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

Abstract

Source: Yang, S. et al., Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster. J. Vis. Exp. (2019)

In this video, we demonstrate the cytopathic effect assay to determine the tissue culture infective viral dose of Drosophila C virus on Drosophila S2* cells. A range of viral concentrations are used to infect the cultured cells. The proportion of cells exhibiting structural changes is determined, and this data is used to calculate the tissue culture infective dose.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 ml Microcentrifuge tubes	Brand	352070
10 cm cell culture dish	Sigma	CLS430167	Cell culture
Cell Incubator	Sanyo	MIR-553
Centrifuge	Eppendorf	5810R
Centrifuge	Eppendorf	5424R
Drosophila Incubator	Percival	I-41NL	Rearing Drosophila
FBS	Invitrogen	12657-029	Cell culture
Schneider's Insect Medium	Sigma	S9895	Cell culture
Statistical software	GraphPad Prism 7
Flat bottom 96-well-plate	Sigma	CLS3922	Cell culture
Cell Incubator	Sanyo	MIR-553
Microscope	Olympus	CKX41