Name: in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection
Uploaded: 2014-11-27T13:00:00.000+00:00
Duration: 10 min 39 s
Description: Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

Tutti gli esperimenti sugli animali sono stati approvati dalla cura e l&#39;uso Comitato animali presso l&#39;Università del Texas Medical Branch. 1. Isolamento delle DC da topi non infetti e WNV infettate <ol><li> Per età e per sesso partita 6-10 settimane di età, wild-type C57BL / 6 (B6) e MyD88 - / - mice. Inoculare per via intraperitoneale (ip) con 500 unità formanti placca (PFU) di WNV NS4B- P38G mutante. Al giorno 3 dopo l&#39;infezione, eutanasia B6 e MyD88 - / - mice con CO 2. Utilizzare i topi non infetti di entrambi i gruppi come controlli. Utilizzare tre topi da ciascun gruppo. </li><li> Pelliccia bagnato sul lato sinistro del mouse sacrificato con etanolo al 70% e tagliato via il pelo lungo il lato sinistro del mouse, circa a metà strada tra le gambe anteriori e posteriori. Tagliare aprire la cavità del corpo. Rimuovere la milza utilizzando le pinze. Posizionare la milza in una capsula di Petri con RPMI. </li><li> Isolare DC milza utilizzando sfere magnetiche anti-CD11c secondole istruzioni del produttore. </li></ol> 2. Purificazione e etichettatura delle cellule T di OTII topi transgenici <ol><li> Harvest una milza da un ingenuo del mouse OTII come descritto al punto 1.2 di cui sopra e omogeneizzare la milza tra le estremità smerigliato di due slitte. Trasferire la sospensione cellulare in una provetta conica da 15 ml, aggiungere mezzo RPMI fino a 14 ml. Sia la sospensione riposare per 5 min e 13 ml di trasferimento ad una nuova provetta conica da 15 ml. </li><li> Isolare le cellule CD4 + T spleniche utilizzando un kit di isolamento di cellule CD4 + T secondo le istruzioni del produttore. </li><li> Trasferire le cellule in una provetta da 50 ml, lavare due volte con PBS. Risospendere le cellule in PBS con albumina sierica bovina 0,5% (BSA) a 1 x 10 6 cellule per ml, e aggiungere 0,5 mmol / ml di CFSE. Incubare a 37 ° C per 10 minuti, al riparo dalla luce. </li><li> Aggiungere 5 ml terreno completo a freddo (RPMI-1640 con siero fetale bovino 10% di calore-inattivato, 1/100 volantibiotici / antimicotici, 1/100 vol L-glutammina e 1 / 1.000 vol di 1.000 x 2-mercaptoetanolo). </li><li> Incubare in ghiaccio per 5 min. Lavare le cellule una volta con PBS, fix 0.2 x 10 6 etichettati cellule CD4 + T in 2% PFA e acquisire su un citofluorimetro. Utilizzare questo esempio per determinare il livello basale di CFSE. Re-sospende il resto delle cellule etichettate in terreno completo. </li></ol> 3. Co-cultura OTII T cellule con DC di topi infettati da WNV <ol><li> Cultura 2 x 10 5 purificati cellule CD4 + T di topi OTII solo o con DC purificate di wild-type o MyD88 - / - mice (2 x 10 4) in un piatto ben 24, con o senza residui OVA 323-339 (1 g / ml) in 1 ml di mezzo completo per pozzetto. </li><li> Incubare le cellule a 37 ° C per 5 giorni. Celle di raccolta, lavare due volte in tampone FACS (PBS con 1% FBS) e fissare in 0,25 ml 2% PFA. Vortex immediatamente. </li></ol> 4. Citometria a flusso Analisi di In Vitro &lt;/ Em&gt; T cellulare adescamento <ol><li> Per l&#39;acquisizione, fare doppio clic sull&#39;icona del software di citometria a flusso. Per una trama densità lineare FSC-A vs linear SSC-A, selezionare i canali da 0 a 200.000 in FSC-A e 0 a 200.000 su SSC-A. Quindi creare un cancello su cellule vive (P2; vedi Figura 1A). </li><li> Per analizzare l&#39;intensità di fluorescenza di CFSE, aprire l&#39;istogramma del canale FL1 e acquisire 20.000 eventi sulla popolazione gated. Impostare un marcatore su campioni di cellule in coltura OTII sola (Figura 1A). </li><li> Acquisire i campioni per le cellule OTII co-coltura con wild-type DC (Figura 1B) o MyD88 - / - DC (Figura 1C) in modo simile. </li></ol> 5. Isolamento e stimolazione di splenociti per citochine saggi <ol><li> Infettare B6 e MyD88 - / - mice con WNV NS4B-P38G mutante utilizzando la stessa procedura descritta in step 1.1. Al giorno 30 dopo l&#39;infezione, ri-sfida superstite topi con 2.000 PFU del wild-type WNV ceppo ip A giorni 8 e 21 di infezione WNV post-primaria o al giorno 4 dopo l&#39;infezione secondaria, eutanasia topi con CO 2 per la raccolta di milza . NOTA: Abbiamo usato 2-3 topi per gruppo, per ogni punto di tempo. </li><li> Fare una sospensione singola cella di splenociti come sopra descritto al punto 1.2 e 2.2 passo. Contare le cellule utilizzando un emocitometro e li ri-sospendere in 10 ml di media completo. </li><li> Diluire le cellule con mezzo completo a 2,5 x 10 6 cellule / ml. Aggiungere 1 ml di splenociti in un tubo micro-centrifuga 1.5 ml. </li><li> Per simulare cellule CD8 + T, diluire NS4B ed E peptidi specifici WNV (SSVWNATTA e IALTFLAV) a 1 mg / ml in DMSO. Per la stimolazione di cellule CD4 + T, diluire NS3 ed E peptidi specifici WNV (RRWCFDGPRTNTILE e PVGRLVTVNPFVSVA) a 1 mg / ml in DMSO. Aggiungere 10 ml di peptidi alle cellule followed da 1 ml di Brefeldina A soluzione. Utilizzare le cellule senza trattamento peptidi come controlli. Mescolare le cellule. </li><li> Punzone due fori con un ago da 18 G nel tappo della provetta. Incubare le cellule a 37 ° C per 5 ore. </li></ol> 6. intracellulare di citochine colorazione <ol><li> Trasferire le cellule in un nuovo tubo micro-centrifuga. Spin celle per 5 minuti a 300-400 xg e versare il surnatante. Aggiungere 1 ml FACS tampone, rotazione 5 min a 300-400 xg e risospendere cellule pipettando con 120 microlitri FACS buffer. </li><li> Aggiungere 2 ml Fc bloccante e incubare cellule a temperatura ambiente per 10 min. Aggiungere 1 ml FACS buffer per ogni provetta. Spin 5 min a 300-400 x g. </li><li> Risospendere le cellule in 300 ml FACS tampone e dividerli in tre provette (circa 0,8 x 10 6 cellule per provetta). Come mostrato nella Tabella 1, la riserva di primo tubo di ciascuna condizione di coltura per ratto IgG-PE colorazione. Per il secondo tubo, aggiungere 3 ml di anti-CD4 APC per CD4 peptidi-trattati cellule, 3 μl di anti-CD8 FITC alle cellule CD8 peptidi-trattati o entrambi gli anticorpi alle cellule senza trattamento peptidi. Utilizzare il terzo tubo di risarcimento colorazione. Per ogni condizione cultura, utilizzare tre provette di cellule colorate con APC-coniugato CD4, CD8 FITC coniugato o in PE etichettati anticorpi CD3 soli (3 ml per provetta) come controlli di compensazione per FL1, FL2 e canali FL4. </li><li> Cellule Vortex brevemente. Lasciare in ghiaccio per 20 minuti e proteggere dalla luce. Aggiungere 1.4 ml di tampone FACS. Spin 5 min a 300-400 xg e versare il surnatante. </li><li> Agitare per disturbare pellet (o brevemente vortex). Risospendere pellet cellulare in 250 ml di fissaggio / permeabilizzazione solutionby pipettaggio. Incubare a temperatura ambiente per 20 min e proteggere dalla luce. </li><li> Aggiungere 1,2 ml di tampone FACS. Spin 5 min a 300-400 XG e risospendere le cellule in 300 microlitri di buffer FACS. NOTA: A questo punto, le cellule possono essere trasferiti ad un laboratorio BL2 per ulteriore trasformazione o conservata in frigorifero e protetto dalla luce per up per tre giorni. </li><li> Aggiungere 500 microlitri FACS buffer. Spin 5 min a 300-400 XG e risospendere le cellule in 500 l di 1x tampone di permeabilizzazione / lavaggio. Spin 5 min a 300-400 x g. </li><li> Risospendere le cellule in 100 microlitri 1x Perm / Wash, aggiungere 3,5 microlitri anti-IFNγ-PE al tubo precedentemente aggiunto con anticorpi per CD4 e / marcatori cellulari o CD8 T in fase 6.3 e 3.5 ml topo IgG-PE nel tubo riservata per il controllo IgG per 25 min in ghiaccio e proteggere dalla luce. Lavare le cellule aggiungendo 1,4 ml di 1x tampone di permeabilizzazione / lavaggio. Spin 5 min a 300-400 x g. Ripetere la fase di lavaggio con l&#39;aggiunta di 1,4 ml di 1x tampone di permeabilizzazione / lavaggio. </li><li> Spin 5 min a 300-400 xg e decantare il surnatante. Lavare le cellule aggiungendo 1,4 ml di tampone FACS e risospendere in 400 microlitri di tampone FACS per acquisizione finale. </li></ol> 7. Citometria a flusso Analisi delle citochine intracellulari colorazione <ol><li> Utilizzare le stesse impostazioni di acquisizione come al punto 4.1. Create un cancello su cellule vive (P2; Figura 2A). Regolare le tensioni con i tubi di compensazione per ogni gruppo. </li><li> Aperti due nuovi dot plot, una per visualizzare i dati raccolti per FL1 vs. FL2 (CD8 vs. IFN-γ), e l&#39;altra è per visualizzare i dati raccolti per FL4 vs. FL2 (CD4 vs. IFN-γ). Acquisire 50.000 eventi per la popolazione gated di ogni campione. </li></ol>

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WNV è un patogeno BL3. A causa delle norme di sicurezza, test immunologici con i campioni WNV-infetti sono spesso limitati dalla disponibilità di attrezzature in BL3 strutture o più lungo e noioso per eseguire. In un recente studio, abbiamo utilizzato due metodi basati citometria a flusso per studiare cellule risposte immunitarie mediate durante l&#39;infezione WNV 5. In entrambi i test, le cellule infettate da WNV sono stati trattati con 1-2% direttamente o con una soluzione di fissaggio / permeabilizzazi...

Virus West Nile (WNV), una neurotropo, plus-rilevati flavivirus, è una minaccia per la salute pubblica emergente. Attualmente, vaccini sono stati approvati per uso umano 1. Un ceppo WNV attenuato, che ha una sostituzione P38G nel non strutturale (NS) di proteine ​​4B, è noto per indurre non letalità nei topi ma citochine superiore innate e le risposte delle cellule T nei topi wild-type di WNV NY99 ceppo 2. I topi immunizzati con il mutante NS4B-P38G stati tutti protetti da una sfida secondario con letale wild-type WNV. Questo suggerisce che il mutante NS4B-P38G ha caratteristiche adatte per un vaccino candidato ideale. I meccanismi con cui il mutante NS4B-P38G induce protezione ad alta immunità adattativa non sono chiaramente ancora capito. Toll-like receptors (TLR), che riconoscono pattern molecolari associati ai patogeni, svolgono un ruolo fondamentale nell&#39;avvio di immunità innata alle infezioni virali. Il percorso di segnalazione nucleo TLR utilizza mieloide risposta differenziazione primaria geNE 88 (MyD88) come scheda primaria 3,4. In un recente studio, la segnalazione MyD88 ha dimostrato di giocare un ruolo importante nello sviluppo delle cellule mediata durante WNV NS4B- P38G infezione nei topi mutanti 5. Le cellule dendritiche (DC) sono una delle più importanti cellule presentanti l&#39;antigene espositrici la capacità unica di avviare le risposte delle cellule T primarie durante l&#39;infezione virale 6,7. Cellule T CD4 + e CD8 + entrambi contribuiscono all&#39;immunità protettiva di lunga durata e sono importanti per la sopravvivenza di accoglienza a seguito di tipo selvaggio WNV infezione 8,9. Due saggi immunologici sono stati usati in questo studio per valutare le funzioni di queste cellule nei topi mutanti infettati NS4B-P38G. In primo luogo, un dosaggio priming di cellule T in vitro è stato utilizzato per confrontare la capacità presentanti l&#39;antigene di DC di wild-type WNV-infetti e MyD88 - / - mice. Per aumentare la sensibilità del test, ingenuo CD4 &lt;sup&gt; + cellule T sono stati isolati da OTII topi transgenici che esprimono uno specifico Vα2 / Vβ5 TCR per l&#39;ovoalbumina pollo (OVA) peptide 323 - 339. DC di WNV topi infetti sono stati purificati, e co-coltura con carboxyfluorescein succinimidil Pasqua (CFSE ) -labeled cellule CD4 + T in presenza di OVA peptide. Dopo 5 giorni di co-coltura, le cellule sono state raccolte e fissate con paraformaldeide (PFA) e analizzati mediante citometria a flusso. Saggi proliferativa, sono state tradizionalmente effettuata mediante incorporazione di 5-bromo-2&#39;-deossiuridina (BrdU) o triziata deoxyriboside timina (3 HTdr) 10. Tuttavia, questi test sono o radioattivi e / o che necessitano di apparecchiature speciali a livello di biosicurezza (BL) 3 strutture, dove vengono condotti studi WNV. L&#39;analisi citofluorimetrica della proliferazione dei linfociti dimezzando seriale dell&#39;intensità di fluorescenza del colorante vitale CFSE è diventato più comunemente usato in saggi immunologici, come il colorante è più stabilee uniformemente incorporati in cellule, rilevata facilmente mediante citometria di flusso, ed è non radioattivo 11. Il test ha anche la capacità di valutare il numero di divisioni cellulari. Uno dei principali vantaggi di questo test in studi WNV è che la fissazione delle cellule infettate con 1-2% PFA possa inattivare WNV 12, che consentirà l&#39;acquisizione dei campioni con un citometro a flusso in un laboratorio BL2. Successivamente, una procedura modificata intracellulare di citochine colorazione (ICS) è stato utilizzato per studiare il ruolo di MyD88 segnalazione nella regolazione del WNV specifiche risposte delle cellule T in NS4B-P38G topi mutanti-infettati. In questo saggio, splenociti isolati da topi infetti sono stati trattati in vitro con WNV peptidi specifici. Brefeldina A inserito mantenere le citochine all&#39;interno della cellula. Dopo 5 ore di incubazione, le cellule sono state raccolte, lavate e colorate per sottogruppi di cellule T. Le cellule sono state poi fissati in PFA, permeabilizzate e colorate per interferone (IFN) -γ e analizzate mediante citometria a flusso.Come con altri flusso-citometria saggio basato, una volta che le cellule vengono trattate con fissazione e tampone di permeabilizzazione contenente PFA, campioni infetti possono essere trasferiti ad un laboratorio BL2 per l&#39;ulteriore elaborazione e analisi. In diversi studi pubblicati, abbiamo usato ICS per misurare le funzioni effettrici delle cellule T in WNV-infetti topi 13,14. Anche se è ben consolidata, uno svantaggio principale di questo saggio è che la procedura è molto lunga e potrebbe essere più tempo quando eseguita all&#39;interno di strutture BL3. Qui, un metodo basato ICS-tube micro-centrifuga ha dimostrato di essere più fattibile, facile procedere e richiede meno tempo quando eseguita all&#39;interno di un laboratorio BL3.

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>11875</td><td>warm up at 37 &amp;deg;C</td></tr><tr><td>anti-CD11c magnetic beads</td><td>Miltenyi Biotec</td><td>130-052-001</td><td>follow the manufacturer&amp;rsquo;s instructions</td></tr><tr><td>anti-CD4 magnetic beads</td><td>Miltenyi Biotec</td><td>130-095-248</td><td>follow the manufacturer&amp;rsquo;s instructions</td></tr><tr><td>CFSE</td><td>Invitrogen</td><td>C34554</td><td></td></tr><tr><td>OVA residue 323-339</td><td>Genscript Corporation</td><td>RP10610</td><td></td></tr><tr><td>Peptides</td><td>Proimmune</td><td>PC0AD-D</td><td></td></tr><tr><td>Brefeldin A solution</td><td>BD Bioscience</td><td>555029</td><td></td></tr><tr><td>Mouse Fc Blocker</td><td>e-Bioscience</td><td>14-0161-85</td><td></td></tr><tr><td>APC-conjugated CD4</td><td>e-Bioscience</td><td>17-0041-81</td><td></td></tr><tr><td>FITC-conjugated CD8</td><td>e-Bioscience</td><td>11-0081-82</td><td></td></tr><tr><td>Fixation/Permeabilization Solution</td><td>BD-Bioscience</td><td>554722</td><td></td></tr><tr><td>Permeabilization/wash buffer</td><td>BD-Bioscience</td><td>554723</td><td></td></tr><tr><td>anti-IFN&amp;gamma;-PE</td><td>e-Bioscience</td><td>12-7311-82</td><td></td></tr><tr><td>Accuri flow cytometer</td><td>BD Bioscience</td><td></td><td></td></tr></tbody></table>

in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

Un virus attenuato West Nile (WNV), una non strutturale (NS) 4B-P38G mutante, indotta citochine superiore innata e le risposte delle cellule T rispetto al wild-type WNV nei topi. Recentemente, mieloide fattore di differenziazione 88 (MyD88) Segnalazione ha dimostrato di essere importante per priming di cellule T iniziale e lo sviluppo delle cellule di memoria T durante l&#39;infezione WNV mutante NS4B-P38G. In questo studio, due flusso metodi basati citometria - una in vitro delle cellule T saggio di adescamento e una citochina colorazione intracellulare (ICS) - sono stati utilizzati per valutare le cellule dendritiche (DC) e funzioni delle cellule T. Nella cella T adescamento test, la proliferazione cellulare è stato analizzato mediante citometria a flusso dopo co-coltura di cellule dendritiche da entrambi i gruppi di topi con estere carboxyfluorescein succinimidil (CFSE) - etichettato cellule CD4 + T di OTII topi transgenici. Questo approccio ha fornito una determinazione accurata della percentuale di cellule proliferanti CD4 + T con migliorata significativamente sensibilità globale rispetto alla tradizioneal saggi con reagenti radioattivi. Un sistema provetta è stato utilizzato in entrambe le procedure di coltura cellulare e citochine colorazione del protocollo ICS. Rispetto al tradizionale sistema basato piastra di coltura tissutale, questa procedura modificata era più facile per eseguire a livello di biosicurezza (BL) 3 strutture. Inoltre, le cellule infette WNV- sono state trattate con paraformaldeide in entrambi i test, che ha consentito un&#39;ulteriore analisi fuori strutture BL3. Nel complesso, questi in vitro test immunologici possono essere utilizzati per valutare in modo efficace risposta immunitaria cellulo-mediata durante l&#39;infezione da WNV.

Nel saggio T cellule adescamento, le cellule CD4 + T CFSE etichettati sono state coltivate con DC purificate dalla wild-type-mutante infetto NS4B-P38G e MyD88 - / - mice in presenza o assenza di peptidi OVA. Cellule T etichettati coltivate solo con o senza OVA per 5 giorni sono stati usati come controlli negativi. Come mostrato nella Figura 1A, le cellule T sono state gated totale per l&#39;analisi di fluorescenza sul canale FL1. Il marcatore è stato istituito sulla base delle app...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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Immunology and Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

8.2K Views.  The University of Texas Medical Branch. Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Video: In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

Misura della γHV68 infezione nei topi

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

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Immunologia e infezioni

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissezione Innate Immune Signaling in Evasion virale di produzione di citochine

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Studi del Virus di riossidazione A in un modello murino di infezione

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Valutazione delle risposte a cellula T Zika Virus-specifici in organi di nuova di Ifnar1 infetti- / - Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Infezioni da arbovirus come strumenti di Screening per l'identificazione di fattori virali immunomodulatori e Host antivirale

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Istituzione dell'infezione virale e analisi dell'interazione ospite-Virus in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

Isolamento e quantificazione del virus zika da più organi in un mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

Rilevamento del ceppo Wolbachia wAlbB nelle linee cellulari Aedes albopictus

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

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