果蝇病毒感染的建立及宿主病毒相互作用分析
1The Joint Center for Infection and Immunity, Guangzhou Institute of Pediatrics,Guangzhou Women and Children’s Medical Center, 2CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai,Chinese Academy of Sciences, 3CAS Key Laboratory of Infection and Immunity (CASKLII), Institute of Biophysics,Chinese Academy of Sciences, 4Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences
Summary
该方案介绍了如何建立病毒感染在果蝇体内使用纳米注射方法和基本技术, 以分析病毒宿主相互作用。
Abstract
病毒传播是传染病的主要原因。因此, 了解病毒与宿主之间的相互作用对于扩大我们预防和治疗病毒感染的知识非常重要。果蝇被证明是最有效和最有生产力的模型生物之一, 以筛选抗病毒因子和调查病毒-宿主相互作用, 由于强大的遗传工具和高度保守的先天免疫信号通路。这里描述的程序演示了一种纳米注射方法, 以建立病毒感染, 并诱导在成年苍蝇的系统抗病毒反应。这种方法对病毒注射剂量的精确控制可实现较高的实验重现性。本研究描述的方案包括苍蝇和病毒的制备、注射方法、存活率分析、病毒负荷测量和抗病毒途径评估。本文提到了苍蝇背景对病毒感染的影响。这种感染方法易于执行, 可定量重复;它可用于筛查与病毒宿主相互作用有关的宿主病毒因子, 并对先天免疫信号与其他生物途径在病毒感染反应中的串扰进行解剖。
Introduction
新出现的病毒感染, 特别是基孔肯雅病毒 1、登革热病毒、黄热病病毒2和寨卡病毒3等弓形虫病毒, 通过引起大流行病, 对公众健康构成巨大威胁4. 因此, 更好地了解病毒与宿主的相互作用对于流行病控制和人类病毒性疾病的治疗变得越来越重要。为此, 必须建立更适当和更有效的模型, 以调查病毒感染的机制。
果蝇,果蝇 (d. 黑色素母细胞), 为研究病毒宿主相互作用5,6提供了一个强大的系统, 并已被证明是研究人类病毒性疾病的最有效的模型之一7,8,9. 高度保守的抗病毒信号通路和无与伦比的遗传工具使苍蝇成为一个很好的模型, 能够产生对人类抗病毒研究具有真正影响的显著结果。此外, 苍蝇在实验室维护起来容易, 价格低廉, 在感染期间, 很方便地大规模筛选新的调节因子6,10和宿主。
最近, 在嗜酸性粒细胞中, 研究了四种主要的高度保守的抗病毒途径 (例如 rna 干扰 (rnai) 通路11、JAK-STAT 通路12、nf-b 通路和自体吞噬通路13)年6。rnai 途径是一种广泛的抗病毒机制, 可以抑制大多数类型的病毒感染6,14。由双宁-2 (dcr-2)或 Argonaute 2 (ago2 ) 等基因突变破坏这一途径可导致病毒滴度和宿主死亡率增加 15, 16,17.jak-stat 途径与控制来自双星科和黄曲霉家族的病毒感染昆虫有关,例如, 16岁苍蝇中的果蝇 c 病毒 (dcv) 和西尼罗河病毒 (wnv)并且登革热病毒在蚊子18,19。五音龙(同源于人类的 nf-b 途径) 和免疫缺陷 (imd) 途径 (类似于人类 nf-b 和 tnf 途径) 都参与了病毒入侵20,21, 22. 自食其力是另一种在病毒感染的调节中被保护的机制, 在德罗菲拉23,24中有很好的特点。因此, 可以很容易地识别这些途径的新调控因素, 并在这些抗病毒信号转接和其他生物途径 (如代谢、衰老、神经反应等) 之间进行解剖系统。
虽然果蝇中最成熟的病毒感染模型是由 rna 病毒诱导的, 但无脊椎动物的虹彩病毒 6i (iv-6) 和 kallithea 病毒的感染表明了研究苍蝇中 dna 病毒的潜力25 26岁此外, 该病毒也可以修改, 以允许感染嗜德维塔, 如流感病毒9。这极大地扩大了食人鱼筛选平台的应用。在这个过程中, 我们使用 dcv 作为一个例子来描述如何在嗜德维拉开发病毒感染系统。dcv 是一种正感单链 rna 病毒, 约9300核苷酸, 编码 9蛋白 27。dcv 作为d. 黑色素瘤的一种天然病原体, 被认为是研究宿主在宿主-病毒相互作用和共同进化过程中生理、行为和基础免疫反应的合适病毒28。此外, 它在野生苍蝇感染后的快速死亡率使 dcv 有助于筛选宿主29中的耐药或易感基因。
然而, 在研究嗜德维拉病毒感染时, 有几个方面值得关注。例如, 共生细菌wolbachia有能力抑制不同谱的 rna 病毒在德洛菲拉和蚊子30,31,32。最近的证据表明,沃尔巴奇亚通过宿主33中甲基转移酶 mt2 表达的提高阻断了 sindbis 病毒 (sinv) 感染的一种可能机制.此外, 昆虫的遗传背景对病毒感染也至关重要。例如,松柏基因中的自然多态性决定了五合34、35 对 dcv感染的易感性, 而ubc-e2h和cg892的位点分别参与了板球麻痹病毒 (crpv) 和羊群屋病毒 (fhv) 感染 36.
建立苍蝇中病毒宿主相互作用的特殊方法, 必须根据研究目的进行选择, 例如对果蝇细胞系 37、38、口服中的宿主细胞成分进行高通量筛选感染研究肠道特异性抗病毒反应22,39,40, 针刺41,42或纳米注射通过上皮屏障, 以刺激全身免疫反应。纳米注射能精确控制病毒剂量, 诱导受控抗病毒反应和生理病变43, 从而保证高实验重现性44。在这项研究中, 我们描述了一种纳米注射方法来研究果蝇中的病毒-宿主相互作用, 强调了苍蝇的背景效应的重要性。
Protocol
注: 在开始实验之前, 所使用的细胞系和苍蝇库存不得受到其他病原体的污染, 特别是对于 dcv、fhv、果蝇 x 病毒 (dxv) 和禽流感病毒 (anv) 等病毒。理想情况下, rna 测序或更简单的基于 pcr 的识别用于检测污染 10,45。如果发生污染, 细胞系和苍蝇库存不应再使用, 直到它们完全净化46。 1. 病毒和飞行准备 病…
Representative Results
本节的结果是在 d. 黑色素瘤dcv 感染后获得的。图 1显示了嗜德维拉病毒感染的流程图。苍蝇被注射胸腔内, 然后采集样本测量病毒 tcid 50 和基因组 rna 水平 (图 1).病毒感染可诱导细胞裂解, 感染后3天观察到 cpe (图 2a)。cpe 检测仪测量的病毒负载与 qpcr 测量的病毒负载一致 (图 2b)。如图 3所示,沃尔巴奇亚抑制…
Discussion
在这篇文章中, 我们提出了一个详细的程序, 如何建立一个病毒感染系统的成人果蝇使用纳米注射。这些协议包括准备适当的飞线和病毒储备、感染技术、评估感染指标和测量抗病毒反应。虽然 dcv 被用作病毒病原体的例子, 但数十种不同的病毒已成功地应用于食人鱼系统的研究。此外, 通过对苍蝇进行大规模筛查, 还发现了数百种监管因素, 无论是病毒还是宿主。因此, 这种方法适应性强, 不?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢 ips 中的整个泛实验室。Cas。我们感谢王兰峰博士 (中国科学院 ips) 的实验协助和戈纳洛·科多娃·斯泰格博士 (斯普林格自然)、杰西卡·瓦尔加斯博士 (ips, 巴黎) 和朱生博士 (ips, 巴黎) 的评论。这项工作得到了中国科学院战略优先研究方案对 l. p (xda13010500) 和 h. t (xdb29030300)、中国国家自然科学基金至 l. p (338887 和 31870887) 和 j. y (31870887) 的资助。l. p 是中科院青年创新促进会会员 (2012083)。
Materials
| 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 |
Riferimenti
- Rahim, M. A., Uddin, K. N. Chikungunya: an emerging viral infection with varied clinical presentations in Bangladesh: Reports of seven cases. BMC Research Notes. 10, 410 (2017).
- Douam, F., Ploss, A. Yellow Fever Virus: Knowledge Gaps Impeding the Fight Against an Old Foe. Trends in Microbiology. , (2018).
- Santiago, G. A., et al. Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses. Nature Communications. 9. 9, 1391 (2018).
- Gould, E., Pettersson, J., Higgs, S., Charrel, R., de Lamballerie, X. Emerging arboviruses: Why today?. One Health. 4, 1-13 (2017).
- Hughes, T. T., et al. Drosophila as a genetic model for studying pathogenic human viruses. Virology. 423, 1-5 (2012).
- Xu, J., Cherry, S. Viruses and antiviral immunity in Drosophila. Developmental & Comparative Immunology. 42, 67-84 (2014).
- Shirinian, M., et al. A Transgenic Drosophila melanogaster Model To Study Human T-Lymphotropic Virus Oncoprotein Tax-1-Driven Transformation In Vivo. Journal of Virology. 89, 8092-8095 (2015).
- Adamson, A. L., Chohan, K., Swenson, J., LaJeunesse, D. A Drosophila model for genetic analysis of influenza viral/host interactions. Genetica. 189, 495-506 (2011).
- Hao, L., et al. Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature. 454, 890-893 (2008).
- Webster, C. L., et al. The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster. Plos Biology. 13, e1002210 (2015).
- Heigwer, F., Port, F., Boutros, M. RNA Interference (RNAi) Screening in Drosophila. Genetica. 208, 853-874 (2018).
- West, C., Silverman, N. p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila. PLOS Pathogens. 14, e1007020 (2018).
- Liu, Y., et al. STING-Dependent Autophagy Restricts Zika Virus Infection in the Drosophila Brain. Cell Host & Microbe. 24, 57-68 (2018).
- Wang, X. H., et al. RNA interference directs innate immunity against viruses in adult Drosophila. Science. 312, 452-454 (2006).
- van Rij, R. P., et al. The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster. Genes & Development. 20, 2985-2995 (2006).
- Deddouche, S., et al. The DExD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila. Nature Immunology. 9, 1425-1432 (2008).
- Chotkowski, H. L., et al. West Nile virus infection of Drosophila melanogaster induces a protective RNAi response. Virology. 377, 197-206 (2008).
- Paradkar, P. N., Trinidad, L., Voysey, R., Duchemin, J. B., Walker, P. J. Secreted Vago restricts West Nile virus infection in Culex mosquito cells by activating the Jak-STAT pathway. Proceedings of the National Academy of Sciences of the United States of America. 109, 18915-18920 (2012).
- Souza-Neto, J. A., Sim, S., Dimopoulos, G. An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense. Proceedings of the National Academy of Sciences of the United States of America. 106, 17841-17846 (2009).
- Zambon, R. A., Nandakumar, M., Vakharia, V. N., Wu, L. P. The Toll pathway is important for an antiviral response in Drosophila. Proceedings of the National Academy of Sciences of the United States of America. 102, 7257-7262 (2005).
- Costa, A., Jan, E., Sarnow, P., Schneider, D. The Imd pathway is involved in antiviral immune responses in Drosophila. PLoS One. 4, e7436 (2009).
- Ferreira, A. G., et al. The Toll-dorsal pathway is required for resistance to viral oral infection in Drosophila. PLOS Pathogens. 10, e1004507 (2014).
- Moy, R. H., et al. Antiviral autophagy restrictsRift Valley fever virus infection and is conserved from flies to mammals. Immunity. 40, 51-65 (2014).
- Shelly, S., Lukinova, N., Bambina, S., Berman, A., Cherry, S. Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus. Immunity. 30, 588-598 (2009).
- Bronkhorst, A. W., et al. The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery. Proceedings of the National Academy of Sciences of the United States of America. 109, E3604-E3613 (2012).
- Palmer, W. H., Medd, N. C., Beard, P. M., Obbard, D. J. Isolation of a natural DNA virus of Drosophila melanogaster, and characterisation of host resistance and immune responses. PLOS Pathogens. 14, e1007050 (2018).
- Jousset, F. X., Bergoin, M., Revet, B. Characterization of the Drosophila C virus. Journal of General Virology. 34, 269-283 (1977).
- Gupta, V., Stewart, C. O., Rund, S. S. C., Monteith, K., Vale, P. F. Costs and benefits of sublethal Drosophila C virus infection. J Evol Biol. 30, 1325-1335 (2017).
- Yang, S., et al. Bub1 Facilitates Virus Entry through Endocytosis in a Model of Drosophila Pathogenesis. Journal of Virology. , (2018).
- Teixeira, L., Ferreira, A., Ashburner, M. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. Plos Biology. 6, e2 (2008).
- Ferguson, N. M., et al. Modeling the impact on virus transmission of Wolbachia-mediated blocking of dengue virus infection of Aedes aegypti. Science Translational Medicine. 7, 279ra237 (2015).
- Hedges, L. M., Brownlie, J. C., O’Neill, S. L., Johnson, K. N. Wolbachia and virus protection in insects. Science. 322, 702 (2008).
- Bhattacharya, T., Newton, I. L. G., Hardy, R. W. Wolbachia elevates host methyltransferase expression to block an RNA virus early during infection. PLOS Pathogens. 13, e1006427 (2017).
- Magwire, M. M., et al. Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila melanogaster. PLOS Genetics. 8, e1003057 (2012).
- Cao, C., Cogni, R., Barbier, V., Jiggins, F. M. Complex Coding and Regulatory Polymorphisms in a Restriction Factor Determine the Susceptibility of Drosophila to Viral Infection. Genetica. , 2159-2173 (2017).
- Martins, N. E., et al. Host adaptation to viruses relies on few genes with different cross-resistance properties. Proceedings of the National Academy of Sciences of the United States of America. 111, 5938-5943 (2014).
- Moser, T. S., Sabin, L. R., Cherry, S. RNAi screening for host factors involved in Vaccinia virus infection using Drosophila cells. Journal of Visualized Experiments. , (2010).
- Zhu, F., Ding, H., Zhu, B. Transcriptional profiling of Drosophila S2 cells in early response to Drosophila C virus. Journal of Virology. 10, 210 (2013).
- Ekstrom, J. O., Hultmark, D. A Novel Strategy for Live Detection of Viral Infection in Drosophila melanogaster. Scientific Reports. 6, 26250 (2016).
- Durdevic, Z., et al. Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2. EMBO Reports. 14, 269-275 (2013).
- Chrostek, E., et al. Wolbachia variants induce differential protection to viruses in Drosophila melanogaster: a phenotypic and phylogenomic analysis. PLOS Genetics. 9, e1003896 (2013).
- Gupta, V., Vale, P. F. Nonlinear disease tolerance curves reveal distinct components of host responses to viral infection. Royal Society Open Science. 4, 170342 (2017).
- Chtarbanova, S., et al. Drosophila C virus systemic infection leads to intestinal obstruction. Journal of Virology. 88, 14057-14069 (2014).
- Merkling, S. H., van Rij, R. P. Analysis of resistance and tolerance to virus infection in Drosophila. Nature Protocols. 10, 1084-1097 (2015).
- Goic, B., et al. RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila. Nature Immunology. 14, 396-403 (2013).
- Ashburner, M., Golic, K., Hawley, R. S. . Drosophila: A Laboratory Handbook. , (2011).
- . Biochemical and Biophysical Research Communications: 1991 index issue. Cumulative indexes for volumes 174-181. Biochemical and Biophysical Research Communications. , 1-179 (1991).
- Cotarelo, M., et al. Cytopathic effect inhibition assay for determining the in-vitro susceptibility of herpes simplex virus to antiviral agents. Journal of Antimicrobial Chemotherapy. 44, 705-708 (1999).
- Reed, L. J. M., H, A simple method of estimating fifty percent endpoints. The American Journal of Hygiene. 27, 493-497 (1938).
- Khalil, S., Jacobson, E., Chambers, M. C., Lazzaro, B. P. Systemic bacterial infection and immune defense phenotypes in Drosophila melanogaster. Journal of Visualized Experiments. , e52613 (2015).
- Schwenke, R. A., Lazzaro, B. P. Juvenile Hormone Suppresses Resistance to Infection in Mated Female Drosophila melanogaster. Current Biology. 27, 596-601 (2017).
- Sabin, L. R., Hanna, S. L., Cherry, S. Innate antiviral immunity in Drosophila. Current opinion in immunology. 22, 4-9 (2010).
- Yin, J., Zhang, X. X., Wu, B., Xian, Q. Metagenomic insights into tetracycline effects on microbial community and antibiotic resistance of mouse gut. Ecotoxicology. 24, 2125-2132 (2015).
Citazione di questo articolo
Yang, S., Zhao, Y., Yu, J., Fan, Z., Gong, S., Tang, H., Pan, L. Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster. J. Vis. Exp. (145), e58845, doi:10.3791/58845 (2019).