Name: ex vivo infection of murine epidermis with herpes simplex virus type 1
Uploaded: 2015-08-24T15:00:00.000+00:00
Duration: 11 min 56 s
Description: Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

Ringraziamo Peter Staeheli per la fornitura di topi B6.A2G-MX1 e semre Özcelik consulenza tecnica. Questo lavoro è stato sostenuto dalla Fondazione per la Ricerca tedesco attraverso SFB829 e KN536 / 16, e la Colonia Fortune Programma / Facoltà di Medicina e Chirurgia, Università di Colonia.

Dichiarazione Etica. La preparazione di fogli epidermici di animali sacrificati viene effettuata in stretta conformità con le raccomandazioni della Guida di Landesamt für Natur, Umwelt e Verbraucherschutz, Nord Reno-Westfalia (Germania). Lo studio è stato approvato dal LANUV NRW (Numero 8.84-02.05.20.13.018). 1. Preparazione degli strumenti e cultura dei media <ol><li> Coltivare fogli epidermica in mezzo modificato Eagle Dulbecco (DMEM) / alto glucosio contenente glutamina dipeptide, il 10% FCS, 100 UI / ml di penicillina, e 100 mg / ml di streptomicina (di seguito denominato &quot;DMEM&quot;). </li><li> Per la preparazione di fogli epidermiche, sciogliere accuratamente dispasi II polvere (5 mg / ml) in PBS riscaldata (fino a 37 ° C). Filtrare la soluzione attraverso un filtro di 0,22 micron e utilizzarlo direttamente per il trattamento. </li><li> Per la preparazione di epidermica intero monta 13, preparare il blocco appassionatoer con 0,5% di latte in polvere, 0,25% di gelatina dall&#39;acqua fredda pelle di pesce e 0,5% Triton X-100 in TBS. Consentire sostanze per sciogliere incubando la miscela per almeno 2 ore a RT su un agitatore rotante. Anche preparare 0,2% Tween 20 in PBS come tampone di lavaggio. Per la fissazione, preparare 3,4% e 4% formaldeide in PBS. </li><li> Per la immunocolorazione di criosezioni, preparare un tampone bloccante con 5% di siero di capra normale e 0,2% Tween 20 in PBS. Per la fissazione, preparare 0,5% di formaldeide in PBS. </li></ol> 2. Preparazione di fogli epidermico da pelle murino <ol><li> Preparazione di epidermide da pelle posteriore neonato per gli studi di infezione <ol><li> Collocare un mouse decapitati (da 1 a 3 giorni dopo la nascita) in un piatto dissezione e tagliare le estremità e la coda vicino al torso per consentire la rimozione efficiente della pelle dal torso completa. Durante la rimozione delle estremità, cercare di mantenere i tagli più piccolo possibile di diametro. </li><li> Fissare il torso curvo con una pinza sottilend spostare delicatamente le forbici punta smussa sotto la pelle dal cranio caudale a lungo la parte posteriore per separare la pelle dal busto. Incidete la pelle lungo la parte posteriore. </li><li> Per l&#39;analisi FACS, isolamento di cheratinociti o la preparazione di RNA, staccare la pelle completo dal tronco con pinze per fissare il tessuto e le forbici punta smussata per spingere fuori il busto. Per la preparazione di criosezioni o supporti interi prendere solo pezzi di pelle indietro. </li><li> Tritate la pelle torna con un bisturi in 1-2 pezzi di circa 10 x 10 mm. Mettete i pezzi in un pozzetto di un 6-pozzetti con ~ 1 ml di PBS sterile. Assicurarsi che il lato dermico affaccia sul fondo. </li><li> Sostituire PBS da 1,5 ml dispasi II (5 mg / ml PBS) e incubare per entrambi 30 minuti a 37 ° C (per i supporti interi) o O / N a 4 ° C. Lavare 3 volte con PBS. Rimuovere delicatamente l&#39;epidermide come un foglio intatto utilizzando una pinza per riparare il derma sottostante e le altre pinze per sollevare l&#39;epidermide. Utilizzare un binocolo a fare questo passo easa. Trasferire il foglio epidermica in DMEM con il lato basale verso il fondo. Utilizzare immediatamente le lenzuola per gli esperimenti di infezione. NOTA: Utilizzare la preparazione di epidermide dalla pelle appena nato principalmente per l&#39;analisi FACS, isolamento di cheratinociti o la preparazione di RNA. A causa della sua fragilità, la preparazione di fogli infettati per criosezioni o supporti interi è meno adatto. </li></ol></li><li> Preparazione di epidermide da pelle coda adulto per gli studi di infezione <ol><li> Euthanize i topi all&#39;età di 1 a 3 mesi per dislocazione cervicale. Prendere coda invece di pelle indietro da allora i follicoli dei capelli lunghi integrati sul retro ostacolano separazione dei fogli epidermico intatte. Tagliare la coda di topi sacrificati e fessura nel senso della lunghezza con un bisturi. </li><li> Staccare la pelle dalle ossa e tritarlo con un bisturi in pezzi di circa 5 x 5 mm. Mettere fino a 4 pezzi in un unico bene e procedere con l&#39;incubazione in dispasi II come descritto nella sezione 2.1.5 NOTA: Utilizzare tai adultil pelle per preparare criosezioni o supporti interi. In alternativa l&#39;uso immediato, code possono essere immagazzinati o trasportati per circa 24 ore a 4 ° C senza perdere significativo l&#39;efficienza dell&#39;infezione. Se esce croce sono mantenuti fino a 48 ore, lenzuola epidermici potrebbero essere difficilmente infettati con HSV-1. </li></ol></li><li> Dissociazione dell&#39;epidermide per analisi FACS NOTA: Il rilevamento di superficie espressione della proteina mediante analisi FACS richiede adeguate tecniche di dissociazione cellulare che non danneggiano la superficie della cellula e la proteina di interesse. <ol><li> Incubare fogli epidermica appena nati in un 6-pozzetti con la parte basale a 1,5 ml / soluzione dissociazione cella ben ricombinante basato tripsina-per 15 minuti a RT e per 15 minuti a 37 ° C. </li><li> Fermare l&#39;incubazione con l&#39;aggiunta di 3 ml di DMEM. Dissociarsi cheratinociti utilizzando un curva pinza sottile per muovere delicatamente l&#39;epidermide in 6 pozzetti in modo che i cheratinociti si dissolvono. Raccogliere la sospensione cellulare e ripetere l&#39;operazione 3 voltesempre utilizzando DMEM fresco. NOTA: Questa procedura di dissociazione è opportuno rilevare nectin-1 sulla superficie dei cheratinociti. </li><li> In alternativa, incubare le lenzuola epidermiche di soluzione dissociazione cella priva di enzima (CDS) per 15 minuti a temperatura ambiente e 15 minuti a 37 ° C. Incubare altri 15 minuti a temperatura ambiente per irrigare delicatamente i cheratinociti pipettando CDS su e giù. </li><li> Raccogliere la sospensione cellulare e ripetere il passaggio di risciacquo 3 volte utilizzando DMEM fresco. NOTA: Questa dissociazione è atto a rilevare l&#39;espressione superficie HVEM. Lo svantaggio della soluzione CDS è che meno cellule sono dissociate che con soluzione ricombinante dissociazione cellulare basato tripsina. Oltre alla superficie espressione, espressione nucleare o citoplasmatica come espressione ICP0 può essere analizzato mediante FACS dopo dissociazione dei fogli epidermica infetti con soluzione ricombinante dissociazione cellulare basato tripsina. </li></ol></li><li> La dissociazione di epidermide per isolare cheratinociti o extratto RNA <ol><li> Mettere fogli epidermiche neonato in una piastra da 6 pozzetti con il lato basale verso il fondo del piatto che contiene 1,5 ml / soluzione ben ricombinante dissociazione cellulare basato tripsina. Incubare per 30 minuti a temperatura ambiente. Aggiungere 3 ml di DMEM e dissociare cheratinociti utilizzando un curva pinza sottile per spostare delicatamente l&#39;epidermide nel piatto. </li><li> Raccogliere la sospensione cellulare e ripetere l&#39;operazione 3 volte utilizzando DMEM fresco. Utilizzare questa sospensione cellulare per estrarre l&#39;RNA o alla cultura primari cheratinociti murini. </li></ol></li></ol> 3. L&#39;infezione di fogli epidermico con HSV-1 <ol><li> Preparare una soluzione di HSV-1 wt ceppo 17 11 in non meno di 500 microlitri DMEM e sostituire il mezzo dalla soluzione virus cui i fogli epidermici sono flottanti. Questo punto di tempo viene definito come tempo 0 12. Eseguire infezione di un foglio epidermica in un pozzetto di una piastra da 24 pozzetti a 37 ° C. Il calcolo del titolo virale è basato sul numero stimatodelle cellule nello strato basale per foglio epidermica (circa 2 x 10 5 cellule per foglio 5 x 5 mm). </li><li> Infettare con HSV-1 a circa 100 unità formanti placca (PFU) / cellulare e sostituire il medio virus contenente in DMEM a 1 ora pi NOTA: Nel caso di fogli epidermica dalla pelle neonato, tenere a mente che i fogli cominciano a dissociarsi durante l&#39;incubazione. </li></ol> 4. Visualizzazione delle cellule infettate <ol><li> Epidermal intero Monti 13 <ol><li> Fissare fogli epidermico con il 3,4% di formaldeide o per 2 ore a temperatura ambiente o O / N a 4 ° C. Lavare due volte con PBS e bloccare con 0,5% di latte in polvere, 0,25% di gelatina dall&#39;acqua fredda pelle di pesce e 0,5% Triton X-100 in TBS per 1 ora a RT 14. </li><li> Incubare le lenzuola O / N con topo anti-ICP0 (anticorpo monoclonale 11060) 15 diluito 1:60 in tampone di bloccaggio a temperatura ambiente. Per visualizzare la rete di filamenti intermedi incubare contemporaneamente policlonale di coniglio anti-Mouse cheratina 14 (100 ng / ml). </li><li> Lavare 4 a 5 volte con PBS-Tween 20 0,2% durante 4 ore a temperatura ambiente. Incubare O / N con AF488-coniugato anti-IgG di topo (1 mg / ml), IgG anti-coniglio AF555-coniugato (1 mg / ml), e DAPI (4 &#39;, 6-diamidino-2-fenilindolo) (100 ng / ml) in tampone di bloccaggio a RT. </li><li> Lavare con PBS-Tween 20 0,2% per 4 ore a temperatura ambiente. Fogli epidermico Mount con la loro parte basale sulla cima di un vetrino, incorporare nel mezzo di montaggio fluorescente e coprire con coprioggetto. </li></ol></li><li> Epidermal criosezioni <ol><li> Fogli epidermiche di età sono stati fissati in formaldeide al 4% in PBS per 20 minuti a RT e lavate 2 volte con PBS. Fogli fissi Incorpora in tessuto di congelamento medio e congelare in azoto liquido. Tagliare 8-10 sezioni micron con un criomicrotomo. </li><li> Fissare nuovamente le sezioni con 0,5% di formaldeide in PBS per 10 minuti a RT, lavare 2 volte con PBS, blocco con 5% di siero di capra normale e 0,2% Tween 20 in PBS per 30 minuti a RT, e quindi macchiare per 60 min con mOuse anti-ICP0 (anticorpo monoclonale 11060) 15 diluito 1:60 in tampone bloccante, seguito da 3 tempi di lavaggio con il tampone di bloccaggio. </li><li> Poi incubare con AF488-coniugato anti-IgG di topo (1 mg / ml) e DAPI (100 ng / ml) in tampone bloccante per 45 minuti a RT e lavare 3 volte. Sezioni incorporare nel mezzo di montaggio fluorescente e coprire con coprioggetto. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Quando i fogli epidermica di pelle adulta da C57BL / 6 sono infettati con HSV-1 a circa 100 PFU / cellula, osserviamo l&#39;infezione in quasi tutte le cellule dello strato basale dell&#39;epidermide interfollicolari mentre dosi virali inferiori correlano con le cellule meno infette e una più lenta progresso dell&#39;infezione. In generale, i follicoli piliferi mostrano un numero variabile di cellule infettate; mentre la maggior parte dei piccoli piuttosto cheratinociti che rivestono i follicoli dei capelli in via di s...

Herpes simplex virus (HSV) può causare una serie di malattie negli esseri umani da lievi lesioni mucocutanee semplici alle infezioni pericolose per la vita. HSV di tipo 1 (HSV-1) è prevalentemente associata a infezioni orofacciale e l&#39;encefalite, mentre HSV di tipo 2 (HSV-2) più probabile causa di infezioni genitali 1. Mentre non vi è un progresso significativo nella comprensione di come HSV entra nelle cellule in coltura, avvia l&#39;infezione e produce progenie virale, sappiamo poco del percorso virale invasione (s) nel tessuto a livello cellulare 2. Per gli studi di HSV infezioni della pelle o della mucosa, topi, conigli e cavie sono stati utilizzati come modelli animali. Infezione della pelle è stato istituito con iniezione intradermica o graffiare la pelle in presenza del virus, e lo sviluppo della malattia è stata correlata con la produzione di virus. Questi metodi hanno aiutato a comprendere vari aspetti della patogenesi della malattia, e sono utilizzate per valutare farmaci antivirali. Per studiare infezione da HSV al tissue livello, sono stati applicati modelli organotipica di pelle umana. Poiché il tasso di infezione è limitato in queste culture zattera, solo un numero limitato di studi che hanno valutato l&#39;infezione, diffusione virale e gli effetti dei componenti antivirali sono stati pubblicati 3-6. Al fine di caratterizzare i determinanti cellulari che svolgono un ruolo durante HSV-1 nell&#39;epitelio intatto, abbiamo stabilito un protocollo per la ex vivo studi infezioni dell&#39;epidermide murini 7. Pelle è stato preparato da neonati o dalle code di topi adulti. Dal momento che HSV-1 non è riuscito a infettare campioni di pelle completi, che sono stati immersi in mezzo contenente il virus, abbiamo separato l&#39;epidermide dal derma da dispasi trattamento II. Dopo variabile dei fogli epidermica su terreno contenente il virus, cellule infettate possono essere visualizzati nello strato epidermico basale in vari momenti postinfection (pi) 7. Per visualizzare l&#39;inizio di infezione in cellule singole prima replica virale unnd produzione di virus, abbiamo macchiato con un anticorpo contro la proteina delle cellule infettate 0 (ICP0), che è una delle prime proteine ​​espresse durante HSV-1. La localizzazione cellulare di ICP0 passa attraverso fasi distinte durante l&#39;infezione precoce. Mentre ICP0 è presente in foci nucleari in una fase precoce di espressione genica virale, rilocalizzazione di ICP0 al citoplasma indica una successiva fase di infezione 8. Abbiamo usato la ex vivo di infezione test di fogli epidermica da diversi modelli murini per testare il potenziale ruolo di diversi fattori cellulari durante l&#39;infezione. Per affrontare l&#39;impatto di Rac1 come un regolatore chiave delle dinamiche di actina, abbiamo infettati l&#39;epidermide di topi con delezione specifica per cheratinociti del gene Rac1 9. Questo modello ha permesso di studiare le conseguenze delle carenti Rac1 sull&#39;efficienza di HSV-1 negli strati epidermici cheratinociti. Il confronto di epidermide infette del controllo littermates ririvelò alcuna differenza significativa, indicando che l&#39;assenza di Rac1 non ha avuto effetto sulla apertura di infezione nello strato basale dell&#39;epidermide 7. L&#39;utilizzo di ulteriori modelli murini ci ha permesso di affrontare le quali recettori cellulari mediano ingresso nell&#39;epidermide. Infettare fogli epidermica sia da nectin-1 o topi HVEM-deficienti con HSV-1 ha rivelato che l&#39;ingresso del virus iniziale in tessuto dipende fortemente dalla presenza di nectin-1 10. Inoltre, i nostri risultati dimostrano che HVEM può anche servire come recettore nell&#39;epidermide murini, anche se meno efficiente rispetto nectin-1 10. Per affrontare la distribuzione spaziale delle cellule infettate negli strati epidermici, visualizziamo espressione ICP0 in sezioni di tessuto e epidermiche monti interi (Figura 1). In criosezioni di pelle completa, nessuna cellula-ICP0 esprimono vengono rilevati (Figura 1). Al contrario, criosezioni di fogli epidermiche dimostrano citoplasmaticaEspressione ICP0 nello strato basale a 3 ore già pi (Figura 1). In tempi più recenti, la diffusione virale di soprabasali strati può essere visualizzato. La distribuzione spaziale delle cellule infette nello strato basale può essere facilmente seguita in epidermiche supporti interi (Figura 1). Al momento l&#39;infezione da HSV-1 a 100 PFU / cellula, circa il 50% dei cheratinociti basali dell&#39;epidermide interfollicolari mostrano espressione ICP0 a 1,5 ore pi A questo punto momento cellule più infettate esprimono ICP0 nucleare. La rilocalizzazione della ICP0 nel citoplasma che indica una fase successiva dell&#39;espressione genica precoce è presente in quasi tutte le cellule a 3 ore pi (Figura 1). Queste modalità di visualizzazione cellule infette su ex vivo HSV-1 forniscono un potente test per studiare l&#39;effetto degli inibitori o di componenti cellulari mutate cancellati / su ingresso del virus e la diffusione nel tessuto.

<table><tbody><tr><td>DMEM/high glucose/GlutaMAX</td><td>Life Technologies</td><td>31966047</td><td>needed for cultivation of epidermal sheets</td></tr><tr><td>dispase II powder</td><td>Roche</td><td>4942078001</td><td>has to be solved in heated PBS</td></tr><tr><td>enzyme-free cell dissociation solution</td><td>Sigma</td><td>C5914</td><td>needed for very gentle dissociation of epidermal sheets</td></tr><tr><td>TrypLE select cell dissociation solution</td><td>Life Technologies</td><td>12563-029</td><td>needed for dissociation of epidermal sheets</td></tr><tr><td>chelex 100 resin</td><td>Bio-Rad</td><td>142-2832</td><td>needed for chelation of polyvalent metal ions from the fetal calf serum</td></tr><tr><td>gelatin from cold water fish skin&amp;nbsp;</td><td>Sigma</td><td>G7765</td><td>needed for minimization of non-specific antibody binding</td></tr><tr><td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td><td>Covance</td><td>PRB-155P</td><td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td></tr><tr><td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (conc.: 2 mg/ml)</td><td>Life Technologies</td><td>A-11029</td><td>used as secondary antibodies</td></tr><tr><td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate (conc.: 2 mg/ml)</td><td>Life Technologies</td><td>A-21429</td><td>used as secondary antibodies</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (conc.: 0.1 mg/ml)</td><td>Sigma</td><td>36670</td><td>used to counterstain the nucleus</td></tr></tbody></table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

Per inserire il suo ospite umano, il tipo di virus herpes simplex 1 (HSV-1) deve superare la barriera della mucosa superfici, pelle, o cornea. HSV-1 obiettivi cheratinociti durante l&#39;inserimento iniziale e stabilisce una infezione primaria nell&#39;epitelio, che è seguita da infezione latente di neuroni. Dopo la riattivazione, i virus possono diventare evidente a siti mucocutanee che appaiono come vescicole cutanee o ulcere della mucosa. Come HSV-1 invade la pelle o le mucose e raggiunge i suoi recettori è poco conosciuta. Per studiare il percorso invasione HSV-1 nel tessuto epidermico a livello cellulare, abbiamo stabilito un modello ex vivo infezione dell&#39;epidermide murine, che rappresenta il sito di infezione primaria e ricorrente in pelle. Il saggio comprende la preparazione della pelle murina. L&#39;epidermide è separato dal derma mediante trattamento dispasi II. Dopo galleggiante i fogli epidermica su terreno contenente il virus, il tessuto è fisso e l&#39;infezione può essere visualizzato in diversi momenti postinfection bycolorazione delle cellule infettate con un anticorpo contro la HSV-1 immediato proteina ICP0 presto. ICP0-esprimenti cellule può essere osservato nello strato basale cheratinociti già a 1,5 ore postinfection. Con tempi più lunghi di infezione, le cellule infettate vengono rilevati in strati soprabasali, indicando che l&#39;infezione non si limita ai cheratinociti basali, ma il virus si diffonde ad altri livelli nel tessuto. Utilizzando fogli epidermico di vari modelli di mouse, il protocollo di infezione consente di determinare il coinvolgimento delle componenti cellulari che contribuiscono alla HSV-1 invasione nei tessuti. Inoltre, il saggio è adatto a testare inibitori nel tessuto che interferiscono con le fasi iniziali di entrata, diffusione cellula-cellula e la produzione di virus. Qui, descriviamo il protocollo ex vivo infezione in dettaglio e presentiamo i nostri risultati con i topi Nectin-1 o HVEM-carenti.

La sfida del metodo è quello di preparare fogli epidermici in cui HSV-1 possono penetrare dallo strato basale. La fase critica è la separazione dell&#39;epidermide dal derma mediante trattamento dispasi II, che, a seconda del ceppo di topo, deve essere adattato. La concentrazione di dispasi II può variare da 2.5 a 5 mg / ml, e il tempo di incubazione da 20 a 45 min. La colorazione del filamento intermedio proteina cheratina 14 permette facilmente predire se lo strato epidermico basale può essere infettati o se mostr...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

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 ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Research

JoVE Journal

Immunology and Infection

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

11.1K Views.  University of Cologne. 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.

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Imaging ex vivo di cellule T nel Fette murini Linfonodo con grandangolari e microscopi confocale

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Ricerca

Immunologia e infezioni

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Dissezione Host-virus Interazione in replica litico di un Herpesvirus modello

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

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

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

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 ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Ricorrente cheratite erpetica Stromal in Mouse, un modello per lo studio HSK umano

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Ex Vivo Modello organotipiche corneale di acuta epiteliali Herpes simplex virus di tipo I Infezione

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

Neuroscienze

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

La rilevazione del genoma e Trascrizioni di un virus DNA persistente in tessuti neuronali da fluorescente In situ Ibridazione In combinazione con Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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.

L'elettroporazione di siRNA alla modulazione dell'autofagia nell'herpes Simplex Virus Tipo 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 ...

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Espianto di tessuto corneale suino per studiare l'efficacia degli antivirali Herpes Simplex Virus-1

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

Plaquing di herpes simplex virus

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells

Source: D&#252;thorn, A., et al. <a href="https://app.jove.com/v/60190">siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells.</a> J. Vis. Exp. (2019).
This video demonstrates a small interfering RNA (siRNA)-based technique to interfere with autophagic flux. A siRNA targeting FIP200 mRNA is introduced into monocyte-derived immature dendritic cells (iDCs) using electroporation, causing the knockdown of FIP200 expression and leading to the inhibition of nuclear lamin disruption via autophagy. Upon adding herpes simplex virus (HSV), the nuclear egress of viral progeny is inhibited due to the absence of lamin disruption via autophagic flux.

Inibizione dell'autofagia basata su siRNA in cellule dendritiche immature infettate da virus dell'herpes simplex

Monocyte-derived DCs were generated from leukapheresis products of healthy donors. For this, a positive vote from the local ethics committee has been ...