JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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면역학 및 감염병학

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

Published: April 28, 2019
doi:
10.3791/59132
, , , , , , ,
1Graduate Institute of Medical Sciences, College of Medicine,Taipei Medical University, 2Department of Microbiology and Immunology, School of Medicine, College of Medicine,Taipei Medical University, 3Center of Infectious Disease and Signaling Research,National Cheng Kung University

Summary

在这里,我们提出了一个协议,创建一个免疫能力ICR(癌症研究所)的中枢神经系统感染的鼠模型,以显示神经病变的发展。通过相同的疾病评分监测急性病毒性脑病,可以显示体内登革热病毒引起的神经病变。

Abstract

登革热病毒(DENV)是一种由蚊子传播的节肢传播病毒,可引起称为登革热出血热的严重疾病,其特征是血浆泄漏、腹水、胸膜渗漏、呼吸窘迫等致命并发症,严重出血和器官损伤。少数DENV感染病例有神经表现;然而,研究没有进一步探讨DENV引起的神经发病机制。在这项研究中,我们提出了一个协议,使用免疫能力超种ICR(癌症研究所)小鼠来研究中枢神经系统(CNS)感染DENV的诱导,然后急性病毒性脑炎类似进展疾病。

Introduction

DENV是Flaviviridae家族的节肢传播病毒,含有一个正义RNA基因组,该基因组编码三种病毒结构蛋白(细胞、前膜和包络)和七种病毒非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B),和 NS5)。DENV(DENV1-4)的四种血清型每年感染约3.9亿人,造成全球负担,尽管各国政府已大力控制蚊媒和疾病控制1。目前,保护性疫苗和治疗性抗病毒药物正在研制中,需要进一步的长期验证2。在临床实践中,虽然感染中枢神经系统的登革热患者很少,但还需要进一步探讨,以了解登革热发展的多样性。需要进一步调查和验证;值得注意的是,世界卫生组织(WHO)将中枢神经系统损伤(如认知障碍、抽搐、脑病和脑炎)纳入严重登革热3、4的分类。构建DENV感染的动物模型对于探索DENV感染的神经发病机制是必不可少的。

为了产生DENV的CNS感染,几项研究已经执行了不同的DENV感染途径,包括(1)接受4 x103斑块形成单位(PFU)的C57BL/6小鼠的脑内接种,6、 (2) 接受7 x 104 PFU的体外神经适应DENV47的BALB/c小鼠的腹腔内接种,(3)接受体内神经适应DENV18 1x 105 PFU的瑞士小鼠的脑内接种,以及(4)接受1 x 106 PFU的不适应DENV29的ICR吸吮小鼠的脑内和腹腔内共接种。根据这些研究的结果,小鼠的DENV感染导致病毒复制,导致急性病毒脑炎样综合征,行为变化伴随肢体瘫痪和姿势不稳定,中枢神经系统神经毒性和炎症,一般和局部血浆泄漏通过血脑屏障(BBB),和致命性。这些研究的所有结果5,6,7,8,9表明DENV感染CNS和诱导急性病毒性脑炎的能力感染后的疾病。

根据我们目前的发现9,10,11,12,13,14,15,我们已经创造了一个DENV感染的鼠模型作为体内平台,以检查靶向剂/因子对病毒复制的疗效,以及神经毒性。在这里,我们报告用于创建免疫能力的杂交ICR小鼠的协议,用于研究CNS感染,并监测由DENV引起的不同神经病变的发展。结果表明,在DENV感染的小鼠中,脑炎样疾病以时间依赖性的方式显著进展。

Protocol

根据台湾科技部制定的指导方针,动物研究实验规程经国防医学中心动物护理与用户委员会批准(IACUC编号:16-261)。 1. 感染程序 准备不适应的DENV2(应变PL046)库存9(最初从台湾疾病控制中心获得,范围从2.5 x 107到1 x 109 PFU/mL)。 与罗斯威尔公园纪念研究所 (RPMI) 1640 介质稀释至 1 x 106 PFU,总体积为 40 μL。 用10μL(2.5 x 105…

Representative Results

登革热发病机制患者已报告出现严重的登革热相关神经并发症。虽然这些病例在临床上很少见,但创建 DENV 感染的免疫能力小鼠模型不仅可用于研究免疫发病机制,还可用于探索中枢神经系统感染、神经炎症、神经毒性和急性病毒脑炎样疾病。在这项研究中,根据我们目前的模型9,10,11,12,13,14,15,7天大的ICR吸吮小鼠接种</…

Discussion

在3、17日,在重症登革热患者中枢神经系统中检测到DENV感染,表明在登革热发病过程中出现急性病毒性脑炎的可能性。在这里,我们报告一个在体内的DENV感染模型,以研究中枢神经系统功能障碍在严重登革热的介入,特别是侧重于DENV引起的急性病毒性脑炎样疾病。与以前的模型相比,特别是单道感染(仅限静脉注射、仅限腹腔内或仅内脑),DENV 的极高滴度(1 x 10<s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了科技部(MOST107-2321-B-038-001)和106TMU-CIT-01-2助学金的支持, 台北,台湾。

Materials

Roswell Park Memorial Institute 1640 medium (RPMI) Gibco 11875-085 Diluting virus
0.3-mL Insulin Syringe  BD Ultra-Fine­II 328838 Intracerebral and intraperitoneal injection
Microbalance METTLER TOLEDO's LabX AL104 Weight mouse
Non-adapted DENV2 (strain PL046) The Centers for Disease Control of Taiwan – Infect mouse
Institute of Cancer Research (ICR) suckling mouse BioLASCO Taiwan Co., Ltd – Our murine model
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References

  1. Guzman, M. G., Gubler, D. J., Izquierdo, A., Martinez, E., Halstead, S. B. Dengue infection. Nature Reviews Disease Primers. 2, 16055 (2016).
  2. Katzelnick, L. C., Coloma, J., Harris, E. Dengue: knowledge gaps, unmet needs, and research priorities. Lancet Infectious Diseases. 17 (3), e88-e100 (2017).
  3. Carod-Artal, F. J., Wichmann, O., Farrar, J., Gascon, J. Neurological complications of dengue virus infection. Lancet Neurology. 12 (9), 906-919 (2013).
  4. Geneva: World Health Organization. . Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. , (2009).
  5. Amaral, D. C., et al. Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice. Journal of Neuroinflammation. 8, 23 (2011).
  6. de Miranda, A. S., et al. Dengue-3 encephalitis promotes anxiety-like behavior in mice. Behavioural Brain Research. 230 (1), 237-242 (2012).
  7. Velandia-Romero, M. L., Acosta-Losada, O., Castellanos, J. E. In vivo infection by a neuroinvasive neurovirulent dengue virus. Journal of Neurovirology. 18 (5), 374-387 (2012).
  8. Despres, P., Frenkiel, M. P., Ceccaldi, P. E., Duarte Dos Santos, C., Deubel, V. Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses. Journal of Virology. 72 (1), 823-829 (1998).
  9. Tsai, T. T., et al. Microglia retard dengue virus-induced acute viral encephalitis. Scientific Reports. 6, 27670 (2016).
  10. Cheng, Y. L., et al. Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-alpha production by mononuclear phagocytes. Scientific Reports. 6, 32000 (2016).
  11. Ho, M. R., et al. Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways. Scientific Reports. 7 (1), 6910 (2017).
  12. Jhan, M. K., et al. Anti-TNF-alpha restricts dengue virus-induced neuropathy. Journal of Leukocyte Biology. 104 (5), 961-968 (2018).
  13. Kao, J. C., et al. The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR. PLoS Neglected Tropical Diseases. 12 (8), e0006715 (2018).
  14. Tsai, T. T., Chen, C. L., Tsai, C. C., Lin, C. F. Targeting heat shock factor 1 as an antiviral strategy against dengue virus replication in vitro and in vivo. Antiviral Research. 145, 44-53 (2017).
  15. Jhan, M. K., et al. Dengue virus infection increases microglial cell migration. Scientific Reports. 7 (1), 91 (2017).
  16. Benskey, M. J., Manfredsson, F. P. Intraparenchymal Stereotaxic Delivery of rAAV and Special Considerations in Vector Handling. Methods in Molecular Biology. , 199-215 (2016).
  17. Fong, C. Y., et al. Mild encephalitis/encephalopathy with reversible splenial lesion (MERS) due to dengue virus. Journal of Clinical Neuroscience. 36, 73-75 (2017).
  18. Sarathy, V. V., et al. A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease. Journal of Virology. 89 (2), 1254-1266 (2015).
  19. Schul, W., Liu, W., Xu, H. Y., Flamand, M., Vasudevan, S. G. A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs. The Journal of Infectious Diseases. 195 (5), 665-674 (2007).
  20. Tyler, K. L. Acute Viral Encephalitis. New England Journal of Medicine. 379 (6), 557-566 (2018).
  21. Yauch, L. E., Shresta, S. Mouse models of dengue virus infection and disease. Antiviral Research. 80 (2), 87-93 (2008).
  22. Assir, M. Z., Jawa, A., Ahmed, H. I. Expanded dengue syndrome: subacute thyroiditis and intracerebral hemorrhage. BMC Infectious Diseases. 12, 240 (2012).
  23. Kumar, R., Prakash, O., Sharma, B. S. Intracranial hemorrhage in dengue fever: management and outcome: a series of 5 cases and review of literature. Surgical Neurology. 72 (4), 429-433 (2009).
  24. Simanjuntak, Y., Liang, J. J., Lee, Y. L., Lin, Y. L. Repurposing of prochlorperazine for use against dengue virus infection. Journal of Infectious Diseases. 211 (3), 394-404 (2015).
  25. Rocha, B. A. M., et al. Dengue-specific serotype related to clinical severity during the 2012/2013 epidemic in centre of Brazil. Infectious Disease Poverty. 6 (1), 116 (2017).

Tags

Murine ModelDengue VirusAcute Viral EncephalitisNeuropathogenesisVirus InfectionVirus InoculationDisease ProgressionBody Weight ChangesSurvival Rates

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Shen, T., Jhan, M., Kao, J., Ho, M., Tsai, T., Tseng, P., Wang, Y., Lin, C. A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease. J. Vis. Exp. (146), e59132, doi:10.3791/59132 (2019).
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