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).

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</s...

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 ...

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, Drosophilamelanogaster (D. melanogaster), provides a powerful system to investigate virus-host interaction5,6 and has proven to be one of the most efficient models to study human viral diseases7,8,9. Highly conserved antiviral signaling pathways and incomparable genetic tools make flies a great model to produce significant results with real implications for human antiviral studies. In addition, flies are easy and inexpensive to maintain in the laboratory and are convenient for large-scale screening of novel regulatory factors6,10 in the virus and the host during infection.
Four major highly conserved antiviral pathways (e.g., the RNA interference (RNAi) pathway11, the JAK-STAT pathway12, the NF-&#954;B pathway, and the autophagy pathway13) are well studied in Drosophila in recent years6. The RNAi pathway is a broad antiviral mechanism that can suppress most kinds of virus infection6,14. Disruption of this pathway by mutation in genes like Dicer-2 (Dcr-2) or Argonaute 2 (AGO2) can lead to increased virus titer and host mortality15,16,17. The JAK-STAT pathway has been implicated in control of infection by a virus from the Dicistroviridae family and the Flaviviridae family in insects, e.g., Drosophila C virus (DCV) in flies16 and West Nile virus (WNV) and Dengue Virus in mosquito18,19. The Drosophila Toll (homologous to the human NF-&#954;B pathway) and Immune deficiency (IMD) pathways (similar to the human NF-&#954;B and TNF pathway) are both involved in defending virus invasion20,21,22. Autophagy is another conserved mechanism involved in the regulation of viral infection, which is well characterized in Drosophila23,24. Thus, identification of novel regulatory factors of these pathways and dissecting crosstalk between these antiviral signaling and other biological pathways, such as metabolism, aging, neural reaction and so on, can be easily set up in the Drosophila system.
Although most well-established viral infectious models in Drosophila are induced by RNA viruses, infection by the Invertebrate iridescent Virus 6I (IV-6) and Kallithea viruses have shown the potential for study of DNA viruses in flies25,26. Moreover, the virus can also be modified to allow infection of Drosophila, such as the influenza virus9. This has greatly expanded the application of the Drosophila screening platform. In this procedure, we use DCV as an example to describe how to develop a viral infectious system in Drosophila. DCV is a positive-sense single stranded RNA virus of approximately 9300 nucleotides, encoding 9 proteins27. As a natural pathogen of D. melanogaster, DCV is considered as a suitable virus to study host physiological, behavioral and basal immune response during host-virus interaction and co-evolution28. Additionally, its rapid mortality rate following infection in wild type flies makes DCV useful to screen for resistant or susceptible genes in the host29.
However, there are several aspects of concern when studying viral infections in Drosophila. For example, symbiotic bacteria Wolbachia have an ability to inhibit a wide spectra of RNA virus proliferation in Drosophila and mosquito30,31,32. Recent evidence shows a possible mechanism in which Wolbachia blocks Sindbis virus (SINV) infection through the upregulation of methyltransferase Mt2 expression in the host33. Additionally, the genetic background of insects is also critical for viral infection. For instance, the natural polymorphism in the gene, pastrel (pst), determines the susceptibility to DCV infection in Drosophila34,35, whilst the loci of Ubc-E2H and CG8492 are involved in Cricket paralysis virus&#160;(CrPV) and Flock house virus (FHV) infection, respectively36.
The particular ways to establish the virus-host interaction in flies, must be chosen according to research purposes such as a high-throughput screen for host cellular components in Drosophila cell lines37,38, oral infection to study gut-specific antiviral response22,39,40, needle pricking41,42 or nano-injection by passing epithelial barriers to stimulate systemic immune responses. Nano-injection can precisely control the viral dose to induce a controlled antiviral reaction and a physiological lesion43, thus guaranteeing high experimental reproducibility44. In this study, we describe a nano-injection method to study virus-host interactions in Drosophila, highlighting the importance of the flies&#39; background effects.

<table>
	<tbody>
		<tr>
			<td>0.22um filter</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge tubes</td>
			<td>Brand</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml RNase free Microcentrifuge tubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>Sigma</td>
			<td>CLS430167</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>100 Replacement tubes</td>
			<td>Drummond Scientific</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>ABI 7500 qPCR system</td>
			<td>ABI</td>
			<td>7500</td>
			<td>qPCR</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Sanyo</td>
			<td>MIR-553</td>
			<td></td>
		</tr>
		<tr>
			<td>Centriguge</td>
			<td>Eppendof</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centriguge</td>
			<td>Eppendof</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>151858</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>DEPC water</td>
			<td>Sigma</td>
			<td>95284-100ML</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Percival</td>
			<td>I-41NL</td>
			<td>Rearing Drosophila</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>12657-029</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>flat bottom 96-well-plate</td>
			<td>Sigma</td>
			<td>CLS3922</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Lysis buffer (RNA extraction)</td>
			<td>Thermo Fisher</td>
			<td>15596026</td>
			<td>TRIzol Reagent</td>
		</tr>
		<tr>
			<td>Lysis buffer (liquid sample RNA extraction)</td>
			<td>Thermo Fisher</td>
			<td>10296028</td>
			<td>TRIzol LS Reagent</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td>Nanoject II Variable Volume (2.3 to 69 nL) Automatic Injector with Glass Capillaries (110V)</td>
		</tr>
		<tr>
			<td>Optical Adhesive Film</td>
			<td>ABI</td>
			<td>4360954</td>
			<td>qPCR</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>qPCR plate</td>
			<td>ABI</td>
			<td>A32811</td>
			<td>qPCR</td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Insect Medium</td>
			<td>Sigma</td>
			<td>S9895</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>statistical software</td>
			<td>GraphPad Prism 7</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TransScript Fly First-Strand cDNA Synthesis SuperMix</td>
			<td>TransScript</td>
			<td>AT301</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>IKA</td>
			<td>VORTEX 3</td>
			<td>RNA extraction</td>
		</tr>
	</tbody>
</table>

establishment of viral infection and analysis of host-virus interaction in drosophila melanogaster

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.

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...

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

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.

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

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 ...

This method can help answer key questions in the virus-host interaction field about how to effectively establish a viral infection in Drosophila melanogaster. This nanoinjection method allows a precise control of the infection dose of the virus and can be easily applied to infections with other microbial pathogens. Begin by growing one times 10 to the seventh viable S2 star cells per milliliter as loose, semi-adherent monolayer in a 10 centimeter cell culture dish with 10 milliliters of complete Schneider's Drosophila Medium without additional carbon dioxide at 25 degrees Celsius for one hour.During the incubation, resuspend freshly thawed Drosophila C Virus or DCV to a multiplicity of infection of 0.01 in one milliliter of complete medium and add virus solution to the cell culture dishes. Then return the plate to the 25 degrees Celsius incubator for three to five days. The virus is ready for collection when the cell morphology looks blurry and the culture medium is full of black particles indicative of cell debris.Mix the supernatant by pipetting a few times before transferring the entire cell culture to a 15 milliliter tube for lysis of the host cells at minus 80 degrees Celsius. To generate working concentrations of virus, thaw the cell suspension in a 25 degrees Celsius water bath with constant shaking before pelleting the cell debris by centrifugation. Then transfer the supernatant into a new sterile 15 milliliter tube for vortexing and aliquot up to 200 microliters of virus solution per tube.To determine the average virus tissue culture infective dose, first seed one times 10 to the five S2 star cells in 100 microliters of complete medium into eight wells per column in 12 rows of a 96 well plate. Then return the plate to the 25 degrees Celsius incubator. Randomly select five tubes of virus from minus 80 degrees Celsius storage.While the cells are settling, fill 10 sterile 1.5 milliliter microcentrifuge tubes with 450 microliters of sterile complete medium and add 50 microliters of virus stock to the first 1.5 milliliter tube containing 450 microliters of sterile complete medium diluting the suspensions 10-fold per step to the 12th well. At the end of the incubation, add 50 microliters of each dilution to the appropriate well in each column for that tube of virus and add 50 microliters of culture medium without virus to one well per column as the negative control well. Then place the plate in the 25 degrees Celsius incubator for three days and assess the cytopathic effect of each virus stock under a Brightfield microscope at a 20X magnification daily.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. Before breeding, thoroughly mix 400 microliters of 50 micrograms per milliliter of Tetracycline with four grams of fresh standard cornmeal fly food and place the food at four degrees Celsius overnight to evaporate the ethanol. The next morning, warm the food to room temperature and place the food and 20 female and 10 male newly eclosed adult flies into a breeding vial for a three to four-day breeding incubation at 25 degrees Celsius and 60%humidity under a normal light/dark cycle.When enough eggs have been laid, collect the newly eclosed adult flies under a light flow of carbon dioxide and breed the Drosophila again two more times as just demonstrated. After three generations of Tetracycline treatment, collect five flies under a light flow of carbon dioxide and homogenize the flies with 250 microliters of double distilled water and a few 0.5 millimeter sterile ceramic beads. After one minute, add 250 microliters of 2X Buffer A to the sample for vortexing before freezing the samples at minus 80 degrees Celsius.Next, quickly thaw samples from minus 80 degrees Celsius storage in a 25 degrees Celsius water bath, followed by a 30-minute incubation in a 70 degrees Celsius water bath before extracting the genomic DNA and amplifying the DNA by polymerase chain reaction according to standard protocols. Confirm the absence of Wolbachia 16 small RNA and wsp in each fly homogenate by gel electrophoresis according to standard protocols. Then rear the Wolbachia-free fly stock on standard cornmeal fly food as demonstrated.For viral infection, first use a stereomicroscope and thin forceps to break the tip of a glass capillary needle to the appropriate diameter for nanoinjection. To assemble the injector, place the ceiling O-ring and a white spacer onto the metal plunger of the injector with the large dimple facing outwards. Use a syringe equipped with a 30 gauge needle to fill the glass needle with mineral oil and place the needle through the collar.Place the larger O-ring around the base of the collar about one millimeter from the blunt end of the needle and mount the needle onto the plunger of the injector. Secure the needle onto the plunger and press the empty button to extend the plunger of the microinjector until an audible signal is heard. Now press fill to retract the plunger five millimeters and dip the needle into a 100 plaque-forming unit viral suspension.Gently shake one or at least three vials of 20 Wolbachia-free male Drosophila onto the injection dish. Male endowed flies are preferred during mating and reproduction may influence females. Then inject the thorax of each fly with 50.6 nanoliters of virus solution at the slightly lighter colored region between the mesopleura and the pteropleura and measure the DCV load by cytopathic effect assay and quantitative RT PCR from ground flies as just demonstrated.After the injection, carefully transfer the flies to a fresh vial and place the vial in a horizontal position to prevent the flies from sticking to the medium while recovering from the anesthesia. The injection is time consuming but the DCV replication is very rapid so it is very important to write down the exact time on the tube once all of the flies from each vial have been injected. Virus infection can induce cell lysis and cytopathic effects are observed at three days post-infection.Wolbachia 16s rRNA and wsp primers can be used to detect the presence of Wolbachia and Drosophila as demonstrated. Wolbachia-free flies exhibit a significantly decreased survival rate after DCV infection and in a dose-dependent manner. DCV activates antiviral signaling pathways in the host that are critical for antiviral infection in Drosophila as evidenced by the decreased survival rate and increased viral load in Dicer-2 mutant flies.While attempting this procedure, it's important to remember that contamination with Wolbachia genotype may affect the susceptibility of the Drosophila melanogaster flies to the DCV infection. Following this procedure, other methods like a larger scale genetic screen can be performed in order to answer additional questions about under defined host genes required for viral infection or antiviral responses. After its development, this technique paved the way for researchers in the field of human viral disease to explore the mechanisms underlying outbreaks of endemic human viral infections in Drosophila melanogaster model.Don't forget that working with virus can be extremely hazardous and that precautions such as wearing the appropriate protective equipment should always be taken while performing this procedure.

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

Abstract