Ringraziamo SIGA Technologies (Corvallis, OR) per la fornitura di ST-246. DKR è stato sostenuto da una borsa di formazione NIH in immunologia alla Boston University (5T32AI 7309). Questo lavoro è stato sostenuto in parte da P41 086.180, NIH RO1AI1096159-01, e RO3 (a JHC).

1. Cellule piastra <ol><li> Dissociare cellule HeLa dalla piastra e diluire in terreni di crescita (DMEM, 2 mM L-glut, 10% FBS) a circa 2,0 x 10 5 cellule / ml. Dispensare 100 microlitri / pozzetto in una, chiara piastra a 96 pozzetti a fondo piatto nero parete (20.000 cellule / pozzetto). </li><li> Incubare le cellule per 24 ore fino confluenti in un incubatore a 37 ° C + 5% di CO 2. </li></ol> 2. Infettare le cellule <ol><li> Diluire virus e infettare le cellule. <ol><li> Virus fluorescenti e PLV (Promoter-less Venere) e disaggregare con sonicazione per 5 minuti; Thaw TrpV (Early Venere, Intermedio mCherry, Dopo TagBFP virus Triple). In alternativa, le riserve di virus grezzo può essere miscelato 1:1 con 0,25 mg / ml di tripsina in mezzi di infezione e incubate a 37 ° C per 30 min. </li><li> Diluire le riserve di virus greggio in 37 ° C mezzi di infezione (DMEM, 2 mM L-glut, 2% FBS). Per le infezioni alto MOI (10 PFU / cellulari), diluire virus stock da 1,0 x 10 7 PFU / ml ipotizzando 5 x 10 4cellule / pozzetto e un volume di inoculo di 50 microlitri. Piano di infettare 3 pozzi repliche per ogni trattamento con TrpV e altri 3 pozzi ripetute per lo stesso trattamento con PLV per tenere conto di fluorescenza di fondo specifico per ogni trattamento. </li><li> Infettare pozzi aggiungendo 50 microlitri virus / ben diluito in mezzi di infezione. Questo è definito come infezione time = 0 h palo (0 HPI). </li></ol></li><li> Diluire composti trattamenti desiderati in supporti di infezione. Per tutti i trattamenti una singola diluizione master mix 2x dovrebbe essere fatto e applicato per replicare pozzetti (es. 350 microlitri volume totale per sei 96-pozzetti con 50 ml ciascuna). <ol><li> Diluire composti sperimentali in supporti di infezione. </li><li> Diluire veicolo-solvente di controllo (ad esempio PBS, DMSO) in supporti infezione. Nota: concentrazioni finali giudizio di solventi veicolo di controllo dovrebbero essere usati come richiesto per i composti sperimentali (ad esempio, se il tuo magazzino IBT è fatto con DMSO e utilizzato in un concentratio finalens di 1 ml / ml, quindi utilizzare DMSO solo a 1 microlitri / ml come il controllo del veicolo). </li><li> Diluire composti di controllo in supporti di infezione. Composti di controllo consigliati sono: 3.6 micron (1 mg / ml) 1-β-D-Arabinofuranosylcytosine (AraC), 50 pm (11,7 mcg / ml) Isatin β-thiosemicarbazone (IBT), 60 mM (50 mg / ml) rifampicina, o 5 micron (1,9 mg / ml) ST-246 8,15,16. Vedi i Risultati Rappresentante per gli effetti attesi. Nota: rendono questa diluizione al doppio (2x) la concentrazione finale desiderata poiché ciò viene aggiunto in un rapporto 1:1 a volume già nel pozzo. </li></ol></li><li> Subito dopo l&#39;aggiunta di virus, aggiungere 50 ml contenenti supporti di infezione trattamenti e controlli desiderati come diluito in fase 2.2. Nota: Per saggiare l&#39;inibizione dell&#39;espressione prima fase iniziale, il composto (s) può essere aggiunto direttamente al inoculo virus diluito (punto 2.1.2) o cellule ospiti prima dell&#39;infezione (prima del punto 2.1.3). </li><li> Incubare 6-24 ore in un # 37 &amp;176, C incubatore + 5% di CO 2. </li></ol> 3. Fissare Celle <ol><li> Fix cellule aggiungendo 100 microlitri 8% paraformaldeide al supporto infezione già in ciascun pozzetto. Incubare a temperatura ambiente per 15 minuti al riparo dalla luce. Nota: Si raccomanda di aggiungere 2x PFA (8%) direttamente ai media di infezione per evitare aerosol di vaccino infettiva che può verificarsi se supporti ad alta titolo è invertito direttamente nel piatto rifiuti in attesa di fissazione. </li><li> Rimuovere fissativo invertendo piatto nel piatto rifiuti. </li><li> Aggiungere 100 microlitri di PBS a temperatura ambiente. </li><li> Sigillare piastra con pellicola adesiva otticamente trasparente. Nota: Le piastre possono essere conservati a 4 ° C, se non di leggere immediatamente. Essere sicuri di tornare piatti sigillati a temperatura ambiente prima di leggere per evitare la condensa di distorsione misurazioni spettrofotometriche. </li></ol> 4. Quantificare crescita Virus <ol><li> Misurare endpoint fluorescenza a 515:530 per Venus, 587:610 per mCherry, e 415:457 fo TagBFP (eccitazione: d&#39;onda di emissione in nm). Quattro le misure devono essere mediate per bene usando impostazioni di guadagno ottimizzate per ogni canale. Nota: La lunghezza d&#39;onda di emissione del caso può essere diversa a seconda delle caratteristiche specifiche del modello e del filtro del vostro lettore per piastra. Questo può essere determinato empiricamente effettuando una scansione della lunghezza d&#39;onda di emissione per determinare il punto in cui vi è la più grande differenza tra TrpV e PLV per ciascun canale. </li><li> Normalizzare i dati grezzi per facilitare il confronto tra esperimenti. <ol><li> Per ogni canale, determinare la media di replicare TrpV e pozzi PLV. </li><li> Sottrarre i media misurazioni del fondo PLV-infettati dalle misure sperimentali medi TrpV-infettati per ogni trattamento. </li><li> Normalizzare i dati dividendo ogni valore di fondo sottratto dal valore unico veicolo-bene. </li><li> Una volta che un esperimento è stato ripetuto più volte, l&#39;analisi a senso unico della varianza (ANOVA) prova e più cONFRONTO post-test può essere eseguito per determinare se uno dei trattamenti riflette una differenza statisticamente significativa dal trattamento solo veicolo per ciascun canale. </li></ol></li></ol> . 5 Protocollo Alternativa: saggi piastra Kinetic <ol><li> Eseguire tutti i passaggi attraverso l&#39;aggiunta di trattamenti (punto 2.3). </li><li> Piastra di tenuta con la pellicola adesiva e posto in camera di lettore di piastre equilibrato a 37 ° C. Nota: Particolare cura deve essere presa per mantenere piastre costantemente a 37 ° C per evitare lo stress delle cellule eccessivo e condensazione. Per i saggi cinetici, è particolarmente importante utilizzare un mezzo tamponato (bicarbonato di sodio e HEPES) per mantenere il corretto pH senza aggiunta di 5% di CO 2. </li><li> Set lettore di piastre ottenere manualmente per evitare la saturazione di punti temporali successivi. Nota: accurata ottimizzazione del guadagno può essere ottenuto eseguendo un endpoint test in condizioni simili. </li><li> Acquisire letture orarie per 8-24 HPI e normalizzare utilizzando simiprocedura lar come nel saggio finale (punto 4.2). </li><li> Punti temporali individuali possono essere analizzati per la significatività statistica utilizzando metodi simili, come il saggio endpoint descritto in precedenza. </li></ol>

<ol>
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Gli autori non hanno nulla da rivelare e senza brevetti sono in corso per individuare eventuali virus o metodi di screening.

Qui, abbiamo descritto l&#39;uso pratico di un multi-stadio di virus del vaiolo vaccino reporter (TrpV), che fornisce riproducibili, misure di feedback in tempo reale di replicazione del virus. Utilizzando inibitori poxvirus ben definite, siamo stati in grado di dimostrare che TrpV risponde in modo coerente con il meccanismo capito di azione per ciascun inibitore. Mentre il virus TrpV fornisce le informazioni più complete sulla progressione fase virus, due stadi (IREV, LREV) e monostadio (EV, IV, LV) virus possono esse...

Tradizionalmente, l&#39;espressione virus viene studiata utilizzando tecniche di biologia molecolare (ad esempio blotting settentrionale, occidentale blotting, ibridazione microarray, ecc) 1. Mentre questi metodi sono in grado di fornire informazioni dettagliate riguardo al categorizzare cambiamenti di espressione di singoli mRNA o proteine, non sono in genere riconducibili a processi real-time e high-throughput. Approcci alternativi che utilizzano i giornalisti a base di fluorescenza sono state applicate in precedenza quando si lavora con poxviruses; tuttavia, il loro sviluppo e il loro utilizzo è stato motivato da finalità diverse. Diversi di questi metodi sono stati progettati per la selezione di virus ricombinanti 2,3. Queste tecniche esprimono EGFP sulla corretta costituzione e l&#39;espressione del DNA esogeno da cloni vaiolo vaccino ricombinanti. Analogamente, diversi ceppi vaccinia esprimono stabilmente EGFP solubile o proteine ​​GFP-tag espresse sotto un promotore nativo vaccinia sono stati ampiamente utilizzati. Questi sono tipcamente utilizzato per quantificare ingresso virus e replica durante test di neutralizzazione a base di anticorpi, inibizione chimica, o il confronto di efficacia antivirale 4-6. Mentre questi virus si sono dimostrati utili, essi sono limitati nella loro capacità di fornire informazioni dettagliate sul punto di inibizione a causa del loro utilizzo di un unico promotore ambiguo anticipo / ritardo virale. Metodi precedenti hanno anche fatto uso di proteina EGFP con caratteristiche ottimali segnale-rumore. A causa della complessità della replicazione poxvirus e la mancanza di strumenti disponibili per saggiare tempo reale i cambiamenti nell&#39;espressione genica virale, abbiamo sviluppato una serie di virus singolo e multi giornalista fase 7,8. Come descritto in precedenti pubblicazioni, questi virus esprimono uno, due, o tre proteine ​​fluorescenti spettralmente distinti da promotori vaiolo vaccino indigene durante le prime fasi, tappe intermedie, o tardive dell&#39;infezione. Questi virus possono essere noied indicatori di progresso replicazione del virus utilizzando un microscopio a fluorescenza e sono ugualmente adatti per high-throughput piastra-e-reader basato saggi. Questi virus sono facili da usare al posto di tipo selvatico del virus, in crescita con cinetiche simili per raggiungere i titoli equivalenti 7. Durante la progettazione e la realizzazione di questi virus, molta cura è stata posta nella scelta fluorofori che presentano elevate caratteristiche segnale-fondo con efficienza superiore pieghevole per facilitare la quantificazione affidabile con un feedback rapido ai cambiamenti di espressione (Venere, mCherry e TagBFP). Inoltre, le combinazioni di promotori virali sono stati scelti che producono alta fedeltà, espressione inequivocabile stadio-specifica, che fornisce informazioni sulla gamma completa di precoce (promotore C11R), intermedio (promotore G8R) e (promotore F17R) espressione genica tardi, in contrasto ambiguo promotori anticipo / ritardo. Questi virus possono essere usati come strumento per investigating il ciclo di vita del poxvirus prototipo, virus del vaiolo vaccino. Molto non è ancora noto circa l&#39;interazione ospite-virus nonostante la sua lunga storia di studio. Vaccinia è complesso, producendo oltre 200 proteine ​​uniche, molte delle quali sono immunomodulante ospitano antagonista. Dopo l&#39;infezione, il virus del vaiolo vaccino comincia subito all&#39;inizio del mRNA di trascrizione. Ciò è facilitato da una RNA polimerasi e fattori di trascrizione che sono stati caricati sul genoma virale durante il confezionamento virione e tenuti in uno stato di pausa fino a successiva infezione. Questo primo espressione produce principalmente proteine ​​necessarie per la soppressione del sistema immunitario dell&#39;ospite (mRNA decapping, dsRNA sequestro, e proteine ​​recettori decoy così come inibitori di apoptosi, risposta allo stress, e Toll, IL, e segnalazione NF-kB) e la replicazione del genoma. Espressione precoce produce anche fattori di trascrizione necessari per l&#39;espressione intermedio. Espressione intermedio comprende l&#39;espressione di fattori di trascrizione fase tardiva. Questoespressione cascata porta alla produzione di proteine ​​virali strutturali ed enzimatiche durante ultimi stadi di infezione, che sono necessarie per completare l&#39;assemblaggio del virione maturo vaccinia. La nostra serie di virus reporter fluorescente consente la rapida compiere progressi nella comprensione della biologia poxvirus. Uno dei metodi più comuni e che richiede tempo nel campo della virologia è il saggio di crescita. Ciò comporta tipicamente infettare le cellule, che introduce una serie di trattamenti, virus raccolta mediante lisi cellule infettate, e quantificare il titolo virale risultante da saggio di placca. Usando i virus giornalista qui descritti consente la misurazione in tempo reale della crescita virale che può essere facilmente dosato e confrontata tra numerosi trattamenti eseguiti in parallelo. Prevediamo questa serie di reagenti sarà utilizzato in vari protocolli per identificare alterazioni dell&#39;espressione genica virale in risposta al trattamento farmacologico, RNAi knockdown, o la restrizione campo ospite. Questo metodo consente anche l&#39;analisi ad alto contenuto su scala più ampia rispetto al passato, consentendo di high-throughput schermi di farmaci anti-virali per le fasi di destinazione specificamente definite di interesse. Mentre sono stati identificati numerosi potenziali trattamenti per combattere l&#39;infezione da poxvirus, l&#39;unico approvato dalla FDA terapie efficaci per il trattamento delle infezioni da poxvirus sono il nucleoside aciclico fosfonato, cidofovir, ed il trattamento con vaccino immunitario globulina 9,10. Nonostante l&#39;eradicazione del vaiolo nel 1977 11, poxviruses rimangono una grave minaccia per la salute umana 12. Cessazione della vaccinazione diffusa contro il virus del vaiolo ha portato ad un aumento della suscettibilità alle altre poxviruses 13. Ad esempio, regioni dell&#39;Africa centrale precedentemente protetti dalla vaccinazione stanno vivendo un aumento di infezioni da virus vaiolo delle scimmie 14. Preoccupazione significativa è stata anchesollevato in merito alla predisposizione latente di rilascio intenzionale di virus Variola. A causa delle limitate terapie attualmente disponibili, vi è una necessità urgente per lo sviluppo di nuovi trattamenti. Questi virus giornalista consentono una rapida e high-throughput di screening piccolo inibitore della molecola per l&#39;inibizione di una determinata fase di replicazione virale. Identificazione di inibitori che mirano fasi di espressione virali attualmente l&#39;obiettivo di inibizione faciliterà lo sviluppo di terapie combinate con una maggiore potenza. Molto può essere spigolato su vaccino biologia cellulare osservando i cambiamenti nell&#39;espressione genica di ogni fase. Attenuazione da chimica o manipolazione genetica di una cellula ospite infettata è solitamente espressa in termini di titoli virali ridotte. Tuttavia, comparando le variazioni di ogni fase della cascata di espressione virale, si può ottenere una comprensione più completa di come idoneità virus è impattoEd da un particolare trattamento. Questi dati hanno dimostrato di correlare bene con la tradizionale uscita titolo virale, ma fornire informazioni più dettagliate meccanicistica nonché capacità high throughput 7.

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	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

Poxviruses sono una famiglia di virus a DNA doppio stranded che comprendono gli agenti patogeni attivi umani, come il vaiolo delle scimmie, mollusco contagiousum e virus Contagalo. La famiglia comprende anche il virus del vaiolo, Variola. A causa della complessità della replicazione poxvirus, molte domande rimangono ancora quanto alla strategia espressione genica. In questo articolo descriviamo la concettualizzazione e l&#39;utilizzo di virus vaccinia ricombinanti che consentono la misurazione in tempo reale di fasi singole e multiple di espressione genica virale in un formato ad alta velocità. Ciò è reso possibile attraverso l&#39;uso di proteine ​​fluorescenti spettralmente distinti come reporter per ciascuna delle tre fasi di replicazione virale. Questi virus forniscono un elevato rapporto segnale-rumore, pur mantenendo i modelli fase specifica espressione, consentendo analisi basate piastra-e osservazioni microscopiche di propagazione dei virus e replicazione. Questi strumenti hanno usi per la scoperta antivirale, studi di interazione virus-ospite, e bio evolutivalogia.

Come un esempio di utilizzo tipico di questi virus, il virus tripla fluorescenza-reporter è stato utilizzato per confrontare il punto di inibizione di diversi inibitori poxvirus ben definiti. Cellule HeLa sono state piastrate in coltura tissutale trasparente trattato piastre a 96 pozzetti a fondo piatto nero a pareti e incubate tutta la notte. Monostrati confluenti sono state infettate ad una molteplicità di infezione (MOI) di 10 utilizzando virus giornalista tripla (TrpV; Tabella ...

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Descriviamo l&#39;utilizzo di un virus vaccinia reporter fluorescente che consente la misurazione in tempo reale di infettività virale e l&#39;espressione genica attraverso l&#39;espressione specifica fase di fluorofori giornalista spettralmente distinti. Abbiamo dettaglio un metodo basato piastra per identificare con precisione la fase in cui la replicazione del virus è influenzato in risposta ad inibizione piccola molecola.

Vaccinia Reporter virus per quantificare Funzione virale in tutte le fasi di espressione genica

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Research

JoVE Journal

Immunology and Infection

11.2K Views.  Boston University School of Medicine.  Descriviamo l'utilizzo di un virus vaccinia reporter fluorescente che consente la misurazione in tempo reale di infettività virale e l'espressione genica attraverso l'espressione specifica fase di fluorofori giornalista spettralmente distinti. Abbiamo dettaglio un metodo basato piastra per identificare con precisione la fase in cui la replicazione del virus è influenzato in risposta ad inibizione piccola molecola. 

Video: Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generazione di Multivirus-cellule T specifiche per prevenire / curare infezioni virali dopo trapianto allogenico di cellule staminali ematopoietiche

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Ricerca

Immunologia e infezioni

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Phage &#39;Bioluminescent&#39; Reporter per il rilevamento di batteri patogeni di categoria A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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

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

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

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

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

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Costo-efficace metodo per il rilevamento di origine microbica Utilizzando specifici virus umani e animali

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolamento di varianti di virus RNA Fidelity e caratterizzazione di frequenza Virus Mutation

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Mouse fetale Fegato Cultura di sistema per sezionare Funzioni gene bersaglio nelle fasi precoci e tardivi del Terminal eritropoiesi

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Sviluppo di un virus System Reporter dell&#39;epatite B per monitorare la fasi iniziali del ciclo di replicazione

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...