使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应
Summary
Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.
Abstract
Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.
Introduction
据世界卫生组织（WHO），流感病毒感染的成年人的5％-10％，每年儿童的20-30％，并导致300万-500万严重病例和全世界1到500,000人死亡。对流感疫苗接种每年仍然以防止疾病的最佳选择。像世卫组织全球行动计划的努力，以降低发病率和季节性流感暴发2相关的死亡率增加了季节性流感疫苗的使用，疫苗生产能力，以及研究和开发更有效的疫苗策略。抗病毒药物像神经氨酸酶抑制剂（ 例如 ，扎那米韦和奥司他韦）是在一些国家可用以及缓解症状已被证明有效，当发病3，4，5的第一48小时内给药。尽管全球努力，季节性流感遏制欧tbreaks保持此时一个严峻的挑战，因为流感病毒抗原漂移往往超过适应病毒6的改变基因组中的电流的能力。疫苗策略靶向病毒的新菌株必须提前开发，有时由于在类型菌株，在一个流感季节最终占优势不可预见的变化呈现小于最佳效果。由于这些原因，有一个明确需要制定包含感染和死亡率降低替代治疗策略。通过实现更好的理解在主机病毒相互作用，有可能开发新的抗流感药物和辅助疗法7,8。
人宿主A型流感病毒（IAV）相互作用是复杂的。人类IAV感染的几种动物模型为了深入了解宿主病毒相互作用，大型的源码已经开发ING小鼠，豚鼠，棉鼠，仓鼠，雪貂，猕猴和9。同时提供具有增强宿主IAV动力学的理解的重要数据，每个模式生物拥有必须尝试将结果转化为人类医学时，应考虑显著的缺点。例如，小鼠，这是最广泛使用的模型，不容易与人流感分离株9感染时开发IAV诱导感染症状。这是因为小鼠缺乏对人流感的天然趋向性分离自小鼠上皮细胞表达α-2,3-唾液酸连接，而不是对人上皮细胞10表示的α-2,6唾液酸连接。存在于人类IAV的血凝素蛋白的分离从优结合并进入宿主细胞到通过受体介导的内吞作用9，11α-2,6唾液酸连接， </s达> 12，13。因此，现在已被接受，在发育的小鼠模型的人流感，必须小心，以与流感的适当应变配对鼠标的适当应变，以达到疾病表型概括的人类疾病的各个方面。与此相反，在雪貂的上呼吸道上皮细胞具有类似于人类细胞14α-2,6唾液酸连接。感染雪貂共享许多在人类疾病中观察到的病理和临床特征，包括人类和禽流感病毒14的致病性和透射性，15。它们也是高度适合于疫苗功效试验。然而，对于人类流感鼬模型有几个缺点，主要与它们的大小和饲养成本，使统计上显的收购具有挑战性着数据。此外，雪貂以前已经显示在药代动力学，生物利用度和毒性，使测试功效很难区别。例如，雪貂表现出毒性M2离子通道抑制剂金刚烷胺16。因此，很显然，在选择的动物模型来研究有关人类IAV感染的问题，必须考虑其固有的优点和局限性，并在主机病毒相互作用是受调查的方面是非常重要的。
斑马鱼， 斑马鱼是动物模型，提供了独特的机会，研究微生物感染，宿主免疫反应，和潜在的药物治疗17，18，19，20，21，22，23，<SUP类=“外部参照”> 24，25，26，27，28。 α-2,6-连接的唾液酸在斑马鱼细胞的表面上存在表明其易感性IAV，其在感染的研究证明了，用IAV 19的荧光报道菌株在体内成像。在IAV感染斑马鱼，抗病毒ifnphi1和MXA转录物的表达增加表明先天免疫应答已经刺激，并通过IAV感染斑马鱼，包括水肿和组织破坏显示的病理，是类似于在人类流感感染观察。此外，IAV抗病毒药神经氨酸酶抑制剂扎那米韦有限死亡率和在斑马鱼19减少病毒复制。
在这份报告中，对于引发体系协议在斑马鱼胚胎IC IAV感染描述。使用扎那米韦临床相应剂量的作为证明的原则，对于抗病毒活性的化合物筛选斑马鱼这种IAV感染模型的效用是证明。此外，用于产生局部的，上皮IAV感染在斑马鱼的协议鱼鳔，被认为是在解剖学和功能上类似于哺乳动物的肺21，29，30，31之间的一个器官，进行说明。使用这种局部IAV感染模型，中性粒细胞募集至感染部位可以跟踪，使调查中性粒细胞生物学在IAV感染和炎症中的作用。这些斑马鱼模型补充人类IAV感染的现有的动物模型，并且用于测试的小分子和由于增强了S的可能性免疫细胞应答特别有用tatistical功率，容量为中到高通量测定法，和能力来跟踪免疫细胞的行为和功能的光显微镜。
Protocol
Representative Results
Discussion
为了最大限度地使用小动物到人类宿主 – 病原体相互作用的模型获得的利益，框架研究问题，并在测试假说，关于模型系统的固有优势利用是很重要的。作为人类IAV感染模型，斑马鱼有几个优势，包括高繁殖力，光学清晰度，顺从于药物筛选，以及该标签的免疫细胞嗜中性粒细胞一样的转基因株系的可用性。斑马鱼已经发展成为日益强大的替代鼠标模型系统的炎症和先天免疫方面的研究。因为他?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Mark Nilan for zebrafish care and maintenance and Meghan Breitbach and Deborah Bouchard for propagating NS1-GFP and determining IAV titers. This research was supported by NIGMS grant NIH P20GM103534 and the Maine Agricultural and Forest Experiment Station (Publication Number 3493).
Materials
| Instant Ocean | Spectrum Brands | SS15-10 | |
| 100 x 25 mm sterile disposable Petri dishes | VWR | 89107-632 | |
| Transfer pipettes | Fisherbrand | 13-711-7M | |
| Tricaine- S (MS-222) | Western Chemical | ||
| Borosilicate glass capillary with filament | Sutter Instrument | BF120-69-10 | |
| Flaming/Brown micropipette puller | Sutter Instrument | P-97 | |
| Agarose | Lonza | 50004 | |
| Zanamivir | AK Scientific | G939 | |
| Dumont #5 forceps | Electron Microscopy Sciences | 72700-D | |
| Microloader tips | Eppendorf | 930001007 | |
| Microscope immersion oil | Olympus | IMMOIL-F30CC | |
| Microscope stage calibration slide | AmScope | MR095 | |
| MPPI-3 pressure injector | Applied Scientific Instrumentation | ||
| Stereo microscope | Olympus | SZ61 | |
| Back pressure unit | Applied Scientific Instrumentation | BPU | |
| Micropipette holder kit | Applied Scientific Instrumentation | MPIP | |
| Foot switch | Applied Scientific Instrumentation | FSW | |
| Micromanipulator | Applied Scientific Instrumentation | MM33 | |
| Magnetic base | Applied Scientific Instrumentation | Magnetic Base | |
| Phenol red | Sigma-Aldrich | P-4758 | |
| Low temperature incubator | VWR | 2020 | |
| SteREO Discovery.V12 | Zeiss | ||
| Illuminator | Zeiss | HXP 200C | |
| Cold light source | Zeiss | CL6000 LED | |
| Glass-bottom multiwell plate, 24 well | Mattek | P24G-0-13-F | |
| Confocal microscope | Olympus | IX-81 with FV-1000 laser scanning confocal system | |
| Fluoview software | Olympus | ||
| Prism v6 | GraphPad | ||
| Influenza A/PR/8/34 (H1N1) virus | Charles River | 490710 | |
| Influenza A X-31, A/Aichi/68 (H3N2) | Charles River | 490715 | |
| Influenza NS1-GFP | Referenced in Manicassamy et al. 2010 | ||
| Tg(mpx:mCherry) | Referenced in Lam et al. 2013 | ||
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