This study investigates the infection process of Drosophila C virus (DCV) in Drosophila melanogaster. It details the methods for determining the average virus tissue culture infective dose using serial dilutions and cytopathic effect assessment.
Drosophila C virus, or DCV, a positive single-stranded RNA virus, commonly infects Drosophila melanogaster.
During infection, DCV uses host cell machinery to replicate the viral genome, eventually producing virus particles. Infected cells exhibit cytopathic effects, CPE, and undergo lysis or die without lysis due to their inability to divide.
To determine the average virus tissue culture infective dose, the DCV concentration at which half the infected cells display CPE, begin with serially diluted DCV suspensions.
Transfer to a multi-well plate containing semi-adherent Drosophila S2* cells derived from late-stage embryos of Drosophila melanogaster, a natural DCV host. Wells containing medium and cells serve as negative controls.
Incubate for the virus to infect the cells. Virus-infected cells display CPE via cell damage and death. Post-incubation, observe the wells under the microscope.
Identify CPE-positive and negative wells across dilutions. Use a suitable method to determine the average virus tissue culture infective dose of DCV.
To determine the average virus tissue culture infective dose, first, seed 1 x 105 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-degree 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 tenfold 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-degree Celsius incubator for three days, and assess the cytopathic effect of each virus stock under a bright field microscope at a 20 times 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.
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