Gli autori desiderano ringraziare i membri passati e presenti nei laboratori di Drs. Peter Palese e Adolfo García-Sastre per lo sviluppo di NDV invertire tecniche di genetica e per l&#39;assistenza tecnica. La ricerca in virus della malattia di Newcastle in laboratorio AG-S è parzialmente finanziato dalla NIAD concedere R01AI088770 e dal Dipartimento di Centro di Eccellenza per la Sicurezza Nazionale Scienza e Tecnologia per emergenti e malattie zoonotiche animali (CEEZAD, numero award 2010-ST-061-AG001). La ricerca in laboratorio LM-S è finanziato da sovvenzioni NIH RO1 AI077719, R21NS075611-01, R03AI099681-01A1, i Centri NIAID di eccellenza per la ricerca e l&#39;Influenza Surveillance (HHSN266200700008C), e l&#39;Università di Rochester Centro per Biodefense immunitario Modeling (HHSN272201000055C) .

1. Preparazione di cellule di mammifero (Figura 4A, 1 ª giornata) Split HEp-2 o cellule A549 il giorno prima della trasfezione in piastre da 6 pozzetti. Densità delle cellule dovrebbe raggiungere il 80-90% di confluenza il giorno seguente. Di solito, un confluente 100 millimetri piatto può essere suddiviso in 8 pozzetti (circa 1 x 10 6 cellule per pozzetto). Per ogni virus di essere recuperata, 2-4 diversi pozzetti devono essere incluse, nonché 2 pozzetti supplementari per i controlli pCAGGS-GFP e pCITE-GFP 18, volti a monitorare trasfezione e MVA-T7 efficienza di infezione, rispettivamente. 2. L&#39;infezione di cellule di mammifero con il virus del vaiolo vaccino ricombinante Modified Ankara Esprimendo il batteriofago T7 RNA polimerasi (MVA-T7) (Figura 4A, 2 ª giornata) <ol><li> Warm up PBS 1x/BA/PS e dei media a 37 ° C. </li><li> Lavare le cellule due volte con 1 ml di PBS 1x/BA/PS. </li><li> Infettare le cellule di mammifero coltivate con il virus MVA-T7 ad una molteplicità di infezione (moi) Di 1 pfu / cellule in un volume finale di 200 microlitri di PBS / BA / PS per 1 ora a temperatura ambiente. </li><li> Durante l&#39;infezione virale incubazione, preparare la miscela di trasfezione come descritto in 3. </li></ol> 3. Trasfezione di cellule di mammifero (Figura 4A, 2 ª giornata) <ol><li> Preparazione del Lipofectamine: Mescolare 1-2 mg di LPF2000 con 250 ml di OptiMEM per tentativo di salvataggio e incubare per 5 minuti a temperatura ambiente. </li><li> Preparazione del DNA: Fare un cocktail plasmide per ogni salvataggio in 50 ml di OptiMEM. Aggiungere 0,4 pg di pTM1-NP, 0,2 mg di pTM1-P, e 0,2 mg di pTM1-L per provetta. Aggiungere 1 mg del full-length cDNA clone di NDV essere salvato ad ogni provetta. Anche preparare due transfezioni di controllo, una con 2 mg di pCAGGS-GFP (per verificare l&#39;efficienza di trasfezione) e l&#39;altra con 2 mg di pCITE-GFP (per controllare T7 polimerasi guidato espressione del MVA-T7). </li><li> Dopo che la soluzione LPF2000-OptiMEM è stato preincubato per 5 min (passo 3.1), aggiungere 250microlitri della soluzione ai tubi DNA (passo 3.2) e incubare per 20-30 min a temperatura ambiente. </li><li> Dopo l&#39;incubazione con MVA-T7, rimuovere l&#39;inoculo del virus mediante aspirazione e sostituire con la miscela di trasfezione di DNA-LPF2000 (passo 3.3). </li><li> Aggiungere 1 ml di DMEM 10% FBS / PS per ogni piatto. </li><li> Incubare le cellule infette / transfettate per 6-8 ore (o tutta la notte) a 37 ° C, 5% CO 2 e quindi sostituire il supporto trasfezione con 1,5 ml di DMEM fresco 10% FBS / PS. </li><li> A 24 ore dopo la trasfezione, pozzetti di controllo possono essere osservati sotto un microscopio a fluorescenza per valutare l&#39;efficienza di trasfezione (pCAGGS-GFP) e l&#39;attività della polimerasi T7 (pCITE-GFP) (Figura 5A). Procedere con la co-coltura delle cellule infette / trasfettate con fibroblasti aviaria come descritto di seguito. </li></ol> 4. Co-coltura di cellule di mammifero con le cellule aviaria (Figura 4A, 3 ª giornata) Di solito, un confluente 100 millimetri di tessuto cultura piatto di pollo (CEFS) or duck (defs) fibroblasti di embrione viene utilizzato per due pozzi transfettate. Assicurati di preparare, in anticipo, abbastanza 100 millimetri di coltura tissutale piatti a base di cellule aviarie per tutti i tentativi di salvataggio. Per salvataggio efficiente del virus, DMEM 10% FBS / PS supporto è completato con 5% di liquido allantoidea 30 mM MgCl 2. A questo punto, 24 hr pi, cellule di mammifero possono iniziare a mostrare effetto citopatico (CPE) a causa di infezione MVA-T7, asevident in figura 5A. <ol><li> Warm up 1x PBS e media a 37 ° C. </li><li> Lavare le cellule di mammifero, 2x, con 1 ml di PBS 1x. </li><li> Trypsinize cellule di mammifero con 0,2 ml di EDTA-tripsina fino a quando non indipendente. Risospendere le cellule in 1 ml di DMEM 10% FBS / PS, 5% fluido allantoideo, 30 mM MgCl 2 e trasferimento in un piatto di coltura tissutale a 100 mm. Aggiungere 3 ml dello stesso supporto. </li><li> Lavare le cellule aviaria, due volte, con 4 ml di PBS 1x. </li><li> Trypsinize cellule aviarie con 1 ml di EDTA-tripsina e incubare per 1-2 minuti a 37 ° C finché le cellule si staccano. Risospendere thcelle e aggiungendo 8 ml di DMEM 10% FBS / PS, 5% fluido allantoideo, 30 mM MgCl 2 e aggiungere 4 ml delle cellule trypsinized alle cellule di mammifero in 100 mm al piatto di coltura tissutale co-coltura per un volume totale di 8 ml (4 ml di mammifero, 4 ml cellule aviarie). </li><li> Agitare delicatamente a mano le cellule co-coltivate nel piatto a 100 mm in modo da avere una distribuzione uniforme e poi metterli in incubatore a 37 ° C per 3-4 giorni. I criteri per infettare uova 3 o 4 giorni dopo co-coltura di cellule di mammifero e aviarie dipende dalla quantità di effetto citopatico (arrotondamento cella, morte, distacco dalla superficie, ecc.) Si osserva nella infezione virale MVA-T7. </li></ol> 5. L&#39;infezione di uova embrionate di pollo (4A, Giorni 6-7) <ol><li> Dopo 3-4 giorni di co-coltura di cellule di mammifero e aviaria, rimuovere 1 ml del supernatante di coltura tissutale e aggiungere in una provetta Eppendorf. </li><li> Centrifugare il surnatante di coltura tissutale per 1 min a 12.000 rpm in un bancocentrifuga per rimuovere i detriti cellulari. </li><li> Candela le uova utilizzando una scatola speratura luce per vedere l&#39;interfaccia tra la sacca d&#39;aria e la cavità allantoidea. Fare un segno sul confine interfaccia evitando la localizzazione dei vasi sanguigni. Spray etanolo al 70% sopra le uova per stabilire condizioni sterili. Fare delicatamente un foro nel guscio sul punto marcato e inoculare 500 ml di supernatante utilizzando un insulina (1 ml) siringa, puntando l&#39;ago verticalmente e direttamente nella cavità allantoica. (Figura 4B). </li><li> Sigillare il nick in guscio d&#39;uovo con cera fusa o paraffina. </li><li> Incubare le uova per 2-3 giorni a 37 ° C. </li></ol> 6. Raccolta di liquido allantoico da Infected uova embrionate di pollo (4A, giorni 8-10) <ol><li> Incubare le uova infette per 2-4 ore o tutta la notte a 4 ° C, al fine di uccidere l&#39;embrione di pollo e coagulare il sangue. </li><li> Spruzzare le uova con il 70% di etanolo (per mantenere condizioni di sterilità). </li><li> Toccare attentamente la sezione apicale dell&#39;uovo, sopra la cavità d&#39;aria, con un cucchiaio. Una volta che il guscio è incrinato, rimuovere i frammenti con una pinza e pienamente esporre la membrana allantoidea. </li><li> Esporre la cavità allantoica da asportare la membrana allantoico con le pinze e forbici, evitando di danneggiare i vasi sanguigni e il tuorlo. </li><li> Spingere delicatamente verso il basso l&#39;embrione con una spatola e raccogliere il liquido allantoico upflowing (8-12 ml per uovo) con una pipetta 10 ml. Evitare di danneggiare la membrana tuorlo. Trasferire il liquido allantoico ad un tubo da centrifuga da 15 ml sul ghiaccio (utilizzare un tubo per uovo). </li><li> Chiarire il liquido allantoico mediante centrifugazione per 5 min a 1.500 giri. Trasferire il surnatante in una nuova provetta senza disturbare il pellet. </li><li> Conservare i campioni a 4 ° C per un massimo di 1 settimana, fino a quando non controllando la presenza di NDV mediante test di emoagglutinazione. </li></ol> 7. Emoagglutinazione (HA) Assay La presenza del virus nel allantfluido OIC da uova infette può essere determinata macroscopicamente dalla loro capacità di hemagglutinate tacchino globuli rossi (RBC). Nel caso di NDV, circa 10 6 unità formanti placca (pfu) per ml è richiesto di dare un segnale positivo nel saggio HA. Saggi HA sono effettuate in piastre da 96 pozzetti V-bottom. Negativo (PBS 1x, liquido allantoico infetto), così come positivo (liquido allantoico da qualsiasi virus NDV) campioni di controllo devono essere sempre inclusi in qualsiasi dosaggio HA per convalidarlo. Per eseguire un saggio HA: <ol><li> Pipettare 50 ml di PBS 1x per pozzetto in una piastra a 96 pozzetti V-bottom. </li><li> Pipettare 50 ml di allantoici campioni di liquido nei pozzetti sulla prima colonna della piastra. Effettuare diluizioni seriali di 2 volte per il resto del piatto ed eliminare l&#39;ultimo, extra 50 microlitri dai pozzi nell&#39;ultima colonna. </li><li> Dispensare 50 ml di 0,5% tacchino RBC in PBS 1x per bene. Agitare delicatamente la piastra toccando. </li><li> Incubare la piastra a 4 ° C (o ghiaccio) fo 30-45 minuti o fino a quando un pellet chiaro si forma nei pozzetti di controllo negativi. </li></ol>

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Virol. 84, 781-788 (2003).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At+least+six+nucleotides+preceding+the+AUG+initiator+codon+enhance+translation+in+mammalian+cells.">At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells.</a> J. Mol. Biol. 196, 947-950 (1987).</li><li>Calain, P., Roux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rule+of+six,+a+basic+feature+for+efficient+replication+of+Sendai+virus+defective+interfering+RNA.">The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.</a> J. Virol. 67, 4822-4830 (1993).</li><li>Wyatt, L. S., Moss, B., Rozenblatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-deficient+vaccinia+virus+encoding+bacteriophage+T7+RNA+polymerase+for+transient+gene+expression+in+mammalian+cells.">Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells.</a> Virology. 210, 202-205 (1995).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+review.+New+mammalian+expression+vectors.">Product review. New mammalian expression vectors.</a> Nature. 348, 91-92 (1990).</li><li>Conzelmann, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetics+of+mononegavirales.">Reverse genetics of mononegavirales.</a> Curr. Top. Microbiol. Immunol. 283, 1-41 (2004).</li></ol>

Adolfo García-Sastre è un inventore di brevetti sul virus della malattia di Newcastle ricombinanti che sono di proprietà della Scuola di Medicina di Icahn al Monte Sinai.

Diversi fattori devono essere considerati per ottenere buoni risultati, mentre il salvataggio NDV. In primo luogo, il costrutto intera lunghezza cDNA da utilizzare deve essere progettato per consentire l&#39;integrazione funzionale dei nuovi transgeni / modifiche nel genoma NDV. Ciò significa, come detto sopra, che le sequenze (i) end appropriato gene (GE), intergenica (IG) e inizio gene (GS) devono essere aggiunti, se necessario, (ii) non vi sono sequenze putative GE o GS nel estera gene, e (iii) il genoma ricombinant...

Virus della malattia di Newcastle (NDV), un paramyxovirus aviario appartenente al genere Avulavirus 1, è un agente di zoonosi economicamente rilevante e quindi ampiamente studiato e sorvegliato, che può influenzare fortemente l&#39;allevamento di pollame in tutto il mondo. Pur non essendo un agente patogeno umano, NDV è stato anche studiato a fondo al di là del campo veterinario sia come modello paramyxovirus e grazie alle sue molto interessanti, proprietà naturali oncolitici 6. Ricerca su NDV notevolmente beneficiato lo sviluppo di tecniche di genetica inversa per, non segmentati virus a singolo filamento di RNA negativo-senso, in primo luogo descritti per virus della rabbia da Conzelmann e coleagues 2. Una varietà di NDVs geneticamente modificati, portatori di geni estranei o modifiche del loro tipo selvatico genoma sono stati ampiamente studiati da allora. Lavorare con questi virus ricombinanti è stato fondamentale per individuare i diversi fattori di virulenza non solo di NDV, ma anche di altri huma pertinenten agenti patogeni come virus dell&#39;influenza A 7 - o l&#39;emergente Nipah virus 8. Inoltre, diversi studi hanno esplorato l&#39;uso di queste tecniche per migliorare l&#39;attività antitumorale innata NDV 6,9,10, soprattutto migliorando le proprietà immunostimolanti del virus. Altro settore di ricerca sulla NDVs ricombinanti è stata la generazione di candidati vaccini contro altre malattie virali come l&#39;influenza 5,11,12, 13 HIV, morbillo 14, SARS 15, o che ha causato dal virus sincytial respiratorio (RSV) 16. Tra i vari notevoli vantaggi forniti dal NDV sono la mancanza di immunità preesistente nella popolazione umana, la stabilità degli inserti estere genetiche, una mancanza di attività recombinatory e nel complesso un elevato profilo di sicurezza combinato con le suddette proprietà immunostimolanti naturali 17. E &#39;inoltre degno di nota il potenziale uso di vaccini bivalenti ricombinanti in poultry, l&#39;influenza aviaria di protezione contro sia NDV e altamente patogeno virus 11,12. Questo può essere un ottimo modo per diminuire le probabilità di quest&#39;ultimo diffusione da selvatici animali domestici, quindi anche aiutando a prevenire un eventuale salto inter-specifica della temuta influenza aviaria agli esseri umani. Infine, sono stati utilizzati reporter esprimere NDV per la valutazione della risposta immunitaria innata come pure l&#39;identificazione di interferone antagonista codificata dal virus multipli 18-27. Il processo di salvataggio di un ricombinante, non segmentato, virus RNA negativo filamento costituito essenzialmente da forzare artificialmente un ciclo di replicazione virale in una cellula che produce trasfettando cDNA codificanti il macchinario molecolare minimo infettiva, noto come ribonucleoproteina o RNP (Figura 2). Le RNP consistono polimerasi virale (P e proteine ​​L), la nucleoproteina (NP) e l&#39;intera lunghezza antigenomic RNA del virus. Questo RNA + antigenome è ªe modello richiesto per la generazione dei genomi RNA complementari, che, associati anche con il resto delle proteine ​​della RNP virale, ricapitola stesso complesso infettiva che un virus naturale libererebbe il citoplasma della cellula dopo l&#39;infezione (Figura 2A ). Da questo punto in poi, il ciclo virale può procedere in modo naturale e virioni ricombinanti, encapsidating i genomi modificati, verrà generato (Figura 2B). Sorprendentemente, trasfezione del cDNA genomico invece del cDNA antigenomic compromette notevolmente o abolisce efficienza salvataggio 2,28-30 completamente. Anche quando cDNA antigenomic viene trasfettato, l&#39;efficienza del incapsidamento del RNA ricombinante in RNPs in cellule trasfettate è probabilmente molto bassa. A causa di questo, protocolli di salvataggio per NDV spesso includono diversi passaggi per l&#39;amplificazione delle particelle virali pochi rilasciate dalle cellule trasfettate originariamente da essi coculturing con cellule permissive e / o dalinfezione di uova embrionate. Prima del salvataggio, il cDNA può essere manipolato da procedure di clonazione standard per generare le modifiche desiderate. Mentre mutazioni specifiche dei diversi prodotti genici e sequenze regolatrici del virus possono essere direttamente raggiunti in questo modo, molti dei lavori pubblicati coinvolge ricombinante NDV ha richiesto l&#39;aggiunta di una nuova unità trascrizionale nel genoma NDV. Come altri membri della famiglia paramyxovirus, il genoma NDV codifica otto proteine ​​differenti in sei unità trascrizionali, che sono differenzialmente espressi in funzione della loro posizione rispetto alla estremità 3 &#39;in un gradiente critico per il ciclo di vita virale 1 discendente. A causa di questo, la posizione della nuova unità trascrizionale all&#39;interno del genoma va scelto opportunamente per raggiungere un equilibrio tra l&#39;espressione del transgene e compromissione della replicazione virale. Inserimento tra P e M geni è stato usato più, tHough altri siti sono stati anche testati 13,31. Qualunque sia l&#39;inserto, la clonazione in NDV cDNA deve seguire alcune regole per generare un costrutto salvabile: (i) qualsiasi nuovo gene da inserire nel genoma NDV deve essere sotto il controllo dei segnali appropriati per il virale RNA-dipendente-RNA polimerasi. Queste sequenze devono essere aggiunti a monte della nuova fase di lettura aperta (ORF) per la polimerasi possono riconoscere la fine del gene precedente (GE) e l&#39;inizio del nuovo transgene (GS), distanziati di un singolo nucleotide sequenza intergenica (IG) . Aggiunta di un valido Kozak (K) sequenza di migliorare traduzione eucariotico ribosomiale è consigliato anche per una migliore espressione proteina estranea 32; (ii) replica efficiente di NDV, come per la maggior parte dei membri della famiglia Paramyxoviridae, dipende dalla lunghezza del genoma essere multiplo di sei 33, pertanto, qualsiasi inserimento nella NDV deve seguire questa &quot;regola del sei&quot;. Se necessario, required nucleotidi possono essere aggiunti a valle della nuova ORF, e (iii) la sequenza del transgene dovrebbe essere controllata per trovare possibili GE e GS come sequenze che potrebbero compromettere l&#39;efficienza di salvataggio, l&#39;espressione del transgene e / o vitalità del virus. Se presente, queste sequenze devono essere rimossi mediante mutagenesi silenzioso. La generazione di cDNA ricombinante piena lunghezza seguenti norme suddette è il primo passo per produrre efficientemente un NDV geneticamente modificato come qui descritto. Nel sistema tutti i costrutti di DNA sono sotto il controllo del promotore T7 RNA polimerasi (Figura 3). Questa polimerasi citoplasmatica è fornito in trans da coinfezione con un modificato vaccinia virus ricombinante Ankara (MVA-T7) 34. Figura 3A mostra il plasmide pNDV-B1, che codifica l&#39;intera lunghezza cDNA antigenomic 5. Figura 3B mostra plasmidi codificanti pTM1 NP, P e L ORF. Plasmidi pCITE-GFP, che codifica, under il promotore T7, la proteina fluorescente verde (GFP), e pCAGGS GFP 18, che codifica lo stesso ORF sotto il promotore actina di pollo beta 35, sono usati come controlli. In questo protocollo mostriamo la procedura per salvare ricombinante NDV dal cDNA del lentogeno NDV ceppo Hitchner B1 5 (Figura 4).

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

rescue of recombinant newcastle disease virus from cdna

Virus della malattia di Newcastle (NDV), il membro prototipo del genere Avulavirus della famiglia Paramyxoviridae 1, è un non-segmentato, negativo-senso, a singolo filamento, avvolta virus RNA (Figura 1), con potenziali applicazioni come vettore per la vaccinazione e trattamento di malattie umane. L&#39;esplorazione in profondità di queste applicazioni è diventato possibile solo dopo l&#39;istituzione della genetica inversa tecniche per salvare i virus ricombinanti da plasmidi che codificano i loro genomi completi come cDNA 2-5. CDNA virale può essere convenientemente modificati in vitro utilizzando procedure di clonazione standard per alterare il genotipo del virus e / o di inserire nuove unità trascrizionali. Salvataggio di tali virus geneticamente modificati, fornisce un prezioso strumento per comprendere i fattori che incidono più fasi dell&#39;infezione, così come consente lo sviluppo e il miglioramento di vettori per l&#39;espressione e la consegna degli antigeniper la vaccinazione e la terapia. Qui si descrive un protocollo per il salvataggio di NDVs ricombinanti.

Salvataggio di NDV è una procedura consolidata, eseguita di routine nei laboratori che hanno accesso al cDNA completa del virus. Tuttavia, la natura intrinseca stocastico del metodo rende difficile raggiungere il 100% di efficienza salvataggio. Monitoraggio prime fasi del processo, specialmente l&#39;efficienza di trasfezione e l&#39;infezione con MVA-T7, aiuta a individuare eventuali problemi. Figura 5a mostra trasfezione standard ed efficienze di trasfezione / infezione che sono abbastanza per un suc...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

Virus della malattia di Newcastle (NDV) è stata ampiamente studiata negli ultimi anni per sviluppare nuovi vettori per la vaccinazione e terapia, tra gli altri. Questi studi sono stati possibili a causa di tecniche di salvataggio virus ricombinante da cDNA, come quelle descritte.

Salvataggio di virus ricombinante malattia di Newcastle da cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

rescue-of-recombinant-newcastle-disease-virus-from-cdna

Research

JoVE Journal

Immunology and Infection

19.2K Views.  Icahn School of Medicine at Mount Sinai.  Virus della malattia di Newcastle (NDV) è stata ampiamente studiata negli ultimi anni per sviluppare nuovi vettori per la vaccinazione e terapia, tra gli altri. Questi studi sono stati possibili a causa di tecniche di salvataggio virus ricombinante da cDNA, come quelle descritte. 

Video: Rescue of Recombinant Newcastle Disease Virus from cDNA

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

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

Espressione dei funzionali ricombinante emoagglutinina e neuraminidasi proteine ​​del virus Novel H7N9 Influenza utilizzando l&#39;espressione sistema Baculovirus

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalabile High Throughput Selection Da sintetici Biblioteche Antibody fagi-visualizzato

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

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 ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Salvataggio e caratterizzazione del virus ricombinante da un nuovo mondo Clone infettivo del virus Zika

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

Rilevamento e rimozione di nucleasi contaminazione durante la purificazione del prototipo ricombinanti Virus schiumoso integrasi

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

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

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

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

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

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Valutazione delle risposte a cellula T Zika Virus-specifici in organi di nuova di Ifnar1 infetti- / - Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificazione di Coding e classi di RNA Non codificanti espressa in suina sangue intero

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...