デング熱蚊の感染症（A.ネッタイシマカ）と感染症の表現型決定のためのプロトコル

I am George Dimopoulos, and now we will show you a procedure on how to infect the Aidas GTI mosquito with dengue virus. And the first thing we'll need to do is to propagate the virus in an Aidas Aus cell line. My name is Jose Luis Ramirez, and I am a PhD Student in the ous Lab.

For this procedure, we'll need a BSL two Facility with a tissue culture hood, a CO2 incubator set at 37 degree Celsius. We need 50 ml plastic conical tubes. We need human serum, we need a water bath, a 37 degrees Celsius, a centrifuge.

We need 96 well plates and other standard molecular biology and biochemistry, reagents And consumables. So this is a regular tissue culture Hood in a BSL two facility, and the first step is to take out the cells that have grown to an 80%co fluency in a 75 square centimeter flask. This is an MEM media with 10%heat, heat and activated fetal bovine serum supplemented with 1%L-glutamine and non-essential amino acids.

Okay, now we will add the Dengue virus to the cells. We have taken out the virus stock from the minus 80 freezer and we had thaw it on a 37 degree incubator. We add about 0.5 milliliters stock virus to the cells to achieve a concentration of about 1.5 virus particles per cell.

The cells are then placed on a shaker for about 15 minutes at a slow shaking rate. Next, the cells are placed in a CO2 incubator for about 45 minutes. At 37 degrees Celsius, CO2 concentration is 5%After the 45 minute incubation, we will take out the cells and add 30 ml of medium, and then we will place the cells in the incubator Again and incubate for another five days.

Now we'll prepare the virus Particles for infection. First, we need to detach the cells from flask with a scraper and then transfer them together with a media to a 50 ml conical tube. Now we have centrifuge cells at 12, 000 RPMs for 10 minutes to pellet them.

At the bottom of this bottle, you can see the pellet. Next, we will remove the supernatant and only leave one ml with the cells in order to release the virus from the cells. We will freeze them.

That will rupture the cells, and then we will thaw them and then freeze them again and we will repeat this procedure three to four times and the freezing is done on dry ice. After freezing, the cell pellet is placed on a 37 degree water bath to thaw them, and then they're freezed again on the dry eyes between each freezing step, WEP suspend the cells through vortexing. Now we are collecting the virus containing supernatant and adding it to the supernatant from the previous centrifugation, which also contains virus particles.

This virus preparation is now ready to be combined with an equal amount of whole human blood And serum. Here we have Combined the virus preparation with whole human blood supplemented with 15%human serum. This blood virus solution is going to be used now for feeding the mosquitoes and infecting them.

We are going to add this blood into a membrane feeder and this membrane feeder will be placed on a cup containing the mosquitoes and we will let the mosquitoes feed on this blood for about 25 minutes. We have placed a clip on the membrane feeder in order to stabilize it. It weights it down basically on the net, and we will now add the blood into the feeder.

In some occasions, this experiment may work better when it's done in dark since naturally mosquitoes frequently feed in dark. This depends of course on how the mosquito colony has been conditioned. The mosquitoes have now fed for about 25 to 30 minutes, and we can remove the membrane feeder and place it in a safe container for decontamination.

After the blood fitting, the unfit mosquitoes have to be removed from the Fed ones. To do this, we knocked down the mosquitoes by putting the small cups containing the blood fed mosquitoes in cold room for five to 10 minutes. As we can see, the mosquitoes are already down in the cold.

We open the lid and transfer the mosquitoes in a small petrol dish, which is kept on a ice bucket so that the mosquitoes are kept in cold all through and they cannot escape. Remember, these are infected mosquitoes, so all care must be taken not to let them go loose. Once the mosquitoes are down in the cold room, which transfer the mosquitoes to the normal insectory, which is maintained at the room temperature, but we put the mosquitoes always in the petro dish, which is kept in the ice bucket, so they're there so that they're always down.

You can see how the Fed mosquitoes can be removed from the UNFI ones. The Fed ones have the blood in them. They're fully engorged.

Some of them have fed on blood partially so they're not fully engorged. They have a small blood in their abdomen, but others have a lot of blood in their abdomen. The UNFI ones do not have any blood in their abdomen, and so you can easily, easily distinguish between the Fed and the unfit ones.

So we'll transfer these mosquitoes from the petrol dish to the wax line cardboard Cups. The next step Is to assay the dengue virus infection in the mosquito. If we want to assay the virus infection in the mosquito midgut, we should dissect the mosquitoes at around day seven after they have taken the blood meal.

And if you want to assay the virus infection in the sali gland, we should dissect the mosquitoes at around 14 days after blood feeding, Jose will now show you how to dissect the gut from the mosquito. The mosquitoes have now been anesthetized on ice, and we will kill them through an incubation in 70%ethanol for about one minute. Next, we transfer the mosquitoes to two successive water baths.

One minute each. This is to wash away the ethanol from the mosquitoes. Now we'll dissect the mosquitoes in PBS on microscope slides.

The best way of dissecting out the midgut from an gyp time mosquito is to place the mosquito upside down on its wings and grab the abdominal part with one forcep and the thorax with another forcep, and then pull the mosquito. Now Jose is pulling the mosquito apart and the gut will appear between the abdomen and the thoracic part of the mosquito. Now the gut is the semi transparent tissue that has appeared on the upper part of the mosquito.

Now we will transfer the dissected gut into a cell line medium and bring it to the tissue culture hood to isolate the virus particles from the gut. We bring the dissected mid guts into the tissue culture room and we homogenize with a pestle in order to release the virus particles. After the midgut homogenization, we add medium to the homogenate to achieve a one to 100 dilution of the virus particles.

Then we continue to dilute this further to one to 10, one to 101 to 1000. In the 96 well plate, we have prepared Alba picto cell line to 80%co fluency in a 24 well plate. Then we remove the sup natant from the hiatus alba picto cells and to these cells, we add the diluted virus particles in order to determine the concentration of virus particles in each dilution.

This will allow us to determine the infection level of the virus. In the mosquito midgut, the cells are then shaked for 15 minutes at the room temperature slowly. Then we place the cells for 45 minutes in the 37 degree CO2 incubator.

Next we add one ml of 1%methyl cellulose overlay to each well, and the plate is then incubated again at 37 degrees CO2 Incubator for five days. After the five day incubation, We add one ml of an acetone methanol fixative to each well. Then we incubate the plate at four degrees Celsius in a refrigerator for one hour.

Next, we remove the fixative and wash each well one time with PBS. Next, we make a one to 1000 dilution of the anti dengue primary antibody in a 5%blood solution. Next, we add 200 microliters of this antibody solution.

To each, well Move your head.Thanks. Next, we incubate the plate for one hour at 37 degrees Celsius. Next we remove the hyperimmune fluid and we wash each well with one XPBS.

Next, we repeat the procedure in exactly the same way with a secondary antibody. We are prepared 20 ml of the substrate according to the protocol, and we add it to a container. Then we add 200 microliters of the substrate to each well.

Next we stop the reaction by removing the substrate and we add one ml of distilled water to each well. Then we discard the water from the plate and we let the plate dry. Each color dot represents a plaque of viruses, and by counting these plaques, we can estimate the number of virus particles that were in the original midgut of the mosquito that we Homogenized.

We have just shown you the Procedure of how to perform a dengue virus infection in aegypti mosquito. This protocol involves the production of virus in the mosquito cell line. The virus particles are then purified from the cell line and isolated, and they are mixed with human whole blood and fed to the mosquitoes.

The virus will infect the mosquitoes and once infection has been established, we have shown you how to isolate the midgut tissue of the mosquito and how to determine the infection level of this mid gut. We basically homogenize the midgut and use the procedure to release the virus particles from this midgut. Then we use this virus particles to infect the mosquito cell line again in order to determine the infection level of the midgut, the number of virus particles that have infected the mosquito me gut.