This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

Tutti i suini utilizzati per la raccolta dei seguenti dati sono stati protetti ai sensi della Istituzionale Animal Care and Use Committee Ohio State University (uso animale numero di protocollo 2009A0134-R1). 1. Preparazione di trasporto virale media e provate Fiale Collection <ol><li> Aggiungere 37 g di Brain Heart Infusion a 900 ml di acqua purificata e mescolare accuratamente con un ancoretta e agitatore magnetico mentre il riscaldamento a 70 ° C per sciogliere completamente la polvere. </li><li> Autoclavare il brodo a 121 ° C per 15 min. Raffreddare sul banco fino brodo raggiunge la temperatura ambiente. Mettete in frigorifero a 4 ° C durante la notte se necessario. </li><li> Sciogliere, mescolando con ancoretta magnetica, 6,02 g di sale sodico Penicillina G e 10 g di solfato di streptomicina in 50 ml di acqua depurata a temperatura ambiente. Aggiungere acqua purificata supplementare per portare il volume totale di 100 ml. Filtrare la soluzione attraverso una membrana polietersulfone 0,22 micron in una bottiglia sterile. </li><li> Asetticamente aggiungere la penicillina filtrato e la soluzione di streptomicina nella raffreddato Brain Heart Infusion Broth e mescolare bene (o ancoretta pallone ricciolo mano) per rendere il mezzo di trasporto virale. </li><li> Asetticamente testare il pH del mezzo di trasporto virale; se necessario, regolare il pH a 7,4 ± 0,2 con HCl o NaOH. </li><li> Eseguire test di garanzia della qualità del mezzo di trasporto virale da test per l&#39;influenza A proteina di matrice del virus da rRT-PCR, come descritto da Zhang e Harmon. 22 </li><li> Dispensare 5 ml di mezzo di trasporto virale in bottiglie di polietilene ad alta densità sterili con capacità da 8 ml. Etichetta le fiale per uso nel settore. </li><li> Conservare le fiale pronti per l&#39;uso in crio-scatole standard a -20 ° C fino a quando la raccolta del campione. </li></ol> 2. Raccolta di nasale Salviette da Swine <ol><li> Scongelare il numero appropriato di flaconcini immediatamente prima del prelievo; Sarà necessario un flaconcino per suino campionato. Scongelamento in cameratemperatura richiede circa 30-45 minuti. Tenere le fiale scongelate raffreddare su impacchi di ghiaccio fino al momento dell&#39;uso. </li><li> Don adeguati dispositivi di protezione personale come dettate dalla situazione (ad esempio, tuta, calzari, respiratore, tappi per le orecchie, ecc). </li><li> Entra nell&#39;area animale. Se necessario, limitare il maiale ad una piccola area ma senza frenare. Non svegliare riposo animali. </li><li> Indossare un paio di guanti monouso di esame. Fare attenzione a non contaminare i guanti da maiali toccando, persone o oggetti inanimati. </li><li> Usare una mano guantata per rimuovere una sterile 5,08 cm × 5,08 cm (2 in. × 2 in.) Tampone di garza di cotone dal suo involucro. Garze sterili devono essere usati quando possibile. Garze confezionati singolarmente sono più comodi da usare rispetto a quelli avvolto con due tamponi di garza per collo. </li><li> Tenere il tampone di garza di cotone con la punta delle dita di una mano esporre come gran parte del pad come possibile per il campionamento. </li><li> Pulire il tampone di garza attraverso il muso del maiale, presaattenzione per entrare nelle narici esterne, se possibile. Raccogliere visibili secrezioni nasali (circa 1 ml) con lo stesso tampone di garza. </li><li> Utilizzando la stessa mano, piegare la garza su se stesso per facilitare il posizionamento nel flaconcino contenente mezzo di trasporto virale. </li><li> Utilizzando invece, rimuovere il tappo dal flacone e posizionare la garza piegata nel flaconcino. Richiudere la fiala e agitare il flaconcino per garantire la miscelazione del garza e mezzo di trasporto virale. </li><li> Togliere i guanti e posto in un contenitore adeguato. Cambiare i guanti tra ogni suino. </li><li> Verificare l&#39;identificativo fiala. Numero record di identificazione maiale, età, sesso, segni clinici, e altre note ritenute necessarie da investigatore. </li><li> Raffreddare i campioni immediatamente dopo il prelievo. Considerare la possibilità aliquote del campione prima del congelamento per evitare più cicli di congelamento-scongelamento. Non appena possibile dopo la raccolta, posizionare i campioni in ghiaccio secco per il trasporto al laboratorio. Conservare le fiale a -80° C fino all&#39;avvio test. </li></ol> 3. Rilevamento di virus influenzale A Nucleic Acid <ol><li> Scongelare i campioni a 37 ° C bagno branello secco per 5 min e quindi completare scongelamento i campioni sul banco a temperatura ambiente per 20-30 min. L&#39;aggiunta contemporanea di più flaconi alle perline causerà la vasca al calore; si deve prestare attenzione per non surriscaldare i flaconi. </li><li> Togliere 100 ml di mezzo di trasporto virale per l&#39;estrazione dell&#39;RNA. <ol><li> Utilizzare un high-throughput (96 formato pozzetti) piattaforma perla magnetica per l&#39;estrazione di RNA da un gran numero di campioni. 31 </li></ol></li><li> Utilizzare in tempo reale analisi della trascrittasi inversa PCR per il rilevamento rapido di influenza virus. 31 </li></ol> 4. L&#39;isolamento del virus influenzale A da nasali Salviettine <ol><li> Scongelare i campioni come descritto in precedenza nella sezione 3.1 </li><li> Trattare i campioni scongelati con gentamicina solfato (1.000mcg / ml), amfotericina B (22,5 mg / ml), e kanamicina solfato (325 mcg / ml). </li><li> Seminare in Madin-Darby Canine Kidney (MDCK) le cellule epiteliali, come descritto in precedenza. 32 </li></ol>

<ol>
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The authors have nothing to disclose.

La raccolta di campioni da suini utilizzando tamponi nasali di poliestere con la punta si è dimostrato utile nel condurre sorveglianza IAV; Tuttavia, l&#39;uso della procedura di tampone nasale ostacola gli sforzi di sorveglianza dovuti all&#39;uso richiesto di un laccio per contenzione. Salviette nasale rappresentano un affinamento delle attuali tecniche di campionamento suina per minimizzare lo stress sulle persone e maiali durante la raccolta del campione. Mentre il metodo è stato messo a punto e validato in contes...

<html> I virus influenzali A (IAV) causano malattie respiratorie in molte specie, tra cui uccelli domestici, suina, e gli esseri umani. A causa riassortimento del genoma segmentato IAV rapida evoluzione virale possono verificarsi e nuovi ceppi IAV emergere frequentemente. Maiali sono una specie che può servire come un vaso di miscelazione per riassortimento di IAV da più specie ospiti. 1 Al momento non ci sono tre principali sottotipi di IAV comunemente circolano tra nordamericano suina (H1N1, H1N2, H3N2), ma più introduzioni IAV da esseri umani hanno ha portato alla vasta diversità IAV all&#39;interno di tali sottotipi. 2 rapida evoluzione della IAV infettare suina è evidente dal 1998, quando un reassortant tripla IAV contenente segmenti di geni provenienti da virus umani, aviari e suini 3 si diffuse tra i maiali negli Stati Uniti. 4 Il gene interna segmenti di quel triplice reassortant IAV rimangono altamente diffusa tra IAV attualmente infettare suina. 5 &quot;&gt; In tutto il mondo, IAV è una causa importante di malattia respiratoria dei suini in cui i segni clinici tipici comprendono febbre, anoressia, letargia, tosse, respiro affannoso, starnuti, scolo nasale e scarso aumento di peso. IAV può essere particolarmente onerosa seminare aziende agricole in cui riproduttiva fallimento a causa di IAV febbre indotta e suinetti deboli di origine sono stati documentati. 6,7 Negli Stati Uniti, IAV è comunemente individuato in allevamenti suini commerciali e l&#39;ampia diversità antigenica e genomica e continua evoluzione tra IAV suina infettare ha ostacolato il controllo di questa virus. 8-11 Salute pubblica teme la comparsa di un ceppo IAV pandemia derivante dal riassortimento nei suini sono stati realizzati nel 2009, quando un IAV suina lignaggio contenente segmenti di geni della stirpe suina nordamericana triplo riassortanti e il lignaggio suina aviaria-come eurasiatico ha causato una pandemia a livello mondiale negli esseri umani. 12 La virus pandemico (A (H1N1) pdm09) ha dalriassortito con suina endemica IAV ceppi 13,14 e alcuni di questi ceppi di nuova riassortito sono stati trasmessi di nuovo agli esseri umani. 15 La frequenza di eventi riassortimento e comparsa di nuovi ceppi IAV con potenziale pandemico rende sorveglianza attiva di far circolare i virus nei suini IAV imperativo, soprattutto all&#39;interfaccia suina umana. L&#39;interfaccia suina-umano è importante per interspecie bidirezionale trasmissione di IAV. Human-to-suina trasmissione che si verificano nella produzione suina commerciale è responsabile di una grande quantità di diversità IAV attualmente presenti nella popolazione suina. Fiere agricole sono le impostazioni per la comingling di persone e suina negli Stati Uniti più grandi e sono noti siti per la trasmissione zoonotica di IAV. 15-21 Nel 2012, durante l&#39;epidemia di una variante H3N2 IAV, il 93% dei casi segnalati partecipazione a una fiera agricola, nei giorni precedenti l&#39;inizio della malattia. 15 Analisi genomicadi isolati virali da suini espositivi rispetto al isolati umani confermato trasmissione zoonotici. 21 Exhibition suina infetta da IAV spesso non presentavano segni clinici di malattia, 21-23 che indicano la necessità di test diagnostici diretti. Il campionamento dei suini visibilmente malati da solo non identificare con successo IAV prevalenza nei suini e non può essere invocata per individuare nuovi ceppi di IAV emergenti tra maiali. La sorveglianza attiva è assolutamente necessario per il rilevamento di ceppi emergenti di IAV in suini e per valutare la loro minaccia sia per la suina e della salute pubblica. La maggior parte delle attività di sorveglianza IAV sono volontarie e, pertanto, sono necessari metodi minimamente dirompenti. Le tre principali procedure di raccolta del campione ante mortem per IAV infettare suina sono: tamponi nasali, fluidi orali, e salviette nasali. Le attuali raccomandazioni per il campionamento dei suini individuale per rilevare lista IAV l&#39;inserimento di fibre sintetiche ribaltati tamponi nelle narici come il metodo preferitoraccogliere le secrezioni nasali e le cellule epiteliali. 24,25 Poiché suini possono cercare di evitare questa procedura, una squadra di personale addestrato deve trattenere suini manualmente o con un laccio seconda della taglia dell&#39;animale. 26 Il processo di ritenuta è laborioso per il personale e stressante per i maiali. Inoltre, mostra suini sono spesso coinvolti in molteplici competizioni in una fiera così la percezione di stress aggiunto su un animale di concorrenza può offrire proprietari resistenti agli sforzi di sorveglianza. Con probabilità di rilevazione IAV che vanno dal 80-100% in IAV allevamenti infetti, fluidi orali sono diventati una alternativa popolare per nasale tamponi per la diagnosi molecolare di IAV nelle popolazioni di suini. 27,28 Inoltre, fluidi orali possono fornire una finestra più ampia di rilevamento IAV di tamponi nasali dopo l&#39;infezione iniziale. Tuttavia, IAV isolamento da fluidi orali è stato problematico con solo il 50% dei virus tentativi di isolamento con conseguente IAV recupero. 29 Utilizzando salviette nasali in luogo di tamponi nasali durante IAV sorveglianza nei suini supera i limiti di cui sopra. Salviette nasali non richiedono l&#39;uso di un laccio di ritegno e possono essere eseguite senza stress agli animali o testimoni della procedura. Formazione tecnica minima è necessaria per raccogliere salviette nasali, che consente ai professionisti non veterinari, compresi i proprietari dei suini, per raccogliere i campioni di sorveglianza. Salviette nasali sono stati precedentemente confrontati con tamponi nasali per la rilevazione e l&#39;isolamento di virus influenzale A 30 e il protocollo dettagliato per questo metodo non invasivo di campionamento è descritto di seguito.

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			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">211059</td>
			<td style="width:356px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
			<td style="width:119px;">021194537</td>
			<td>1500 u/mg</td>
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			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
			<td style="width:119px;">0382</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Gentamicin Solution</td>
			<td style="width:360px;">Mediatech, Inc.</td>
			<td style="width:119px;">30-005-CR</td>
			<td>50 mg/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B Solution</td>
			<td style="width:360px;">Fisher</td>
			<td style="width:119px;">S 24 25</td>
			<td>250 ug/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin Sulfate</td>
			<td style="width:360px;">Teknova</td>
			<td style="width:119px;">K2105</td>
			<td>5000 ug/ml</td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">TPP Rapid Filtermax System</td>
			<td style="width:360px;">TPP Techno Plastic Products AG&#160;</td>
			<td style="width:119px;">99150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nalgen Diagnostic Bottles</td>
			<td style="width:360px;">Thermo Scientific</td>
			<td style="width:119px;">342002-9025</td>
			<td>HDPE with white PP closure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Dermacea Gauze Sponge, 8 ply</td>
			<td style="width:360px;">Covidien</td>
			<td style="width:119px;">441211</td>
			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrile Exam Gloves</td>
			<td style="width:360px;">Saftey Choice</td>
			<td style="width:119px;">19-170-010 (A-D)</td>
			<td></td>
		</tr>
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nasal wipes for influenza a virus detection and isolation from swine

Sorveglianza di virus dell&#39;influenza A in suina è fondamentale per la salute umana e animale perché virus evolve rapidamente nella popolazione suina e nuovi ceppi sono continuamente emergendo. Swine sono in grado di essere infettati da diversi lignaggi di virus influenzale che li ospita importanti per la formazione e il mantenimento di nuova influenza A ceppi di virus. Campionamento suini in contesti diversi, quali aziende agricole suini, fiere agricole e mercati di animali vivi è importante fornire una visione completa di ceppi attualmente in circolazione IAV. La corrente ante mortem tecnica di campionamento gold-standard (ossia la raccolta di tamponi nasali) è laborioso perché richiede contenzione fisica dei suini. Salviette nasale coinvolgere strofinando un pezzo di tessuto attraverso il muso del maiale con minima o nessuna moderazione dell&#39;animale. Il nasale wipe procedura è semplice da eseguire e non richiede personale con veterinaria professionale o formazione trattamento degli animali. Wentre leggermente meno sensibile di tamponi nasali, tassi di rilevazione di virus e l&#39;isolamento sono sufficienti per fare nasale pulisce una valida alternativa per il campionamento singoli suini quando sono richiesti bassi di stress metodi di campionamento. Il protocollo procedura delinea i passi necessari per raccogliere un nasale valida wipe da un maiale individuo.

Uso di successo di questo metodo produce rRT-PCR risulta che, accompagnato con l&#39;uso di un controllo interno durante l&#39;estrazione di RNA e rRT-PCR, mostrano campioni non contenevano inibitori della PCR da eventuali detriti ambientali prelevati durante il campionamento. Dopo il campione inoculazione, virus sacche di isolamento dovrebbe essere libero di detriti visibile ambientale dal campione. Vi è ragionevole concordanza tra i risultati rRT-PCR e risultati di isolamento del virus con la consapevolezza che la PC...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K Views.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

Video: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

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, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificazione per affinità di Complessi virus influenzali ribonucleoproteina dalla cromatina delle cellule infettate

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-immunoprecipitazione della proteina mouse Mx1 con il virus nucleoproteina Influenza A

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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 ...

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 ...

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.