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...

<ol>
	<li>Xia, H., Wang, Y., Atoni, E., Zhang, B., Yuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito-associated+viruses+in+China.">Mosquito-associated viruses in China.</a> Virologica Sinica. 33 (1), 5-20 (2018).</li><li>Atoni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dataset+of+distribution+and+diversity+of+mosquito-associated+viruses+and+their+mosquito+vectors+in+China.">A dataset of distribution and diversity of mosquito-associated viruses and their mosquito vectors in China.</a> Scientific Data. 7 (1), 342 (2020).</li><li>Atoni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+and+global+distribution+of+novel+mosquito-associated+viruses+in+the+last+decade+(2007-2017).">The discovery and global distribution of novel mosquito-associated viruses in the last decade (2007-2017).</a> Reviews in Medical Virology. 29 (6), 2079 (2019).</li><li>Xia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+metagenomic+profiling+of+viromes+associated+with+four+common+mosquito+species+in+China.">Comparative metagenomic profiling of viromes associated with four common mosquito species in China.</a> Virologica Sinica. 33 (1), 59-66 (2018).</li><li>Ong, O. T. W., Skinner, E. B., Johnson, B. J., Old, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito-borne+viruses+and+non-human+vertebrates+in+Australia:+A+review.">Mosquito-borne viruses and non-human vertebrates in Australia: A review.</a> Viruses. 13 (2), 265 (2021).</li><li>Agboli, E., Leggewie, M., Altinli, M., Schnettler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito-specific+viruses-transmission+and+interaction.">Mosquito-specific viruses-transmission and interaction.</a> Viruses. 11 (9), 873 (2019).</li><li>Halbach, R., Junglen, S., van Rij, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito-specific+and+mosquito-borne+viruses:+evolution,+infection,+and+host+defense.">Mosquito-specific and mosquito-borne viruses: evolution, infection, and host defense.</a> Current Opinion in Insect Science. 22, 16-27 (2017).</li><li>Bolling, B. G., Olea-Popelka, F. J., Eisen, L., Moore, C. G., Blair, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+dynamics+of+an+insect-specific+flavivirus+in+a+naturally+infected+Culex+pipiens+laboratory+colony+and+effects+of+co-infection+on+vector+competence+for+West+Nile+virus.">Transmission dynamics of an insect-specific flavivirus in a naturally infected Culex pipiens laboratory colony and effects of co-infection on vector competence for West Nile virus.</a> Virology. 427 (2), 90-97 (2012).</li><li>Baidaliuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-fusing+agent+virus+reduces+arbovirus+dissemination+in+Aedes+aegypti+mosquitoes+in+vivo.">Cell-fusing agent virus reduces arbovirus dissemination in Aedes aegypti mosquitoes in vivo.</a> Journal of Virology. 93 (18), 00715-00719 (2019).</li><li>Atoni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+virome+analysis+of+Culex+mosquitoes+from+Kenya+and+China.">Metagenomic virome analysis of Culex mosquitoes from Kenya and China.</a> Viruses. 10 (1), 30 (2018).</li><li>Xia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+isolation+and+characterization+of+a+group+C+Banna+virus+(BAV)+from+Anopheles+sinensis+mosquitoes+in+Hubei,+China.">First isolation and characterization of a group C Banna virus (BAV) from Anopheles sinensis mosquitoes in Hubei, China.</a> Viruses. 10 (10), 555 (2018).</li><li>Shi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+the+virome+in+lab-+and+field-collected+Aedes+albopictus+mosquitoes+across+different+developmental+stages+and+possible+core+viruses+in+the+publicly+available+virome+data+of+Aedes+mosquitoes.">Stability of the virome in lab- and field-collected Aedes albopictus mosquitoes across different developmental stages and possible core viruses in the publicly available virome data of Aedes mosquitoes.</a> mSystems. 5 (5), 00640 (2020).</li><li>Zhou, M., Chu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook for Classification and Identification of Main Vectors. , (2019).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+main+mosquito+species+in+China+based+on+DNA+barcoding.">Identifying the main mosquito species in China based on DNA barcoding.</a> Plos One. 7 (10), (2012).</li><li>Ratnasingham, S., Hebert, P. D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bold:+The+Barcode+of+Life+Data+System+(www.barcodinglife.org).">Bold: The Barcode of Life Data System (www.barcodinglife.org).</a> Molecular Ecology Notes. 7 (3), 355-364 (2007).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+characterization+of+a+new+strain+of+mosquito+Flavivirus+derived+from+Culicoides.">In vitro and in vivo characterization of a new strain of mosquito Flavivirus derived from Culicoides.</a> Viruses. 14 (6), 1298 (2022).</li><li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+Tanay+virus+isolated+from+Anopheles+sinensis+mosquitoes+in+Yunnan,+China.">Characterization of a novel Tanay virus isolated from Anopheles sinensis mosquitoes in Yunnan, China.</a> Frontiers in Microbiology. 10, 1963 (1963).</li><li>Ren, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+reassortment+Tibet+orbivirus+isolated+from+Culicoides+spp.+in+Yunnan,+PR+China.">Characterization of a novel reassortment Tibet orbivirus isolated from Culicoides spp. in Yunnan, PR China.</a> Journal of General Virology. 102 (9), 001645 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;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 cm2 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>CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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> 
			 
			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 &#176;C)</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td>Instrument for cell culture</td>
		</tr>
		<tr>
			<td>incubator (37 &#176;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 &#176;C)</td>
			<td>sanyo</td>
			<td>MDF-U54V</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute&#160; 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>&#160;FCT010005</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile pestles</td>
			<td>Tiangen</td>
			<td>OSE-Y004</td>
			<td>Consumables&#160; 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 ...

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. Using thin lines to isolate viruses in Biosafety level 2 laboratory is safer and more efficient. Unlike other virus isolation procedures, such as cycling red inoculation with varying fluid, this technique does not necessitate animal experimentation or ethical assessment.The contamination from samples and the cells drawing during the incubation can lead to the failure of the experiment. Hence, it is necessary to add 2%Penicillin-Streptomycin-Amphotericin into the medium to prevent contamination. Begin with the addition of three-millimeter ceramic beads, 1.5 milliliters of RPMI medium supplemented with 2%Penicillin-Streptomycin-Amphotericin B Solution in a two-milliliter sterile tube placed on ice and then transfer 50 mosquitoes in it.Next, homogenize the mosquitoes with a low temperature tissue homogenizer by grinding for 30 seconds at 70 hertz and 4 degrees Celsius for 3 cycles. Centrifuge the homogenate at 15, 000 G for 30 minutes at 4 degrees Celsius and transfer the supernatants to the new tubes. To remove the mosquito debris completely, centrifuge the supernatant again at 15, 000 G for 10 minutes at 4 degrees Celsius.Aliquot the 200 microliters of supernatant P0 into a two-milliliter screw cap storage tube, and store it at minus 80 degrees Celsius. For the virus amplification, use Aedes albopictus RNAi-deficient mosquito cell line C6/36 and a vertebrate cell line, baby hamster kidney, or BHK-21 from a 75-centimeter square flask and seed them in 24 well plates separately. Place the seeded 24 well plate overnight at 28 or 37 degrees Celsius.The next day, observe the cells under the microscope to confirm 80 to 90%confluency in each well. Prepare 500 milliliters of the cell culture maintenance medium supplemented with antibiotics. Remove the cell culture medium from the 24 well plates and add 100 microliters of the cell culture maintenance medium to each well.Then inoculate the cells with 100 microliters of the supernatant of the mosquito homogenate or P0.Incubate the plates at 28 or 37 degrees Celsius for 60 minutes and gently shake the plates after every 15 minutes to prevent the drying of the cells. Once done, remove the supernatant and rinse each well with 600 microliters of cell culture maintenance medium to remove the debris completely. Next, add 800 microliters of cell culture maintenance medium to each well before placing the plates in a 5%carbon dioxide humidified incubator at 28 or 37 degrees Celsius for 7 days.Daily monitor the state of the cells of each well under the microscope. On the seventh day, collect the supernatant of cells known as P1 and store it at minus 80 degrees Celsius. Repeat the procedure to collect P2 and P3 viral supernatants.Due to the cytopathogenic effect, or CPE, cells from some of the wells die and detach from the surface. To greatly amplify the viruses Collect 300 to 400 microliters of the supernatant from wells showing the CPE effect and transfer it to a new six-well plate having 80 to 90%cell confluency of culture cells. Finally, collect and transfer 500 microliters of the supernatant from the wells of the six-well plate to two-milliliter screw cap storage tubes before storing them at minus 80 degrees Celsius.The inoculation of C6/36 cells with the mosquito homogenates P0 exhibited a wide intercellular space in exfoliated cells at 120 hours post-infection period compared to the uninoculated or control cells. Incubation of BHK21 cells with the P3 viral supernatants demonstrated a visible CPE at 48 hours post-infection, displaying the death of the cell and detachment from the surface in contrast to the control cells. Commercially available universal primers or Flaviviruses, Alphaviruses and Bunyaviruses were used to generate the PCR products for the viral supernatant.A PCR product for the Bunyavirus, Ebinur Lake virus, was set as a positive control. The PCR amplification bans in lanes belonging to Bunyaviruses and the positive control highlighted the possibility of the presence of Bunyavirus in the supernatants. To improve the success rate of virus isolation, keep a low temperature during sample transportation, isolation, and grinding.Add antibiotics when homogenizing the mosquitoes and the culture itself. And handle the culture rapidly during incubation and the removing the culture medium. In addition to this procedure, the grinding solution can be injected into a cycling rat or chicken embryo.However, the affirmation procedure will raise ethical concerns and increase the expense of the experiment.

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1.6K vistas.  Chinese Academy of Sciences. Este protocolo es un enfoque útil para aislar virus asociados a mosquitos y puede ayudar en futuras investigaciones sobre la distribución de patógenos relacionados con los mosquitos y el control de enfermedades transmitidas por mosquitos. El uso de líneas delgadas para aislar virus en el laboratorio de nivel 2 de bioseguridad es más seguro y eficiente. A diferencia de otros procedimientos de aislamiento del virus, como la inoculación roja ciclista con fluidos variables, esta técnica no...