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>

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</ol>

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.

<table><tbody><tr><td>BBL Brain Heart Infusion</td><td>Becton, Dickinson and Company</td><td>211059</td><td></td></tr><tr><td>Penicillin G Sodium Salt</td><td>MP Biomedicals, LLC</td><td>021194537</td><td>1500 u/mg</td></tr><tr><td>Streptomycin Sulfate</td><td>AMRESCO LLC</td><td>0382</td><td></td></tr><tr><td>Gentamicin Solution</td><td>Mediatech, Inc.</td><td>30-005-CR</td><td>50 mg/ml</td></tr><tr><td>Amphotericin B Solution</td><td>Fisher</td><td>S 24 25</td><td>250 ug/ml</td></tr><tr><td>Kanamycin Sulfate</td><td>Teknova</td><td>K2105</td><td>5000 ug/ml</td></tr><tr><td>TPP Rapid Filtermax System</td><td>TPP Techno Plastic Products AG&amp;nbsp;</td><td>99150</td><td></td></tr><tr><td>Nalgen Diagnostic Bottles</td><td>Thermo Scientific</td><td>342002-9025</td><td>HDPE with white PP closure</td></tr><tr><td>Dermacea Gauze Sponge, 8 ply</td><td>Covidien</td><td>441211</td><td>5.08 cm &amp;times; 5.08 cm (2 in. &amp;times; 2 in)&amp;nbsp;</td></tr><tr><td>Nitrile Exam Gloves</td><td>Saftey Choice</td><td>19-170-010 (A-D)</td><td></td></tr></tbody></table>

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

9.4K 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

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Analisi quantitativa di tutti influenza di tipo A Hemagglutinins e neuraminidasi virale utilizzando anticorpi Universale di saggi semplice Slot Blot

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Ricerca

Immunologia e infezioni

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, ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

Rilevamento di virus da Bioaerosols utilizzando resina a scambio anionico

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Ambiente

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizzando le risposte dell'anticorpo al virus A(H3N2) in sieri umani di Microneutralization l'Influenza di misurazione analisi utilizzando cellule MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

La formazione immagine interazione cellula nella Mucosa trachea durante l'infezione da Virus influenzale usando la microscopia Intravital due-fotone

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

Un'analisi di inibizione (HI) ottimizzato emagglutinazione per quantificare i titoli dell'anticorpo specifico dell'Influenza

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Panni nasale per l'influenza A rilevazione antivirus e isolamento da Suina

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