Noi riconosciamo con gratitudine gli importanti contributi di Drs. John Anderson, Andrew principale e Shirley Tirrell a questo studio. Questo lavoro è stato sostenuto in parte da sovvenzioni provenienti dal Centers for Disease Control and Prevention (U50/CCU116806-01-1) e il Dipartimento dell&#39;Agricoltura degli Stati Uniti (58-6615-1-218, CONH00768 e CONH00773).

1. Mosquito smistamento e identificazione <ol><li> Le seguenti procedure vengono eseguite su un banco in un laboratorio aperto dedicato biosicurezza di livello-2 (BSL-2) da personale di laboratorio che sono addestrati a lavorare con le zanzare dal vivo. Una &quot;catena del freddo&quot; è mantenuta per tutta la procedura. </li><li> Anestetizzare vivono zanzare adulte mettendo borsa zanzara raccolta in un congelatore -20 ° per 5 minuti. Trasferimento sacca di raccolta in un contenitore isolato contenente ghiaccio secco. Chiudere il coperchio ed esporre le zanzare di anidride carbonica per 1-3 minuti per garantire la completa knock-down. </li><li> Toccare le zanzare anestetizzato su un pre-raffreddata padella posta su un letto di ghiaccio secco. Separare le zanzare individuale femminile con una pinza sottile e il luogo in bicchieri di plastica parte. Coprire con coperchio e coppe posto sul ghiaccio bagnato. </li><li> Identificare le specie di zanzare usando i tasti tassonomica descrittiva basata sulla morfologia zanzara esterno con l&#39;ausilio di un microscopio stereo dissezione (10-60X zoom) montato su un tavolo freddo elettronico. </li><li> Combina identificate specie di zanzare in pool di ≤ 50 persone in 2 ml con tappo a scatto fiale contenenti un BB rame. I flaconi sono etichettati con un identificatore univoco. </li><li> A seguito di identificazione di tutte le zanzare, le fiale sono posti in sacchetti etichettati e tenuto in un dispositivo di raffreddamento di polistirolo contenente ghiaccio secco. </li><li> Conservare piscine zanzara in un congelatore ° -80 C fino a quando le analisi virologiche. </li></ol> 2. Considerazioni di Biosicurezza per isolare virus zanzare <ol><li> Le seguenti procedure vengono eseguite in un biosicurezza di livello 3 (BSL-3) di laboratorio da personale che sono addestrati a lavorare a questo livello di contenimento 1. Strutture di laboratorio, apparecchiature, procedure e prassi sono soggetti al controllo istituzionali, statali e federali e di ispezione. Cercate la formazione e l&#39;approvazione prima di provare i seguenti protocolli. </li><li> Indossare la adeguati dispositivi di protezione individuale (DPI) per ogni procedimento come camici monouso, guanti, visiere e respiratori. </li><li> Disinfettare le superfici di lavoro con il 70% di alcol prima e dopo il completamento delle procedure. </li><li> Colture cellulari e materiale potenzialmente infetto sono trattati in II classe biosicurezza cabinet (BSC). </li><li> Piscine zanzara sono omogeneizzati in un mixer-mulino che viene inserito all&#39;interno di una BSC. </li><li> Omogenati di zanzara vengono centrifugati in una tenuta di aerosol microcentrifuga. Se questa procedura non può essere eseguita all&#39;interno di una BSC, l&#39;operatore deve indossare un respiratore durante il funzionamento e il recupero di campioni dalla centrifuga. </li><li> Pipette e puntali movimentazione dei materiali nel BSC (biosicurezza armadio) sono imbevuti di candeggina al 10% prima della sterilizzazione in autoclave. Tutti i rifiuti di laboratorio è decontaminati in autoclave prima dello smaltimento. </li></ol> 3. Preparazione di cellule Vero <ol><li> Terreni di coltura decantare da un gran fiasco coltura dei tessuti (175 cm 2) di confluenti cellule Vero (Clone E6) in coppa rifiuti contenenti candeggina. </li><li> Lavare le cellule Vero con l&#39;aggiunta di un&#39;aliquota di 5 ml di PBS e agitare oltre strato di cellule e lungo i lati del pallone avendo cura di coprire accuratamente monostrato intero. </li><li> Decantare PBS in coppa dei rifiuti. </li><li> Aggiungere 2,5 mL di tripsina-EDTA e agitare oltre strato di cellule avendo cura di coprire accuratamente monostrato intero. </li><li> Incubare pallone in incubatore per 5 minuti a 37 ° C, 5% di CO 2. </li><li> Scuotere e pallone turbolenza per rimuovere le cellule dalla superficie inferiore. Le cellule devono sfilare facilmente; incubare un ulteriore 1-5 minuti se le cellule non facilmente scivolare. </li><li> Aggiungere 5 ml di Minimal Essential Media (MEM), 5% di siero fetale bovino (FBS), utilizzando una pipetta sierologica e pipetta up-and-down 10-20 volte per rompere i grumi di cellule. </li><li> Aggiungere un ulteriore 15 ml MEM, 5% FBS per un volume totale di 22,5 ml e mescolare bene. </li><li> Posto 40 piccole fiasche coltura dei tessuti (25 cm 2) in posizione verticale in due rack (20 palloni / rack). </li><li> Collocare il rack contenente fiaschi all&#39;interno dell&#39;armadio biosicurezza e rimuovere i tappi fiasco, mettendo tappi di fronte fiaschi. </li><li> Dispensare 4 ml di MEM, 5% FBS in ogni pallone con una pipetta sierologica. </li><li> Dispensare 0,5 ml di cellule sospese in ogni pallone (~ 01:06 rapporto di splittaggio) Sostituire tappi pallone. </li><li> Aggiungere 50 ml di MEM, 5% FBS nuovo al primo pallone grandi colture di tessuti contenenti la sospensione di cellule rimanenti (~ 2,5 ml) e tornare pallone per l&#39;incubatrice. Lo stesso grande pallone di colture di tessuti possono essere riutilizzati fino a 5 passaggi e poi un pallone nuovo dovrebbe essere di set-up per sostituirlo. </li><li> Coltura tissutale piccolo fiaschi in pile sul vassoio. Agitare il vassoio per assicurarsi che i media copre uniformemente il fondo del flaks. </li><li> Luogo piccoli palloni in incubatrice a 37 ° C, 5% di CO 2 e crescere durante la notte fino a 80-90% confluenti. </li></ol> 4. Preparazione di piscine zanzara <ol><li> Tenere piscine zanzara freddo durante le procedure di collaudo. Utilizzare pre-refrigeratorack congelatore e mixer-mulino cassette, ghiacciata reagenti e una centrifuga refrigerata durante l&#39;elaborazione di piscine zanzara. </li><li> Tubi contenenti luogo le zanzare in una pre-raffreddata a rack freezer e aggiungere 1-1,5 ml di PBS-G a ciascuna provetta di zanzare. </li><li> Recupera mixer-mulino cassette dal congelatore. Posto 24 tubi in ogni cassetto e poi sicuro nel mixer-mulino. </li><li> Omogeneizzare i campioni per 4 minuti a 25 cicli / secondo. Il mulino mixer scuote i tubi ad alta velocità e un BB in metallo all&#39;interno di ciascun tubo distrugge il tessuto zanzara. </li><li> Centrifugare i tubi per 6 minuti a 7.000 giri al minuto. </li><li> Provette per la schiena in rack congelatore fino inoculati in cellule Vero. </li></ol> 5. Inoculazione di cellule Vero con omogenati piscina zanzara <ol><li> Controllare almeno un pallone colture di tessuti di ciascuna serie e sotto microscopio invertito per assicurare un&#39;adeguata salute confluenza e cellule; aspettare circa 80-90% di copertura. </li><li> Allineare 20 piccole fiasche di coltura tissutale in un rack e fiaschi etichetta con i numeri corrispondenti adesione di ciascun gruppo di zanzara. </li><li> Allentare i cappucci e decantare la maggior parte dei media da fiasche di coltura in coppa dei rifiuti. Lasciare i supporti abbastanza nel pallone in mano completamente monostrato cellulare. </li><li> 100μL un&#39;aliquota del surnatante di ciascun gruppo zanzara trattati nel pallone corrispondente coltura di tessuti. </li><li> Sostituire il tappo e serrare fiasco cultura. </li><li> Lay fiaschi cultura piatto su agitatore per 5 minuti (temperatura ambiente) a 35-40 giri al minuto. </li><li> Ritorno 20 fiasche coltura dei tessuti in posizione verticale in un rack e posto nella BSC. </li><li> Uncapping 3 fiasche coltura dei tessuti in una sola volta, dispensare 4 media di crescita ml in ogni pallone con una pipetta sierologica, sostituire tappi. </li><li> Assicurarsi che la punta della pipetta non contatta il collo del matraccio cultura o delle labbra. Cambia puntali come necessario se viene a contatto con il pallone. </li><li> Incubare cultura fiasche tessuto a 37 ° C, 5% di CO 2. </li></ol> 6. Screening cellule Vero per la crescita dei virus <ol><li> Schermo inoculato fiasche coltura dei tessuti per i segni di infezione virale, effetto citopatico (CPE), da 3-7 giorni dopo l&#39;inoculazione. </li><li> Ispezionare visivamente colture cellulari per la CPE e / o contaminazione inclinando la fiaschi ed esaminando i media che pozze sul fondo del pallone. I media appaiono torbidi come le cellule si deteriorano durante CPE oppure a causa della crescita del lievito, funghi, o di contaminazione batterica. </li><li> Riesaminare colture di cellule che mostrano i media nuvoloso sotto un microscopio invertito. I virus si indurre le cellule a deformarsi e staccarsi dalla cultura fiaschi. Colture cellulari con contaminanti visibili, per esempio lievito, altri batteri, funghi o detriti zanzara pesanti, sono ri-testati passando la piscina zanzara originale attraverso un filtro a siringa 0.22μM. </li><li> Asetticamente dispensare 1-2 ml di supporti da virus positivo colture cellulari in cryotubes etichettati utilizzando una pipetta sierologica. </li><li> Culture del virus sono conservati a -80 ° C fino identificato con il test diagnostico appropriato. </li></ol> 7. Preparazione dei reagenti <ol><li> MEM, 5% FBS. Sciogliere 37,6 g di MEM in polvere in un volume finale di 4 L di H 2 0 gg. Dispensare soluzione in 500 ml di aliquote in flaconi media e autoclave. Quando la soluzione si è raffreddata, aggiungere asetticamente 26 inattivato al calore ml di siero fetale bovino, 5,2 mL L-Glutammina, 5,2 anti-biotic/mycotic mL e 10,4 mL di bicarbonato di sodio 7,5% per ogni bottiglia. Conservare a 4 ° C. </li><li> PBS-G. Sciogliere 16 g di NaCl, 0,4 g di KCl, 2,3 g di Na 2 HPO 4, 0,4 g di KH 2 PO 4, e 4 mL di fenolo rosso allo 0,5% in 1000 mL gg H 2 0. Regolare il pH a 7,1-7,4 con 2 N NaOH (se necessario). In un recipiente a parte, il caldo 600 ml di H 2 0 a ebollizione. Rimuovi dalla piastra, aggiungere 10 g di gelatina, e si dissolvono. Aggiungere la gelatina sciolta soluzione alla soluzione PBS e regolare il volume finale a 2 L con dd H 2 O. Dispensare 100 ml di soluzione in bottiglie e autoclave. Quando la soluzione si è raffreddata, aggiungere asetticamente 42,8 ml di siero siero di coniglio e 1,4 anti-biotic/mycotic ml. Conservare a 4 ° C. </li><li> PBS cellula lavaggio. Aggiungere 50 ml di PBS 10X Dulbecco a 400 ml gg H 2 O. Regolare il pH a 7,1 con NaOH 2 N. Regolare il volume finale a 500 ml con dd H 2 O. Filtro soluzione con unità 0.2uM filtro vuoto. Dispensare in 5 aliquote ml in 8 ml con tappo a vite tubi. Conservare a 4 ° C. </li></ol> 8. Rappresentante Risultati Nove diversi di virus da 4 famiglie tassonomico sono stati recuperati dalle zanzare raccolti durante la sorveglianza continua in tutto lo stato del Connecticut 1997-2010 (tabella 1). Cinque di questi virus sono noti per causare la malattia umana, i patogeni più importanti sono il virus del Nilo occidentale e orientale virus dell&#39;encefalite equina. Nuove scoperte sono: il primo isolamento del WNV nel zanzare raccolti inNord America nel 1999 2, il primo isolamento del virus Potosi nel nord-est degli Stati Uniti nel 2000 3, e il primo isolamento del virus La Crosse nel New England nel 2005 4. <table border="1"><tbody><tr><td> Virus </td><td> Famiglia </td><td> No. isola </td><td> No. posizioni </td><td> No. anni rilevato </td><td> Malattie umane </td><td> Refs. </td></tr><tr><td> Virus del Nilo occidentale </td><td> Flaviviridae </td><td> 1002 </td><td> 94 </td><td> 12 </td><td> Da moderata a grave, febbre, encefalite </td><td> 2,5 </td></tr><tr><td> Encefalite equina orientale virus </td><td> Togaviridae </td><td> 292 </td><td> 41 </td><td> 10 </td><td> Gravi, encefalite </td><td> 6,7 </td></tr><tr><td> Highlands J virus </td><td> Togaviridae </td><td> 178 </td><td> 39 </td><td> 11 </td><td> Non nota </td><td> 6 </td></tr><tr><td> Jamestown Canyon virus </td><td> Bunyaviridae </td><td> 305 </td><td> 74 </td><td> 14 </td><td> Da lieve a moderata, la febbre, la meningite </td><td> 8 </td></tr><tr><td> Potosi virus </td><td> Bunyaviridae </td><td> 259 </td><td> 76 </td><td> 4 </td><td> Non nota </td><td> 3 </td></tr><tr><td> Cache Valley virus </td><td> Bunyaviridae </td><td> 114 </td><td> 51 </td><td> 8 </td><td> Da lieve a grave, la febbre, la malattia neurologica in due casi documentati </td><td></td></tr><tr><td> Trivittatus virus </td><td> Bunyaviridae </td><td> 83 </td><td> 21 </td><td> 11 </td><td> Non nota </td><td></td></tr><tr><td> La Crosse virus </td><td> Bunyaviridae </td><td> 1 </td><td> 1 </td><td> 1 </td><td> Da moderata a grave, l&#39;encefalite </td><td> 4 </td></tr><tr><td> Fiandre virus </td><td> Rhabdoviridae </td><td> 4 </td><td> 4 </td><td> 2 </td><td> Non nota </td><td></td></tr></tbody></table> I virus Tabella 1. Rilevato in campo raccolti zanzare mediante test di coltura cellulare Vero. Le zanzare sono stati raccolti durante la sorveglianza in tutto lo stato in Connecticut dal 1997-2010.

<ol>
	<li>Chosewood, L. C., Wilson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2007).</li><li>Anderson, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+West+Nile+virus+from+mosquitoes,+crows,+and+a+Cooper's+hawk+in+Connecticut.">Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.</a> Science. 286, 2331-2333 (1999).</li><li>Armstrong, P. M., Andreadis, T. G., Anderson, J. F., Main, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+West+Nile+virus+in+Connecticut,+USA:+a+five+year+analysis+of+mosquito+data+1999-2003.">Epidemiology of West Nile virus in Connecticut, USA: a five year analysis of mosquito data 1999-2003.</a> Vector Borne Zoonotic Dis. 4, 360-378 (2004).</li><li>Andreadis, T. G., Anderson, J. F., Tirrell-Peck, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+isolations+of+eastern+equine+encephalitis+and+highlands+J+viruses+from+mosquitoes+(Diptera:+Culicidae)+during+a+1996+epizootic+in+southeastern+Connecticut.">Multiple isolations of eastern equine encephalitis and highlands J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut.</a> J Med Entomol. 35, 296-302 (1998).</li><li>Armstrong, P. M., Andreadis, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eastern+equine+encephalitis+virus+in+mosquitoes+and+their+role+as+bridge+vectors.">Eastern equine encephalitis virus in mosquitoes and their role as bridge vectors.</a> Emerg Infect Dis. 16, 1869-1874 (2010).</li><li>Andreadis, T. G., Anderson, J. F., Armstrong, P. M., Main, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolations+of+Jamestown+Canyon+virus+(Bunyaviridae:+Orthobunyavirus)+from+field-collected+mosquitoes+(Diptera:+Culicidae)+in+Connecticut,+USA:+a+ten-year+analysis,+1997-2006.">Isolations of Jamestown Canyon virus (Bunyaviridae: Orthobunyavirus) from field-collected mosquitoes (Diptera: Culicidae) in Connecticut, USA: a ten-year analysis, 1997-2006.</a> Vector Borne Zoonotic Dis. 8, 175-188 (2008).</li><li>Beaty, B. J., Calisher, C. H., Shope, R., Lennette, E. H., Lennette, D. A., Lennette, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11.">Chapter 11.</a> Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. , 189-212 (1995).</li><li>Karabatsos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Catalogue of Arboviruses Including Certain Other Viruses of Vertebrates. , (1985).</li><li>Lanciotti, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+west+nile+virus+from+human+clinical+specimens,+field-collected+mosquitoes,+and+avian+samples+by+a+TaqMan+reverse+transcriptase-PCR+assay.">Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay.</a> J Clin Microbiol. 38, 4066-4071 (2000).</li><li>Lambert, A. J., Martin, D. A., Lanciotti, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+North+American+eastern+and+western+equine+encephalitis+viruses+by+nucleic+acid+amplification+assays.">Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.</a> J Clin Microbiol. 41, 379-385 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

Test su cellule Vero cultura serve come un metodo efficace per lo screening sul campo raccolte zanzare per una varietà di virus. Questo contrasta con metodi molecolari che specificamente di mira uno o alcuni virus di interesse. Inoltre, abbiamo trovato che il Vero saggio coltura cellulare è altrettanto se non più sensibile di RT-PCR 7 e ci fornisce isolati di virus che sono memorizzati nella nostra collezione di riferimento per studi futuri. Tuttavia, i sistemi di coltura cellulare non può essere opportun...

<table border="1"><tbody><tr><th> Nome del reagente / attrezzature </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Siero fetale bovino </td><td> Gibco - Invitrogen </td><td> 16140 </td></tr><tr><td> Anti-biotic/mycotic </td><td> Gibco - Invitrogen </td><td> 15240 </td></tr><tr><td> Bicarbonato di sodio NaHCO 3, 7,5% Soln. </td><td> Gibco - Invitrogen </td><td> 25080 </td></tr><tr><td> L-Glutammina 100x 200mm </td><td> Gibco-Invitrogen </td><td> 25030 </td></tr><tr><td> Polvere terreno minimo indispensabile (MEM) </td><td> Gibco - Invitrogen </td><td> 11700 </td></tr><tr><td> 10X PBS Dulbecco </td><td> Gibco - Invitrogen </td><td> 14080 </td></tr><tr><td> Tripsina-EDTA </td><td> Gibco - Invitrogen </td><td> 15400 </td></tr><tr><td> Siero di coniglio </td><td> Gibco - Invitrogen </td><td> 16120 </td></tr><tr><td> Le cellule Vero (Clone E6) </td><td> ATCC </td><td> CRL-1586 </td></tr><tr><td> Tessuto di piccole fiasche cultura, tappi ventilati, 25 cm 2 </td><td> Falcon - Becton Dickinson </td><td> 353112 </td></tr><tr><td> Grandi fiaschi del tessuto culturale, tappi ventilati, 175 cm 2 </td><td> Falcon - Becton Dickinson </td><td> 353108 </td></tr><tr><td> Rame rivestita di BB, 4,5 millimetri </td><td> Crosman </td><td> 0767 </td></tr><tr><td> Mixer Mill </td><td> Retsch </td><td> MM300 </td></tr><tr><td> IsoPack congelatore rack </td><td> Eppendorf </td><td> 022510240 </td></tr></tbody></table>

detection of infectious virus from field-collected mosquitoes by vero cell culture assay

Le zanzare trasmettono un certo numero di diversi virus tra cui importanti patogeni umani come il virus West Nile, virus dengue, virus e chickungunya. Molti di questi virus hanno intensificato nelle loro gamme endemiche e esteso a nuovi territori, che richiedono efficaci programmi di sorveglianza e di controllo per rispondere a queste minacce. Una strategia per monitorare l&#39;attività dei virus prevede la raccolta di un gran numero di zanzare da siti endemiche e il loro test per l&#39;infezione virale. In questo articolo viene descritto come gestire, elaborare e schermo campo raccolti per le zanzare virus infettivo mediante test Vero colture cellulari. Le zanzare sono ordinati in base alla località trappola e specie, e raggruppati in vasche contenenti ≤ 50 individui. I campioni in pool sono omogeneizzati in soluzione salina tamponata con un mixer-mulino e la fase acquosa viene inoculata in colture cellulari confluenti Vero (Clone E6). Le colture cellulari sono monitorati per effetto citopatico da 3-7 giorni post-inoculazione e qualsiasi virus coltivato in colture cellulari sono identificati dal test diagnostico appropriato. Utilizzando questo approccio, abbiamo isolato 9 virus diversi da zanzare raccolte nel Connecticut, Stati Uniti, e tra questi, 5 sono noti per causare la malattia umana. Tre di questi virus (West Nile virus, virus Potosi, e La Crosse virus) rappresentano i nuovi record per il Nord America o la regione del New England dal 1999. La capacità di rilevare un&#39;ampia gamma di virus è fondamentale per il monitoraggio sia stabilita e virus emergenti nella popolazione di zanzare.

Watch this Scientific Journal Video about Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay at JoVE.com

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Descriviamo un metodo per elaborare e schermo campo raccolti zanzare per una varietà di virus mediante test Vero colture cellulari. Utilizzando questa tecnica, abbiamo rilevato 9 virus diversi da 4 famiglie tassonomiche nelle zanzare raccolte nel Connecticut.

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

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Immunology and Infection

18.3K Views.  The Connecticut Agricultural Experiment Station. Descriviamo un metodo per elaborare e schermo campo raccolti zanzare per una varietà di virus mediante test Vero colture cellulari. Utilizzando questa tecnica, abbiamo rilevato 9 virus diversi da 4 famiglie tassonomiche nelle zanzare raccolte nel Connecticut.

Video: Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Imaging cellulare dal vivo di Alphaherpes Virus Trasporti anterograda e Diffusione

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Ricerca

Immunologia e infezioni

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoidi come modello per le malattie infettive: cultura dei diritti dell&#39;uomo e organoidi stomaco murini e microiniezione dell&#39;Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus System infettiva coltura cellulare e la<em&gt; In Vitro</em&gt; Effetto profilattico di interferoni

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

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

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

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

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

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

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 ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

Identificazione dei Mouse e dei repertori di anticorpo umano mediante sequenziamento di nuova generazione

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Rilevamento di immunoglobulina Anti-acquaporina-4 G di siero umano dall'analisi Cell-based

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Purificazione dei preparati vescicoli extracellulari ad alto rendimento lontano dal virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Analisi di competenza vettoriale sulle zanzare Aedes aegypti utilizzando il virus Zika

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...