Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Summary

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Abstract

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Introduction

Emerging viral infections, especially by arboviruses, such as the Chikungunya virus¹, the Dengue virus, the Yellow fever virus² and the Zikavirus³, have been a huge threat to public health by causing pandemics⁴. Thus, a better understanding of virus-host interaction has become increasingly important for epidemic control and treatment of viral diseases in humans. For this goal, more appropriate and efficient models must be established to investigate the mechanisms underlying virus infection.

The fruit fly, Drosophilamelanogas....

Protocol

NOTE: Before starting experiment, the cell lines and fly stocks used must not be contaminated by other pathogens, especially for viruses such as DCV, FHV, Drosophila X virus (DXV), and Avian nephritis virus (ANV). Ideally, RNA sequencing or a simpler PCR-based identification are used to detect the contamination^10,45. If contamination occurred, the cell lines and fly stocks should not be used any more until they are decontaminated completely⁴⁶

Discussion

In this article, we present a detailed procedure on how to establish a viral infectious system in adult Drosophila melanogaster using nano-injection. The protocols include the preparation of appropriate fly lines and virus stock, infection techniques, the evaluation of infectious indicators and the measurement of the antiviral response. Although DCV is used as an example of a viral pathogen, tens of different kinds of virus have been successfully applied for study in the Drosophila system. In addition, hundreds .......

Materials

Name	Company	Catalog Number	Comments
0.22um filter	Millipore	SLGP033RS
1.5 ml Microcentrifuge tubes	Brand	352070
1.5 ml RNase free Microcentrifuge tubes	Axygen	MCT-150-C
10 cm cell culture dish	Sigma	CLS430167	Cell culture
100 Replacement tubes	Drummond Scientific	3-000-203-G/X
15 ml tube	Corning	352096
ABI 7500 qPCR system	ABI	7500	qPCR
Cell Incubator	Sanyo	MIR-553
Centriguge	Eppendof	5810R
Centriguge	Eppendof	5424R
Chloroform	Sigma	151858	RNA extraction
DEPC water	Sigma	95284-100ML	RNA extraction
Drosophila Incubator	Percival	I-41NL	Rearing Drosophila
FBS	Invitrogen	12657-029	Cell culture
flat bottom 96-well-plate	Sigma	CLS3922	Cell culture
Fluorescence microscope	Olympus	DP73
Isopropyl alcohol	Sigma	I9516	RNA extraction
Lysis buffer (RNA extraction)	Thermo Fisher	15596026	TRIzol Reagent
Lysis buffer (liquid sample RNA extraction)	Thermo Fisher	10296028	TRIzol LS Reagent
Microscope	Olympus	CKX41
Nanoject II Auto-Nanoliter Injector	Drummond Scientific	3-000-204	Nanoject II Variable Volume (2.3 to 69 nL) Automatic Injector with Glass Capillaries (110V)
Optical Adhesive Film	ABI	4360954	qPCR
Penicillin-Streptomycin, Liquid	Invitrogen	15140-122	Cell culture
qPCR plate	ABI	A32811	qPCR
Schneider’s Insect Medium	Sigma	S9895	Cell culture
statistical software	GraphPad Prism 7
TransScript Fly First-Strand cDNA Synthesis SuperMix	TransScript	AT301	RNA extraction
Vortex	IKA	VORTEX 3	RNA extraction