アルファウイルス形質導入システム：蚊で感染を可視化するためのツール

Alpha viruses are mosquito born RNA viruses that persistently infect susceptible mosquito cells with minimal cytopathology. This protocol shows the utility of infectious alpha virus transducing systems that express fluorescent reporters for detection of vector infection and virus transmission. First feed female mosquitoes a blood meal containing the alpha virus of interest, then determine mosquito infection efficiency, and select only mosquitoes expressing the marker gene.

Next, harvest saliva from the infected mosquitoes and demonstrate transmissibility by infecting cultured mosquito cells with collected saliva. Results obtained by epi fluorescent microscopy. Visualize a report of protein like GFP and thus track vector infection and virus transmission by eighties or QE species of mosquitoes.

Alpha virus expression systems or ATSs can be used to rapidly assess gene expression and vector mosquitoes for both arbovirus studies as well as prior to doing more difficult mosquito manipulations such as mosquito transgenesis. Demonstrating these procedures will be Dr.Irma Sanchez Vargas, Dr.Eric Mossel, and Aaron Phillips from Dr.Ken Olson's laboratory. In order to produce a TS virus in Bhk 21 cells, use a symbis recombinant virus that expresses the GFP reporter following in vitro transcription, electro rate the genomic alpha virus transducing system RNA into bhk 21 cells.

Then rescue and amplify the virus before the infection. Prime mosquitoes for efficient blood feeding from an artificial feeder by food deprivation. Remove sugar 24 hours and water 12 hours prior to the feed.

Mixed commercially obtained defibrillated or citrated animal blood with the prepared cell culture derived a TS virus Suspension for efficient midgut infection of the mosquitoes. Formulate 10 to the seven plaque forming units per milliliter virus titer for the blood meal to stimulate the mosquitoes to engorge. Add a TP to a final concentration of 0.02 molar.

Place a pore sign intestinal membrane over the mouth of the water jacketed glass feeder. Next, set up the feeders with circulating 37 degrees Celsius water. Then pipette the meal into the chamber and place feeders securely on the carton.

After feeding the mosquitoes for 10 to 30 minutes, sort cold anesthetize mosquitoes for the blood engorged specimens to allow replication and dissemination of a TS virus in the selected mosquitoes. Incubate them at 28 degrees Celsius and 75%relative humidity for eight to 10 days. In order to immobilize the mosquitoes.

Place the paper holding carton at four degrees Celsius for approximately 15 minutes. Now, transfer five to 10 mosquitoes to a chill table that is adapted for use on a fluorescent microscope. Examine and sort the mosquitoes based on presence or absence of fluorescence.

Return selected mosquitoes to the paper carton for use as desired. At this point, determine infection efficiency using fluorescent intensity as a proxy. Finally, dissect tissues such as mid guts, SIV glands, and the fat bodies, and monitor for fluorescence.

These organs and tissues can be dissected at earlier times if desired. Deprive mosquitoes of sugar source for 24 hours prior to the feed. Prepare a 50 microliter capillary tube that has been heated, pulled and snipped with three to five microliters of Cargill.

Type B immersion oil to ensure the mosquitoes don't escape. Remove their legs and wings. Now insert the probos into the tube.

Carefully monitor the droplets of saliva that exude from the probos into the immersion oil. If necessary, place a drop of 1%pilocarpine on the mosquito thorax to increase salivation after 60 to 90 minutes, remove the mosquitoes and store them for future virus isolation. Now to harvest solution from the capillary tube, expel the saliva oil mixture into a tube containing 500 microliters of 20%F-B-S-P-B-S sterile.

Filter the inoculum through a 0.2 micron syringe filter, and then use this inoculum to infect cultured cells in mosquito cells. The efficacy of infection with five prime DS MRE 16 GFP virus is evaluated by expression of GFP. Over time, these cells were infected with virus at 0.01 multiplicity of infection.

An epi fluorescence microscope allows whole body view of GFP in the infected mosquito as well as in the different body parts here at 10 days post-infection. Organ selective expression of GFP is indicative of a disseminated infection. Mid guts usually have distinct foci infection early after ingesting a blood meal containing virus that spreads throughout the posterior midgut at later times post infection.

These events are signaled by the GFP and DS RED reporters. The transmission assay indicates that five prime DS MRE 16 GFP virus stably expresses GFP throughout the extrinsic incubation period. In the mosquito here, GFP and mosquito saliva is detected as early as eight days post infection.

After watching this video, you should have good understanding of how to orally infect an A TS virus to a mosquito, how to visually assess virus infection in a mosquito, and how to assess virus transmission from the mosquito.