The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

NOTA: Osservazioni generali: eseguire tutte le procedure che comportano la manipolazione delle uova in condizioni sterili, e una tecnica sterile deve essere utilizzato di conseguenza. Pre-pulire tutte le apparecchiature con il 70% di etanolo prima dell&#39;uso. In generale, l&#39;inoculazione di virus influenzale e la raccolta devono essere condotti in un laboratorio BSL-2. Tuttavia, se si utilizza questo protocollo per propagare molto più patogeno virus influenzali (ad esempio, ceppi pandemici e pre-pandemia, i ceppi che rappresentano un pericolo per pollame e bestiame, i ceppi di influenza aviaria ad alta patogenicità, 1918 H1N1 ceppo), i livelli di biosicurezza più alti e in alcuni casi, sono necessarie precauzioni di biosicurezza e le autorizzazioni. Il Comitato Istituzionale Biosicurezza locale dovrebbe approvare tutte le ricerche che coinvolgono il virus dell&#39;influenza. 1. Preparazione di uova per Inoculation <ol><li> Ottenere uova siero (SPF) esenti da organismi patogeni appena fecondato aviaria da un fornitore adatto. Tipicamente, si usano uova di gallina. Le uova devono essere noied immediatamente all&#39;arrivo. </li><li> Al posto All&#39;arrivo uova in una incubatrice dell&#39;uovo umidificato con un tornitore automatico uovo per ruotare le uova regolarmente. Se un tornitore automatica uovo non è disponibile, ruotare manualmente le uova almeno 2-3 volte al giorno (assicuratevi di indossare guanti e tenere tutto il più sterile possibile). </li><li> Incubare le uova a 37 ° C e 55-60% di umidità per 10-11 giorni. </li></ol> Speratura 2. Egg <ol><li> Utilizzare un candler uovo per esaminare l&#39;uovo contro una luce in una stanza buia per controllare le uova per l&#39;infertilità da speratura dopo circa 7 o 8 giorni di incubazione. <ol><li> Pulire la candler uovo con il 70% di etanolo. Cambiare i guanti spesso e disinfettare con il 70% di etanolo per minimizzare il rischio di contaminazione da patogeni. </li><li> Rimuovere le uova dal termostato e metterli in cartoni delle uova vuote. </li><li> Spegnere le luci in sala. </li><li> Tenere l&#39;estremità grande ogni uovo uno alla volta contro l&#39;candler. </li><li> Osservare l&#39;uovo al determine se è fertile o infertile. NOTA: i vasi sanguigni sottili che portano a un embrione a forma di fagiolo deve essere chiaramente visibile. Uova non fecondate appariranno come una piccola macchia di sangue con un tuorlo d&#39;uovo visibile. </li></ol></li><li> Eliminare le uova che sono non fecondato, e restituire le uova vitali per l&#39;incubatrice. Non lasciare uova di fuori dell&#39;incubatore per più di 30 min. Se lo stato di un uovo non può essere confermata, segnare, e osservare in seguito. </li></ol> 3. Preparazione di Virus Inoculum <ol><li> Diluire virus dell&#39;influenza stock da circa marzo 10 - aprile 10 pfu / ml in PBS sterile contenente HEPES 10 mM (pH 7.4). Passaggio fondamentale: Diluire lo stock di virus immediatamente prima dell&#39;uso e conservare il virus diluito su ghiaccio per tutto il tempo. </li></ol> 4. Influenza Virus inoculazione mediante la Strada Allantoic <ol><li> Il giorno di inoculazione del virus (il giorno 10 o 11), candela di nuovo le uova ed eliminare eventuali embrioni morti. Distinguere embrioni vivi percercando movimento all&#39;interno dell&#39;uovo. </li><li> Rimuovere le tre uova dal termostato, e organizzare le uova in un supporto separato con il sacco aria fino. </li><li> Segnare lo spazio aereo delle uova con un pennarello indelebile. </li><li> Lavare il guscio d&#39;uovo sopra lo spazio aereo con il 70% di etanolo. </li><li> Mettere le uova con lo spazio aereo fino in un cartone di uova e in una cappa di biosicurezza (BSL-2). </li><li> Utilizzare un ago 18G sterili per perforare un piccolo foro nel guscio sopra la sacca d&#39;aria di ciascun uovo. Fare attenzione a non inserire l&#39;ago troppo profondamente per evitare di pugnalare l&#39;embrione o il tuorlo. </li><li> Redigere il virus dell&#39;influenza diluita in una siringa sterile da 1 ml e inserire un ago 22G. </li><li> Avanzare con cautela la siringa con l&#39;ago a un angolo nella cavità allantoica 45 °. </li><li> Iniettare 0,2 ml di virus dell&#39;influenza diluito. </li><li> Ripetere l&#39;iniezione con le altre due uova, e poi scarta la siringa e l&#39;ago. </li><li> Sigillare il foro con una goccia di colla da una pistola per colla (o paraffina fusa). Be attenzione che la colla non perde dentro l&#39;uovo. </li><li> Mettere le uova di nuovo nel incubatore uovo con lo spazio aereo puntato verso l&#39;alto. </li><li> Ripetere i passaggi 4,2-4,12 finché non sono stati inoculati tutte le uova. </li></ol> 5. Incubazione <ol><li> Incubare le uova infette senza girare a 37 ° C e ~ 60% di umidità. </li><li> Scegliere tempo ottimale di incubazione a seconda del tipo di virus dell&#39;influenza. Incubare influenza A per 48 ore; incubare l&#39;influenza B e C per 72 ore. </li></ol> 6. Influenza Virus Harvest <ol><li> Dopo il periodo di incubazione, le uova raffreddare a 4 ° C per un minimo di 4 ore (o O / N) per uccidere l&#39;embrione e costrizione dei vasi sanguigni per ridurre il rischio di contaminazione del liquido allantoico infetto di sangue. Questa operazione consente di evitare che i globuli rossi di assorbire il virus, che riduce il rendimento. In alternativa, le uova freddo per 30 min a -20 ° C; tuttavia questo aumenta il rischio di contaminazione del sangue. </li><li> Dopo che le uova sono state sufficientemente raffreddata, trasferire le uova di una cappa di biosicurezza. Mettere l&#39;uovo in un supporto stabile con la sac di aria verso l&#39;alto. Pulire la superficie dell&#39;uovo con il 70% di etanolo. </li><li> Usare le forbici sterili per aprire il guscio sopra la sacca d&#39;aria. Togliere il guscio intorno al sacco aereo facendo attenzione a non distruggere la membrana corioallantoidea. </li><li> Aprire la membrana corioallantoidea con una pinza smussato sterili. Spostare delicatamente a parte l&#39;embrione e il sacco vitellino con una piccola spatola o un cucchiaio. Fare attenzione a non rompere il tuorlo. </li><li> Usando una pipetta Eppendorf 1 ml, raccogliere con cura il liquido allantoico. Se necessario, inclinare l&#39;uovo un po &#39;di lato per raccogliere quanto più fluido possibile. </li><li> Combinare il liquido allantoico da tutte le uova in un tubo di plastica conica 50 ml. Tenere tubi sul ghiaccio in qualsiasi momento durante la vendemmia virus. I rendimenti Un uovo 5-10 ml di un liquido leggermente giallognolo. </li><li> Spin il liquido allantoico contenente il virus a 1.000 xg per 10 min a 46; C a pellet detriti. Trasferire il liquido chiaro in un nuovo tubo da 50 ml tenuta in ghiaccio. Smaltire le uova in un contenitore di rifiuti BSL-2. </li></ol> 7. Conservazione <ol><li> Un&#39;aliquota immediatamente il liquido chiarificato allantoico (ad esempio, 50, 100 o 500 aliquote ul) e snap-congelare in azoto liquido. Trasferire le riserve di virus congelati a -80 ° C per una conservazione a lungo termine. </li></ol>

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+deaths+associated+with+seasonal+influenza+---+United+States,+1976-2007.">Estimates of deaths associated with seasonal influenza --- United States, 1976-2007.</a> MMWR. Morbidity and mortality weekly report. 59 (33), 1057-1062 (2010).</li><li>Neumann, G., Noda, T., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+pandemic+potential+of+swine-origin+H1N1+influenza+virus.">Emergence and pandemic potential of swine-origin H1N1 influenza virus.</a> Nature. 459 (7249), 931-939 (2009).</li><li>Tumpey, T. M., Garcia-Sastre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+Influenza+Viruses+with+Genes+from+the+1918+Pandemic+Virus:+Functional+Roles+of+Alveolar+Macrophages+and+Neutrophils+in+Limiting+Virus+Replication+and+Mortality+in+Mice.">Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice.</a> J Virol. 79 (23), 14933-14944 (2005).</li><li>Doherty, P. C., Turner, S. J., Webby, R. G., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+and+the+challenge+for+immunology.">Influenza and the challenge for immunology.</a> Nat Immu. 7 (5), 449-455 (2006).</li><li>Webster, R. G., Govorkova, 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=Continuing+challenges+in+influenza.">Continuing challenges in influenza.</a> Ann N Y Acad Sci. 1323 (1), 115-139 (2014).</li><li>Szretter, K. J., Balish, A. L., Katz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+15G.1+Influenza:+propagation,+quantification,+and+storage.+.">Unit 15G.1 Influenza: propagation, quantification, and storage. .</a> Current protocols in microbiology. , (2006).</li><li>Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M., Kuiken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+influenza-induced+acute+respiratory+distress+syndrome.">Pathogenesis of influenza-induced acute respiratory distress syndrome.</a> Lancet Infect Dis. 14 (4), 57-69 (2014).</li><li>Ito, T., Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sialic+acid-galactose+linkages+in+the+chicken+egg+amnion+and+allantois+influence+human+influenza+virus+receptor+specificity+and+variant+selection.">Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.</a> J Virol. 71 (4), 3357-3362 (1997).</li><li>Robertson, J. S., Nicolson, C., Major, D., Robertson, E. W., Wood, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+amniotic+passage+in+the+egg-adaptation+of+human+influenza+virus+is+revealed+by+haemagglutinin+sequence+analyses.">The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.</a> J Gen Virol. 74 (Pt 10), 2047-2051 (1993).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells).">Replication and plaque assay of influenza virus in an established line of canine kidney cells).</a> Appl Microbiol. 16 (4), 588-594 (1968).</li></ol>

The authors have nothing to disclose.

Influenza provoca un grande carico globale di malattia, e il lavoro continua nella comprensione della patogenesi del danno polmonare 7. Per la ricerca facilitato in questa malattia mortale, vari metodi sono stati sviluppati per diffondere il virus dell&#39;influenza 6. Qui, descriviamo una tecnica per la produzione di virus dell&#39;influenza in uova di gallina. Il vantaggio di questo metodo è che è altamente riproducibile e si traduce in grandi quantità ad alto titolo di riserve di virus influe...

Influenza A continua ad essere una grave minaccia per la salute umana. Si tratta di una malattia respiratoria potenzialmente devastante con un grande carico globale provocando fino a 500.000 morti nel mondo ogni anno 1. I virus influenzali sono nella famiglia Orthomyxoviridae e portano a 8 negativo-sense singolo filamento di RNA nel loro genoma 1,2. L&#39;elevata mutevolezza (cioè, &quot;drift&quot; antigenico) del genoma virale impedisce immunità a lungo termine. Inoltre, l&#39;influenza è sempre più resistenti ai farmaci anti-virali 3. Il 2009 H1N1 pandemia influenzale ha evidenziato tutti i problemi difficili associati alla malattia influenzale (ceppo pandemico, la resistenza anti-virale, la produzione di vaccini in ritardo). La formulazione del vaccino è determinato annualmente dalla Organizzazione Mondiale della Sanità per i ceppi influenzali più probabile patogeni (uno ciascuno per H1N1, H3N2 e influenza B) 4. Poiché questo metodo si basa su ceppo influenzale previstos, i ceppi patogeni sono a volte erroneamente identificati e l&#39;efficacia del vaccino si riduce drasticamente. Inoltre, la comparsa di nuovi ceppi di influenza può aggirare questi programmi di prevenzione che causano pandemie 2. La creazione di un vaccino influenzale universale è stato sfuggente 5. Pertanto, la ricerca deve continuare nella comprensione della patogenesi del danno polmonare. Per struttura di ricerca di laboratorio, diversi metodi sono stati sviluppati per l&#39;isolamento virale e la propagazione 6. Virus influenzali umani possono essere amplificati in una varietà di substrati cellulari di mammifero. Tuttavia, generando virus ad alto titolo in grandi quantità sono più raggiunti in uova embrionate. Il protocollo che segue descrive una tecnica per l&#39;influenza A la propagazione del virus e lo stoccaggio delle scorte di virus esistenti.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:225px;">Company</td>
			<td style="width:128px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Phosphate buffered saline (PBS)</td>
			<td style="width:225px;">Cellgro&#160;</td>
			<td style="width:128px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serum pathogen-free (SPF) fertilized chicken eggs&#160;</td>
			<td>VALO BioMedia</td>
			<td style="width:128px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">humidified egg incubator&#160;</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">automatic egg turner</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg candler</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3300&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1 ml syringe</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">18G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">20G / 22G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305176 / 305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">HEPES</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol, &#62;99.5%</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">459844</td>
			<td>diluted to 70% using water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue gun &#34;Ad tech Hi Temp Project Pro&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>Model 1105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue &#34;Ad tech MINI SIZE, Multi Temp&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>220-34ZIP30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MDCK cells</td>
			<td style="width:225px;">ATCC</td>
			<td>CRL-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Washed Pooled Turkey Red Blood Cells, 10%</td>
			<td style="width:225px;">Lampire Biological Laboratories</td>
			<td>724908</td>
			<td></td>
		</tr>
	</tbody>
</table>

influenza virus propagation in embryonated chicken eggs

Infezione influenzale è associata con circa 36.000 morti e più di 200.000 ricoveri ogni anno negli Stati Uniti. L&#39;emergere continuo di nuovi ceppi di virus influenzale dovute alla mutazione e ri-assortimento complica il controllo del virus e richiede lo sviluppo permanente di nuovi farmaci e vaccini. Lo studio in laboratorio di influenza richiede un metodo affidabile e conveniente per la propagazione del virus. Qui, un protocollo globale è costituita per influenza A propagazione dei virus in uova di gallina fertili, che produce costantemente le scorte virali ad alto titolo. In breve, esenti da organismi patogeni (SPF) uova di gallina fecondate siero sono incubate a 37 ° C e 55-60% di umidità per 10 - 11 giorni. In questo periodo, lo sviluppo dell&#39;embrione può essere facilmente monitorato mediante un Candler uovo. Virus inoculazione viene effettuata mediante iniezione del virus magazzino nella cavità allantoidea utilizzando un ago. Dopo 2 giorni di incubazione a 37 ° C, le uova sonorefrigerata per almeno 4 ore a 4 ° C. Il guscio d&#39;uovo sopra la sacca d&#39;aria e la membrana corioallantoidea sono poi accuratamente aperto, e il liquido allantoico contenente il virus è raccolto. Il fluido viene cancellato da detriti mediante centrifugazione, aliquotato e trasferito a -80 ° C per la conservazione a lungo termine. La grande quantità (5-10 ml di virus contenenti liquido per uovo) titolo e precisa di virus che di solito si ottiene con questo protocollo ha reso l&#39;utilizzo di uova per la preparazione virus nostro metodo favorevole, in particolare per gli studi in vitro, che richiedono grandi quantità del virus in cui sono necessari dosaggi elevati di virus stesso magazzino.

Più metodi sono stati sviluppati per titolo virale influenzale. Una considerazione quando si sceglie un metodo appropriato è che alcuni determinano particelle totali, indipendentemente della redditività (ad esempio, test emoagglutinina), mentre altri si basano su infettività, che valuterà virioni vitali (ad esempio, test placca) 6. Qui, il titolo virale di liquido allantoico raccolti da uova di pollo inoculate con un virus influenzale mouse adattato (A / PR / 8/34) è stata determinata ...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

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 ...

influenza-virus-propagation-in-embryonated-chicken-eggs

Research

JoVE Journal

Immunology and Infection

42.7K Views.  Cedars-Sinai Medical Center. 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.

Video: Influenza Virus Propagation in Embryonated Chicken Eggs

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

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 ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

Propagazione di<em&gt; Virus-01 Homalodisca coagulata</em&gt; Via<em&gt; Homalodisca vitripennis</em&gt; Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

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 ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Misurazione Attaccamento e internalizzazione del virus A in cellule A549 mediante citometria di flusso

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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.

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 ...

Methods Article

Influenza Virus Propagazione in embrionate di pollo Uova