Questo lavoro è sostenuto in parte A1064826 sovvenzioni NIH R01-01, U19 AI062627-01, NO1-HHSN26600500032C (a SM). Ringraziamo i nostri membri di laboratorio per la discussione e aiuto tecnico, Tina Heil per la revisione critica del manoscritto. Vorremmo ringraziare David Bina, Milwaukee Dipartimento di Sanità Laboratorio per la cultura virus e PCR.

<ol>
	<li>Hirst, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+agglutination+of+red+cells+by+allantoic+fluid+of+chick+embryos+infected+with+influenza+virus.">The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus.</a> Science. 94, 22-23 (1941).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Kalter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinating+Behavior+of+Mouse+and+Egg-adapted+Type+A+(PR8)+Influenza+Virus.">Hemagglutinating Behavior of Mouse and Egg-adapted Type A (PR8) Influenza Virus.</a> Science. 110, 184-184 (1949).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Plaques+in+Monolayer+Tissue+Cultures+by+Single+Particles+of+an+Animal+Virus.">Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus.</a> Proc. Natl. Acad. Sci. U. S. A. 38, 747-752 (1952).</li><li>Deyde, V. M., Nguyen, T., Bright, R. A., Balish, A., Shu, B., Lindstrom, S., Klimov, A. I., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+markers+of+antiviral+resistance+in+influenza+A+(H5N1)+viruses+using+a+pyrosequencing+method.">Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.</a> Antimicrob. Agents Chemother. 53, 1039-1047 (2009).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Rahman, M., Kieke, B. A., Vandermause, M. F., Mitchell, P. D., Greenlee, R. T., Belongia, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+Directigen+flu+A+B+enzyme+immunoassay+and+direct+fluorescent+assay+for+detection+of+influenza+infection+during+the+2004-2005+season.">Performance of Directigen flu A+B enzyme immunoassay and direct fluorescent assay for detection of influenza infection during the 2004-2005 season.</a> Diagnostic Microbiology and Infectious Disease. 58, 413-418 (2007).</li><li>Landry, M. L., Ferguson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboptimal+detection+of+influenza+virus+in+adults+by+the+Directigen+Flu+A+B+enzyme+immunoassay+and+correlation+of+results+with+the+number+of+antigen-positive+cells+detected+by+cytospin+immunofluorescence.">Suboptimal detection of influenza virus in adults by the Directigen Flu A+B enzyme immunoassay and correlation of results with the number of antigen-positive cells detected by cytospin immunofluorescence.</a> J. Clin. Microbiol. 41, 3407-3409 (2003).</li><li>Herrmann, B., Larsson, C., Zweygberg, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+typing+of+influenza+viruses+A+and+B+by+a+nested+reverse+transcription-PCR:+comparison+to+virus+isolation+and+antigen+detection+by+immunofluorescence+and+optical+immunoassay+(FLU+OIA).">Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA).</a> J. Clin. Microbiol. 39, 134-138 (2001).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Epidemiol. 27, 493-497 (1938).</li><li>Doerr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+into+the+infrared.">Fluorescent proteins: into the infrared.</a> Nat. Meth. 6, 482-483 (2009).</li><li>Shu, B., Wu, K. H., Emery, S., Villanueva, J., Johnson, R., Guthrie, E., Berman, L., Warnes, C., Barnes, N., Klimov, A., Lindstrom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+the+CDC+real-time+reverse+transcriptase+PCR+swine+flu+panel+for+detection+of+2009+A+(H1N1)+pandemic+influenza+virus.">Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.</a> J. Clin. Microbiol. 49, 2614-2619 (2011).</li></ol>

Non abbiamo nulla da rivelare.

Lo scoppio recente epidemia di influenza aviaria nel Sud-Est asiatico e la paura globale della pandemia di influenza suina impongono enormi preoccupazioni per la salute e la sicurezza pubblica. Attenzione critica è stata dedicata nel generare vaccini per i ceppi esistenti e nuovi di virus influenzale. Disponibilità di rapide tecnologie high-throughput per la rilevazione precisa ed accurata calcoli titolo virale sarà migliorare enormemente il trattamento. Le tecniche più comunemente utilizzate per il calcolo impiegati titolo virale comprendono saggi emoagglutinazione la formazione della placca e l&#39;influenza. Il saggio la formazione della placca è stato inizialmente sviluppato per stimare i titoli batteriofagi ed è stato adottato da Renato Dulbecco per calcolare animali titoli virali 4. Per eseguire questo saggio, 10-diluizioni di archivio di influenza o di esemplari di animali, quali fluidi BAL sono preparati e 0,1 ml sono inoculate sul monostrato di cellule MDCK in 6-pozzetti. Il virus è permesso di adsorbire sulle cellule MDCK seguite da l&#39;annunciodizione di un mezzo nutriente e agarosio. Durante l&#39;incubazione, le celle originali infette rilasciare progenie virale che si diffuse solo per le cellule vicine. L&#39;effetto citopatico dell&#39;infezione influenzale virale produce una zona circolare di cellule infettate chiamato una targa. Ogni placca viene presumibilmente formata dopo infezione di una singola cella con una particella virus singolo seguito da replicazione del virus. Contrasto tra le cellule viventi e delle targhette possono essere arricchito da coloranti come la violetta cristallo. Un inconveniente principale di questo saggio è la mancanza di riproducibilità e di enormi variazioni all&#39;interno di esperimenti. Un&#39;altra dosaggio utilizzato per determinare il titolo virale influenzale è il dosaggio emoagglutinazione. La presente proteina emoagglutinina sulla superficie del virus influenzale si lega in modo efficiente per N-acetilneuraminico-proteine ​​contenenti su mammiferi e aviaria globuli rossi 1. Questa interazione tra il virus influenzale e gli eritrociti conduce al agglutinazione in forma di un reticolo. Il lEvel di agglutinazione viene utilizzato per stimare il titolo del virus dell&#39;influenza in campioni. Test di agglutinazione degli eritrociti viene utilizzata solo per determinare la titolazione di un virus noto e non può essere utilizzato a fini di identificazione estensimetri. Dal momento che questo test non fa differenza fra la vita e la morte dei virus, è difficile ottenere un calcolo preciso di titoli virali. Attuali diagnosi cliniche di infezioni influenzali si basano su test sierologici, reazione a catena della polimerasi, immunofluorescenza diretta del campione e la cultura delle cellule 1,6. Il nuovo metodo descritto qui ha anche clinico e di laboratorio potenziale diagnostico. Influenza rilevamento kit Directigen FLU-A (BD Biosciences, San Jose, CA) e BinaxNOW (Binax, Inc., Scarborough, ME) immunocromatografia test utilizzare per rilevare l&#39;antigene virale, come nucleoproteina 7. L&#39;utilizzo di questi kit, il rilevamento di virus possono essere raggiunti entro 15 minuti, tuttavia, tutti i campioni negativi devono essere ulteriormente screened dal metodo coltura cellulare. Diversi studi hanno anche riportato bassa sensibilità di questi kit, in particolare, quando l&#39;infezione era lieve o basso 8. Nel nostro metodo, abbiamo costantemente rilevare a partire da 100 PFU di virus. Questo suggerisce la nostra tecnica è utile per rilevare virus H1N1 in campioni clinici, anche quando l&#39;infezione è mite. Un metodo basato sulla fluorescenza per rilevare antigeni virali è stata riportata, in cui sia campioni di aspirato nasofaringeo sono stati analizzati direttamente o sono state inoculate su un monostrato di cellule sensibili alla moltiplicazione del virus e le analisi successive 9. Tuttavia, questi metodi sono limitati per scopi diagnostici e non sono adatti per calcoli titolo virale. Q-PCR aiuta a identificare la presenza di RNA virale e non stimare la infettività di virus vivi. Rispetto a questi saggi, IR dye-based sistema di analisi aiuterà nella ricerca e la conta titolo virale in campioni clinici e di laboratorio. In caso di focolaio di influenza, ècritico per diagnosticare migliaia di campioni in un periodo di tempo breve. Il nostro metodo basato su una piastra a 384-bene e scanner IR possibile eseguire la scansione 6-piastre alla volta (&gt; 2.000 campioni), offrendo pertanto un vantaggio maggiore rispetto ad altri metodi diretti fluorescenti. Fattibilità di un trattamento rapido di migliaia di campioni clinici potrebbero ridurre la variabilità di prova, la durata del tempo di saggio e di costi. L&#39;uso di un M2-specifico anticorpo offre il vantaggio di essere indipendente di differenze di ceppi in proteine ​​H e N. La proteina M2 è relativamente conservata nella maggior parte dei ceppi influenzali che infettano gli esseri umani. Se gli anticorpi specifici per macchie circolanti sono combinati con il nostro sistema uno può misurare sia il titolo di virus e identificare il ceppo. Tuttavia, poiché M2 è l&#39;obiettivo per i farmaci come amantadina, i mutanti possono nascere che potrebbe cambiare il sito di legame per l&#39;anticorpo monoclonale usato qui. Questo rappresenta una potenziale limitazione di questo sistema negli individui che vengono trattati con questi farmaci. Multiplosaggi vengono utilizzati per enumerare i titoli influenzali nei campioni di prova. Per calcolare virus titolo, 50% della dose coltura tissutale infettiva (TCID 50) e uova allantoici fluido-saggi basati sono ampiamente utilizzati 1,10. Tuttavia, i requisiti di più giorni rendono questi metodi meno affidabile. Inoltre, in questi metodi calcoli basati sulla variazione 50% nel il numero di placche fornire titolazioni virali teoriche ma non accurate. La formazione della placca analisi può essere eseguita entro 96 ore, tuttavia, la mancanza di riproducibilità è un problema importante nei calcoli titolo virale. La presente tecnica descritta qui ha diversi vantaggi. In primo luogo, essa è una tecnica altamente riproducibile con esiti coerenti. In secondo luogo, i calcoli titolo virale si basano sulla quantificazione semplice intensità di fluorescenza che vengono confrontati con le curve standard definiti. Terzo, due o più anticorpi primari può essere utilizzato per diagnosticare sierotipi multipli in un dato campione. Inoltre, IR dye-anticorpi coniugati sono minimal autofluorescenza e sono stati utilizzati per cella l&#39;imaging 11. UV o visivo radiazioni (300-600 nM) risultati in alta autofluorescenza cellulare dovuta alla presenza di elevati quantistica fluorofori rendimento endogene. Questi includono gli amminoacidi aromatici, lipo-pigmenti, nucleotidi pyridinic (NADPH), elastina, collagene e coenzimi Flavin. Questa proprietà intrinseca di cellule rende difficile utilizzare queste fonti di radiazioni come sonde per quantificare l&#39;espressione di proteine ​​specifiche. Al contrario, near-IR coloranti emozionare ed emettono tra i 670-1000 nm. Molte molecole host cellulari e gli idrocarburi policiclici aromatici non eccitano a questa lunghezza d&#39;onda e grazie alla riduzione della dispersione della luce, emettono fluorescenza di fondo estremamente basso. Inoltre, la finestra tra lo sfondo e rilevazione specifico è molto migliorato a causa di un coefficiente elevato di estinzione delle vicino-IR fluorofori. Fotostabilità, superiore rapporto segnale-rumore, è estremamente rilevante nella quantificazione dei titoli virali. Uso di microflcamere uidic automatizza il processo di screening attraverso controlli robotici, e potrebbe consentire di testare campioni clinici altamente contagiose con facilità. Così, l&#39;utilizzo di vicino-IR coloranti in questi metodi è ideale e altamente affidabile per enumerare titoli virali in campioni di tessuto.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Bovina albumina sierica </td><td> Atlanta Biologicals </td><td> S11750 </td><td></td></tr><tr><td> PR8 virus </td><td></td><td></td><td> Dal Dr Thomas Moran M </td></tr><tr><td> Capra anti-topo IRDye @ 800 </td><td> LI-COR bioscienze </td><td> 926-32210 </td><td> 1:200 diluizione </td></tr><tr><td> LI-COR Odyssey IR scanner </td><td> LI-COR-bioscienze </td><td> 9201-01 </td><td></td></tr><tr><td> 384 pozzetti della piastra di fondo piatto </td><td> Nalge Nunc internazionale </td><td> 164730 </td><td></td></tr><tr><td> 96-pozzetti della piastra di fondo piatto </td><td> Nalge Nunc internazionale </td><td> 165305 </td><td></td></tr><tr><td> DMEM media </td><td> Invitrplasminogeno </td><td> 11965126 </td><td></td></tr><tr><td> Medium RPMI </td><td> Invitrogen </td><td> 11875135 </td><td></td></tr><tr><td> Bicarbonato di sodio </td><td> Invitrogen </td><td> 25080 </td><td></td></tr><tr><td> L-glutammina 100x </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Trypson-EDTA </td><td> Invitrogen </td><td> R001100 </td><td></td></tr><tr><td> Sodio Piruvato </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> Anti-M-2 anticorpo </td><td></td><td></td><td> Dal Dr Thomas Moran M </td></tr><tr><td> Anti-NP mAb </td><td> CDC </td><td> V2S2208 </td><td> Ottenuto con un MTA </td></tr></tbody></table>

high-throughput detection method for influenza virus

Il virus influenzale è un patogeno respiratorio che provoca un elevato livello di morbilità e mortalità ogni anno in più parti del mondo. Pertanto, la diagnosi precisa del ceppo infettante e rapida high-throughput screening di un gran numero di campioni clinici è fondamentale per controllare la diffusione delle infezioni pandemiche. Attuali diagnosi cliniche di infezioni influenzali si basano su test sierologici, reazione a catena della polimerasi, immunofluorescenza diretta del campione e la cultura delle cellule 1,2. Qui, segnaliamo lo sviluppo di una nuova tecnica diagnostica utilizzata per rilevare virus influenzali vivi. Abbiamo usato il mouse adattato umana A/PR/8/34 (PR8, H1N1) virus 3 per testare l&#39;efficacia di questa tecnica utilizzando cellule MDCK 4. Cellule MDCK (10 4 o 5 x 10 3 per pozzetto) sono state coltivate in 96 - o piastre da 384 pozzetti, infettati con PR8 e proteine ​​virali sono stati rilevati utilizzando anti-M2 seguita da uno IR colorante-coniugato secanticorpale secondaria. M2 5 e emoagglutinina 1 sono due proteine ​​marker principali usati in molti diversi saggi diagnostici. Che impiegano anticorpi secondari IR-dye-coniugati minimizzato l&#39;autofluorescenza associata ad altri coloranti fluorescenti. L&#39;uso di anti-M2 anticorpo ci ha permesso di utilizzare l&#39;antigene-specifica intensità di fluorescenza come una metrica diretta di quantità virale. Per enumerare l&#39;intensità di fluorescenza, abbiamo usato il LI-COR Odyssey scanner basato su IR. Questo sistema utilizza due canali laser basati su rilevamenti a raggi infrarossi per identificare fluorofori e differenziarli dal rumore di fondo. Il primo canale eccita a 680 nm ed emette a 700 nm per contribuire a quantificare lo sfondo. Il secondo canale rileva fluorofori che eccitano a 780 nm ed emette a 800 nm. Scansione delle cellule MDCK PR8-infetti nello scanner IR indicato un titolo virale-dipendente fluorescenza. Una correlazione positiva di intensità di fluorescenza a titolo virale a partire dal 10 2 -10 5 PFU potrebbe essere conconsistentemente osservato. Positività Minimal ma rilevabile sempre visto con 10 2 -10 3 PFU PR8 titoli virali dimostrato l&#39;elevata sensibilità delle near-IR coloranti. Il rapporto segnale-rumore è stata determinata confrontando i mock-infettate o cellule MDCK anticorpi isotipo-trattati. Utilizzando le intensità di fluorescenza da 96 - o formati piastra da 384 pozzetti, abbiamo costruito curve di titolazione standard. In questi calcoli, la prima variabile è il titolo virale mentre la seconda variabile è l&#39;intensità di fluorescenza. Pertanto, abbiamo usato la distribuzione esponenziale per generare una curva-fit per determinare la relazione polinomiale tra i titoli virali e intensità fluorescenza. Collettivamente, possiamo concludere che IR dye-based sistema di rilevazione delle proteine ​​può aiutare a diagnosticare infettare i ceppi virali e precisamente enumerare il titolo degli agenti patogeni infettanti.

Watch this Scientific Journal Video about High-throughput Detection Method for Influenza Virus at JoVE.com

High-throughput Detection Method for Influenza Virus

Questo metodo descrive l&#39;utilizzo di Infrared sistema colorante di imaging based per il rilevamento di H1N1 in bronchioalveolar lavaggio (BAL) fluido di topi infettati ad una sensibilità elevata. Questa metodologia può essere eseguita in uno 96 - o 384-bene piastra, richiede &lt;10 volume pl di materiale di prova e ha il potenziale per lo screening concomitante di agenti patogeni multipli.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

high-throughput-detection-method-for-influenza-virus

Research

JoVE Journal

Immunology and Infection

26.0K Views.  Blood Research Institute. Questo metodo descrive l'utilizzo di Infrared sistema colorante di imaging based per il rilevamento di H1N1 in bronchioalveolar lavaggio (BAL) fluido di topi infettati ad una sensibilità elevata. Questa metodologia può essere eseguita in uno 96 - o 384-bene piastra, richiede 

Video: High-throughput Detection Method for Influenza Virus

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Generazione di influenza da virus ricombinante del DNA plasmidi

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Ricerca

Immunologia e infezioni

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX rilevazione colorimetrica su un DNA microarray per l&#39;influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza Virus Propagazione in embrionate di pollo Uova

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Panni nasale per l&#39;influenza A rilevazione antivirus e isolamento da Suina

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro Smontaggio del virus influenzale A capsids per centrifugazione in gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Detection High-throughput respiratorie patogeni nei campioni animali da Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Studi del Virus di riossidazione A in un modello murino di infezione

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generazione di varianti di fuga di neutralizzare l'Influenza Virus anticorpi monoclonali

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...