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. ....

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