Assay to Determine the Virus Infectious Dose of Drosophila C Virus in Cultured Insect Cells

1. Drosophila C virus load measure by CPE assay

1. Pick 5 random virus-containing tubes to determine the average virus tissue culture infective dose (TCID₅₀) by a cytopathic effect (CPE) assay (1 unit of TCID₅₀= 0.7 plaque-forming unit [PFU]).
   NOTE: CPE refers to structural changes in host cells caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to reproduce.
2. On day 1, collect prepared S2* cells by pipetting. Count the cell number. Centrifuge cells for 300 x g for 4 min and discard the supernatant. Dilute the cells to the density of 1 x 10⁶ cells/mL with a complete medium for S2* cells.
3. Seed 1 x 10⁵ S2*cells (100 μL) per well to the flat-bottom 96-well-plate (~30% confluency).
4. Perform serial 10-fold dilutions of the DCV stock with complete medium for S2* cells. Add 50 µL dilutions into the well. Add 50 µL of culture medium without virus to the well classified as the negative control well.
   NOTE: For each dilution rate, 8 wells were used. As the typical DCV yield is around 10⁸-10⁹ PFU/mL, the dilution should at least cover ~10⁻⁵-10⁻¹⁰.
5. On day 4, observe CPE with a bright-field microscope with a 20X or 40X objective. 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".
6. Mark CPE positive or negative wells with + or –, respectively. Calculate the virus TCID₅₀ by the Reed and Muench method.