A. propager le virus dans la ligne de C6/36 cellule. <ol><li> Les cellules se développent à 80% de confluence dans flacon de 75 cm2; </li><li> Retirez le support avec 3-4 ml restant dans le ballon; </li><li> Prenez 0,5 ml stock de virus et d&#39;ajouter dans la cellule ci-dessus avec la multiplicité d&#39;infection de 1,5 particules virales par cellule. Secouer le flacon pendant 15 minutes lentement à la température ambiante. </li><li> Incuber pendant 45 minutes à 5% de CO 2 et 37 ° C </li><li> Ajouter 30 ml de milieu, et incuber pendant 5 jours. </li></ol> B. Préparer le mélange de virus et de sang <ol><li> Détachez la cellule avec le racloir et le transfert de la cellule et les médias pour les 50 ml tubes coniques; </li><li> Centrifugat à 800g pendant 10 minutes; recueillir le surnageant, mais laissez 1ml de surnageant avec le culot cellulaire; </li><li> Gel de la cellule au-dessus de granulés de CO 2 sec puis dégel dans l&#39;eau bain à 37 ° C, répéter deux à trois fois; </li><li> Centrifugat à 800g pendant 10 minutes, prenez le surnageant, et de combiner avec le surnageant recueilli de l&#39;étape 3; </li><li> Collecter le sang total humain avec la même procédure (étape 3) pour la préparation de la culture des gamétocytes pour infecter des moustiques Anophèles par Plasmodium falciparum; </li><li> Combinez la même quantité de sang humain total ci-dessus avec le surnageant de virus de l&#39;étape 4, et ajouter le sérum humain (10% de l&#39;ensemble du volume). </li><li> Incuber le mélange ci-dessus du sang humain et le virus de la dengue pendant 30 minutes à 37 ° C bain d&#39;eau </li></ol> Nourrir les moustiques C. Cette procédure est presque identique à ce qui a été décrit dans la section pour les infections à Plasmodium falciparum chez les moustiques. Nous dosage de l&#39;infection intestin moyen au jour 7, et l&#39;infection salivaires à 14 jours après le sang maternel. Tout le matériel ayant été en contact avec le virus sont d&#39;abord traitées avec de l&#39;éthanol 75% puis à l&#39;eau de Javel à 10%. D. Assay le titre du virus dans les tissus des moustiques <ol><li> Deux ou trois jours avant la dissection des tissus de moustiques, de grandir dans une cellule de C6/36 plaque à 24 puits de telle sorte que la cellule atteindre 80% de confluence le jour lorsque le dosage est effectué; </li><li> Les moustiques sont anesthésiés à 4 ° C, sacrifiés et surface stériles en les trempant dans de l&#39;éthanol 75% pendant 1 minute. Puis les moustiques ont été lavés avec de l&#39;eau stérile à deux fois avant la dissection des glandes salivaires ou de l&#39;intestin moyen sous la loupe binoculaire. A partir de cette étape, toute la procédure ci-dessous doit être menée dans un environnement stérile, comme une enceinte de sécurité biologique. Ces tissus sont transférés dans un tube contenant 150 ul des médias et homogénéisé avec un moteur Kontes granulés pilon pendant 90 secondes; </li><li> Faire une dilution de 1:100 en ajoutant 10 pl de l&#39;homogénat dans les médias 990 ul; </li><li> Faire encore 1: 10, 1:100 et 1:1000 dilution avec le milieu dans la plaque de 96 puits; </li><li> Retirez le support de la plaque de 24 puits, et ajouter 100 ul de chacune de ces quatre dilutions dans chaque puits correspondants; </li><li> Agiter la plaque doucement pendant 15 minutes à la température ambiante, puis incuber pendant 45 minutes à 5% de CO 2 et 37 ° C; </li><li> Ajouter 1ml de méthycellulose superposition (1%) dans chaque puits; </li><li> Incuber les plaques à 37 ° C avec 5% de CO 2 pendant 5 jours; </li><li> Les étapes à partir d&#39;ici n&#39;ont pas besoin d&#39;un environnement stérile, mais comme avec tous les soins d&#39;autres agents pathogènes doivent être prises à tout moment. Jeter superposition méthycellulose de chaque plaque et éponger pour enlever l&#39;excès des médias </li><li> Ajouter 1 ml de méthanol / acétone fixatif (1:1) à chaque puits. Conserver à 4 ° C pendant 1 h; </li><li> Verser le fixateur, et se laver une fois avec du PBS 1X; </li><li> Faire dilution de 1:1000 à l&#39;anticorps primaire contre le virus avec 5% blotto; </li><li> Ajouter 200 pi de liquide hyperimmun dilué dans chaque puits et incuber à 37 ° C pendant 1 heure; </li><li> Retirer du liquide hyperimmun et la plaque de lavage avec PBS 1 X </li><li> Préparer une dilution 1:1000 de chèvre anti-souris conjugué (Cat # 074-1806 KPL) dans 5% BLOTTO (lait écrémé en poudre 5g dans 100 ml d&#39;une X PBS), puis ajouter 200 ul à chaque puits et incuber à 37 ° C pendant 1 h; </li><li> Préparer substrat comme suit: ajouter une tablette de 3&#39;-diaminobenzidine terrahydrochloride (Cat Sigma # D5905) dans 20 ml de PBS 1 X, après dissolution, puis ajouter 8 l de peroxydase hydrogène à 30% </li><li> Ajouter 200 ul de substrat ci-dessus pour chaque puits. Laisser reposer à température ambiante pendant 10 minutes </li><li> Retirer substrat et arrêter la réaction en ajoutant 1 ml d&#39;eau distillée dans chaque puits </li><li> Verser l&#39;eau et des assiettes blot pour sécher </li><li> Comptez le nombre de particules virales </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with 5% CO2, 37oC</td>
</tr>
<tr>
<td>50 mL conical tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>plastic</td>
</tr>
<tr>
<td>human serum and blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>O+</td>
</tr>
<tr>
<td>pipette tip</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture </td>
</tr>
<tr>
<td>pipettman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipetts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass, sterile</td>
</tr>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>37C</td>
</tr>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>parafilm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>circulating water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass membrane feeders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with rubber tubing to fit feeders</td>
</tr>
<tr>
<td>mosquito cups</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75% ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue-culture plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>6-well, 24-well and 96-well plates</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>fine-tip forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X, sterile</td>
</tr>
<tr>
<td>dissecting microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>C6/36 cells</td>
<td>cells</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For virus propagation</td>
</tr>
<tr>
<td>C6/36 medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>MEM with 10% heat inactivated FBS, 1% L-glutamine and non-essential amino acid and 1% (v/v) Penicillin-Streptomycin.</td>
</tr>
<tr>
<td>3&#x2019;-Diaminobenzidine terrahydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>1 tablet in 20 ml of 1 X PBS, dissolve and then add 8 &#181;l of 30% hydrogen peroxidase</td>
</tr>
<tr>
<td>30% hydrogen peroxidase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. aegypti</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquito</td>
</tr>		</tbody>
		</table>
		</div>

protocol for dengue infections in mosquitoes (a. aegypti) and infection phenotype determination

Le but de cette procédure est d&#39;infecter le moustique Aedes avec virus de la dengue dans un état de laboratoire et d&#39;examiner le niveau d&#39;infection et dynamique du virus dans les tissus des moustiques. Ce protocole est couramment utilisé pour étudier les interactions moustiques-virus, en particulier pour l&#39;identification des facteurs de l&#39;hôte roman qui sont en mesure de déterminer la compétence vectorielle. L&#39;expérience entière doit être menée dans un laboratoire BSL2. Similaire à Plasmodium falciparum, une tenue correcte, y compris des gants et une blouse de laboratoire doit être porté en tout temps. Après l&#39;expérience, tous les matériaux qui sont entrés en contact avec le virus doivent être traités avec de l&#39;éthanol 75% et blanchi avant de procéder à un lavage normal. Tous les autres matériaux doivent être autoclavés avant de les jeter.

Watch this Scientific Journal Video about Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination at JoVE.com

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Une fois qu&#39;un gène est identifié comme étant potentiellement réfractaires pour le virus de la dengue, il doit être évalué pour son rôle dans la prévention des infections virales dans le moustique. Ce protocole illustre la façon dont l&#39;étendue des cas de dengue de moustiques peuvent être dosés. Les techniques pour cultiver le virus en culture, l&#39;alimentation à membrane moustiques sang humain, et en dosant les titres viraux dans l&#39;intestin moyen des moustiques sont démontrés.

Protocole pour la dengue dans les moustiques (Aedes aegypti) et détermination de phénotype d&#39;infection

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

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JoVE Journal

Biology

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

17.4K vues.  Johns Hopkins University. Une fois qu'un gène est identifié comme étant potentiellement réfractaires pour le virus de la dengue, il doit être évalué pour son rôle dans la prévention des infections virales dans le moustique. Ce protocole illustre la façon dont l'étendue des cas de dengue de moustiques peuvent être dosés. Les techniques pour cultiver le virus en culture, l'alimentation à membrane moustiques sang humain, et en dosant les titres viraux dans l'intestin moy...

Vidéo: Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Microinjection d&#39;embryons aegypti a. Pour obtenir les moustiques transgéniques

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

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Biologie

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Protocole pour les infections à Plasmodium falciparum dans la détermination Phénotype moustiques et les infections

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

Dissection des glandes salivaires de l&#39;intestin moyen et Ae. aegypti

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocole pour des essais de l&#39;ARNi dans les moustiques adultes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

Prévenir la propagation du paludisme et la dengue utilisant moustiques génétiquement modifiés

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

Bâtir un meilleur moustique: l&#39;identification des gènes de résistance Activation fièvre malaria et la dengue chez A. gambiae et A. aegypti

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

Inactivation génique médiée par ARNi et<em&gt; In Vivo</emEssai diurèse&gt; Femmes adultes<em&gt; Aedes aegypti</em&gt; Les moustiques

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Un protocole de vidéo infections rétrovirales dans intestinale primaire Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Maintenir les moustiques Aedes aegypti infectés par Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocoles pour tester la toxicité des nouvelles chimies insecticides aux moustiques

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...