La pubblicazione di questo articolo è stato sostenuto da una borsa di studio presso l&#39;Ufficio di Ricerca presso l&#39;Università del Tennessee. Gli autori ringraziano la precedente generazione di studenti e ricercatori che hanno lavorato per affinare queste procedure. Ricerca è stata sostenuta dalle concessioni dal National Science Foundation (NSF-0851113, NSF-0825405 e NSF-0550485).

1. Ultrafiltrazione di acqua di mare per generare acqua &quot;virus-free&quot; (Wilhelm &amp; Poorvin 2001) <ol><li> Circa 20L di acqua di mare / lakewater è raccolto come in modo asettico possibile. </li><li> L&#39;acqua è in serie prefiltrata attraverso 142-mm di diametro filtri di policarbonato 0,8 micron, che possono essere conservati a -20 ° C per l&#39;analisi della comunità. Filtri più grandi dimensioni dei pori possono essere utilizzati prima di questo passaggio per sistemi molto produttivo. </li><li> Per ottenere acqua ultrafiltrato da un sistema M12 Amicon (Millipore) viene utilizzato un cut-off 30 kDa-cartuccia spirale di escludere tutti i virus, anche piccolo virus RNA. </li><li> I campioni vengono processati e concentrati a velocità ~ 25% ~ 15-16 kPa di contropressione. </li><li> Il campione rimanente di circa 500 ml di acqua conterrà una comunità concentrata virus (che può essere salvato per altri esperimenti), mentre il restante 19,5 L di virus acqua verrà utilizzata per le prove di produzione virale. </li><li> Dopo ogni giorno di utilizzo, il Amicon M12 sistema deve essere pulito per evitare di danneggiare la membrana del filtro a cartuccia. </li><li> Se si lavora con acqua di mare, lavare la membrana con almeno 6L di Milli-Q acqua seguita da un lavaggio con soluzione di NaOH 0.1N per 30-45 minuti. </li><li> Anche in questo caso lavare la cartuccia con almeno 6L di acqua Milli-Q. </li><li> Al termine usando il sistema di M12, la cartuccia a spirale deve essere conservato in un 0.05MH 3 PO 4 soluzione a 4 ° C. </li></ol> 2. Virus Metodo di riduzione per la produzione virale (Wilhelm et al. 2002) <ol><li> Fino a 500 ml di acqua di mare / lakewater campione sia con ospite e virus si ottiene e posto in una unità sterifilter con un 0,2 micron nominale dei pori di dimensioni a basso legame proteico filtro (ad esempio, Durapore) posto su di esso. </li><li> Il campione viene delicatamente vuoto sotto pressione a &lt;200 mmHg, mentre continuamente risospendere il campione usando una pipetta sterile trasferimento di inibire le cellule batteriche di concentrarsi sul filtro. </li><li> Lentamente tre volumi di ultrafiltrato si aggiungono alla sospensione batterica per ridurre significativamente il numero di virus libero nel campione. </li><li> La frazione batterica viene diluito torna a 500 ml con acqua priva di virus e divisa in tre repliche da 150 ml ciascuno e sono collocati in chiaro 250ml bottiglie in policarbonato. </li></ol> 3. Portata di filtrazione tangenziale (TFF) Metodo di produzione virale (Weinbauer et al. 2002, Winget et al. 2005) TFF rappresenta un approccio alternativo al metodo di riduzione virale. <ol><li> Circa 500 ml di campione naturale viene raccolto come sopra descritto. </li><li> Questo esempio è concentrata con un 0,2 micron nominale dei pori di dimensioni tangenziale sistema di filtraggio del flusso. </li><li> Quando la frazione batterica è ridotta a circa 10-15 ml, ultrafiltrato, senza virus viene aggiunta acqua e distribuito come sopra. </li><li> Le bottiglie vengono incubate a replicare in situ utilizzando camere ambientali. <ol><li> I livelli di luce sono alterati alle condizioni della superficie utilizzando azzurrata acrilico acrilico o deselezionare con lo screening rete per diminuire l&#39;intensità della luce. </li><li> Temperature di superficie ambiente sono spesso ottenute utilizzando un mazzo di acqua di mare che scorre incubatrice. </li></ol></li><li> I campioni per la stima dell&#39;abbondanza batteriche e virali vengono prese al tempo 0, con una concentrazione finale di 2,0-2,5% glutaraldeide sterile aggiunto in cryovials. Questi campioni sono immediatamente istantaneamente congelati con l&#39;azoto liquido e conservati congelati fino elaborati. <ol><li> Se l&#39;azoto liquido non è disponibile, diapositive microscopia possono essere preparati e trasformati immediatamente (vedere la procedura di seguito) </li></ol></li><li> Sottocampioni sono raccolti ogni 2,5 ore per almeno 10 ore con il metodo sopra descritto. <ol><li> In questo momento l&#39;acqua possono essere raccolti per l&#39;analisi PCR quantitativa. Fino a 5 ml del campione può essere aggiunto a una provetta Microbank ™ con nessun agente fissativo con il congelamento immediato il flash in azoto liquido. </li></ol></li></ol> 4. Produzione Microscopia virale (Noble &amp; Fuhrman 1998, Wen et al. 2004) <ol><li> Campioni congelati da 0.02-micron filtrata per microscopia devono essere scongelati su ghiaccio. </li><li> Preparare una soluzione stock di SYBR Green diluendo la soluzione stock 1:10 con acqua sterile. Successivamente, dalla soluzione madre, preparare una soluzione di lavoro con l&#39;aggiunta di 1 ml di soluzione madre a 39 ml di acqua sterile. Un glicerolo 50%, 50% di soluzioni tampone fosfato salino (PBS, 0,05 M Na 2 HPO 4, 0,85% NaCl, pH 7,5) e fresco soluzione madre al 2,5% del p-fenilendiammina dovrebbe anche essere preparato prima di iniziare. Tenere il glycerol/50% 50% soluzione di PBS a 4 ° C e il p-phenylenediammagazzino ine a -20 ° C al buio fino a quando partire. A destra prima di filtrare aggiungere il p-fenilendiammina alla PBS 50% glycerol/50% ad una concentrazione finale del 0,1% per essere utilizzato come soluzione Antifade. </li><li> Mettere un 25 millimetri 0.02-micron filtro Anodisc sulla cima di un 0.45-micron MicronStep, filtro di supporto cellulosico. Aggiungere 850 ml di campione fissato alla parte superiore del Anodisc e vuoto a 20 pKa fino a completa essiccazione. Mettere 100 ml di soluzione SYBR Green lavorando ad una piastra di Petri sterile e, con il vuoto ancora, rimuovere con attenzione il Anodisc dalla torre di filtro e luogo on the Green SYBR. Incubare i campioni al buio a temperatura ambiente per 20 minuti. Rimuovere con cautela il filtro dalla soluzione SYBR Green e stoppino sul retro del filtro con un Kimwipe per rimuovere tutti i residui di colorante. Se lo si desidera, ritorno il filtro alla torre e passare fino a 800 ml di 0.02-micron acqua filtrata o supporti sterile attraverso il filtro a sciacquare via macchia in eccesso. </li><li> Aggiungere una piccola goccia di soluzione Antifade di un vetrino da microscopio e luogo uno slittamento di copertura sulla parte superiore. Rimuovere la lista di coperchio e aggiungere il filtro essiccato al vetrino da microscopio bagnato con la soluzione Antifade. Ancora una volta, aggiungere una piccola quantità di soluzione Antifade al vetrino e lentamente posto sulla parte superiore del filtro, avendo cura di eliminare eventuali bolle che si possono formare. </li><li> Immediatamente congelare i campioni a -20 ° C fino al momento (che dovranno essere utilizzati entro pochi mesi per prevenire lo scolorimento e abbassato conta virus) </li><li> I virus sono enumerati utilizzando la microscopia a fluorescenza (nel nostro caso un microscopio Leica DMRXA), con un ampio set di filtro blu (λ = Ex 450-490 nm, Em λ = 510 nm con un filtro di soppressione a λ = 510 nm). Ogni filtro avrà almeno 20 campi di vista contati, facendo attenzione a quantificare virus totale da ogni griglia campo per garantire un&#39;equa distribuzione dei virus attraverso la membrana del filtro. </li><li> I tassi medi di ripetersi virus da tre indipendenti replica vengono poi calcolati e una deviazione standard è determinato dai tassi di produzione. </li></ol> 5. Rappresentante Risultati I dati grezzi raccolti dal ricercatore richiede un numero minimo di elaborazione matematica per generare tassi di ricorrenza di abbondanza virus. Il set di dati primari risultante da questo studio è il tasso di ricorrenza di abbondanza virus nel sottocampioni dal incubazioni. Questi i risultati del modulo regressioni indipendente dal tempo contro virus abbondanza per ognuno dei campioni. Per ogni campione le incubazioni individuo agisce in un unico trattamento, in modo da replicare completando tre-incubazioni il ricercatore in grado di calcolare i tassi così come una stima della variazione (ad esempio, la deviazione standard) (vedi Figura 1). Un avvertimento di questo processo è che la riduzione invariabile virus abbondanza porta ad una riduzione della cellule ospiti nel campione che portano il peso del virus. Per compensare questa perdita, enumerazione di abbondanza batterica sia dalla sorgente (acqua di mare filtrata o di lago) e la T = 0 campione di incubazione sono necessari. Queste informazioni possono essere utilizzate per tenere conto della percentuale di cellule perse: ipotizzando che il processo di riduzione abbondanza virus non è selettiva a favore o contro qualsiasi membro della comunità microbica, questo fattore può quindi essere utilizzata per stimare il tasso di produzione in loco di virus . <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="Figura 1" /> Figura 1. La produzione di campioni particelle virus-simili nel corso di un incubazione di 10 ore in condizioni in situ tramite microscopia in epifluorescenza. Sono stati raccolti nel corso di una fioritura di fitoplancton al largo della costa della Nuova Zelanda, nel settembre del 2008. <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="Figura 2" /> Figura 2. Schema del processo di flusso di lavoro per analizzare la produzione di virus. Il processo inizia con l&#39;ultrafiltrazione di acqua campione di generare virus acqua. Questo è completato con un sistema di ultrafiltrazione. Nei campioni di acqua parallelo sono raccolti dal sito stesso e il virus liberi passare attraverso un filtro, mentre la comunità microbica (contenente una miscela di cellule infette e non infette) viene mantenuto. Questa comunità è poi risospeso in acqua privo di virus e incubate in condizioni in situ. Il tasso di ricomparsa del virus viene poi monitorato per le prossime 10 ore per determinare il tasso di produzione di virus.

<ol>
	<li>Higgins, J. L., Kudo, I., Nishioka, J., Tsuda, A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+the+virus+community+to+a+mesoscale+iron+fertilization+in+the+sub-Arctic+Pacific+Ocean.">The response of the virus community to a mesoscale iron fertilization in the sub-Arctic Pacific Ocean.</a> Deep-Sea Res II. 56, 2788-2795 (2009).</li><li>Mioni, C. E., Poorvin, L., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+and+siderophore-mediated+transfer+of+available+Fe+between+heterotrophic+bacteria:+characterization+using+a+Fe-specific+bioreporter.">Virus and siderophore-mediated transfer of available Fe between heterotrophic bacteria: characterization using a Fe-specific bioreporter.</a> Aquat Microb Ecol. 41, 233-245 (2005).</li><li>Noble, R. T., Fuhrman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Parada, V., Herndl, G., Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+burst+size+of+heterotrophic+prokaryotes+in+aquatic+systems.">Viral burst size of heterotrophic prokaryotes in aquatic systems.</a> J Mar Biol Assoc U K. 86, 613-621 (2006).</li><li>Poorvin, L., Rinta-Kanto, J. M., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+release+of+Fe+and+its+bioavailability+to+marine+plankton.">Viral release of Fe and its bioavailability to marine plankton.</a> Limnol Oceanogr. 49, 1734-1741 .</li><li>Rowe, J. M., Saxton, M. A., Cottrell, M. T., DeBruyn, J. M., Berg, G. M., Kirchman, D. L., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constraints+on+virus+production+in+the+Sargasso+Sea+and+North+Atlantic.">Constraints on virus production in the Sargasso Sea and North Atlantic.</a> Aquat Microb Ecol. 52, 233-244 (2008).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+prokaryotic+viruses.">Ecology of prokaryotic viruses.</a> FEMS Microbiol Rev. 28, 127-181 (2004).</li><li>Weinbauer, M. G., Peduzzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency,+size+and+distribution+of+bacteriophages+in+different+marine+morphotypes.">Frequency, size and distribution of bacteriophages in different marine morphotypes.</a> Mar Ecol Prog Ser. 108, 11-20 (1994).</li><li>Weinbauer, M. G., Winter, C., H&amp;ouml, f. l. e., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsidering+transmission+electron+microscopy+based+estimates+of+viral+infection+of+bacterioplankton+using+conversion+factors+derived+from+natural+communities.">Reconsidering transmission electron microscopy based estimates of viral infection of bacterioplankton using conversion factors derived from natural communities.</a> Aquat Microb Ecol. 27, 103-110 (2002).</li><li>Wen, K., Ortmann, A. C., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+estimation+of+viral+abundance+by+epifluorescence+microscopy.">Accurate estimation of viral abundance by epifluorescence microscopy.</a> Appl Environ Microbiol. 70, 3862-3867 (2004).</li><li>Wilhelm, S. W., Brigden, S. M., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dilution+technique+for+the+direct+measurement+of+viral+production:+a+comparison+in+stratified+and+tidally+mixed+coastal+waters.">A dilution technique for the direct measurement of viral production: a comparison in stratified and tidally mixed coastal waters.</a> Microb Ecol. 43, 168-173 (2002).</li><li>Wilhelm, S. W., Matteson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+and+marine+virioplankton:+a+brief+overview+of+commonalities+and+differences.">Freshwater and marine virioplankton: a brief overview of commonalities and differences.</a> Freshw Biol. 53, 1076-1089 (2008).</li><li>Wilhelm, S. W., Poorvin, L., Paul, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantification of algal viruses in marine samples. , 53-66 (2001).</li><li>Wilhelm, S. W., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+and+nutrient+cycles+in+the+sea.">Viruses and nutrient cycles in the sea.</a> Bioscience. 49, 781-788 (1999).</li><li>Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Jeffrey, W. H. <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+sunlight+in+the+removal+and+repair+of+viruses+in+the+sea.">The role of sunlight in the removal and repair of viruses in the sea.</a> Limnol Oceanogr. 43, 586-592 (1998).</li><li>Winget, D. M., Williamson, K. E., Helton, R. R., Wommack, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tangential+flow+diafiltration:+an+improved+technique+for+estimation+of+virioplankton+production.">Tangential flow diafiltration: an improved technique for estimation of virioplankton production.</a> Aquat Microb Ecol. 41, 221-232 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Una componente fondamentale nella comprensione come i virus dell&#39;influenza comunità microbiche marine è quello di determinare la velocità con cui vengono prodotte particelle di virus. Dato che abbondanze sono (più o meno) statici nella maggior parte dei sistemi (Wilhelm &amp; Suttle 1999, Weinbauer 2004), e che i virus sono stati rimossi o resi non infettivi rapidamente in sistemi acquatici (Wilhelm et al. 1998), allora i tassi di produzione deve essere rispetto rapida per sostituire particelle perso. La stima del virus le cause di mortalità per la comunità microbica richiede una conoscenza di quanti virus vengono prodotti ogni volta che un virus lisa una cella (la &quot;dimensione burst&quot;). Le dimensioni del virus in campioni di scoppio naturale può variare notevolmente. Burst dimensioni possono essere determinate direttamente al microscopio elettronico a trasmissione (ad esempio, Weinbauer e Peduzzi 1994), ma questo è spesso oltre le capacità di un laboratorio dato o non sempre pratico. In situazioni in cui non può essere empiricamente determinato, i valori della letteratura di 24 virus per evento lisi può essere utilizzato per sistemi marini e 34 per i sistemi di acqua dolce (Parada et al. 2006). Se il tasso di produzione di virus è diviso per questo numero, il risultato è l&#39;abbondanza di cellule per volume distrutto da virus su base giornaliera. I microbi lisato valore può essere diviso per il magazzino in piedi di abbondanza batterica conseguente mortalità del virus indotto per il sistema in questione: già esistenti le stime variano da pochi punti percentuali a quasi tutta la popolazione e sono spesso dipende da altri fattori del sistema in questione (Wilhelm &amp; Matteson 2008). Per determinare la percentuale di mortalità totale è spesso questo numero moltiplicato per due (lavorativi dal presupposto che il 50% delle cellule andare a riprodursi e il 50% delle cellule si perdono, Weinbauer 2004). Dato che la biodisponibilità dei nutrienti ed elementi traccia (per esempio, N, P, Fe) può limitare il tasso di produttività primaria, e come tale flusso del carbonio, attraverso sistemi acquatici, e la comprensione del ruolo del virus-driven mortalità microbica in questo processo è diventato di interesse per geochimici marino. Alcune stime indicano che ora esistono virus rilasciare un concentrazioni significative di elementi nutritivi di nuovo alla colonna d&#39;acqua su base giornaliera (Rowe et al. 2008, Higgins et al. 2009) e che questi elementi sono rapidamente assimilate dalla comunità microbica (Poorvin et al. 2004, Mioni et al. 2005). Il tasso di flusso di nutrienti per l&#39;ambiente può essere determinato moltiplicando il numero di cellule distrutte dalla quantità di nutrienti per cella (indicato &quot;quote&quot;). Queste informazioni possono fornire una componente fondamentale per la nostra comprensione di come le catene alimentari microbiche funzione tutti i sistemi acquatici. Sviluppi in corso: gli sforzi attuali per una serie di gruppi di ricerca riguardano l&#39;adeguamento della strategia di cui sopra per enumerare virus specifici all&#39;interno della comunità e, come tale, per determinare come gli organismi specifici sono influenzate dalla attività dei virus. Per fare questo i ricercatori utilizzano il quantitativo di reazione a catena della polimerasi (qPCR) per stimare l&#39;abbondanza di particolari gruppi di virus o famiglie in parallelo alle stime della comunità virus totale. I risultati sono quindi applicati direttamente per fornire stime di mortalità del virus, ricambio nutritivo, ecc per specifici gruppi di plancton. Questo approccio nuovo e potente permetterà ai ricercatori nei prossimi anni a scavare molto più a fondo i processi associati con l&#39;ecologia del virus e, per la prima volta, di quantificare le interazioni di specifici virus ospitano comunità oltre i vincoli dei sistemi di laboratorio.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>2.5% p-phenylenediamine</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>130575000</td>
<td>Stock for Antifade</td>
</tr>
<tr>
<td>Amicon Proflux M12 system</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>N/A</td>
<td>Any ultrafiltration device may be used for this step</td>
</tr>
<tr>
<td>SYBR Green I nucleic acid gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-7563</td>
<td>&nbsp;</td>
<tr>
<td>Helicon S10 30kDa Filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>CDUF010LT</td>
<td>&nbsp;</td>
<tr>
<td>Pelicon XL filters 0.22 &mu;m</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>PXGVPPC50</td>
<td>&nbsp;</td>
<tr>
<td>GE 0.2 &mu;m PCTE membrane filters (47mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-732-35</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Labscale Tangential Flow Filtration System</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>XX42LSS11</td>
<td>Other TFF systems may be used for this step</td>
</tr>
<tr>
<td>0.45 &mu;m Micronstep, Cellulosic, white plain filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E04WP02500</td>
<td>&nbsp;</td>
<tr>
<td>GE Whatman 0.02 &mu;m Anodisc filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>68-09-6002</td>
<td>&nbsp;</td>
<tr>
<td>Leica DMRXA microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Any epifluorescence microscope with a blue filter set may be used</td>
</tr>
<tr>
<td>20L polycarbonate carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP329-500, S640-500</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151-1</td>
<td>&nbsp;</td>
<tr>
<td>Corning 2 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-71</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td>Corning 5 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-74</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td> 85% H3PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A242-212</td>
<td>Spiral Membrane storage</td>
</tr>
<tr>
<td>NaOH pellets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S318-3</td>
<td>M12 cleaning</td>
</tr>
<tr>
<td>Graduated Cylinders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopore 0.8-&mu;m pore-size membrane filter (142mm)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ATTP14250</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>YY30 090 00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

estimating virus production rates in aquatic systems

I virus sono componenti pervasivo dei sistemi marini e d&#39;acqua dolce, e sono noti per essere agenti significativo di mortalità microbica. Sviluppare stime quantitative di questo processo è fondamentale come si può poi sviluppare migliori modelli di struttura delle comunità microbiche e della funzione così come migliorare la nostra comprensione di come i virus lavorano per alterare acquatici cicli biogeochimici. La tecnica di riduzione del virus permette ai ricercatori di stimare la velocità con cui vengono rilasciate particelle virali da parte della comunità microbica endemica. In breve, l&#39;abbondanza di liberi (extracellulare) virus si riduce in un campione, mentre la comunità microbica è mantenuta a vicino concentrazione ambiente. La comunità microbica è poi incubate in assenza di virus libero e la velocità con cui i virus ripresentarsi nel campione (attraverso la lisi dei membri già infetti della comunità) può essere quantificato mediante microscopia in epifluorescenza o, nel caso di virus specifici, quantitativa PCR. Questi tassi possono poi essere utilizzati per stimare il tasso di mortalità microbica a causa di virus-mediata lisi cellulare.

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

Il tasso di turnover dei virus in sistemi marini e d&#39;acqua dolce può essere stimato da una riduzione e tecnica ripetersi. I dati permettono ai ricercatori di dedurre i tassi di virus-mediata mortalità microbica nei sistemi acquatici.

Stimare i tassi di produzione del virus nei sistemi acquatici

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

estimating-virus-production-rates-in-aquatic-systems

Research

JoVE Journal

Immunology and Infection

12.6K Views.  University of Tennessee. Il tasso di turnover dei virus in sistemi marini e d'acqua dolce può essere stimato da una riduzione e tecnica ripetersi. I dati permettono ai ricercatori di dedurre i tassi di virus-mediata mortalità microbica nei sistemi acquatici.

Video: Estimating Virus Production Rates in Aquatic Systems

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Ricerca

Immunologia e infezioni

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Efficienti di produzione Parvovirus ricombinante con l&#39;aiuto delle Adenovirus sistemi derivati

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems (IVIS) il rilevamento di un neuro-invasiva Virus encephalitic

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Valutazione della efficacia e la tossicità degli RNA targeting HIV-1 Produzione per l&#39;uso in gene terapia farmacologica

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

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 ...

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 ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

Misurazione dei tassi di Mistranslazione micobatterica specifica con sistemi reporter di guadagno di funzione

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Test ELISA in vitro per valutare la potenza del vaccino contro la rabbia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Analisi delle interazioni tra endobiotici e microbiota intestinale umana utilizzando sistemi di fermentazione bagno in vitro

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...