Ringraziamo i revisori per i loro suggerimenti premurosi che hanno contribuito a migliorare questo manoscritto. Questo lavoro è stato supportato da sovvenzioni a favore di MVC (NS21072, HD23315), ACW (GM61139, S10RR017970) e IM (AI073898, GM056927) dal NIH. MK è stata sostenuta in parte da una formazione sovvenzione del NIH (5T32 AI007180).

<ol>
	<li>Camarena, V., Kobayashi, M., Kim, J. Y., Roehm, P., Perez, R., Gardner, J., Wilson, A. C., Mohr, I., Chao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+duration+of+growth+factor+signaling+through+receptor+tyrosine+kinases+regulates+HSV-1+latency+in+neurons.">Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons.</a> Cell Host &#38; Microbe. 8, 320-330 (2010).</li><li>Johnson, M. I., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+sympathetic+ganglia.">Primary cultures of sympathetic ganglia.</a> Protocols for Neural Cell Culture. , 71-94 (2001).</li><li>Letourneau, P. C., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+substrata+for+in+vitro+culture+of+neurons.">Preparation of substrata for in vitro culture of neurons.</a> Protocols for Neural Cell Culture. , 245-254 (2001).</li><li>Price, J. P., Brewer, G. J., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+media+for+neural+cell+cultures.">Serum-free media for neural cell cultures.</a> Protocols for Neural Cell Culture. , 255-264 (2001).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+family+Herpesviridae:+A+brief+introduction.">The family Herpesviridae: A brief introduction.</a> Fields Virology. 2, 2381-2397 (2001).</li><li>Price, R. W., Rubenstein, R., Khan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+infection+of+isolated+autonomic+neurons+in+culture:+viral+replication+and+spread+in+a+neuronal+network.">Herpes simplex virus infection of isolated autonomic neurons in culture: viral replication and spread in a neuronal network.</a> Arch. Virol. 71, 127-140 (1982).</li><li>Tomishima, M. J., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+alpha-herpesvirus+protein+necessary+for+axonal+localization+of+viral+membrane+proteins.">A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.</a> J. Cell Biol. 154, 741-752 (2001).</li><li>Ch'ng, T. H., Flood, E. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+primary+and+transformed+neuronal+cells+for+studying+pseudorabies+virus+infection.">Culturing primary and transformed neuronal cells for studying pseudorabies virus infection.</a> Methods Mol. Biol. 292, 299-316 (2005).</li><li>Wang, F., Tang, W., McGraw, H. M., Bennett, J., Enquist, L. W., Friedman, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+glycoprotein+E+is+required+for+axonal+localization+of+capsid,+tegument,+and+membrane+glycoproteins.">Herpes simplex virus type 1 glycoprotein E is required for axonal localization of capsid, tegument, and membrane glycoproteins.</a> J. Virol. 79, 13362-13372 (2005).</li><li>Benboudjema, L., Mulvey, M., Gao, Y., Pimplikar, S. W., Mohr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+herpes+simplex+virus+type+1+us11+gene+product+with+the+cellular+kinesin+light-chain-related+protein+PAT1+results+in+the+redistribution+of+both+polypeptides.">Association of the herpes simplex virus type 1 us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides.</a> J. Virol. 77, 9192-9203 (2003).</li><li>Blaho, J., Morton, E. R., Yedowitz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus:+propagation,+quantification+and+storage.">Herpes simplex virus: propagation, quantification and storage.</a> Curr. Protoc. Microbiol. Chapter 14, Unit 14E.1 (2005).</li><li>Van Zeijl, M., Fairhurst, J., Jones, T. R., Vernon, S. K., Morin, J., LaRocque, J., Feld, B. L., O'Hara, B. L., Bloom, J. D., Johann, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+class+of+thiourea+compounds+that+inhibit+herpes+simplex+virus+type+1+DNA+cleavage+and+encapsidation:+resistance+maps+to+the+UL6+gene.">Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.</a> J. Virol. 74, 9054-9061 (2000).</li><li>Newcomb, W. W., Brown, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+herpes+simplex+virus+replication+by+WAY-150138:+assembly+of+capsids+depleted+of+the+portal+and+terminase+proteins+involved+in+DNA+encapsidation.">Inhibition of herpes simplex virus replication by WAY-150138: assembly of capsids depleted of the portal and terminase proteins involved in DNA encapsidation.</a> J. Virol. 76, 10084-10088 (2002).</li><li>Pesola, J. M., Zhu, J., Knipe, D. M., Coen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+immediate-early+and+early+gene+expression+during+reactivation+from+latency+under+conditions+that+prevent+infectious+virus+production.">Herpes simplex virus 1 immediate-early and early gene expression during reactivation from latency under conditions that prevent infectious virus production.</a> J. Virol. 79, 4516-14525 (2005).</li><li>Arthur, J. L., Scarpini, C. G., Connor, V., Lachmann, R. H., Tolkovsky, A. M., Efstathiou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+promoter+activity+during+latency+establishment,+maintenance+and+reactivation+in+primary+dorsal+root+neurons+in+vitro.">Herpes simplex virus type 1 promoter activity during latency establishment, maintenance and reactivation in primary dorsal root neurons in vitro.</a> J. Virol. 75, 3885-3895 (2001).</li><li>Danaher, R. J., Jacob, R. J., Steiner, M. R., Allen, W. R., Hill, J. M., Miller, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+induce+reactivation+of+herpes+simplex+virus+type+1+in+a+latency-associated+transcript-+independent+manner+in+neuronal+cells.">Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript- independent manner in neuronal cells.</a> J. Neurovirol. 11, 306-317 (2005).</li><li>Terry-Allison, T., Smith, C. A., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxed+repression+of+herpes+simplex+virus+type+1+genomes+in+murine+trigenminal+neurons.">Relaxed repression of herpes simplex virus type 1 genomes in murine trigenminal neurons.</a> J. Virol. 71, 12394-12405 (2007).</li><li>Harris, R. A., Preston, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+latency+in+vitro+by+the+herpes+virus+type+1+mutant+in1918.">Establishment of latency in vitro by the herpes virus type 1 mutant in1918.</a> J. Gen. Virol. 72, 907-913 (1991).</li><li>Wagner, E. K., Bloom, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+herpes+simplex+virus+latency.">Experimental investigation of herpes simplex virus latency.</a> Clin. Microbiol. Rev. 10, 419-443 (1997).</li><li>Strelow, L. I., Laycock, K. A., Jun, P. Y., Rader, K. A., Brady, R. H., Miller, J. K., Pepose, J. S., 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=A+structural+and+functional+comparison+of+the+latency-associated+transcript+promoters+of+herpes+simplex+virus+type+1+strains+KOS+and+McKrae.">A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae.</a> J. Gen Virol. 75, 2475-2480 (1994).</li><li>Stroop, W. G., Banks, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+strain+KOS-63+does+not+cause+acute+or+recurrent+ocular+disease+and+does+not+reactivate+ganglionic+latency+in+vivo.">Herpes simplex virus type 1 strain KOS-63 does not cause acute or recurrent ocular disease and does not reactivate ganglionic latency in vivo.</a> Acta Neuropathol. 87, 14-22 (1994).</li><li>Sawtell, N. M., Poon, D. K., Tansky, C. S., Thompson, 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=The+latent+herpes+simplex+virus+type+1+genome+copy+number+in+individual+neurons+is+virus+strain+specific+and+correlates+with+reactivation.">The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain specific and correlates with reactivation.</a> J. Virol. 72, 5343-5350 (1998).</li><li>Thompson, R. L., Cook, M. L., Devi-Rao, G., Wagner, E. K., Stevens, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+analysis+of+the+avirulent+wild-type+herpes+simplex+virus+type+1+strain+KOS.">Functional and molecular analysis of the avirulent wild-type herpes simplex virus type 1 strain KOS.</a> J. Virol. 58, 203-211 (1986).</li><li>Wilcox, C. L., Smith, R. L., Freed, C. R., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor-dependence+of+herpes+simplex+virus+latency+in+peripheral+sympathetic+and+sensory+neurons+in+vitro.">Nerve growth factor-dependence of herpes simplex virus latency in peripheral sympathetic and sensory neurons in vitro.</a> J. Neurosci. 10, 1268-1275 (1990).</li><li>Roehm, P. C., Camarena, V., Gardner, J. B., Wilson, A. C., Mohr, I., Chao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+vestibular+ganglion+neurons+demonstrate+latent+herpes+simplex+type+I+reactivation.">Cultured vestibular ganglion neurons demonstrate latent herpes simplex type I reactivation.</a> Laryngoscope. 121, 2268-2275 (2011).</li><li>Kuhn, M. A., Nayak, S., Camarena, V., Gardner, J., Wilson, A., Mohr, I., Chao, M. V., Roehm, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+culture+model+of+facial+palsy+resulting+from+reactivation+of+latent+herpes+simplex+virus+type+1.">A cell culture model of facial palsy resulting from reactivation of latent herpes simplex virus type 1.</a> Otology &#38; Neurotology. , (2012).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Questa cultura neurone primario e l&#39;infezione del sistema fornisce un metodo semplice ed efficace per esplorare i meccanismi molecolari alla base HSV-1 la latenza e la riattivazione. Il sistema riprende fedelmente le caratteristiche di latenza accettati definiti in entrambe le infezioni umane ed in live-modelli animali. Quando il virus è latente nelle culture SCG, particelle virali infettive e prodotti genici litici non può essere rilevato. L&#39;unico prodotto del gene virale rilevato in neuroni ...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Catalogo # </td><td> Comments </td></tr><tr><td> 70μm nylon filtro (filtro cella) </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> Salt 1x Hank soluzione equilibrata (HBSS - / -) </td><td> Invitrogen </td><td> 14175 </td><td> w / o CaCl 2 e MgCl 2 </td></tr><tr><td> 1x supporti minimi essenziali (MEM) </td><td> Invitrogen </td><td> 11095-080 </td><td></td></tr><tr><td> 5-Fluoro-2&#39;-desossiuridina </td><td> Sigma </td><td> F0503 </td><td> preparare 20 mM in 1x MEM, da conservare a -20 ° C </td></tr><tr><td> 96 e piastre piane TC ben inferiori </td><td> Corning </td><td> 3599 </td><td></td></tr><tr><td> Acyclovir </td><td> &lt;em&gt; Calbiochem </td><td> 114798 </td><td> preparare 31 mM in DMSO; conservare a -20 ° C </td></tr><tr><td> Afidicolina </td><td> Calbiochem </td><td> 178273 </td><td> preparare stock 10 mM in DMSO; conservare a -20 ° C </td></tr><tr><td> B-27 Supplement </td><td> Invitrogen </td><td> 17504-44 </td><td></td></tr><tr><td> Collagenasi </td><td> Sigma </td><td> C2674 </td><td> preparare i 10 mg / ml in magazzino HBSS - / -; conservare a -20 ° C </td></tr><tr><td> D-(+)-Glucosio </td><td> Sigma </td><td> G6152 </td><td> preparare 40% stock in H 2 O; filtro sterilizzare e conservare a 4 ° C </td></tr><tr><td> L-Glutammina </td><td> Invitrogen </td><td> 25030-081 </td><td></td></tr><tr><td> Laminina </td><td> Sigma </td><td> L2020 </td><td> preparare 1 mg / ml in magazzino H 2O; rapido congelamento 20 plaliquats e conservare a -80 ° C, diluita a 2 ug / ml di lavoro conc. sterile in H 2 O </td></tr><tr><td> Leibovit&#39;z L-15 supporti </td><td> Invitrogen </td><td> 11415 </td><td></td></tr><tr><td> Nerve Growth Factor </td><td> Harlan Laboratories </td><td> BT.5017 </td><td> preparare i 50 mg / ml in magazzino HBSS - / -, da conservare a -80 ° C </td></tr><tr><td> Media Neurobasal </td><td> Invitrogen </td><td> 12348 </td><td></td></tr><tr><td> Acido fosfonoacetico (PAA) </td><td> Sigma </td><td> P6909 </td><td> preparare i 75 mg / ml in magazzino H 2 O; conservare a -20 ° C </td></tr><tr><td> Poly-D-lisina bromidrato </td><td> Sigma </td><td> P0899 </td><td> preparare i 20 mg / ml in magazzino H 2 O; conservare a -20 ° C </td></tr><tr><td> Rat-tail collagene </td><td> Millipore </td><td> 08-115 </td><td>Concentrazione varia con il lotto di fornitura; conservare a 4 ° C e diluire a 0,66 mg / ml conc di lavoro. sterile in H 2 O </td></tr><tr><td> A tricostatina </td><td> Sigma </td><td> T8552 </td><td> preparare 1 mM in DMSO; conservare a -20 ° C </td></tr><tr><td> Tripsina 2,5% </td><td> Invitrogen </td><td> 15090-04 </td><td></td></tr></tbody></table>

a primary neuron culture system for the study of herpes simplex virus latency and reactivation

Herpes simplex virus di tipo 1 (HSV-1) stabilisce una vita lunga infezione latente nei neuroni periferici. Questo serbatoio latente è la fonte di eventi riattivazione ricorrenti che garantiscono la trasmissione e contribuire alla malattia clinica. Antivirali attuali non influenzano il serbatoio latente e non ci sono vaccini. Mentre i dettagli molecolari di replicazione litico sono ben caratterizzati, meccanismi che controllano la latenza nei neuroni rimangono elusivi. La nostra attuale comprensione di latenza è derivato da studi in vivo su modelli animali di piccole dimensioni, che sono stati indispensabili per la definizione dei requisiti dei geni virali e il ruolo delle risposte immunitarie. Tuttavia, è impossibile distinguere effetti specifici sul virus-neurone rapporto da conseguenze più generali di infezione mediati da cellule immunitarie di sostegno o non neuronali in animali vivi. Inoltre, la sperimentazione animale è costosa, richiede tempo, e limitate in termini di opzioni disponibili per la manipolazione di accoglienzaprocessi. Per superare queste limitazioni, un neurone solo sistema è disperatamente necessario che riproduce la vivo nelle caratteristiche di latenza e la riattivazione, ma offre i vantaggi di coltura tissutale in termini di omogeneità e di accessibilità. Qui vi presentiamo un modello in vitro utilizzando colture di neuroni primari dai gangli simpatici rat cervicale superiore (SCG) (Figura 1) per studiare HSV-1 la latenza e la riattivazione che si adatta maggior parte se non tutti i criteri desiderati. Dopo aver eliminato cellule non neuronali, vicino omogenei TrkA + colture neuronali sono infettati con HSV-1 in presenza di acyclovir (ACV) per sopprimere la replicazione litico. Dopo la rimozione ACV, non produttivi infezioni da HSV-1 che presentano caratteristiche fedelmente accettato di latenza sono stabiliti in modo efficiente. In particolare, mRNA litici, proteine ​​e virus infettivo diventa non rilevabile, anche in assenza di selezione, ma latenza associata trascrizione (LAT) expression persiste in nuclei neuronale. Genomi virali sono mantenuti ad un numero medio di copie 25 per neurone e possono essere indotte a replicare produttivamente interferendo con PI3-chinasi / Akt segnalazione o il ritiro semplice del nerve growth factor 1. A ricombinante HSV-1 EGFP codifica fusa alla proteina virale US11 litico fornisce uno funzionale, in tempo reale marcatore per la replica risultante da riattivazione che è facilmente quantificato. In aggiunta ai trattamenti chimici, metodologie genetiche come la RNA-interferenza o il gene di consegna attraverso vettori lentivirali può essere applicato con successo al sistema di autorizzazione gli studi meccanicistici che sono molto difficili, se non impossibile, negli animali. In sintesi, il SCG-based HSV-1 la latenza / riattivazione sistema fornisce un potente strumento necessario per svelare i meccanismi molecolari che controllano HSV1 latenza e riattivazione nei neuroni, un puzzle di lunga data in virologia, la cui soluzione può offrire nuove intuizioni nello sviluppo di nuove terapie che obiettivo tsi riserva herpesvirus latente.

Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Il protocollo descrive un sistema modello efficiente e riproducibile per studiare herpes simplex virus di tipo 1 (HSV-1) latenza e riattivazione. Il dosaggio utilizza omogenei culture neurone simpatico e consente la dissezione molecolare del virus-neurone interazioni utilizzando una varietà di strumenti interferenza tra RNA e l&#39;espressione di proteine ​​ricombinanti.

A primaria del sistema cultura Neuron per lo Studio della latenza del virus Herpes Simplex e la riattivazione

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

a-primary-neuron-culture-system-for-study-herpes-simplex-virus

Research

JoVE Journal

Immunology and Infection

17.2K Views.  New York University School of Medicine. Il protocollo descrive un sistema modello efficiente e riproducibile per studiare herpes simplex virus di tipo 1 (HSV-1) latenza e riattivazione. Il dosaggio utilizza omogenei culture neurone simpatico e consente la dissezione molecolare del virus-neurone interazioni utilizzando una varietà di strumenti interferenza tra RNA e l'espressione di proteine ​​ricombinanti.

Video: A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

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

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in 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 ...

Ricerca

Immunologia e infezioni

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo infezione di Murine epidermide con Herpes Simplex Virus di tipo 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

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 ...

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), ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

Mouse e umano-derivato cultura dello strato monomolecolare epiteliale gastrica primaria per lo studio della rigenerazione

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

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, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Analisi temporale della traslocazione nucleare--citoplasmico di Herpes Simplex Virus 1 proteina mediante microscopia confocale immunofluorescente

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

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

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

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

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

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 ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.