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* These authors contributed equally
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.
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.
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....
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 contamination10,45. If contamination occurred, the cell lines and fly stocks should not be used any more until they are decontaminated completely46
Results of this section are obtained after DCV infection of D. melanogaster. Figure 1 shows the flow chart of viral infection in Drosophila. Flies are injected intra-thoracically, and then the samples are collected for the measurement of the viral TCID50 and the genome RNA level (Figure 1). Virus infection can induce cell lysis and CPE is observed at 3 days post infection (Figure 2A). The virus load measured by the CPE assay is in line with t.......
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 .......
We would like to thank the entire Pan lab in IPS. CAS. We thank Dr. Lanfeng Wang (IPS, CAS) for experimental assistance and Dr. Gonalo Cordova Steger (Springer nature), Dr. Jessica VARGAS (IPS, Paris) and Dr. Seng Zhu (IPS, Paris) for comments. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences to L.P (XDA13010500) and H.T (XDB29030300), the National Natural Science Foundation of China to L.P (31870887 and 31570897) and J.Y (31670909). L.P is a fellow of CAS Youth Innovation Promotion Association (2012083).
....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 |
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