A. Propagate the virus in the C6/36 cell line.
<ol>
<li>Cells grow to 80% confluency in 75 cm2 flask;</li>
<li>Remove the media with 3-4 ml remaining in the flask;</li>
<li>Take 0.5 ml stock virus and add into the above cell with the multiplicity of infection of 1.5 virus particles per cell. Shaking the flask for 15 minutes slowly at the room temperature.</li>
<li>Incubate for 45 minutes at 5% CO2 and 37&deg;C</li>
<li>Add 30 ml media, and incubate for 5 days.</li>
</ol>
B. Prepare the mixture of virus and blood
<ol>
<li>Detach the cell with the scraper and transfer the cell and media to the 50 mL conical tubes;</li>
<li>Centrifugate at 800g for 10 minutes; collect the supernatant, but leave 1ml of supernatant with the cell pellet;</li>
<li>Freeze the above cell pellet&nbsp; in dry CO2 and then thaw in 37&deg;C water bath, repeat two to three times;</li>
<li>Centrifugate at 800g for 10 minutes, take the supernatant, and combine with the supernatant collected from step 3;</li>
<li>Collect whole human blood with the same procedure (step 3) for preparation of gametocyte culture to infect Anopheles mosquito with Plasmodium falciparum;</li>
<li>Combine the equal amount of the above whole human blood with virus supernatant from step 4, and add human serum (10% of the whole volume). </li>
<li>Incubate the above mixture of human blood and dengue virus for 30 minutes at 37&deg;C water bath</li>
</ol>
C. Feeding mosquitoes
This procedure is almost identical to what have been described in the section for Plasmodium falciparum infections in mosquitoes. We assay the midgut infection at day 7, and the salivary infection at day 14 after blood-feeding. All material that came in contact with the virus are treated first with 75% ethanol and then with 10% bleach.
D. Assay the virus titer in mosquito tissue
<ol>
<li>Two or three days before dissection of mosquito tissue, grow C6/36 cell in a 24 well plate such that the cell reach 80% confluency at the day when the assay is conducted; </li>
<li>Mosquitoes are anesthetized at 4&deg;C, sacrificed and surfaced sterile by soaking them in 75% ethanol for 1 minute. Then mosquitoes were washed with sterile water twice before dissection of midgut or salivary gland under the dissecting microscope. From this step, all the procedure below needs to be conducted in a sterile environment such as a biological safety cabinet. These tissues are transferred to a tube containing 150 &micro;l media and homogenized with a Kontes pellet pestle motor for 90 seconds; </li>
<li>Make a 1:100 dilution by adding 10 &micro;l of the homogenate into 990 &micro;l media; </li>
<li>Make further 1: 10, 1:100 and 1:1000 dilution with the medium in the 96 well plate; </li>
<li>Remove the media in the 24 well plate, and add 100 &micro;l of each of the above four dilutions to each corresponding well; </li>
<li>Shake the plate slowly for 15 minutes at the room temperature, then incubate for 45 minutes at 5% CO2 and 37&deg;C; </li>
<li>Add 1ml of methycellulose overlay (1%) to each well; </li>
<li>Incubate plates at 37&deg;C with 5% CO2 for 5 days; </li>
<li>The steps from here on do not require a sterile environment but as with any other pathogen care should be taken at all times. Discard methycellulose overlay from each plate and blot to remove excess media </li>
<li>Add 1ml of methanol/acetone fixative (1:1) to each well. Keep at 4&deg;C for 1 hr; </li>
<li>Pour off the fixative, and wash once with 1X PBS; </li>
<li>Make 1:1000 dilution to the primary antibody against the virus with 5% blotto; </li>
<li>Add 200 &micro;l of diluted hyperimmune fluid to each well, incubate at 37&deg;C for 1 hr; </li>
<li>Remove hyperimmune fluid and wash plate with 1 X PBS </li>
<li>Prepare a 1:1000 dilution of goat anti-mouse conjugate (KPL Cat# 074-1806) in 5% blotto (5g powdered skim milk in 100 ml of 1 X PBS), and then add 200 &micro;l to each well, and incubate at 37&deg;C for 1 hr; </li>
<li>Prepare substrate as follows: add 1 tablet of 3'-Diaminobenzidine terrahydrochloride (Sigma Cat# D5905) in 20 ml of 1 X PBS, after dissolved, and then add 8 &micro;l of 30% hydrogen peroxidase </li>
<li>Add 200 &micro;l of the above substrate to each well. Let stand at room temperature for 10 minutes </li>
<li>Remove substrate and stop reaction by adding 1ml of distilled water to each well </li>
<li>Pour off water and blot plates to dry </li>
<li>Count the virus particle </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

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 the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

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

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

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

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 ...

protocol-for-dengue-infections-mosquitoes-aegypti-infection-phenotype

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17.5K Views.  Johns Hopkins University. Una volta che un gene è identificato come potenzialmente refrattaria per il virus della dengue, deve essere valutato per il suo ruolo nella prevenzione delle infezioni virali all'interno della zanzara. Questo protocollo illustra come il grado di dengue di zanzare possono essere analizzati. Le tecniche per crescere il virus in coltura, l'alimentazione a membrana zanzare sangue umano, e saggio titoli virale nel midgut zanzara sono dimostrati.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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