This method can help answer key questions in the virus-host interaction field about how to effectively establish a viral infection in Drosophila melanogaster. This nanoinjection method allows a precise control of the infection dose of the virus and can be easily applied to infections with other microbial pathogens. Begin by growing one times 10 to the seventh viable S2 star cells per milliliter as loose, semi-adherent monolayer in a 10 centimeter cell culture dish with 10 milliliters of complete Schneider's Drosophila Medium without additional carbon dioxide at 25 degrees Celsius for one hour.
During the incubation, resuspend freshly thawed Drosophila C Virus or DCV to a multiplicity of infection of 0.01 in one milliliter of complete medium and add virus solution to the cell culture dishes. Then return the plate to the 25 degrees Celsius incubator for three to five days. The virus is ready for collection when the cell morphology looks blurry and the culture medium is full of black particles indicative of cell debris.
Mix the supernatant by pipetting a few times before transferring the entire cell culture to a 15 milliliter tube for lysis of the host cells at minus 80 degrees Celsius. To generate working concentrations of virus, thaw the cell suspension in a 25 degrees Celsius water bath with constant shaking before pelleting the cell debris by centrifugation. Then transfer the supernatant into a new sterile 15 milliliter tube for vortexing and aliquot up to 200 microliters of virus solution per tube.
To determine the average virus tissue culture infective dose, first seed one times 10 to the five S2 star cells in 100 microliters of complete medium into eight wells per column in 12 rows of a 96 well plate. Then return the plate to the 25 degrees Celsius incubator. Randomly select five tubes of virus from minus 80 degrees Celsius storage.
While the cells are settling, fill 10 sterile 1.5 milliliter microcentrifuge tubes with 450 microliters of sterile complete medium and add 50 microliters of virus stock to the first 1.5 milliliter tube containing 450 microliters of sterile complete medium diluting the suspensions 10-fold per step to the 12th well. At the end of the incubation, add 50 microliters of each dilution to the appropriate well in each column for that tube of virus and add 50 microliters of culture medium without virus to one well per column as the negative control well. Then place the plate in the 25 degrees Celsius incubator for three days and assess the cytopathic effect of each virus stock under a Brightfield microscope at a 20X magnification daily.
Classify a well in which the cells look blurry and the medium is full of fragments as a positive well and a well in which the cell morphology is normal as a negative well. Before breeding, thoroughly mix 400 microliters of 50 micrograms per milliliter of Tetracycline with four grams of fresh standard cornmeal fly food and place the food at four degrees Celsius overnight to evaporate the ethanol. The next morning, warm the food to room temperature and place the food and 20 female and 10 male newly eclosed adult flies into a breeding vial for a three to four-day breeding incubation at 25 degrees Celsius and 60%humidity under a normal light/dark cycle.
When enough eggs have been laid, collect the newly eclosed adult flies under a light flow of carbon dioxide and breed the Drosophila again two more times as just demonstrated. After three generations of Tetracycline treatment, collect five flies under a light flow of carbon dioxide and homogenize the flies with 250 microliters of double distilled water and a few 0.5 millimeter sterile ceramic beads. After one minute, add 250 microliters of 2X Buffer A to the sample for vortexing before freezing the samples at minus 80 degrees Celsius.
Next, quickly thaw samples from minus 80 degrees Celsius storage in a 25 degrees Celsius water bath, followed by a 30-minute incubation in a 70 degrees Celsius water bath before extracting the genomic DNA and amplifying the DNA by polymerase chain reaction according to standard protocols. Confirm the absence of Wolbachia 16 small RNA and wsp in each fly homogenate by gel electrophoresis according to standard protocols. Then rear the Wolbachia-free fly stock on standard cornmeal fly food as demonstrated.
For viral infection, first use a stereomicroscope and thin forceps to break the tip of a glass capillary needle to the appropriate diameter for nanoinjection. To assemble the injector, place the ceiling O-ring and a white spacer onto the metal plunger of the injector with the large dimple facing outwards. Use a syringe equipped with a 30 gauge needle to fill the glass needle with mineral oil and place the needle through the collar.
Place the larger O-ring around the base of the collar about one millimeter from the blunt end of the needle and mount the needle onto the plunger of the injector. Secure the needle onto the plunger and press the empty button to extend the plunger of the microinjector until an audible signal is heard. Now press fill to retract the plunger five millimeters and dip the needle into a 100 plaque-forming unit viral suspension.
Gently shake one or at least three vials of 20 Wolbachia-free male Drosophila onto the injection dish. Male endowed flies are preferred during mating and reproduction may influence females. Then inject the thorax of each fly with 50.6 nanoliters of virus solution at the slightly lighter colored region between the mesopleura and the pteropleura and measure the DCV load by cytopathic effect assay and quantitative RT PCR from ground flies as just demonstrated.
After the injection, carefully transfer the flies to a fresh vial and place the vial in a horizontal position to prevent the flies from sticking to the medium while recovering from the anesthesia. The injection is time consuming but the DCV replication is very rapid so it is very important to write down the exact time on the tube once all of the flies from each vial have been injected. Virus infection can induce cell lysis and cytopathic effects are observed at three days post-infection.
Wolbachia 16s rRNA and wsp primers can be used to detect the presence of Wolbachia and Drosophila as demonstrated. Wolbachia-free flies exhibit a significantly decreased survival rate after DCV infection and in a dose-dependent manner. DCV activates antiviral signaling pathways in the host that are critical for antiviral infection in Drosophila as evidenced by the decreased survival rate and increased viral load in Dicer-2 mutant flies.
While attempting this procedure, it's important to remember that contamination with Wolbachia genotype may affect the susceptibility of the Drosophila melanogaster flies to the DCV infection. Following this procedure, other methods like a larger scale genetic screen can be performed in order to answer additional questions about under defined host genes required for viral infection or antiviral responses. After its development, this technique paved the way for researchers in the field of human viral disease to explore the mechanisms underlying outbreaks of endemic human viral infections in Drosophila melanogaster model.
Don't forget that working with virus can be extremely hazardous and that precautions such as wearing the appropriate protective equipment should always be taken while performing this procedure.
