Istituto di Scienze Translational UTMB-sovvenzione del NIH 1UL1RR029876-01 e Alisha Prather per il suo aiuto con l&#39;editing video di questo manoscritto.

1. Preparazione degli animali <ol><li> Animal arrivo: All&#39;arrivo al livello animale biosicurezza 2 (ABSL2) servizi, consentire che gli animali un minimo di 2 giorni per acclimatarsi al nuovo ambiente. Dopo questo periodo di riposo, al controllo degli animali per valutare la loro salute e in generale l&#39;aspetto fisico. <ol><li> Quando si utilizza reporter fluorescenti è importante mettere tutti i soggetti animali su una dieta di erba medica libero di limitare la quantità di autofluorescenza GI e segnale di fondo. </li></ol></li><li> Shave animali: Per migliorare il segnale bioluminescente rilevato dal cranio durante l&#39;esposizione; radere le teste di tutti gli animali. Anestetizzare gli animali con una miscela di ossigeno e gas vaporizzato isoflurano. Una volta che gli animali sono completamente anestetizzati, utilizzare tagliatori elettrici a radersi la testa. Una volta finito con rasatura, riportare gli animali nelle loro gabbie e osservare per un completo recupero dagli effetti dell&#39;anestesia. </li><li> Bio Medic datiSystems (BMDS) inserimento transponder (che si terrà dopo la rasatura dei topi, mentre gli animali sono completamente anestetizzati): Per un mezzo meno invasivo per monitorare la temperatura di un impianto pre-programmato BMDS transponder wireless per via sottocutanea nella regione dorsale di ogni topo. Questi transponder permettere di identificare wireless e registrazione della temperatura. Dopo l&#39;impianto del transponder, applicare un veterinario qualità del tessuto adesivo alla ferita. </li><li> Collezione di base: prima infezione, registrare una temperatura basale e il peso per tutti gli animali. Prelevare il sangue per la riduzione della placca prova di neutralizzazione (PRNT) analisi attraverso retro-orbitali (RO) spurgo, mentre i topi sono sotto anestesia. Dopo il prelievo del sangue, applicare pomata antibiotica veterinaria occhio per ridurre il potenziale infezione batterica secondaria. Questa raccolta di base deve essere completato con considerazione il numero di volte che gli animali sono sottoposti in anestesia da stress sui topi. Future collezioni RO should essere completato dopo l&#39;imaging, mentre il mouse è ancora sotto anestesia. Il sangue raccolto da un massimo RO dovrebbe essere di circa 200 pl. </li><li> Infezione: topi posto sotto anestesia come descritto in precedenza. Inoculare per via intranasale utilizzando una dose di 5x10 6 a 5x10 7 pfu TC83-luciferasi in un volume totale di 40 microlitri diluito soluzione tampone fosfato (PBS) attraverso infezione intranasale. A seguito di infezione, posizionare i topi all&#39;interno della loro gabbia di alloggiamento e osservare il recupero. </li></ol> 2. Animal Imaging Disclaimer: Mantenere topi all&#39;interno della loro unità abitativa, l&#39;armadio biosicurezza (BSC), o la scatola di contenimento XIC in ogni momento. Le BSC approvati per questo protocollo sono una classe di tipo II B1 o B2. <ol><li> Iniettare tutti gli animali con 10 ml per grammo di peso corporeo di intraperitoneale (IP) luciferina ad una concentrazione di 15 mg / ml. <ol><li> I topi trattati con Ampligen devono essere injecteIP d alla dose di 12,5 mg / kg di peso corporeo al messaggio infezione -4 ore o 48 ore dopo l&#39;infezione in base al loro gruppo. </li></ol></li><li> Prima dell&#39;iniezione con luciferina, separare i topi in gruppi di tre per garantire un posizionamento adeguato all&#39;interno del XIC-3 scatola di contenimento. <ol><li> Per tutte imaging, utilizzano unguento oculare / lubrificante per impedire l&#39;essiccazione della cornea durante tutta la procedura. </li></ol></li><li> Aprire il software LivingImage e premere Inizializza per preparare la casella di imaging, auto risparmio di serie, tempo di esposizione di auto (tempo di esposizione su auto è una nuova funzione del software LivingImage 3.2 e successivi), vedi campo C, e filtro per aprire; campo di vista si basa sul numero di topi sei imaging; e il filtro può essere ottimizzato come necessario per progetti imaging. </li><li> Verificare che i filtri dell&#39;aria a carbone non è scaduto, il serbatoio è pieno di ossigeno, e il serbatoio viene riempito isoflurano. Mettere piccole strisce di nastro isolante nel XIC-3 icasella di consolazione che aiuta a ridurre il movimento degli animali durante l&#39;esposizione. </li><li> Iniettare i topi con IP luciferina come descritto nella sezione 2.1 e posto all&#39;interno della camera di induzione isoflurano (con coperchio aperto) per 3-5 min. </li><li> Dopo 5 min, chiudere il coperchio della camera di induzione e avviare il flusso di isoflurano. L&#39;anestetico viene utilizzata a circa 3-5% del valore di frequenza di flusso di ossigeno di circa 2,0 L / min. Una volta completamente inconscio attendere un ulteriore 30 sec e trasferire i topi al XIC-3 box di isolamento. Assicurarsi che l&#39;armadio biosicurezza stati utilizzati consente per la sicurezza evacuazione di isoflurano poiché tale procedura comporterà la perdita di isoflurano dalla camera di induzione (l&#39;unità IVIS contiene i propri contenitori passivi Fiera e la XIC-3 scatola di contenimento si collega direttamente alla in / out prese garantire contenute flusso anestetico). <ol><li> Trasferire i topi basate su chip / numero del gruppo nel XIC-3 box di isolamento con il connettore isoflurano allegato und aprire. Posizionare i topi in modo che sono previste in decubito sternale con la testa delicatamente appoggiate sulle strisce di nastro. </li></ol></li><li> Pulire la scatola XIC contenimento con etanolo, le mani a spruzzo e il fondo del XIC-3 con CaviCide, e trasferire alla camera IVIS imaging. Ricollegare l&#39;anestesia alla scatola di contenimento, una volta dentro l&#39;unità di riproduzione. </li><li> Dopo 10 minuti dopo l&#39;iniezione di luciferina, l&#39;immagine dei topi attraverso il software di immagine vivente. </li><li> A seguito di imaging iniziale con un filtro aperto, avviare una DLIT 3-Dimensional immagini utilizzando l&#39;analisi di sequenza guidata di installazione Immagine per bioluminescente luciferasi lucciola. Questo processo di imaging avrà min 4-5 e deve essere eseguita da un campo di vista rilevante per il numero di topi desiderati. <ol><li> Ex-vivo analisi è possibile necroscopia seguente e raccolta del cervello. Mettere il cervello su una piastra di Petri sterile, gocciolamento 50-100 ml di magazzino luciferina sulla parte superiore, e il luogo all&#39;interno del imaging box. </li></ol></li><li> Finalizzare una seconda immagine aperta filtro a 15-17 minuti dopo l&#39;infezione al termine di imaging DLIT. Assicurarsi che l&#39;analisi di sequenza è spento e il campo di vista e impostazioni del filtro Vengono ripristinati i valori precedenti. </li></ol> 3. I topi di ritorno e analisi dei dati <ol><li> Spegnere il vaporizzatore isoflurano e trasferire il recipiente di contenimento XIC torna al mobile biosicurezza. </li><li> Mettere i topi indietro all&#39;interno della loro unità abitativa, chiudere il coperchio superiore, spruzzare l&#39;esterno con un disinfettante appropriato e rimetterlo sul rack custodia. <ol><li> Assicurarsi che i topi hanno pienamente recuperato dall&#39;anestesia. </li></ol></li><li> Ripetere la procedura di cui sopra come l&#39;esperimento è necessario fino a quando l&#39;imaging è stata completata. </li><li> Disinfettare il BSC, spegnere l&#39;apparecchio e trasferire tutti i dati in una posizione laboratorio esterno per ulteriori analisi. </li><li> Analizzare i dati e generare 3 immagini tridimensionali basate su IVIS risultati utinalizzare il software LivingImage. <ol><li> Assicurarsi che l&#39;immagine sia orientato correttamente e illuminazione / bilanciamento del colore è impostato. All&#39;interno della tavolozza degli strumenti le opzioni includono Regolazione immagine, correzioni, informazioni sull&#39;immagine, e Strumenti ROI. </li><li> Utilizzare forme ROI per identificare i punti di forza del segnale specifici basati su posizione. </li><li> Ricostruire la topografia della superficie per evidenziare il corpo del mouse, inizializzare ricostruzione 3D DLIT, e utilizzare in forma lineare per impostare la mappa organo alla ricostruzione topografica. </li></ol></li><li> Ulteriori analisi titolazione virale può essere completato attraverso la raccolta di sangue e di organi. Titolazione in questo studio è stato completato attraverso l&#39;omogeneizzazione del tessuto cerebrale nella modificato aquile medio (MEM). L&#39;omogenato è stato diluito serialmente e abbiamo infettato una piastra ben 6 di cellule Vero per 1 ora. Le cellule sono state coperte con una sovrapposizione agarose/2xMEM 1,5% ed incubate per 2 giorni a 37 gradi. Cellule sono state fissate con formalina al 10% per 30 minuti e trattata concristallo viola. </li></ol>

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	<li>Steele, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Neurovirulence+and+Tissue+Tropism+of+Wild-type+and+Attenuated+Strains+of+Venezuelan+Equine+Encephalitis+Virus+Administered+by+Aerosol+in+C3H/HeN+and+BALB/c+Mice.">Comparative Neurovirulence and Tissue Tropism of Wild-type and Attenuated Strains of Venezuelan Equine Encephalitis Virus Administered by Aerosol in C3H/HeN and BALB/c Mice.</a> Veterinary Pathology Online. 35, 386-397 (1998).</li><li>Ludwig, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+neurovirulence+of+attenuated+and+non-attenuated+strains+of+Venezuelan+equine+encephalitis+virus+in+mice.">Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice.</a> Am. J. Trop. Med. Hyg. 64, 49-55 (2001).</li><li>Charles, P. C., Walters, E., Margolis, F., Johnston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Neuroinvasion+of+Venezuelan+Equine+Encephalitis+Virus+in+the+Mouse.">Mechanism of Neuroinvasion of Venezuelan Equine Encephalitis Virus in the Mouse.</a> Virology. , 208-662 (1995).</li><li>Volkova, E., Gorchakov, R., Frolov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficient+packaging+of+Venezuelan+equine+encephalitis+virus-specific+RNAs+into+viral+particles+is+determined+by+nsP1-3+synthesis.">The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1-3 synthesis.</a> Virology. 344, 315-327 (2006).</li><li>Patterson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+non-invasive+imaging+of+alphaviral+brain+infection:+Reducing+animal+numbers+and+morbidity+to+identify+efficacy+of+potential+vaccines+and+antivirals.">Rapid, non-invasive imaging of alphaviral brain infection: Reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.</a> Vaccine. 29, 9345-9351 (2011).</li><li>Cook, S. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Monkeypox+Viruses+Pathogenesis+in+Mice+by+In+Vivo+Imaging.">Comparison of Monkeypox Viruses Pathogenesis in Mice by In Vivo Imaging.</a> PLoS ONE. 4, e6592 (2009).</li><li>Luker, G. D., Prior, J. L., Song, J., Pica, C. M., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging+Reveals+Systemic+Dissemination+of+Herpes+Simplex+Virus+Type+1+in+the+Absence+of+Interferon+Receptors.">Bioluminescence Imaging Reveals Systemic Dissemination of Herpes Simplex Virus Type 1 in the Absence of Interferon Receptors.</a> J. Virol. 77, 11082-11093 (2003).</li><li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li><li>Kuehne, R. W., Pannier, W. L., Stephen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+mouse+model+for+the+evaluation+of+potential+antiviral+compounds:+results+with+Venezuelan+equine+encephalomyelitis+virus.">Indirect mouse model for the evaluation of potential antiviral compounds: results with Venezuelan equine encephalomyelitis virus.</a> Antimicrob. Agents Chemother. 11, (1977).</li><li>Lukaszewski, R. A., Brooks, T. J. 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Nessun conflitto di interessi dichiarati.

Anche se questo protocollo riguarda gli aspetti di imaging per l&#39;analisi in vivo, è importante riconoscere il vettore bioluminescente come un fattore chiave per gli studi futuri. L&#39;utilizzazione di TC83, un ceppo di vaccino attenuato VeeV, come vettore per l&#39;espressione della luciferasi assicura che grandi quantità dell&#39;enzima si producono a causa della elevata velocità di replicazione del virus nel SNC come precedentemente descritto 1-4. Mentre l&#39;aggiunta di un secondo promoto...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> D-Luciferin </td><td></td><td></td><td></td></tr><tr><td> Isoflurano </td><td></td><td></td><td></td></tr><tr><td> Xenogen IVIS System (Spectrum) </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> XGI-8-gas anestesia sistema </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> XIC-3 Box di contenimento </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> LivingImage 4.0 Software </td><td> Caliper Life Sciences </td><td></td><td></td></tr><tr><td> Telemetria / identificazione chip </td><td> Bio Medic Data Systems </td><td> IPTT-300 </td><td> Animal ID e Temperature </td></tr><tr><td> BD siringa Integra TB 1 ml con 26 gx &quot;ago 3/8 </td><td> Fisher Scientific </td><td> 305279 </td><td></td></tr><tr><td> Vet legame adesivo del tessuto </td><td> Fisher Scientific </td><td> NC9259532 </td><td></td></tr><tr><td> Vetropolycin oftalmica Pomata </td><td> Webster veterinari </td><td> 78444656 </td><td></td></tr><tr><td> Fosfato Dulbecco Buffered Saline 1X </td><td> Invitrogen </td><td> 14190-144 </td><td></td></tr><tr><td> BMDS Chip Reader </td><td> Bio Medic Data Systems </td><td> DAS-7007S </td><td></td></tr><tr><td> DAS-HOST Software </td><td> Bio Medic Data Systems </td><td></td><td> Consente di scaricare le informazioni della sonda </td></tr></tbody></table>

in vivo imaging systems (ivis) detection of a neuro-invasive encephalitic virus

Progressi moderni in tecnologia di imaging incoraggiare l&#39;ulteriore sviluppo e perfezionamento nel modo in cui si compie la ricerca virale. Inizialmente proposto da Russel e Burch nel 3R Hume (sostituzione, riduzione, perfezionamento), l&#39;utilizzo di modelli animali nella ricerca scientifica è costantemente sotto pressione per individuare nuove metodologie per ridurre l&#39;uso degli animali, migliorando la precisione scientifica e velocità. Una sfida importante per principi di Hume tuttavia, è come garantire gli studi sono statisticamente accurate, riducendo la morbilità della malattia degli animali e numeri complessivi. Studi di efficacia vaccino attualmente richiedono un gran numero di animali per essere considerato statisticamente significativo e spesso comportano elevata morbilità e mortalità endpoint per l&#39;identificazione di protezione immunitaria. Abbiamo utilizzato in vivo imaging sistemi (IVIS) in combinazione con un enzima bioluminescente lucciola per monitorare progressivamente l&#39;invasione del sistema nervoso centrale (CNS) da un mentophalitic virus in un modello murino. Tipicamente, la malattia progredisce in modo relativamente lento, tuttavia replicazione del virus è rapido, soprattutto nel SNC, e può portare ad un spesso, esito letale. A seguito di infezione intranasale dei topi con TC83-Luc, un ceppo attenuato equina venezuelana, virus dell&#39;encefalite modificate per esprimere un gene di luciferasi, siamo in grado di visualizzare la replicazione del virus all&#39;interno del cervello, almeno tre giorni prima della comparsa dei sintomi clinici di malattia. Utilizzando invasione del SNC come endpoint chiave encephalitic sviluppo della malattia, siamo in grado di identificare rapidamente terapeutico e protezione del vaccino contro TC83-Luc infezione prima che i sintomi clinici si sviluppano. Con la tecnologia IVIS siamo in grado di dimostrare il test rapido e preciso di terapie farmacologiche e di vaccini, riducendo il numero degli animali e la morbilità.

Con un virus geneticamente modificato, TC83-luciferasi, abbiamo visto un aumento di potenza del segnale bioluminescente la replicazione del virus si sposta dalla regione nasale nel SNC centrali (Figura 1). A causa dell&#39;elevato tasso di replicazione virale, ci aspettiamo di vedere un alto livello di segnale bioluminescente (Figura 2A) dipendenti dal vettore e la risposta immunitaria degli animali al vettore. Ci aspettiamo che questo aumento del segnale di continuare ad un picco, tra ...

Watch this Scientific Journal Video about In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus at JoVE.com

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

Utilizzando luciferasi e sistemi di imaging in vivo (IVIS) come un nuovo mezzo per identificare terminazioni malattia prima sviluppi clinici verificarsi. IVIS ci ha permesso di visualizzare in tempo reale l&#39;invasione di virus encefalitiche più giorni, fornendo un modello di malattia più accurato per lo studio futuro. E ci ha anche permesso di identificare le caratteristiche potenziali di protezione di antivirali e vaccini più veloce rispetto ai modelli animali attualmente utilizzati. La capacità di utilizzare singoli animali più intervalli di tempo multipli assicura condizioni di polizia, i costi ridotti, e morbilità generale per gli animali utilizzati garantire un mezzo più umane e più scientifico di studio della malattia.

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 ...

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Immunology and Infection

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

20.5K Views.  University of Texas Medical Branch. Utilizzando luciferasi e sistemi di imaging in vivo (IVIS) come un nuovo mezzo per identificare terminazioni malattia prima sviluppi clinici verificarsi. IVIS ci ha permesso di visualizzare in tempo reale l'invasione di virus encefalitiche più giorni, fornendo un modello di malattia più accurato per lo studio futuro. E ci ha anche permesso di identificare le caratteristiche potenziali di protezione di antivirali e vaccini più veloce rispetto ai modelli animali attu...

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Imaging in vivo del transgenico parassiti Leishmania in un host live

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

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Immunologia e infezioni

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

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 ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Panni nasale per l&#39;influenza A rilevazione antivirus e isolamento da Suina

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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 ...

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

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

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

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

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

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

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

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

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

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

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una piattaforma di High-throughput per lo Screening della Salmonella spp /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...