Name: small rna transfection in primary human th17 cells by next generation electroporation
Uploaded: 2017-04-13T15:00:00
Description: Watch this Scientific Journal Video about Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation at JoVE.com

This work was supported by the US National Institutes of Health grants (R01HL109102, P01HL107202, U19CA179512, F31HL131361), a Leukemia &amp; Lymphoma Society scholar award (K.M.A.), and the National Institute of General Medical Sciences (NIGMS) Medical Scientist Training Program (Grant #T32GM007618) (M.M.).

Ce protocole est conforme aux directives de l&#39;UCSF pour l&#39;éthique de la recherche humaine. 1. Préparation de T culture cellulaire, l&#39; isolement des cellules T CD4 +, et Th17 Polarisation <ol><li> Le jour 0, le manteau 6 puits des plaques de culture de tissu avec 1,5 ml par puits d&#39;anti-CD3 humain (2 pg / ml) et du CD28 anti-humain (4 pg / mL) dans du PBS avec du calcium...

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Ce protocole fournit un procédé amélioré pour la livraison de petits ARN dans des cellules Th17 humains. Bien que les cellules Th17 humains ont été utilisés ici, cette méthode de électroporation avec de petits ARN peut être utilisé avec d&#39;autres sous-ensembles T auxiliaires humain primaire, comme Th1, Th2 et Treg. Il n&#39;a pas bien fonctionné pour naïfs cellules T CD4 + de sorte que les cellules doivent être activées dans la culture avant la transfection. Pour ce protocole, nous avons opt...

Cellules T CD4 + sont orchestrateurs essentiels de la réponse immunitaire adaptative. Naïf cellules T CD4 + sont capables de se développer en plusieurs différentes cellules T effectrices (par exemple , Th1, Th2, Th17, etc.), chacun avec son propre ensemble de cytokines caractéristiques et des facteurs de transcription, en fonction du micro - environnement local 1. Les décisions de la lignée que les cellules T font sont essentielles tant pour l&#39;immunité protectrice et la tolérance à l&#39;auto. Les cellules Th17 sont un sous - ensemble de cellules T connues pour lutter contre les bactéries extracellulaires et les champignons, mais les réponses inappropriées sont également impliqués dans la pathogenèse de multiples maladies auto - immunes et inflammatoires telles que la sclérose en plaques et le psoriasis 2, 3. Th17 cellules humaines peuvent être générées à partir de cellules naïves T CD4 + in vitro en leur fournissant un environnement approprié de polarisation 4. Vario<html> nous combinaisons ont été utilisées des cytokines IL-1β, IL-23, TGFß, et l&#39;IL-6 pour le développement des cellules Th17 humaines. Th17 cellules humaines expriment CCR6, un récepteur de chimiokine qui est couramment utilisé pour identifier cette population de cellules et sont définies par l&#39;expression de leur facteur de transcription principale, RORγt (codée par RORC) 5, 6. les cellules Th17 ont la capacité d&#39;exprimer des cytokines multiples, mais IL-17A est la cytokine effectrice définissant lignée produite par ces cellules. Nous avons examiné l&#39;expression des trois marqueurs associés Th17 (CCR6, RORγt, IL-17A) pour évaluer la solidité de notre essai de différenciation Th17 in vitro humaine. De plus, nous avons cultivé des cellules T CD4 + humains dans des conditions non-polarisation, où aucune des cytokines ou des anticorps bloquants ont été ajoutés aux milieux de culture à utiliser comme contrôle négatif puisque l&#39; expression de ces marqueurs Th17 devrait être très faible ou absente. ve_content &quot;&gt; Une façon d&#39;étudier le développement normal des cellules T humaines et de la biologie est de manipuler l&#39;expression génique au cours de leur développement. ARN court interférent (ARNsi) sont des petites molécules d&#39;ARN synthétiques qui ciblent les ARNm codant pour des protéines et peuvent être utilisés pour réduire l&#39;expression d&#39;un gène spécifique . Les microARN (miARN) sont de petits ARN non codants endogènes connus pour moduler miARN post-transcriptionnelle l&#39; expression génique. ont été montré à jouer un rôle important dans les deux murin et la biologie des cellules T humaines, y compris dans les cellules Th17 7, 8, 9. Il est essentiel de disposer de méthodes fiables de manipulation petite activité d&#39;ARN dans les cellules T humaines pour étudier leurs effets sur l&#39;expression des gènes et, finalement, sur la biologie des cellules T humaines. ici, nous décrivons un outil facile à utiliser, protocole cohérent et fiable que nous avons développé pour l&#39;introduction les petits ARN et des acides nucléiques verrouillés (LNA synthétiques, des acides nucléiques modifiés chimiquement avec une stabilité accrue)dans les cellules immunitaires, et plus spécifiquement dans les cellules Th17 humaines. Il existe plusieurs méthodes alternatives de l&#39; introduction de petits ARN dans les cellules de mammifères, qui se situent généralement dans des catégories chimiques, biologiques ou physiques 10. Couramment utilisés méthodes chimiques, y compris transfections à base de lipides et transfections calcium-phosphate, reposent sur la création de complexes ADN chimique qui sont plus efficacement absorbé par les cellules. En général, les méthodes chimiques ne sont pas aussi efficaces pour la transfection de cellules T primaires. Le procédé biologique la plus courante consiste à utiliser un vecteur viral (par exemple, retrovirus ou lentivirus), qui insère directement l&#39; ARN étranger dans un hôte en tant que partie de son cycle de replication naturel. transduction virale prend généralement plus de temps, en particulier lorsque l&#39;on tient compte du temps pour le clonage moléculaire des plasmides proviraux. En outre, des vecteurs de transduction viraux peuvent être potentiellement dangereux pour les chercheurs humains. L&#39;électroporation est une physique méthode d&#39;induire la perméabilisation de la membrane en soumettant les cellules à des impulsions à haute tension, ce qui permet d&#39;entrer des acides nucléiques de manière transitoire dans la cellule où ils peuvent agir sur leur cible. Les instruments traditionnels de électroporation ne sont pas efficaces pour transfecter les lymphocytes primaires. Cependant, l&#39;électroporation de la prochaine génération optimisée est avérée être capable de transfecter des cellules T à un rendement très élevé, en particulier lorsque le matériau à transfecter est un petit ARN. Le terme de nouvelle génération est vaguement utilisé pour différencier les deux nouvelles plates - formes (par exemple, Neon, Amaxa) de machines d&#39;électroporation traditionnelles. En outre, cette méthode est facilement extensible pour des écrans de débit modéré avec jusqu&#39;à environ 120 petits ARN en une seule expérience, souvent en utilisant des réactifs de synthèse validés. Il est important, transfections succès peut être atteint en moins de 16 h après l&#39;activation des lymphocytes T. L&#39;inconvénient de cette méthode, cependant, est qu&#39;il ne donne pas lieu à incorporati génomique stablesur, et est donc transitoire. Par conséquent, il vaut l&#39;effort supplémentaire pour créer une construction d&#39;expression stable qui peut être emballé dans un vecteur viral et exprimé avec succès dans les cellules T dans les cas où l&#39;expression à long terme d&#39;un petit ARN est nécessaire. Nous avons utilisé une transfection de prochaine génération (par exemple, le néon) pour délivrer ARN simple brin ou double brin synthétiques ou divers outils oligonucléotides LNA à des fins différentes 11, 12, 13. L&#39;interférence ARN efficace peut être induite chez la souris et les cellules T primaires humains en utilisant l&#39;ARN court interférant double brin (siRNA). Ce protocole décrit les conditions optimales pour l&#39;utilisation de cette technique dans les cellules Th17 humaines. En plus de siARN, imitateurs miARN synthétiques et les inhibiteurs disponibles dans le commerce peuvent être utilisés pour étudier le gain miARN et la perte de la fonction. imite miARN sont des molécules d&#39;ARN double brin très semblables à ARNsi, mais designed avec la séquence de miARN matures endogènes. les inhibiteurs de miARN sont d&#39;ARN modifiés chimiquement et / ou LNA à base d&#39;oligonucléotides simple brin qui se lient aux miRNAs natifs et antagonisent leur fonction. Nous avons constaté que tous ces outils peuvent être utilisés efficacement dans les lymphocytes T primaires en culture, y compris, mais sans s&#39;y limiter, les cellules Th17 humaines.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-human IL-17A PE</td>
			<td>ebioscience</td>
			<td>12-7179-42&#160;</td>
			<td>Clone: eBio64DEC17</td>
		</tr>
		<tr>
			<td>anti-human IFNg FITC</td>
			<td>ebioscience</td>
			<td>11-7319-82</td>
			<td>Clone: 4S.B3</td>
		</tr>
		<tr>
			<td>anti-human CD4 eVolve605</td>
			<td>ebioscience</td>
			<td>83-0047-42</td>
			<td>Clone: SK3</td>
		</tr>
		<tr>
			<td>mouse anti-human CD196 (CCR6) BV421</td>
			<td>BD biosciences</td>
			<td>562515</td>
			<td>Clone: 11A9</td>
		</tr>
		<tr>
			<td>anti-human RORgt AF647</td>
			<td>BD biosciences</td>
			<td>563620</td>
			<td>Clone: Q21-559</td>
		</tr>
		<tr>
			<td>anti-human CD45 eFluor450</td>
			<td>ebioscience</td>
			<td>48-9459-42</td>
			<td>Clone: 2D1</td>
		</tr>
		<tr>
			<td>Foxp3/Trascription Factor Staining Buffer Set</td>
			<td>ebioscience</td>
			<td>00-5523-00</td>
			<td>For intracellular transcription factor flow cytometry staining</td>
		</tr>
		<tr>
			<td>Hu FcR Binding Inhibitor Purified</td>
			<td>ebioscience</td>
			<td>14-9161-71</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoCult-XF T Cell Expansion Medium</td>
			<td>Stemcell Technologies</td>
			<td>10981</td>
			<td>&#34;Serum-Free Base Media&#34;</td>
		</tr>
		<tr>
			<td>MACS CD28 pure functional grade, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-375</td>
			<td>Clone: 15E8</td>
		</tr>
		<tr>
			<td>anti-human CD3 Purified</td>
			<td>UCSF monoclonal antibody core</td>
			<td>N/A</td>
			<td>Clone: OKT-3</td>
		</tr>
		<tr>
			<td>LEAF purified anti-human IL-4</td>
			<td>Biolegend</td>
			<td>500815</td>
			<td>Clone: MP4-25D2</td>
		</tr>
		<tr>
			<td>anti-human IFNg, functional grade purified</td>
			<td>ebioscience</td>
			<td>16-7318-85</td>
			<td>Clone: NIB42</td>
		</tr>
		<tr>
			<td>Recombinant human IL-23</td>
			<td>Peprotech&#160;</td>
			<td>200-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-1&#946;</td>
			<td>Peprotech&#160;</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TGF-&#946;1</td>
			<td>Peprotech&#160;</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-6</td>
			<td>Peprotech&#160;</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>siGENOME Control Pool, Non-targeting #2</td>
			<td>Dharmacon</td>
			<td>D-001206-14-05</td>
			<td></td>
		</tr>
		<tr>
			<td>siGENOME SMARTpool Human RORC</td>
			<td>Dharmacon</td>
			<td>M-003442-00</td>
			<td></td>
		</tr>
		<tr>
			<td>siGENOME SMARTpool Human PTPRC</td>
			<td>Dharmacon</td>
			<td>M-008067-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Human CD4 T Cells Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>11346D</td>
			<td>Human CD4+ T cell Isolation Kit</td>
		</tr>
		<tr>
			<td>Neon Tranfection System</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td>Next generation electroporation instrument</td>
		</tr>
		<tr>
			<td>Neon Tranfection System 10 &#956;l Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Resuspension Buffer T</td>
			<td>ThermoFisher Scientific</td>
			<td>Provided in kit (MPK1096)</td>
			<td>&#34;Transfection Resuspension Buffer&#34;</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>#07801</td>
			<td>Density Gradient Medium</td>
		</tr>
		<tr>
			<td>costar 6-well tissue culture treated plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>flat bottom plates</td>
		</tr>
		<tr>
			<td>costar 48-well tissue culture treated plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>flat bottom plates</td>
		</tr>
		<tr>
			<td>BD Pharm Lyse lysing buffer, 10 X</td>
			<td>BD biosciences</td>
			<td>555899</td>
			<td>Must make 1X solution with distilled water prior to use</td>
		</tr>
	</tbody>
</table>

small rna transfection in primary human th17 cells by next generation electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

La première étape pour l&#39; élaboration d&#39; un système fiable de électroporation avec succès les cellules Th17 humaines était de générer in vitro robuste différenciées des cultures de cellules Th17 humaines. Les cellules T cultivées dans des conditions de polarisation de Th17 ont exprimé le récepteur de chimiokine CCR6 et le facteur de transcription RORγt (figure 1A, gauche). Ces marqueurs ne sont pas exprimés lorsque les lymphocytes T ont ?...

Watch this Scientific Journal Video about Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation at JoVE.com

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Petit ARN primaire dans Transfection Th17 cellules humaines par électroporation Next Generation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

The overall goal of this procedure is to efficiently transfect primary human T cells with small RNA reagents using next generation electroporation. This method can help answer key questions in T cell biology, such as how specific gene products regulate T cell differentiation and immune function. The main advantage of this technique is that it is a highly efficient and reliable method to transfect primary human T cells with sRNAs and other synthetic small RNA reagents.This method can provide insight into the biology of human Th17 cells, but it can also be applied to other systems, such as other lymphocytes from humans or mice. On day zero, coat six well tissue culture plates with 1.5 milliliters per well of antihuman CD3 and antihuman CD28 in PBS with calcium and magnesium for at least two hours at 37 degrees Celsius. Once the cord-blood mononuclear cells, or CBMCs, have been isolated and washed, perform CD4 positive T cell isolation by negative selection using a commercial human CD4 positive T cell kit.Add isolation buffer and re-suspend the mononuclear cells. Transfer the cells to a new five milliliter tube, then add 100 microliters of fetal bovine serum, or FBS, and 100 microliters of the antibody mix per tube, then incubate each tube at four degrees Celsius for 20 minutes on an orbital shaker to mix well. After the incubation, add three to four milliliters of isolation buffer to wash the cells.Centrifuge the cells at 300 times g for eight minutes at four degrees Celsius to pellet the cells. Carefully aspirate the supernatant. Transfer magnetic beads into a new tube and add an equal volume of isolation buffer to wash the beads.Use a one milliliter micropipet to mix the beads well and then place the tube in the magnet for at least one minute. Carefully aspirate the supernatant and re-suspend the magnetic beads in the same volume initially transferred prior to the wash. Add 500 microliters of the isolation buffer to each tube and re-suspend the cells, then add 500 microliters of pre-washed magnetic beads.Incubate the cells with the magnetic beads for 15 minutes on an orbital shaker at room temperature to mix well. After incubation, using a one milliliter micropipet, thoroughly pipet the cells at least 10 times, then add three to four milliliters of isolation buffer and place each tube in the magnet for two minutes. Carefully transfer the negatively selected CD4 positive T cells that are in the supernatant to a new tube.Once CD4 positive T cell isolation is completed, keep the cells on ice and count the cells with a hemocytometer. Wash the antibody coated plates two times with PBS, then add 1.5 milliliters of two X mix of Th17 polarizing media to each well. Next, centrifuge the human CD4 positive T cells to pellet the cells.Carefully aspirate the supernatant and discard it, then re-suspend the cells in serum free base media and plate the cells so that the final volume is three milliliters per well and polarizing cytokines are now at a one X final concentration, then place the plates in 5%carbon dioxide in a 37 degrees Celsius incubator for two days. On day two, coat 48 well tissue culture plates with 250 microliters per well of antihuman CD3 and antihuman CD28 in PBS with calcium and magnesium for at least two hours at 37 degrees Celsius. To prepare small RNAs for transfection, aliquot one microliter of a five micromolar stock solution of siRNA into a 1.5 milliliter microcentrifuge tube for each transfection.Keep all of the tubes on ice. Next, wash the antibody coated plates two times with PBS, then add 500 microliters of one X Th17 polarizing media to each well. After the culture plates and transfection reagents are prepared, re-suspend the cells with a one milliliter micropipet, pipetting gently but ensuring that all the cells are detached from the bottom of the wells.Pool the cells into a conical tube and centrifuge the tubes at 500 times g for five minutes at four degrees Celsius. Carefully aspirate the supernatant and then re-suspend the cells in at least one milliliter of PBS to wash. Count the live Th17 cells with a hemocytometer, using trypan blue exclusion to assess viability, then pellet the cells in a microcentrifuge tube.Carefully aspirate the supernatant and re-suspend the cells using the provided re-suspension buffer from the kit. Keep the cells at room temperature. Add nine microliters of cells to one microliter of small RNA in each microcentrifuge tube.Pipet once to mix the cells and small RNAs for transfection, then load the cells into the provided pipet electrode tip. To ensure optimal transfection, avoid introducing air bubbles into the pipet electrode tip prior to transfection. We add 9.5 microliters of cell suspension to ensure there is enough mixture to prevent creating bubbles in the electroporation pipet.Fill the provided cuvette with three milliliters of room temperature electrolytic buffer. Place the cuvette inside the neon pipet station and then place the pipet into position inside the cuvette. Immediately electroporate each 10 microliter mix of cells and small RNA.After the electroporation is complete, directly add the cell mixture to 500 microliters of one X Th17 polarizing media in prepared wells of a culture plate. Finally, place the plates in a 5%carbon dioxide, 37 degrees Celsius incubator for two more days. Work carefully but quickly to prevent cell death.If there are many samples to electroporate, the cells can be collected and transfected in batches. This contour plot shows that T cells cultured under Th17 polarizing conditions expressed the chemokine receptor, CCR6, and the transcription factor, ROR gamma T, which were not expressed when T cells were cultured under non-polarizing conditions. T cells cultured under Th17 polarizing conditions also produced IL-17A upon re-stimulation, but not under ThN conditions.Th17 differentiation still occurs after transfection with small RNAs, but in some experiments, there is an observed reduction in the frequency of IL-17A production. This demonstrates the importance of having a chemistry matched small RNA control for comparison within each experiment. Viability is maintained after transfection with small RNAs and is typically greater than 70%Transfection of human Th17 cells on day two with an siRNA pool targeting the gene, PTPRC, which encodes CD45, strongly reduced the expression of CD45 compared to a chemistry matched control siRNA.The unimodal population shift in CD45 expression, seen here, indicates near 100%transfection efficiency. Reducing ROR gamma T expression by RNAi had a functional effect, as these cells exhibited reduced IL-17A production compared to cells transfected with a control siRNA. After watching this video, you should have a good understanding of how to transfect in vitro polarized human Th17 cells with small RNAs using next generation electroporation.

Research

JoVE Journal

Immunology and Infection

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

La transfection et la mutagenèse des gènes cibles dans les cellules de moustiques en Fermés d&#39;acide nucléique modifiés Oligonucléotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

Recherche

Immunologie et infection

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

Génération des cultures organotypiques de Raft kératinocytes humains primaires

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

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Génération de cellules T régulatrices induites de l&#39;homme primaires cellules T naïves et mémoire

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

Modification piggyBac système transposon des cellules T humaines primaires

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative<em&gt; In vitro</em&gt; Test pour mesurer l&#39;adhésion des neutrophiles à Activé cellules endothéliales microvasculaires humaines primaires dans des conditions statiques

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

La transfection très efficace des droits de THP-1 par les macrophages nucléofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

Génération des cellules de knock-out primaire et élargi NK humaine à l’aide de Cas9 ribonucléoprotéines

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

Quantification de l’amélioration dépendante des anticorps du Virus dans les cellules humaines primaires Zika

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

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

Identification des souris et des répertoires d’anticorps humain par séquençage de prochaine génération

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

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Imagerie confocale d’ARN bicaténaire et récepteurs de reconnaissance de modèle dans l’infection par le Virus ARN de polarité négative

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...