Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

This protocol can determine whether the guts and the thyroid glands hinder viral spread and can aid in the risk assessments of mosquito-borne viruses transmitted by Aedes aegypti. Our protocol included two infection methods, oral feeding and the intrathoracic injection, which could effectively assess the vector competence of arbovirus. This protocol might be applied to additional arbovirus infections in a variety of mosquito vectors, such as Dengue virus and Zika virus infection in Aedes, and could prove to be a practicable procedure.

Demonstrating the procedure will be Fei Wang, a research assistant from my lab. To perform artificial feeding using the artificial mosquito feeding system, cut the collagen membranes to the appropriate size, and secure them to the reservoirs with the O-rings. Add three milliliters of the virus-blood mixture to the reservoirs, and seal the reservoirs using plastic plugs to prevent leakages.

Put the plastic cups containing mosquitoes into the glove box. Screw the sealed reservoirs into the FU1 feeder, and put one reservoir on one cup. Turn on the power unit, and feed the mosquitoes for one hour.

After feeding, anesthetize the mosquitoes with ice for several seconds until they faint. Pour the anesthetized mosquitoes into a Petri dish placed on ice, and cover the lid quickly. Pick engorged female mosquitoes with forceps, and transfer them to new plastic cups at 50 female mosquitoes per cup.

Wrap the cup with a cut mosquito net mesh, and cover it with a lid with a hole in the middle. Cut the sponge into small pieces, and put them on the cups. Add 8%glucose solution onto the sponge using a plastic disposable dropper.

Place the cups containing female mosquitoes in the incubator at 27 degrees Celsius at 80%humidity for 10 days. Replace a new glucose-saturated sponge every 72 hours. For intrathoracic inoculation, prepare female mosquitoes as described in the text manuscript.

To prepare the infectious virus dilution, remove the virus stock from the 80 degrees Celsius freezer, and thaw them on ice. Dilute virus stock at a 100-nanoliter virus dilution containing 100 to 500 plaque-forming units virus with RPMI 1640 medium containing 10%FBS and 1%penicillin-streptomycin. Place the virus dilution on ice.

Next, prepare the microinjection needles using a Puller 1000 with the parameters in the needle puller program set as heat index to 450, force in g to 110, distance in millimeter to one, and delay in seconds to zero. Cut the tip of a pulled needle with tweezers under a dissecting microscope at 16x magnification. Backfill the needle with mineral oil through a disposable sterile syringe.

Attach the needle into the injector following the instructions on the machine. Insert the needle carefully into a tube containing an infectious virus dilution. Then, press the Fill button to fill approximately four microliters of virus dilution into the needle.

Once the needle is filled, do not touch or damage the needle. Pick approximately 50 female anesthetized mosquitoes with tweezers, and put them on a ice plate. Place the plate under the injector, and insert the needle into the thoracic cavity of the mosquito under a dissecting microscope.

Set the injection volume to 100 nanoliters and the rate to 50 nanoliters per second. Press the Inject button to let the virus solution flow into the mosquito. Upon successful injection, the abdomen bulges slightly.

Put the infected mosquito into a new plastic cup placed on ice. When the number of infected mosquitoes is enough, wrap the cup with a cut mosquito net mesh and cover it with the lid. To collect saliva from the infected female mosquitoes, pour the anesthetized mosquitoes into a Petri dish placed on ice, and close the lid quickly.

Place 10-microliter pipette tips filled with immersion oil side by side on the rubber mud. Pick the 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 to collect the saliva as secreted into the oil by the mosquitoes at room temperature. After 45 to 60 minutes, place the pipette tips in 1.5-milliliter tubes containing 200 microliters of virus diluent medium. Centrifuge the tubes at 5, 000 g for five minutes at four degrees Celsius to expel saliva into the tubes.

Store the saliva-containing tubes at 80 degrees Celsius for subsequent determination of transmission rates. After saliva collection, dissect the infected female mosquitoes. Using tweezers, cut the heads of the mosquitoes, and place each head into an individual tube containing 200 microliters of RPMI 1640 medium.

Grab the thorax with a tweezer. Then, grab the penultimate segment of the abdomen with another tweezer, and pull the gut out. Clean the gut for any stray tissue and organs.

Wash the gut with PBS, and place each gut into an individual tube containing 200 microliters of RPMI 1640 medium. Store these tubes at 80 degrees Celsius for subsequent determination of the virus dissemination and infection rate. In this representative analysis, viral RNAs in the gut, head, and saliva of the female mosquitoes at 10 days post-infection were determined.

The virus titer of Ebinur Lake virus in the guts, heads, and saliva of the intrathoracically inoculated mosquitoes were higher than that in the oral-infected mosquitoes. The infection and dissemination rate for the intrathoracically inoculated mosquitoes was 100%and for the oral-infected mosquitoes was found to be 70%and 38.1%respectively. The transmission rate for the intrathoracically inoculated mosquitoes reached up to 90%but for the oral-infected mosquitoes was only 4.8%The engorged female mosquitoes were picked to maintain consistency, as the feeding amount affects the viral content.