This protocol describes how to establish viral infection in vivo de 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, 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-κB 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-κB pathway) and Immune deficiency (IMD) pathways (similar to the human NF-κB 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 (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' background effects.
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.
1. Virus and Fly Preparation
2. Viral Infection in Drosophila
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 that measured by qPCR (Figure 2B). As shown in Figure 3, Wolbachia inhibits DCV infection in Drosophila. wol-16s rRNA and wsp primers are used to detect Wolbachia presence (Figure 3A) and Wolbachia-free flies show significantly decreased survival rate after DCV infection (Figure 3B). Figure 4 shows how to verify pst polymorphism by gradient PCR using specific primers. Figure 5 shows dose dependent effects of DCV infection in the wildtype fly. The survival rate in Figure 5A and the virus load at 3 days post infection is shown in Figure 5B. Figure 6 demonstrates that DCV infection can activate the Dcr2/RNAi and JAK/STAT antiviral signaling pathways in the host52. The expression level of the reporter gene vago expression of Dcr2/RNAi pathway is elevated 3 days post infection (Figure 6A). The JAK-STAT pathway is also activated upon DCV infection, which is indicated by the expression level of reporter gene vir-1 (Figure 6B). Figure 7 shows that the Dcr2/RNAi pathway is critical for antiviral infection in Drosophila52. Dcr-2 mutant flies have a decreased survival rate (Figure 7A) and an increased virus load (Figure 7B) after DCV infection.
Figure 1: Flow chart of Drosophila infection and virus load analysis.
Establishing Drosophila infection by nano-injection. The schema chart shows that flies are infected intra-thoracically and that virus load are measured by the CPE assay and qRT-PCR. Please click here to view a larger version of this figure.
Figure 2: DCV load analysis.
The virus load measured by the CPE assay is in line with that measured by qPCR. (A) S2* cells are cultured at 25 °C for 3 days with a different virus dilution (50 μL). 1 Unit TCID50 of used virus is 4.29 x 109 (equivalent to 3 x 109 PFU/mL). First panel, 10-3 dilution; second panel, 10-7 dilution; third panel, 10-10 dilution; forth panel, w/o infection. Cells in panel one and two show the typical CPE positive phenotype (white arrows indicate cell debris), and cells in panel three and four show the typical negative phenotype. The scale bar is indicated in the figures. (B) Measurement of DCV load by qPCR. Perform serial 10-fold dilutions of DCV and infect the S2* cell line. The total RNA of infected cells is extracted and qPCR is performed to measure the DCV RNA level. Gradient viral infectious load (determined by CPE assay) in cells is matched and positively related to the measurement by qPCR (r2=0.999). Please click here to view a larger version of this figure.
Figure 3: Viral Infection of flies with or without Wolbachia.
Wolbachia infection contributes to DCV resistance in Drosophila. (A) The Wolbachia presence is detected by wol-16s rRNA and wsp primers. G1, G2 and G3 represented Orer flies (Oregon-R) (Bloomington 5) treated by tetracycline for one, two and three generations respectively. The PCR product size is around 200 bp (wol-16s rRNA) and 600 bp (wsp). (B) Wolbachia free flies are more sensitive to DCV infection. 3-5 day old male adult flies are injected with 100 PFU of DCV. Wolbachia free flies (solid lines) show significantly reduced survival than Wolbachia protected flies (dash lines) upon DCV infection. Two different wildtype lines, Orer and w1118, are used for the survival assay and show similar result. All error bars represent standard error (s.e.) of at least three independent tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant. Kaplan–Meier (B). Please click here to view a larger version of this figure.
Figure 4: Pastrel genotyping by gradient PCR.
pst polymorphism is verified by gradient PCR. Gradient PCR is performed to distinguish pst R (512C) and S (512T) allele in the Drosophila adult. Tm gradients are a: 54 °C, b: 54.7 °C, c: 55.5 °C, d: 58 °C, e: 59.7 °C, f: 62.2 °C, g: 63.7 °C, h: 64 °C. Please click here to view a larger version of this figure.
Figure 5: Survival rate and DCV titer of Orer flies infected with a different dose of DCV.
Orer flies are injected with three different doses of DCV, 10 PFU/fly, 50 PFU/fly and 100 PFU/fly. (A) The flies’ survival rate is significantly lower when infected with higher infectious doses. (B) The DCV RNA level in the whole body of indicated flies is measured by qRT-PCR at the indicated time and normalized to that of 10 PFU/fly inoculation group. All error bars represent standard error (s.e.) of at least three independent tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant. Kaplan–Meier (A) or One-way anova (B). Please click here to view a larger version of this figure.
Figure 6: Dcr2/RNAi and JAK/STAT antiviral signaling pathway activated after DCV infection.
Orer flies were infected with 100 PFU of DCV. Vago (A) and vir-1 (B) mRNA expressed in the whole body are measured by qRT-PCR at indicated time points post infection. The change of expression is normalized to that of basal level before infection. All error bars represent standard error (s.e.) of at least three independent tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant. One-way ANOVA (A, B). Please click here to view a larger version of this figure.
Figure 7: Dcr-2 mutant fly sensitive to DCV infection.
Dcr-2 mutant flies and its genetic control w1118 flies were infected with 100 PFU of DCV. (A) Survival rates of Dcr-2 deficient flies and wild-type (w1118) flies post DCV infection. (B) DCV RNA level in the whole body of indicated flies is measured by qRT-PCR 2 days post infection and normalized to that of w1118. All error bars represent standard error (s.e.) of at least three independent tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant. Kaplan–Meier (A) or student’s T-test (B). Please click here to view a larger version of this figure.
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 of regulatory factors, either of the virus or of the host, have been identified through large-scale screening in flies. Thus, this method is highly adaptable and does not limit to DCV/Drosophila interaction.
To successfully propagate and harvest high titer DCV, a few precautions must be taken. Cells should be healthy, cultured at a suitable density and harvested on time. Reducing cell culture volume does not significantly affect virus titer. Typically, 107 cells infected by DCV at MOI=0.01 will finally yield approximate 108-109 TCID50 of DCV, which can meet the demands of most in vivo and in vitro experiments. The CPE assay and qRT-PCR assay are described in this protocol for the measurement of virus load. The CPE assay is somehow tricky. The appropriate density of cell inoculation and an experienced standard of positive/negative CPE discrimination allow a quick and successful CPE assay. Thus, we provide an easy qRT-PCR assay here to help decide the cut-off dilution point in CPE assay before mastering the CPE assay.
However, DCV is a very potent virus for the adult fly and Drosophila cell line. Only 100 PFU/fly inoculation can kill 50% of wild flies within 5 days. An excess infection dose may shadow the significance between the wild type fly and potential susceptible lines. It is important to apply at least two infection doses when initiating a new screen. Since the massive death of Drosophila may happen in a very short time window because of the high lethality of DCV, recording the death number more frequently in preliminary experiments is strongly recommended. After DCV infection, some flies occasionally have ascites syndrome. Notably, flies that die within 0.5 h post infection should be excluded, which might be caused by inappropriate needle injection injury.
The infectious strength of DCV is also influenced by many host factors, which should be considered before starting an experiment. Wolbachia is a vertical transmit endosymbiont that has great impact on virus infection in flies32. Tetracycline treatment in fly food is a common used method to eliminate Wolbachia infection. However, this method also alters intestinal microbiota composition, which may in turn affect antiviral response53. A few studies have emphasized the importance of genetic variance on virus infection36, such as ubc-e2h, cg8492 and pst. For example, most wild type fly lines have pst S allele and are sensitive to picorna-like virus, while most mutant lines we tested from Bloomington stock center have pst R allele, thus having resistant phenotypes. It is very important to rule out the impact of pst, especially when screening out a resistance gene comparing with wild type control29.
Besides nano-injection to induce systemic virus infection, other methods are available to study virus infection in flies. Drosophila cell lines have proven to be a high-throughput method to screen out virus infection related host cellular component37,38. However, an in vitro system is always accompanied with a high rate of false positives, when confirming in vivo. DCV can also be applied to infect Drosophila orally, whereas it can only trigger a restricted and milder infection. Only 20% of larvae were infected within 12 h and 14% mortality was observed, with just 25% mortality in 20 days in adult flies22. Pricking flies with 0.15-mm-diameter pins is another way to set viral infection but it cannot ensure the accurate infection dose and quantitative repeatability. Overexpressing viral proteins by genetic manipulation in flies is a good way to study molecular interactomes in vivo7. However, it may not portray the reality of physiology and pathology in the host upon infection. Meanwhile, the nano-injection method also has drawbacks, as it is not the natural route of infection and can directly stimulate a strong system immune response just after infection, which bypassed the cuticle, the first antiviral defense line. In sum, the specific research purpose and available experimental condition are the deciding factors in the choice among different methods.
The authors have nothing to disclose.
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).
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 |