This work is supported by the National Institutes of Health (NIH R01 AI46435) and the RMRCE (NIH AI065357).

The following protocol is written for production and use of an ATS based on the Sindbis virus MRE16. The ATS is designed to express GFP in the mosquito vector Aedes aegypti. cDNA Infectious clones of a number of alphaviruses (Sindbis virus, Chikungunya virus, O'nyong-nyong virus, western equine encephalitis virus) are currently available that have been designed as ATS's (Table 1). For best results plasmid DNA is amplified in bacteria such as SURE competent bacteria (Stratagene/Agilent Technologies) to avoid unwanted recombination and loss of the viral cDNA. All infectious clone plasmids have a bacteriophage T7 or SP6 promoter at the immediate 5' end of the viral cDNA for transcription of full-length genomic RNA and a unique restriction endonuclease at the 3' end for runoff transcription. For each ATS, users must use the appropriate bacteriophage DNA dependent RNA polymerase and unique restriction endonuclease for in vitro transcription of the viral RNA genome.
1. Generation of Infectious RNA from cDNA Clone.
<ol>
 <li>Linearize the 5&mu;g of the infectious clone plasmid (p5'dsMRE16/GFP) with 20 units of XhoI restriction enzyme in a 100 &mu;L reaction. Incubate for 2-3h at 37&deg;C</li>
 <li>Purify linearized DNA using Qiagen QIAprep Spin Miniprep Kit alternate purification protocol, eluting DNA from spin column using 50 &mu;L RNase-free water. Plasmid concentration should be approximately 1.0 &mu;g/&mu;L.</li>
 <li>In an RNase-free 0.2 mL PCR tube, set-up a 50 &mu;L transcription reaction using Ambion MAXIscript Kit per protocol as follows. 
 <ul>
 <li>22.5 &mu;L RNase-free dH2O
 <ul>
 <li>5.0 &mu;L Linearized DNA template (1.0 &mu;g/&mu;L)</li>
 <li>2.5 &mu;L 10mM ATP</li>
 <li>2.5 &mu;L 10mM CTP</li>
 <li>2.5 &mu;L 10mM UTP</li>
 <li>2.5 &mu;L 1mM GTP</li>
 <li>2.5 &mu;L 10mM cap analog (m7G(5')ppp(5')G)</li>
 <li>5.0 &mu;L 10X transcription buffer</li>
 <li>5.0 &mu;L T7 or SP6 polymerase mix</li>
 </ul>
 </li>
 <li>50.0 &mu;L Total</li>
 </ul>
 </li>
 <li>Incubate the transcription reaction for 1 hr at 37&deg;C. After incubation, the reaction should be used immediately for electroporation of BHK-21 cells. Preparation of the BHK-21 cells for electroporation should begin during incubation transcription reaction.</li>
</ol>
2. Generation of Virus from Infectious RNA
<ol>
 <li>Trypsinize two 70-80% confluent 150 cm2 (T-150) flask BHK-21 cells per ATS.</li>
 <li>Transfer all cells to a 15 mL conical centrifuge tube.</li>
 <li>Pellet the cells by centrifugation at 2000 rpm, 10 min, 4&deg;C in a conventional tabletop centrifuge.</li>
 <li>Resuspend cells in sterile PBS without Mg++ and Ca++. Repeat steps 2.3 and 2.4 until cells have been washed three times with PBS.</li>
 <li>Resuspend the pelleted cells 0.5 mL MEM containing 10% FBS.</li>
 <li>Dilute 10 &mu;L cells with 90 &mu;L MEM with 10% FBS and count on a hemacytometer. If necessary, dilute cells to 2.5-12.5 x 107 cells/mL with MEM containing 10% FBS.</li>
 <li>To an ice cold 0.2 cm electroporation cuvette, add 400 &mu;L cell suspension and 20 &mu;L transcription reaction. Wipe condensation from cuvette.</li>
 <li>Pulse the cuvette twice: 450V, 1200&Omega;, 150 &mu;F. We use an Electro Cell Manipulator, Harvard Apparatus Model ECM630.</li>
 <li>Place the entire contents of the cuvette into a 25 cm2 tissue culture flask containing 5 mL MEM with 10% FBS.</li>
 <li>Incubate flask(s) at 37C with 5% CO2.</li>
 <li>Monitor the infection daily using an inverted fluorescent microscope with appropriate filter set (e.g. GFP filter: excitation wavelength = 460-490 nm, emission wavelength = 510-550 nm; or dsRed filter: excitation wavelength = 460-560 nm, emission wavelength = >590 nm).</li>
 <li>When fluorescence suggests infection is confluent and/or CPE is visible throughout the flask, collect the contents of the flask, add 30% FBS, aliquot as necessary, and store at -80&deg;C. If desired, cell lysates may be cleared by centrifugation (2000 rpm, 10 min, 4&deg;C) prior to addition of FBS.</li>
 <li>Passage virus at least once through a new flask of cells to increase the virus titer for subsequent blood feeding assays.</li>
</ol>
3. per os Infection of Mosquitoes
<ol>
 <li>Mix together blood (usually purchased de-fibrinated sheep blood) and cell-culture derived virus suspension from step 2.13. Final virus titer in the blood meal typically should be ~1x107 plaque forming units (pfu)/ mL for efficient midgut infection of the mosquito. To stimulate mosquitoes to engorge, adenosine 5'-triphosphate (ATP) can be added to a final concentration of 0.02M.</li>
 <li>Mosquitoes may have to be deprived of water and sugar before infection to stimulate them to efficiently blood feed from an artificial feeder. Duration of deprivation should be previously established to minimize mortality. Times will depend on mosquito species, humidity of the insectary etc. As a starting point, remove sugar 24h and water 12h prior to the feed. This protocol uses water-jacketed glass feeders17 with circulating 37&deg;C water. A porcine intestinal membrane (i.e. thoroughly washed sausage casing available at most grocery stores) is placed over the mouth of the feeder and the meal is pipetted into the chamber. Various designs, membranes and protocols are available for different vector types.</li>
 <li>Mosquitoes are sorted to select only those mosquitoes engorged with blood. Engorged mosquitoes are transferred to a carton with organdy on the top and the mosquitoes are incubated at 28&deg;C, 75-80% relative humidity until processed for GFP expression.</li>
</ol>
4. Intrathoracic Inoculation of Mosquitoes
Intrathoracic inoculation is an alternative method for infecting the arthropod. This method is used when dissemination through the midgut is not required. (Method described below but the technique is not shown in this visual experiment).
<ol>
 <li>A small number of mosquitoes (5-10) is aspirated from the holding cages into plastic holding tubes. The sealed holding tubes will be placed at 4&deg;C for 15 min to chill the mosquitoes.</li>
 <li>2 5 mosquitoes will be spilled from the tube onto a glass petri dish resting on ice. Place the lid on the Petri dish when not in use or if mosquitoes begin to move around. During handling of mosquitoes, care must be taken to count and keep track of the number of mosquitoes being manipulated. As mosquitoes are spilled onto the chill table, the total number will be recorded on a lab counter.</li>
 <li>The needle is prepared by melting capillary tubing using Nanoject specific glass and a needle puller.</li>
 <li>Setup the Nanoject II microinjector per the manufacturer's instructions for delivery of 69 nL inoculum. Care must be exercised when drawing the inoculum into the needle so that the needle is not damaged.</li>
 <li>Using a dissecting microscope, insert the needle into the thorax of the mosquito and activate the Nanoject for inoculation. Care must be exercised when inoculating mosquitoes so that the needle does not completely penetrate the mosquito and exit the other side, resulting in the subsequent death of the mosquito. Check each mosquito to be sure that the inoculum entered before removing it from the needle.</li>
 <li>After inoculation, while the mosquito is still impaled on the needle, place it in a paper holding carton through a small hole in the side that is plugged with cotton or a cork stopper at all other times.</li>
 <li>When the mosquitoes have inoculated and placed into the carton (up to approximately 50 per carton), carefully tape the cork in place to prevent accidental release of the mosquitoes. Place the cartons in a secure holding bin before further incubation in the insectary at 28&deg;C and 80% relative humidity.</li>
</ol>
5. Monitoring Infection of Mosquitoes
<ol>
 <li>Place the paper holding carton at 4&deg;C for approximately 15 minutes to immobilize mosquitoes.</li>
 <li>Transfer a small number of mosquitoes (5-10 at a time) to a chill table adapted for use on a fluorescent microscope.</li>
 <li>Examine and sort mosquitoes based on presence or absence of fluorescence. Additionally, fluorescent intensity may be used as a proxy quantitative measurement of infection. Mosquito tissues such as midguts, salivary glands, fat body can be dissected and monitored for fluorescence.</li>
 <li>If appropriate, return selected mosquitoes to paper carton to continue incubation of infected mosquitoes.</li>
</ol>
6. Transmission Assay (Forced Salivation to Demonstrate Virus Transmission)
<ol>
 <li>Deprive mosquitoes of sugar source for 24 h prior to feed.</li>
 <li>Remove legs and wings</li>
 <li>To a 50 &mu;L capillary tube that has been heated, pulled and snipped at one end, add 3-5 &mu;L of Cargille Type B immersion oil or 10% serum-sucrose solution.</li>
 <li>Insert the proboscis into the tube. If using oil, droplets of saliva will be seen to exude from the proboscis. One &mu;L of a 1% pilocarbine solution in PBS and 0.1% Tween 80 can be applied to the thorax to stimulate.</li>
 <li>After 60-90 minutes, remove mosquitoes and store for virus isolation if required.</li>
 <li>Remove solution from the tube by centrifugation in a tube containing 100 &mu;L 20% FBS-PBS.</li>
 <li>Sterile filter the inoculum through a 0.2 &mu;m syringe filter and then use the filtered the inoculum to infect cultured cells.</li>
</ol>
BIOSAFETY NOTE: This protocol describes a method for generating, identifying, and monitoring infected mosquitoes. Based on your facilities (i.e. a chill table, containment facility, etc.) and IBC approval, you may be limited to examining them only after removing wings and legs to prevent escape. SINV-based ATS's can be generated and used in a BSL2 environment. The use of ATS's based on other alphaviruses such as Chikungunya virus or western equine encephalitis viruses will require BSL3 facilities.
7. Representative Results
An overview of expression of a marker gene (GFP) using the ATS 5'dsMRE16/GFP is shown in Figure 1. Expression of GFP in BHK-21 and C6/36 (Aedes albopictus) cells is shown in Figure 2 at specific times after infection of cells with 5'dsMRE16/GFP virus at 0.01 multiplicity of infection. Figure 3 is an overview of alphavirus and ATS virus infection in the mosquito when the virus is delivered through an infective blood meal. Figure 4 shows preparation of a blood meal with ~107 pfu/mL of ATS virus followed by oral infection of Aedes aegypti. Whole body view of infected mosquito and selected body parts at 10 days post infection using an epifluorescence microscope (Figure 5). Detection of GFP and dsRED in midguts of infected mosquitoes following infection with ATS 5'dsMRE16 viruses expressing each fluorescent marker gene (Figure 6). Transmission assay using mosquitoes infected with 5'dsMRE16/GFP ATS virus (Figure 7).
Table 1. ATS's currently engineered for gene expression in mosquitoes.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp; Alphavirus</td>
 <td>ATS</td>
 <td>Mosquito</td>
 <td>Reference</td>
 </tr>
 <tr>
 <td>Sindbis Virus</td>
 <td>TE/3'2J and TE/5'2J</td>
 <td>Aedes aegypti</td>
 <td>12 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Ochlerotatus triseriatus</td>
 <td>13 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>3'dsMRE16 and 5'dsMRE16</td>
 <td>A. aegypti</td>
 <td>5</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Culex tritaeniorhynchus</td>
 <td>5 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>5'dsTR339</td>
 <td>A. aeg ypti</td>
 <td>2 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chikungunya virus </td>
 <td>3'dsCHIKV and 5'dsCHIKV</td>
 <td>A. aegypti/A. albopictus</td>
 <td>20 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>O'nyong nyong virus </td>
 <td>5'dsONNV </td>
 <td>Anopheles gambiae</td>
 <td>1,9 </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Western equine encephalitis virus </td>
 <td>5'dsWEEV.McMillan</td>
 <td>Culex tarsalis</td>
 <td>Stauft et al., unpublished </td>
 </tr>
</tbody>
</table>
Table 2. Advantages/Disadvantages of using ATS's for gene expression in mosquitoes.
<table border="1">
<tbody>
 <tr>
 <td>Advantages:</td>
 </tr>
 <tr>
 <td>Rapid production of high-titer virus</td>
 </tr>
 <tr>
 <td>Broad host range (SINV)</td>
 </tr>
 <tr>
 <td>High RNA replication rate</td>
 </tr>
 <tr>
 <td>High transgene expression levels</td>
 </tr>
 <tr>
 <td>Relative ease of manipulating genes</td>
 </tr>
 <tr>
 <td>Stable, persistent gene expression in insects</td>
 </tr>
 <tr>
 <td>Minimal pathology in insects</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disadvantages:</td>
 </tr>
 <tr>
 <td>Short-term expression mode in vertebrate cells</td>
 </tr>
 <tr>
 <td>Strong cytopathic effects on vertebrate cells</td>
 </tr>
 <tr>
 <td>Gene size restrictions (&lt;1Kb, ideally)</td>
 </tr>
 <tr>
 <td>Some alphaviruses require BSL3 containment</td>
 </tr>
 </tbody>
</table>
<img src="/files/ftp_upload/2363/2363fig1.jpg" alt="Figure 1" /> 
Figure 1: Overview of ATS virus production in BHK-21 cells using p5'dsMRE/GFP SINV expressing GFP. Following in vitro transcription, genomic ATS RNA is electroporated into BHK-21 cells where virus is rescued. The ATS virus can then be used to infect mosquitoes. SGP = subgenomic promoter. Three ATS RNA species are transcribed in the cells, the genomic, subgenomic 1 and subgenomic 2 RNAs. C (capsid), E2 and E1 glycoproteins are assembled with ATS genome to generate infectious virus.
<img src="/files/ftp_upload/2363/2363fig2.jpg" alt="Figure 2" /> 
Figure 2: Expression of GFP in mosquito (C6/36) cells following infection with SINV 5'dsMRE16/GFP virus.
<img src="/files/ftp_upload/2363/2363fig3.jpg" alt="Figure 3" /> 
Figure 3: Overview of an ATS virus infection in mosquitoes leading to virus transmission.
<img src="/files/ftp_upload/2363/2363fig4.jpg" alt="Figure 4" /> 
Figure 4: ATS SINV 5'dsMRE16 infection by exposing mosquitoes to an infective blood meal.
<img src="/files/ftp_upload/2363/2363fig5.jpg" alt="Figure 5" /> 
Figure 5: ATS SINV 5'dsMRE16/GFP infection at 10 days post infection. Whole body detection of GFP shows that virus has escaped the midgut and disseminated to secondary tissues. Figure also shows GFP expression in selected body parts that also is indicative of a disseminated infection.
<img src="/files/ftp_upload/2363/2363fig6.jpg" alt="Figure 6" /> 
Figure 6: ATS SINV 5'dsMRE16/GFP and 5'dsMRE16/dsRED infection of midguts at specific times post-infection. Midguts usually have distinct foci of infection early after ingesting a blood meal containing virus that spreads throughout the posterior midgut at later times post-infection.
<img src="/files/ftp_upload/2363/2363fig7.jpg" alt="Figure 7" /> 
Figure 7: Detection of ATS 5'dsMRE16/GFP in mosquito saliva as early as 8 days post infection. Assay is designed to show transmission of the ATS virus and shows that the 5'dsMRE16/GFP virus can stably express GFP throughout the extrinsic incubation period in the mosquito. The left panel shows a mosquito salivating into a capillary tube, the center panel shows C6/36 cells infected with saliva at 1 day and right panel 3 days post infection.

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No conflicts of interest declared.

The methods presented here enable researchers to follow the infection of an alphavirus in mosquito cell culture and the adult female mosquito. The advantages and disadvantages of using ATS viruses are summarized in Table 2. An ATS infectious clone can be genetically manipulated to express any GOI and have been used to express single chain antibodies, suppressors of RNAi, antisense RNAs targeting endogenous genes, and pro-apoptotic proteins. If a reporter is not used, then the imaging portion of this method is not relevant. However, many of the same protocols are necessary for ATS virus rescue, propagation, and mosquito infection. There are limitations in the maximal size of the transgene when inserted into an alphavirus transducing system. The size constraints vary depending on the parental strain used to generate the ATS, but are usually about 1kb although larger reporter genes such as firefly luciferase have been used in constructing ATS for use in mosquitoes. Research labs with a modest amount of virology background should be able to use ATS's following these protocols. A number of research labs have already done so using SINV-based ATS's.

<table><tbody><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>MAXIscript Kit</td><td>Ambion</td><td>AB1312</td><td>Be sure to use a kit containing the appropriate polymerase for your construct.</td></tr><tr><td>Cap Analogue (m&lt;sup&gt;7&lt;/sup&gt;G(5&amp;rsquo;)ppp(5&amp;rsquo;)G)</td><td>Ambion</td><td>AM8050</td><td></td></tr><tr><td>Nanoject II nanoliter injector</td><td>Drummond Scientific</td><td>3-000-204</td><td></td></tr><tr><td>Electro Cell Manipulator</td><td>Harvard Apparatus</td><td>ECM 630</td><td></td></tr></tbody></table>

alphavirus transducing system:  tools for visualizing infection in mosquito vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Watch this Scientific Journal Video about Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors at JoVE.com

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

alphavirus-transducing-system-tools-for-visualizing-infection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.7K Views.  Colorado State University. Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Video: Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Developmental Biology

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

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.

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

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Biology

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors

Summary

Explore More Videos

Chapters in this video

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Visualizing Dengue Virus through Alexa Fluor Labeling

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

2D and 3D Chromosome Painting in Malaria Mosquitoes

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

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection