri frammentazione tampone 25X, 5 microlitri di buffer 5X blocco, 9,5 microlitri (o la quantità di normalizzare) di Cy3-etichettati campione, e l&#39;acqua ad un volume totale di 25 microlitri (Agilent) </li><li> Aggiungere 25 ml di tampone 2X ibridazione Gex (Agilent). Spin down brevemente per rimuovere le bolle. </li><li> Carico massimo di 40 ml di campione sul vetrino guarnizione. </li><li> Luogo Agilent stampato Virochip array (Università di California, San Francisco / Agilent) (lato marcato &quot;Agilent&quot; verso il basso) sulla parte superiore della diapositiva guarnizione e fissare stringere. </li><li> Ibridare la notte in forno a 65 ° C, impostazione della velocità a 10 giri. </li><li> Per lavare gli array, preriscaldare tampone 2 (Agilent) a 37 ° C. Smontare guarnizioni scivolo / array subacquea panino in un tampone 1 (Agilent). Lavare in buffer di 1 x 1 min. Lavare in tampone preriscaldato 2 x 1 min. Rimuovere lentamente scivolare dal buffer 2, facendo attenzione ad evitare le bolle (uso kimwipe per favorire l&#39;umidità in più facendo attenzione ad evitare di toccare direttamente lato attivo della matrice) </li><li> Luogo scivolare in supporto diapositive e inserire scanner array. </li></ol> 6. Virochip di scansione e analisi <ol><li> Scansione Cy3 marcato serie a 5 micron o 2 micron secondo il protocollo standard di scansione dello strumento (Figura 2B). </li><li> Analizzare microarray Virochip mediante ispezione visiva, cluster analysis, o il programma di analisi automatica Virochip, E-Predict 11. Da ciascuno di questi tre metodi, il virus dell&#39;influenza sta prontamente rilevate nel campione tampone nasale (da un bambino con sindrome simil-influenzale) (Figura 2C) </li></ol> 7. Rappresentante dei risultati: Quando il protocollo è fatto correttamente, uno striscio dovrebbe essere visto su elettroforesi su gel di agarosio dopo la fase di Rd B (Figura 2A). I microarray Virochip sulla piattaforma Agilent sarà difficile da analizzare visivamente (Figura 2B), ma se un virus è presente dovrebbe essere facilmente identificabili mediante ispezione visiva, cluster analysis, e / o E-Previsione (Figura 1C). I campioni possono essere addizionati con quantità determinata di acido nucleico da un virus conosciuto (ad esempio virus del mosaico del tabacco; batteriofago MS2) per servire come controllo positivo per il test. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Figura 1" /> Figura 1. Schematica del trattamento Virochip microarray e analisi. Estratta acidi nucleici da campioni clinici sono amplificati in modo casuale, etichettato con tinture fluorescenti e ibridato al microarray Virochip. Microarray sono state scansionate ad una risoluzione di 2 micron e analizzati utilizzando una varietà di strumenti di calcolo, tra cui E-prevedere. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Figura 2" /> Figura 2. Passi nel saggio Virochip Dopo l&#39;amplificazione di PCR random, una macchia di 200 -. 1000 bp possono essere visualizzati mediante elettroforesi su gel (A) (B) Tre microarray Virochip su 8 array / vetrino vengono mostrati, con una piccola regione. di un microarray soffiato nella inserto nell&#39;angolo in basso a destra. (C) di analisi automatizzata microarray virale con E-Prevedere rivelare la presenza del virus A nel campione clinico. Il punteggio di somiglianza elevata (s = 0,55) corrisponde ad un valore-p che è vicino a zero, rendendo questa una previsione estremamente significativa.

<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>

Nessun conflitto di interessi dichiarati.

Il protocollo Virochip come descritto qui è complessa e richiede un tecnico di ricerca meticolosa e competente. Concentrazioni dei reagenti corrette e le condizioni per la PCR, l&#39;etichettatura, e l&#39;ibridazione sono critici. Il protocollo Virochip può essere facilmente modificato per ospitare l&#39;analisi dei tessuti malati con l&#39;uso di un metodo di estrazione dei tessuti come TRIzol (Invitrogen) per l&#39;estrazione degli acidi nucleici. Sonde possono essere aggiunti o eliminati come necessario per ottenere lo spettro desiderato e l&#39;ampiezza di copertura, per esempio, sonde per la rilevazione di bersagli non virali come batteri e funghi possono essere progettati e ha aggiunto &quot;on-the-fly&quot;. Alcune applicazioni del test Virochip attualmente in fase di sviluppo includono ampio spettro diagnostica virale nel laboratorio clinico, focolaio, lo screening della purezza di farmaci e vaccini, e la scoperta di nuovi agenti patogeni virali.

<table border="1"><tbody><tr><th> Nome del reagente / materiale </th><th> Nome del reagente / materiale </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 mL) </td><td> 2 microlitri di buffer RT 5X </td></tr><tr><td> 1 microlitri 12,5 mm dNTP (Invitrogen) </td></tr><tr><td> 1 acqua microlitri </td></tr><tr><td> 0,5 microlitri 0.1M DTT </td></tr><tr><td> 0,5 microlitri SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 mL) </td><td> Buffer di 1 microlitri Sequenase 5X </td></tr><tr><td> 3,8 microlitri di acqua </td></tr><tr><td> 0,15 Sequenase microlitri (USB Corporation) </td></tr><tr><td rowspan="5"> Rd B PCR Master Mix (45 mL) </td><td> 5 microlitri di buffer PCR 10X </td></tr><tr><td> 1 microlitri 12,5 mm dNTP (Invitrogen) </td></tr><tr><td> 1 ml 100 pmol / mL Primer B </td></tr><tr><td> 1 ml KlenTaq LA (Sigma) </td></tr><tr><td> 37 microlitri di acqua </td></tr><tr><td rowspan="5"> ° C PCR Master Mix (45 mL) </td><td> 5 uL Buffer PCR 10X </td></tr><tr><td> 1 microlitri 12,5 mm dNTP (Invitrogen) </td></tr><tr><td> 1 ml 100 pmol / mL Primer B </td></tr><tr><td> 1 ml KlenTaq LA (Sigma) </td></tr><tr><td> 37 microlitri di acqua </td></tr><tr><td rowspan="4"> Ibridazione del buffer (25 mL) </td><td> Buffer di 1 microlitri frammentazione 25X (Agilent) </td></tr><tr><td> 5 microlitri di buffer 5X blocco (Agilent) </td></tr><tr><td> 9,5 microlitri (o la quantità di normalizzare) di Cy3 marcato campione </td></tr><tr><td> Aggiungere acqua al volume totale di 25 microlitri </td></tr><tr><td> Agilent Virochip microarray </td><td> in attesa di licenza, disponibile da Chiu laboratorio, UCSF </td></tr></tbody></table>

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

La diagnosi di cause virali di molte malattie infettive è difficile a causa della intrinseca diversità sequenza di virus così come l&#39;emergere continuo di nuovi agenti patogeni virali, come la SARS coronavirus e il 2009 pandemia di H1N1 virus dell&#39;influenza, che non sono rilevabili con i metodi tradizionali. Per affrontare queste sfide, abbiamo precedentemente sviluppato e validato un pan-virale piattaforma microarray chiamato Virochip con la capacità di rilevare tutti i virus conosciuti così come le varianti romanzo sulla base di omologia di sequenza conservati 1. Utilizzando il Virochip, abbiamo identificato l&#39;intero spettro dei virus associati con infezioni respiratorie, compresi i casi di inspiegabili malattia critica nei pazienti ospedalizzati, con una sensibilità pari o superiore a 05/02 convenzionali test clinici. Il Virochip è stato utilizzato anche per identificare nuovi virus, tra cui il coronavirus della SARS 6,7, un clade rinovirus romanzo 5, XMRV (un retrovirus collegato al cancro della prostata) 8, bornavirus aviaria (la causa di una malattia devastante in pappagalli) 9, e un cardiovirus romanzo in bambini con malattie respiratorie e diarrea 10. La versione attuale del Virochip è stato portato su una piattaforma microarray Agilent ed è costituito da ~ 36.000 sonde derivanti da più di ~ 1.500 virus in GenBank a partire dal dicembre del 2009. Qui mostriamo i passaggi necessari per l&#39;elaborazione di un saggio Virochip dall&#39;inizio alla fine (i tempi di consegna ~ 24 ore), tra cui l&#39;estrazione degli acidi nucleici del campione, amplificazione PCR con primer random, incorporazione colorante fluorescente, e l&#39;ibridazione microarray, scansione e analisi.

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

Il Virochip è un pan-virale microarray progettati per rilevare contemporaneamente tutti i virus conosciuti e nuovi virus sulla base di omologia di sequenza conservati. Qui mostriamo come eseguire un test Virochip per analizzare i campioni clinici per la presenza di virus noti e sconosciuti.

Utilizzando un pan-virale Assay Microarray (Virochip) allo schermo campioni clinici di patogeni virali

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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JoVE Journal

Immunology and Infection

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

18.7K Views.  University of California, San Francisco. Il Virochip è un pan-virale microarray progettati per rilevare contemporaneamente tutti i virus conosciuti e nuovi virus sulla base di omologia di sequenza conservati. Qui mostriamo come eseguire un test Virochip per analizzare i campioni clinici per la presenza di virus noti e sconosciuti.

Video: 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.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<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 ...

Ricerca

Immunologia e infezioni

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.

Utilizzando Clicca Chimica per misurare l&#39;effetto dell&#39;infezione virale sulla cellula ospite RNA Sintesi

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.

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

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.

Un miniaturizzato Glycan microarray test per la valutazione avidità e Specificità del virus influenzale A emoagglutinine

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.

Misurare Influenza neuraminidasi Inibizione anticorpo I titoli da Enzyme-linked lectina 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.

Utilizzazione di capsule per la colorazione negativa di campioni virali all&#39;interno del 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.

Anticorpli uccisione test per valutare le risposte immunologiche contro batteri patogeni

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.

Determinazione dell'efficienza dell'inibitore chimico contro la crescita intracellulare del Toxoplasma Gondii utilizzando un saggio di crescita basato su Luciferase

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).

Misurazione di anticorpi che inducono la fagonenza acquisita naturalmente ai parassiti Plasmodium falciparum mediante un'analisi basata sulla citometria del flusso

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

Diagnosi delle infezioni batteriche respiratorie umane mediante amplificazione isotermica

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.

Autore in primo piano: Miglioramento del rilevamento rapido dei patogeni respiratori mediante chip microfluidico 

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.

Rilevamento rapido di agenti patogeni batterici che causano infezioni del tratto respiratorio inferiore tramite amplificazione isotermica mediata da loop basata su chip microfluidico

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