This work was supported by the Wuhan Science and Technology Plan Project (2018201261638501).

1. Mosquito sampling and sorting
<ol>
	<li>Trap the adult mosquitoes through the light traps MXA-02 or carbon dioxide mosquito traps in the field.</li>
	<li>Kill the collected mosquitoes by dipping in liquid nitrogen10,11. Transport them to the laboratory by the cold-chain logistics system12. 
	NOTE: Dry ice was primarily used in the cold-chain logistics system.</li>
	<li>(Optional) If the sample sites were near the lab, directly send the alive mosquitoes in the net traps to the laboratory and euthanize them by freezing at &#8804;-20 &#176;C for 30 min4.</li>
	<li>Make a morphological identification of the collected mosquitoes through the tool book13 for classification and identification of mosquitoes. 
	NOTE: If necessary, DNA barcoding could be used to classify and identify mosquitoes14,15.&#160;</li>
	<li>Based on sampling dates and sites, assign them into different pools,&#160;and then store&#160;them in 2-5&#160;mL tubes.</li>
	<li>Draw a table to record each tube with mosquito species, number, sampling code, date, and sites.</li>
	<li>Print the labels with sampling codes and attach them to the tubes.</li>
	<li>Keep tubes with the mosquito samples at -80 &#176;C until further analysis.</li>
</ol>
2. Mosquito grinding
<ol>
	<li>Place 20-50 mosquitoes into each 2 mL sterile tube with 3 mm ceramic beads with 1.5 mL of Roswell Park Memorial Institute (RPMI) medium containing 2% Penicillin-Streptomycin-Amphotericin B Solution. 
	NOTE: Keep the tubes on ice oron anice tray.</li>
	<li>Homogenize the mosquitoes with a low-temperature tissue homogenizer by grinding for 30 s at 70 Hz at 4 &#176;C for three cycles16.</li>
	<li>Centrifuge the mixtures at 15,000 &#215; g for 30 min at 4 &#176;C and transfer the supernatants to new tubes.</li>
	<li>Centrifuge the supernatants again at 15,000 &#215; g for 10 min at 4 &#176;C to remove the mosquito debris12,17. 
	NOTE: If necessary, the supernatants can be filtered using 0.22 &#956;m filters.</li>
	<li>Aliquot the supernatants (P0) into 2 mL screw cap storage tubes (200 &#181;L per tube) and store them at -80 &#176;C.</li>
</ol>
3. Preparation of cells and cell culture maintenance medium
NOTE: Mosquito cell line C6/36 (Aedes albopictus RNAi-deficient) and vertebrate cell line BHK-21 (baby hamster kidney) were used for the virus amplification and isolation.
<ol>
	<li>Inoculate 1 &#215; 106 C6/36 cells into a 75 cm2 flask with 10 mL of RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 28 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Inoculate 1 &#215; 106 BHK-21 cells into a 75 cm2 flask with 10 mL of Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% FBS and 1% P/S at 37 &#176;C in a 5% CO2 humidified incubator. 
	NOTE: The cells were passaged two or three times per week17,18.</li>
	<li>Seed the cells cultured in a 75 cm2 flask (C6/36 or BHK-21 cells) into the 24-well plates (C6/36 per well: 1.4 &#215; 105; BHK-21 per well: 0.8 &#215; 105).</li>
	<li>Place the 24-well plates at 28 &#176;C or 37 &#176;C overnight.</li>
	<li>Observe the cells under the microscope and confirm whether cell confluency in each well is 80%-90%.</li>
	<li>Prepare the cell culture maintenance medium (500 mL). 
	&#8203;NOTE: For C6/36, the medium was RPMI medium supplemented with 2% FBS and 2% Penicillin-Streptomycin-Amphotericin B Solution. For BHK-21, the medium was DMEM medium supplemented with 2% FBS and 2% Penicillin-Streptomycin-Amphotericin B Solution.</li>
</ol>
4. Virus isolation
NOTE: All steps were carried out in a biosafety level 2 (BSL-2) laboratory. The safety level requirement of the biosafety laboratory was determined by the biosafety risk assessment based on the regulations of different countries and regions. The process must be performed in a biosafety cabinet.
<ol>
	<li>Remove the cell culture medium from the 24-well plates and add 100 &#181;L of the cell culture maintenance medium and 100 &#181;L of the supernatant of the mosquito homogenate (P0) to each well.</li>
	<li>Incubate the plates at 28 &#176;C or 37 &#176;C for 60 min and gently shake them every 15 min to prevent cell drying.</li>
	<li>Remove the supernatant and rinse each well gently with 600 &#181;L of cell culture maintenance medium to remove the debris completely.</li>
	<li>Add 800 &#181;L of cell culture maintenance medium to each well and keep the plates in a 5% CO2 humidified incubator at 28 &#176;C or 37 &#176;C for 7 days.</li>
	<li>Monitor the cell state of each well under the microscope daily15.</li>
	<li>Collect the supernatants of cells (P1 supernatants) on the 7th day and store the supernatants at -80 &#176;C.</li>
	<li>Repeat steps 4.1-4.6 2x to get P2 and P3 supernatants. 
	NOTE: In step 4.3, rinsing each well gently with 600 &#181;L of cell culture maintenance medium was optional for the P2 and P3.</li>
	<li>Depending on which well shows the cytopathogenic effect (CPE) &#160;&#8212;&#160;cells die, pyknosis,&#160;and detach from the surface; fusion with adjacent cells to form syncytia; or the appearance of nuclear or cytoplasmic inclusion bodies &#8212; add 300-400 &#181;L of the supernatant of this CPE well into each well of the new 6-well plates containing cells (the cell confluency was 80%-90%) to greatly amplify the viruses.</li>
	<li>Collect the supernatants and aliquot them into 2 mL screw cap storage tubes (500 &#181;L per tube). Store them at -80 &#176;C. 
	&#8203;NOTE: After three generations of successive passaging in cells, the samples that did not induce CPE were discarded. If necessary, the generations of successive passaging in cells can be increased.</li>
</ol>
5. Detecting viral sequences by RT-PCR
<ol>
	<li>Extract total RNA from 200 &#181;L of the viral supernatant using a viral RNA mini kit16. 
	NOTE: Here, anautomated nucleic acid extraction system was used to extract total RNA following the instrument manufacturer&#39;s instructions.</li>
	<li>Convert the RNA to cDNA following the RT-PCR kit instructions using a PCR instrument.
	<ol>
		<li>Add 15 &#181;L of RNA mixed with 1 &#181;L of random primers into the PCR tube. Place the tube at 70 &#176;C for 5 min and then place it on ice for 5 min.</li>
		<li>Add 5 &#181;L of 5x buffer and 25 &#181;L of mixture (10 mM dNTP, 40 U/&#181;L RNase inhibitor, and 200 U/&#181;L M-MLV) into the tube and place the tube at 42 &#176;C for 60 min and 95 &#176;C for 10 min.</li>
		<li>Store the tube at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Use a PCR kit and universal primers (Table 1) to detect viruses by PCR.
	<ol>
		<li>For arboviruses, set up the PCR reaction system (total 30 &#181;L) with the following components: 3 &#181;L of 10x Taq buffer, 3 &#181;L of dNTP (1mM), 1 &#181;L of forward primer (10 &#181;m), 1 &#181;L of reverse primer (10 &#181;m), 0.3 &#181;L of Taq (10 U/&#181;L), 19.7 &#181;L of ddH2O, and 2 &#181;L of cDNA.</li>
		<li>For the detection of flaviviruses, set up the reaction process as: stage 1: 35 cycles of 94 &#176;C for 30 s, 52 &#176;C for 30 s, and 72 &#176;C for 30 s; stage 2: 72 &#176;C for 10 min.</li>
		<li>For the detection of alphaviruses, set up the reaction process as: stage 1: 35 cycles of 94 &#176;C for 30 s, 50 &#176;C for 30 s, and 72 &#176;C for 30 s; stage 2: 72 &#176;C for 10 min.</li>
		<li>For the detection of bunyaviruses, set up the reaction process as: stage 1: 35 cycles of 94 &#176;C for 30 s, 50 &#176;C for 30 s, and 72 &#176;C for 30 s; stage 2: 72 &#176;C for 10 min.</li>
	</ol>
	</li>
	<li>Detect the PCR amplification products by 1% agarose gel electrophoresis and send them for sequencing analysis. 
	NOTE: If conditions permit, the supernatants can be undergo next generation sequencing analysis to identfy new viruses and obtain the whole viral genome seqeunces.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The objective of this method was to offer a practical way for isolating mosquito-associated viruses using various cell lines. It is critical to add the Antibiotic-Antimycotic (Penicillin-Streptomycin-Amphotericin) to the supernatants of the mosquito homogenates to avoid contamination by bacteria or fungi. Mosquitoes and viral supernatants obtained in the field must be refrigerated at -80 &#176;C to avoid repeated freeze-thaw cycles.
Another critical step in the protocol was grinding. Mosquito ...

Mosquitoes are a group of important pathogenic arthropod vectors. There are approximately 3,500 species of mosquitoes in the family Culicidae1,2. The development of high-throughput sequencing technologies has led to the discovery of many novel, virus-like sequences in mosquitoes from different parts of the world3. Generally, these mosquito-associated viruses can be classified into two main groups: MBVs and MSVs.
MBVs are a group of diverse viruses that are causative agents of many human or animal illnesses, such as yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Rift Valley fever virus (RVFV)4. They have seriously threatened public health by causing severe morbidity and mortality in both humans and animals across the world. MBVs naturally sustain a life cycle between diverse hosts through transmission from an infected mosquito to a na&#239;ve host, as well as from a virus-infected host and to a feeding mosquito5. Therefore, these viruses can infect both mosquito cell lines and vertebrate cell lines in the lab1.
MSVs, which include Yichang virus (YCN), Culex flavivirus (CxFV), and Chaoyang virus (CHAOV), are a subgroup of insect-specific viruses1,6,7. In recent years, there has been a rise in the discovery of novel MSVs, and some of these MSVs have been found to have an impact on the transmission of MBVs. For example, CxFV, which can be a persistent infection in Culex pipiens, could suppress WNV replication at an early stage8. Another insect-specific flavivirus, cell-fusing agent virus (CFAV), has been found to inhibit the propagation of DENV and Zika virus (ZIKV) in Aedes aegypti mosquitoes9. Thus, this protocol is a useful approach for isolating mosquito-associated viruses and can help in further research into the distribution of pathogens related to mosquitoes and the control of mosquito-borne diseases.

<table><tbody><tr><td>0.22 &amp;micro;m membrane filter</td><td>Millipore</td><td>SLGP033RB</td><td>Polymer films with specific pore ratings.To remove cell debris and bacteria.</td></tr><tr><td>24-well plates</td><td>CORNING</td><td>3524</td><td>Containers for cell</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; flasks</td><td>CORNING</td><td>430641</td><td>Containers for cell</td></tr><tr><td>a sterile 2 mL tube with 3 mm ceramic beads</td><td></td><td></td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240-062</td><td>Antibiotic in the medium to prevent contamination from bacteria and fungi</td></tr><tr><td>Automated nucleic acid extraction system</td><td>NanoMagBio</td><td>S-48</td><td></td></tr><tr><td>BHK-21 cells</td><td>National Virus Resource Center, Wuhan Institute of Virology</td><td></td><td></td></tr><tr><td>C6/36 cells</td><td>National Virus Resource Center, Wuhan Institute of Virology</td><td></td><td></td></tr><tr><td>Centrifugal machine</td><td>Himac</td><td>CF16RN</td><td>Instrument for centrifugation of mosquito samples</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s minimal essential medium (DMEM)</td><td>Gibco</td><td>C11995500BT</td><td>medium for vertebrate cell lines</td></tr><tr><td>Ebinur Lake virus</td><td></td><td></td><td>Cu20-XJ isolation</td></tr><tr><td>Feta Bovine Serum (FBS)</td><td>Gibco</td><td>10099141C</td><td>&lt;br/&gt; &lt;br/&gt; Provide nutrition for cells</td></tr><tr><td>high-speed low-temperature tissue homogenizer</td><td>servicebio</td><td>KZ-III-F</td><td>Instrument for grinding</td></tr><tr><td>incubator (28 &amp;deg;C)</td><td>Panasonic</td><td>MCO-18AC</td><td>Instrument for cell culture</td></tr><tr><td>incubator (37 &amp;deg;C)</td><td>Panasonic</td><td>MCO-18AC</td><td>Instrument for cell culture</td></tr><tr><td>PCR tube</td><td></td><td></td><td></td></tr><tr><td>penicillin-streptomycin</td><td>Gibco</td><td>15410-122</td><td>Antibiotic in the medium to prevent contamination from bacteria</td></tr><tr><td>Penicillin-Streptomycin-Amphotericin B Solution</td><td>Gibco</td><td>15240096</td><td></td></tr><tr><td>Refrigerator (-80 &amp;deg;C)</td><td>sanyo</td><td>MDF-U54V</td><td></td></tr><tr><td>Roswell Park Memorial Institute&amp;nbsp; medium (RPMI)</td><td>Gibco</td><td>C11875500BT</td><td>medium for mosiquto cell lines</td></tr><tr><td>Screw cap storage tubes (2 mL)</td><td>biofil</td><td>&amp;nbsp;FCT010005</td><td></td></tr><tr><td>sterile pestles</td><td>Tiangen</td><td>OSE-Y004</td><td>Consumables&amp;nbsp; for grinding</td></tr><tr><td>TGrinder OSE-Y30 electric tissue grinder</td><td>Tiangen</td><td>OSE-Y30</td><td>Instrument for grinding</td></tr><tr><td>The dissecting microscope</td><td>ZEISS</td><td>stemi508</td><td></td></tr><tr><td>the light traps MXA-02</td><td>Maxttrac</td><td></td><td></td></tr><tr><td>The mosquito absorbing machine</td><td>Ningbo Bangning</td><td></td><td></td></tr><tr><td>The pipette tips</td><td>Axygen</td><td>TF</td><td></td></tr><tr><td>The QIAamp viral RNA mini kit</td><td>QIAGEN</td><td>52906</td><td></td></tr><tr><td>Tweezers</td><td>Dumont</td><td>0203-5-PO</td><td></td></tr></tbody></table>

mosquito-associated virus isolation from field-collected mosquitoes

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two main categories of these new mosquito-associated viruses are &#34;mosquito-borne viruses (MBVs)&#34; and &#34;mosquito-specific viruses (MSVs)&#34;. These novel viruses might be pathogenic to both vertebrates and mosquitoes, or they could just be symbiotic with mosquitoes. Entity viruses are essential to confirm the biological characters of these viruses. Thus, a detailed protocol has been described here for virus isolation and amplification from field-collected mosquitoes. First, the mosquito samples were prepared as supernatants of mosquito homogenates. After centrifugation twice, the supernatants were then inoculated into either mosquito cell line C6/36 or vertebrate cell line BHK-21 for virus amplification. After 7 days, the supernatants were collected as the P1 supernatants and stored at -80 &#176;C. Next, P1 supernatants were passaged twice more in C6/36 or BHK-21 cells while the cell status was being checked daily. When cytopathogenic effect (CPE) on the cells was discovered, these supernatants were collected and used to identify viruses. This protocol serves as the foundation for future research on mosquito-associated viruses, including MBVs and MSVs.

After inoculation with the supernatants of the mosquito homogenates (P0), the C6/36 cells exhibited a wide intercellular space, and exfoliated cells were observed at 120 h (Figure 1A) compared to the uninoculated cells (control) at the same time (Figure 1B). After incubating the BHK-21 cells with the P3 supernatants, visible CPE was observed in the BHK-21 cells at 48 h (Figure 1C) in contrast to the control cells (<strong class="xfi...

Watch this Scientific Journal Video about Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes at JoVE.com

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two ...

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1.7K Views.  Chinese Academy of Sciences. Dit protocol is een nuttige aanpak voor het isoleren van muggen-geassocieerde virussen en kan helpen bij verder onderzoek naar de verspreiding van pathogenen gerelateerd aan muggen en de bestrijding van door muggen overgedragen ziekten. Het gebruik van dunne lijnen om virussen te isoleren in bioveiligheidsniveau 2 laboratorium is veiliger en efficiënter. In tegenstelling tot andere virusisolatieprocedures, zoals het fietsen van rode inenting met variërende vloeistof, vereist deze techniek g...

Video: Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

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.

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 ...

Immunology and Infection

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.

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.

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 ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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 ...

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

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 ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

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, ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

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