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

1. Preparation of viruses and mosquitoes
<ol>
	<li>Preparation of viruses 
	NOTE: All processes were carried out in a biosafety level 2 (BSL-2) laboratory. The level of biosafety contaiment used should be&#160;determined by the pathogen&#39;s risk assessment and regulations specific to nations and regions. The process must be performed in a biosafety cabinet.
	<ol>
		<li>Inoculate 1 x 106 C6/36 cells into a T75 culture flask. Fill the flask with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). 
		NOTE: Leibovitz&#39;s L-15 medium (L15) or Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) can also be used to maintain C6/36 cells.</li>
		<li>Maintain the cells in 5% CO2 at 28 &#176;C overnight and remove the cell supernatant the next day when the cells are 80%-90% confluent.</li>
		<li>Add 1 mL of virus dilution (Multiplicity of Infection (MOI) = 0.01) into the flask and incubate the cells for 1 h in 5% CO2 at 28 &#176;C. Ebinur Lake virus (EBIV)15, a mosquito-borne orthobunyavirus, was used as a model virus for this protocol.</li>
		<li>Remove the supernatant and wash the cells 3x with 5 mL of PBS. Fill the flask with 15 mL of new RPMI medium with 2% FBS. Maintain the flask in 5% CO2 at 28 &#176;C.</li>
		<li>Collect the supernatant containing virus when the required viral titer is reached (after nearly 5 days, depending on the virus used). For EBIV, this titer value was 107 plaque forming units (PFU)/mL. Sub-package them into individual 2 mL screw cap storage tubes and store the tubes containing 0.5-1 mL of virus at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Determination of viral titer 
	NOTE: Determine viral titer through the plaque assays16. BHK-21 was used to determine viral titer of EBIV, due to the obvious cytopathic effect (CPE) observed in infected cells.
	<ol>
		<li>Inoculate 1 x 105 BHK-21 cells into each well of 24-well plates. Fill each well with 1 mL of DMEM medium containing 10% FBS and 1% P/S. Maintain the cells in 5% CO2 at 37 &#176;C overnight.</li>
		<li>Make six (10-6) successive 10-fold dilutions of virus-containing supernatant with fresh DMEM medium containing 10% FBS and 1% P/S.</li>
		<li>Add 100 &#181;L of virus diluent into each well of 24-well plates containing the monolayer BHK-21. Discard the virus diluent after incubation for 1 h in 5% CO2 at 37 &#176;C. Add 500 &#181;L of DMEM containing 1.5% methylcellulose to each well.</li>
		<li>Culture cells in 5% CO2 at 37 &#176;C for 48 h (the apparent CPE was found after 48 h for EBIV). Fix the cells overnight with 750 &#181;L of 3.7% formaldehyde.</li>
		<li>Stain them with 200 &#181;L of 2% crystal violet for 15 min. Count the number of plaques to calculate the viral titer.</li>
	</ol>
	</li>
	<li>Mosquito breeding in the lab 
	NOTE: Rear mosquitoes in an arthropod containment level 1 (ACL-1) laboratory.
	<ol>
		<li>For the preparation of the dechlorinated water, fill a 20 L bucket with tap water and allow it to stand for 7-8 days without light. Do not cover the lid.</li>
		<li>Keep eggs and larvae of Ae. aegypti in a mosquito room with the following conditions: 28 &#177; 1 &#176;C with a light/dark cycle of 12 h and a relative humidity of 75% &#177; 5%.</li>
		<li>Maintain nearly 1,000 larvae in a shallow dish (22 cm x 15 cm x 6 cm) containing dechlorinated water after the eggs hatch. Feed them with nearly 1/8 teaspoon of mice feedstuff powder. Refresh the food daily and change the water every 2 days.</li>
		<li>Once the larvae pupate after 5-7 days, use a one-off dropper to transfer pupae to a plastic cup (7 oz) filled with dechlorinated water.</li>
		<li>Put five cups containing pupae (about 200 per cup) into one mesh cage (30 cm x 30 cm x 30 cm) and keep the cages in the insect incubator with the same conditions as in step 1.3.2.</li>
		<li>Put a tiny sponge containing an 8% (m/w) glucose solution on each mesh cage to feed adult mosquitoes. After adult emergence, take out the cups without pupae. Keep the mesh cages with about 1,000 adult mosquitoes per cage in the same insect incubator.</li>
	</ol>
	</li>
</ol>
2. Preparation for oral infection
<ol>
	<li>Prepare female mosquitoes for oral infection. Use the mosquito absorbing machine to collect 5-8-day-old mosquitoes. Anesthetize them at -20 &#176;C for 1 min and check to see whether the mosquitoes are dizzy. If not, anesthetize them for another minute at -20 &#176;C.</li>
	<li>Pour them quickly into the plastic cups (24 oz) at about 200 mosquitoes (female and male) per cup. Cover each cup quickly with a well-cut mosquito net, followed by a lid with a hole (approximately 6 cm in diameter) in the middle.</li>
	<li>Starve the mosquitoes for 24 h before the oral-infection and prepare infectious blood meal as follows.</li>
	<li>In a biosafety cabinet under BSL-2 conditions, take out the virus stock from the -80 &#176;C freezer and thaw them on ice. Dilute the virus stock to a concentration of 2 x 106 PFU/mL with RPMI 1640 medium containing 10% FBS and 1% P/S. Mix the virus dilution with an equal volume of defibrated horse blood. 
	NOTE: The processes were carried out in a biosafety cabinet in an ACL-2 laboratory.&#160;</li>
</ol>
3. Perform oral infection
NOTE: All steps were performed in an ACL-2 laboratory. The process must be performed in the glove box to prevent mosquitoes from escaping.
<ol>
	<li>Perform artificial feeding by using the artificial mosquito feeding system for 1 h.
	<ol>
		<li>Cut the collagen membranes to the appropriate size (approximately 5 cm in diameter), secure them to the reservoirs with o-rings, then add the virus-blood mixture (approximately 3 mL) to the reservoirs. Use plastic plugs to seal reservoirs and prevent leaks. 
		NOTE: These steps were performed in a biosafety cabinet.</li>
		<li>Put the plastic cups (24 oz) containing mosquitoes into the glove box. Screw the sealed reservoirs onto the FU1 Feeder and put one reservoir on one cup, then turn on the power unit. Feed the mosquitoes for 1 h</li>
	</ol>
	</li>
	<li>Anesthetize mosquitoes by CO2 for several seconds until they faint. In brief, place the carbon dioxide spray gun in the middle of the net mesh through the hole on the cup containing mosquitoes, open the value, and allow huge amounts of carbon dioxide to be spewed out to anesthetize mosquitoes.</li>
	<li>Pour them into a Petri dish placed on ice and cover the lid quickly. Pick out the engorged female mosquitoes with forceps. To identify, males have feathery antennae and females have plain antennae. Transfer them to new plastic cups at 50 female mosquitoes per cup. 
	NOTE: The engorged female mosquitoes were picked to maintain consistency as feeding amount affects the viral content.</li>
	<li>Wrap the cup with a cut mosquito net mesh and then cover it with a lid with a hole in the middle. Cut the sponge into a small piece and put it on the cup.</li>
	<li>Add 8% glucose solution onto the sponge using a plastic disposable dropper. Keep the cups containing female mosquitoes in the incubator at 27 &#176;C at 80% humidity for 10 days. Replace a new glucose-saturated sponge every 72 h.</li>
</ol>
4. Preparation for intrathoracic inoculation
<ol>
	<li>Prepare the microinjection needles as follows. Use a PUL-1000 to make microinjection needles (Figure 1A) with the following parameters in the needle puller program: Heat index = 450, force (g) = 110, distance (mm) = 1.00, and delay (s) = 0.</li>
	<li>Cut the tip of a pulled needle with tweezers under the dissecting microscope at 16x magnification (Figure 1B). Do not make the tip too big, otherwise it can harm the mosquitoes.</li>
	<li>Prepare female mosquitoes as in step 2.1. 
	NOTE: The following processes were performed in an ACL-2 laboratory.&#160;</li>
	<li>Prepare the infectious virus dilution as follows:
	<ol>
		<li>Take out the virus stock from the -80 &#176;C freezer and thaw them on ice. Dilute virus to a 100 nL virus dilution containing 100-500 PFU virus with RPMI 1640 medium containing 10% FBS and 1% P/S. Keep the virus dilution on ice.</li>
	</ol>
	</li>
	<li>Backfill the needle with mineral oil through a disposable sterile syringe. Attach the needle into the injector following the instruction on the machine.</li>
	<li>Insert the needle into a tube containing the infectious virus dilution. Do not break the tip of the needle. Press the FILL button to fill the needle with virus solution. The final volume of the virus solution in the needle is approximately 4.0 &#181;L. Once the needle is filled with the virus, be careful not to touch and damage it. 
	NOTE: The steps were performed in a biosafety cabinet.</li>
</ol>
5. Perform viral injection under a dissecting microscope
NOTE: All steps were performed in an ACL-2 laboratory.&#160;
<ol>
	<li>Anesthetize mosquitoes at -20 &#176;C for 1 min. Pour them into a Petri dish placed on ice and cover the lid quickly.</li>
	<li>Pick out nearly 50 female mosquitoes with tweezers and put them on the ice plate. Place the ice plate under the injector and insert the needle into the thoracic cavity of mosquito under the dissecting microscope (Figure 1C).</li>
	<li>Set the injection volume to 100 nL and rate to 50 nL/s. Press the INJECT button to let the virus solution flow into the mosquito. If successful injection occurs, the abdomen will slightly bulge.</li>
	<li>Put the infected mosquito into a new plastic cup (24 oz) placed on ice. When the number of the infected mosquitoes is enough, wrap the cup with a cut mosquito net mesh and then cover it with the lid.</li>
	<li>Keep the cup containing infectious mosquitoes in the incubator at 27 &#176;C at 80% humidity for 10 days. Feed the mosquitoes as in step 3.5.</li>
</ol>
6. Processing of the infected female mosquitoes
NOTE: Three different tissues/organs were dissected from each female mosquito: the gut for calculating the virus infection rate (VIR), the head for calculating the virus dissemination rate (VDR), and saliva for calculating the virus transmission rate (VTR). VIR was defined as the number of mosquitoes with virus-positive guts. VDR was defined as the number of mosquitoes with virus-positive heads divided by the number of mosquitoes with virus-positive guts by 100. VTR was defined as the number of mosquitoes with virus-positive saliva divided by the number of mosquitoes with virus-positive guts by 100.
<ol>
	<li>Collection of saliva from the infected female mosquitoes 
	NOTE: The processes must be performed in an ACL-2 laboratory.&#160;
	<ol>
		<li>Anesthetize the infected female mosquitoes by carbon dioxide anesthetization as in step 3.2. Pour them into a Petri dish placed on ice and close the lid quickly.</li>
		<li>Place 10 &#181;L pipette tips filled with immersion oil side by side on the rubber mud.</li>
		<li>Pick out female mosquitoes with tweezers and put them on an ice plate. Remove their legs and wings with tweezers. Place the mouthpart of one mosquito on each pipette tip.</li>
		<li>Collect the saliva as secreted into the oil by the mosquitoes for the next 45-60 min at room temperature.</li>
		<li>Place the pipette tips in 1.5 mL tubes containing 200 &#181;L of virus diluent medium (RPMI 1640 medium containing 10% FBS and 1% P/S). Centrifuge them at 5,000 x g for 5 min at 4 &#176;C to expel saliva into tubes. Store these tubes at -80 &#176;C for subsequent determination of transmission rates.</li>
	</ol>
	</li>
	<li>Dissect the infected female mosquitoes 
	NOTE: The processes must be performed in an ACL-2 laboratory.&#160;
	<ol>
		<li>Use tweezers to cut the heads of the female mosquitoes after saliva collection. Place each head into an individual tube containing 200 &#181;L of RPMI 1640 medium.</li>
		<li>Grab the thorax with a tweezer, then the penultimate segment of abdomen with another tweezer and pull it out. The gut will be pulled out. Clean the pulled-out gut of any stray tissue and organs.</li>
		<li>Wash the gut with PBS and place each gut into an individual tube containing 200 &#181;L of RPMI 1640 medium. Store these tubes at -80 &#176;C for subsequent determination of the virus infection rate and dissemination rate.</li>
	</ol>
	</li>
</ol>
7. Data analysis
<ol>
	<li>RNA extraction
	<ol>
		<li>Grind the tissues into pieces with a low temperature tissue homogenizer grinding machine set to the following parameters: operating frequency = 60 Hz, operation time = 15 s, pause time = 10 s, cycles = 2, and setting temperature = 4.0 &#176;C.</li>
		<li>Centrifuge the samples at 12,000 x g for 10 min at 4 &#176;C. Extract total RNA of each sample by automated nucleic acid extraction system following instrument instructions.</li>
	</ol>
	</li>
	<li>Performing qRT-PCR
	<ol>
		<li>Use the one-step RT-PCR kit to quantify the viral RNA copies using a quantitative PCR instrument17. Use the following primers for the detection of EBIV S segment F: ATGGCATCACCTGGGAAAG and R: TTCCAATGGCAAGTGGATAGAA. Set up the reaction process as: stage 1: 42 &#176;C for 5 min, 95 &#176;C for 10 s; stage 2: 40 cycles of 95 &#176;C for 5 s and 60 &#176;C for 20 s.</li>
		<li>Determine the lowest virus dose for the infection in the cells through the serial 10-fold dilutions inoculated on cells. Use the BHK-21 cells to determine the lowest virus dose of EBIV, using dilutions till 10-7 as described in step 1.2.</li>
		<li>Calculate the Ct value of the lowest virus dose by qRT-PCR. Use the cutoff value for EBIV-positive by qRT-PCR as &#60; 35. Use the following equation to calculate the copies of EBIV in each sample: 
		y = -4.0312x + 3.384 [x = log (the copies of EBIV), y = Ct value, R2 = 0.9905]</li>
		<li>Calculate the infection rate, dissemination rate and transmission rate as follow: 
		Infection rate (%) = 100 x (the number of virus-positive mosquito guts / the number of total mosquitoes) 
		Dissemination rate (%) = 100 x (the number of virus-positive mosquito heads / the number of virus-positive mosquito guts) 
		&#8203;Transmission rate (%) = 100 x (the number of virus-positive mosquito saliva / the number of virus-positive mosquito guts)</li>
		<li>Analyze the differences in continuous variables and differences in mosquito infection rates, dissemination rates, and transmission rates by using the non-parametric Kruskal-Wallis analysis for multiple comparisons and Fisher&#39;s exact test where appropriate.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The goal of this method was to provide a comprehensive risk assessment of one mosquito-borne virus by evaluating vector competence through oral feeding and intrathoracic inoculation.
In the oral-feeding experiment, engorged-mosquitoes need to be picked out and transferred to a new container, posing a severe risk to the operators. The reason for this is because any mosquito, including uninfected mosquitoes, might be a source of infection19. Consequently, mosquitoes must ...

Mosquito-borne viruses (MBVs), a heterogeneous group of RNA viruses, can persist in mosquito vectors and subsequently spread to vertebrate hosts1. The clinically important MBVs are majorly distributed in four virus families, namely Flaviviridae, Togaviridae, Reoviridae, and Peribunyavividae2,3. In recent decades, these viruses have been reported all across the globe, causing public health issues. As one of the most well-known MBVs, Dengue virus (DENV) has become the most prevalent emerging or re-emerging arbovirus in over 100 countries during the last 20 years4. Since the discovery of Zika virus (ZIKV) inland, almost all tropical and subtropical countries and territories of the continent have reported human ZIKV infections5. In order to assess the risk of virus transmission, numerous studies in recent years have focused on mosquito vector competence for these viruses6,7. As a result, it is critical to effectively prevent and control vector-borne diseases.
Aedes aegypti (Ae. aegypti), one of the most easily reared mosquitoes in the laboratory, is an important vector of DENV, ZIKV, Chikungunya virus (CHIKV), and yellow fever virus (YFV)8. For a long time, Ae. aegypti was solely found in the African continent and in Southeast Asia, but in recent years it has colonized nearly all continents9. Furthermore, the global abundance of Ae. aegypti has been continuously growing, with an estimated 20% increase by the end of the century10. From 2004 to 2009 in China, there was an evident increase in Ae. aegypti vector competence for DENV due to higher day-to-day temperatures11. The status of Ae. aegypti as the pathogenic vector has significantly risen in China. Consequently, to address these challenges, it is necessary to investigate the vector competence of Ae. aegypti's to transmit viruses.
As a haematophagous arthropod, the female mosquito pierces the skin of a vertebrate host and feeds on the blood. Mosquitoes do occasionally acquire viruses from virus-infected hosts and then transfer the viruses to a new host. As such, to determine vector competence, mosquitoes are fed an artificial bloodmeal containing arboviruses through a feeding system in the laboratory setting12. Individual mosquitoes are separated into heads, bodies, and saliva secretions several days after infection. To measure virus infection, dissemination, and transmission rates, virus titers have been detected by quantitative reverse-transcription PCR (qRT-PCR) or plaque assay. However, not all mosquitoes develop midgut infections and the capacity to transfer a virus to the next host following blood feeding. It is linked with mosquitoes' physiological barriers, which prevent pathogens from penetrating the body and play a vital role in their innate immunity13. The midgut barriers, particularly the midgut infection barrier (MIB) and midgut escape barrier (MEB), influence whether the virus could infect the vector systemically and the efficiency with which it spreads. It obstructs the analysis of other tissues' infection, such as salivary glands which also exhibit salivary gland infection and escape barriers13,14. To better characterize the infection of midguts and salivary glands in the vector, a detailed protocol for oral feeding and intrathoracic inoculation of arbovirus in Ae. aegypti is presented herein. This protocol might be applied to additional arbovirus infections in a variety of mosquito vectors, such as DENV and ZIKV infection in Aedes spp., and could prove to be a practicable procedure.

<table><tbody><tr><td>Aedes aegypti&amp;nbsp;</td><td></td><td></td><td>Rockefeller strain</td></tr><tr><td>Automated nucleic acid extraction system&amp;nbsp;</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>Buckets</td><td></td><td></td><td></td></tr><tr><td>C6/36 cells&amp;nbsp;</td><td>National Virus Resource Center, Wuhan Institute of Virology</td><td></td><td></td></tr><tr><td>Carbon dioxide spray gun&amp;nbsp;</td><td>wuhan Yihong</td><td>YHDFPCO2</td><td></td></tr><tr><td>Centrifugal machine</td><td>Himac&amp;nbsp;</td><td>CF16RN</td><td></td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System&amp;nbsp;</td><td>Bio-Rad</td><td>CFX96 Touch</td><td></td></tr><tr><td>Ebinur Lake virus</td><td></td><td></td><td>Cu20-XJ isolation</td></tr><tr><td>Formaldehyde&amp;nbsp;</td><td>Wuhan Baiqiandu</td><td>B0003</td><td></td></tr><tr><td>Glove box&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>Hushi</td><td>10010518</td><td></td></tr><tr><td>Immersion oil&amp;nbsp;</td><td>Cargille</td><td>16908-1</td><td></td></tr><tr><td>Insect incubator</td><td>Memmert</td><td>HPP750T7</td><td></td></tr><tr><td>Low Temperature Tissue Homogenizer Grinding Machine&amp;nbsp;</td><td>Servicebio</td><td>KZ-III-F</td><td></td></tr><tr><td>Magnetic Virus Genome Extraction Kit</td><td>NanoMagBio</td><td>NMG0966-16</td><td></td></tr><tr><td>mesh cages (30 x 30 x 30 cm)</td><td>Huayu</td><td>HY-35</td><td></td></tr><tr><td>methylcellulose</td><td>Calbiochem</td><td>17851</td><td></td></tr><tr><td>mice feedstuff powder&amp;nbsp;</td><td>BESSN</td><td>BS018</td><td></td></tr><tr><td>Microelectrode Puller</td><td>WPI</td><td>PUL-1000</td><td>PUL-1000&amp;nbsp;is a microprocessor controlled horizontal puller for making glass micropipettes or microelectrodes used in intracellular recording, patch clamp studies, microperfusion or microinjection.</td></tr><tr><td>Mosquito net meshes&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Nanoject III Programmable Nanoliter Injector</td><td>Drummond</td><td>3-000-207</td><td></td></tr><tr><td>One Step TB Green PrimeScript PLUS RT-PCR Kit&amp;nbsp;</td><td>Takara</td><td>RR096A</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>C10010500BT</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>151140-122</td><td></td></tr><tr><td>Petri dishes&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Plastic cupes (7 oz)&amp;nbsp;</td><td>Hubei Duoanduo</td><td></td><td></td></tr><tr><td>Plastic cups (24 oz)&amp;nbsp;</td><td>Anhui shangji</td><td>PET32-Tub-1</td><td></td></tr><tr><td>Plastic disposable droppers</td><td>Biosharp</td><td>BS-XG-O3L-NS</td><td></td></tr><tr><td>Refrigerator (-80 &amp;deg;C)</td><td>sanyo</td><td>MDF-U54V</td><td></td></tr><tr><td>Replacement Glass Capillaries</td><td>Drummond</td><td>3-000-203-G/X</td><td></td></tr><tr><td>RPMI medium 1640&amp;nbsp;</td><td>Gibco</td><td>C11875500BT</td><td></td></tr><tr><td>Screw cap storage tubes (2 mL )</td><td>biofil</td><td>&amp;nbsp;FCT010005</td><td></td></tr><tr><td>Shallow dishes&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Sponge</td><td></td><td></td><td></td></tr><tr><td>Sterile defibrillated horse blood</td><td>Wuhan Purity Biotechnology</td><td>CDHXB413</td><td></td></tr><tr><td>T75 culture flask</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>The artificial mosquito feeding system&amp;nbsp;</td><td>Hemotek</td><td>Hemotek PS6</td><td></td></tr><tr><td>The dissecting microscope&amp;nbsp;</td><td>ZEISS</td><td>&amp;nbsp;stemi508</td><td></td></tr><tr><td>The ice plates</td><td></td><td></td><td></td></tr><tr><td>The mosquito absorbing machine&amp;nbsp;</td><td>Ningbo Bangning</td><td></td><td></td></tr><tr><td>The pipette tips&amp;nbsp;</td><td>Axygen</td><td>TF</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Tweezers</td><td>Dumont</td><td>0203-5-PO</td><td></td></tr></tbody></table>

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.

To examine EBIV distribution in the infected mosquitoes via artificial blood feeding (the viral final titer was 6.4 x 106 PFU/mL) and intrathoracic injection (the viral dose was 340 PFU), viral RNAs in saliva, heads, and guts of the mosquitoes at 10 days post infection (dpi) were determined.
For Ae. aegypti, virus titer of EBIV in the guts, heads, and saliva of the intrathoracically inoculated female mosquitoes were much higher than that in the oral-infected female...

Watch this Scientific Journal Video about Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection at JoVE.com

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

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 ...

experimental-viral-infection-adult-mosquitoes-oral-feeding

Research

JoVE Journal

Immunology and Infection

2.2K Views.  Chinese Academy of Sciences. Dit protocol kan bepalen of de darmen en de schildklier de verspreiding van virussen belemmeren en kan helpen bij de risicobeoordelingen van door muggen overgedragen virussen die worden overgedragen door Aedes aegypti. Ons protocol omvatte twee infectiemethoden, orale voeding en de intrathoracale injectie, die de vectorcompetentie van arbovirus effectief konden beoordelen. Dit protocol kan worden toegepast op extra arbovirusinfecties in verschillende muggenvectoren, zoals het Dengue-virus en ...

Video: Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

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 ...

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Environment

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

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicine

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 ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

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 ...

Biology

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Mosquito Feeding Assay: A Method to Evaluate Test Compound Effects on Mortality and Fecundity of Adult Female Mosquitoes

Source: Brito-Sierra, C. A. et al., <a href="https://www.jove.com/v/57768">Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes.</a> J. Vis. Exp. (2019).
This video demonstrates a blood-feeding assay to evaluate the mortality and fecundity of test compounds with novel modes of action on female mosquitoes. This technique helps in screening new classes of insecticides to kill the resistant population of mosquitoes and control disease spread.

Source: Brito-Sierra, C. A. et al., Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes. J. Vis. Exp. (2019).
This video ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

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