从小鼠多个器官分离和定量寨卡病毒
Summary
该协议的目的是通过分离和量化寨卡病毒,从感染后小鼠的多个器官中分离和量化,来展示用于调查病毒性疾病的技术。
Abstract
提出的方法展示了从寨卡病毒感染动物中分离器官和定量病毒载量的实验室程序。该程序的目的是量化小鼠在感染后不同时间点或不同实验条件下的周围和中枢神经系统区域的病毒性色度,以确定调节寨卡病毒感染的病毒学和免疫学因素。所演示的器官分离程序允许对病毒性定位剂进行焦点形成测定定量和定量PCR评估。快速器官分离技术是为保存病毒性皮炎而设计的。通过聚焦形成测定对病毒进行量度定量,可快速评估寨卡病毒的通量。重点形成测定的好处是评估传染性病毒,这种测定的局限性是降低检测极限的器官毒性的潜力。病毒性排名评估与定量PCR相结合,使用重组RNA拷贝控制病毒基因组拷贝数在器官内评估检测限值低。总体而言,这些技术为分析寨卡病毒感染动物的外围寨卡病毒分子和中枢神经系统提供了准确的快速高通量方法,并可用于评估大多数感染动物器官中的病毒性定子病原体,包括登革热病毒。
Introduction
寨卡病毒(ZIKV)是一种属于浮病毒家族的病毒,包括重要的神经侵入性人类病原体,如波瓦桑病毒(POWV)、日本脑炎病毒(JEV)和西尼罗病毒(WNV)1。在隔离和鉴定后,非洲和亚洲定期报告人类ZIKV感染2、3、4、5和中美洲和南美洲的流行病(在参考6。然而,直到最近,ZIKV才被认为会导致严重的疾病7。现在有成千上万的神经系统疾病和出生缺陷与ZIKV感染有关。ZIKV的迅速出现引发了许多问题:为什么疾病严重程度增加,对ZIKV感染的免疫反应是什么,以及是否有病毒和/或免疫介导的病理与神经学的增加有关表现和先天缺陷。现在人们急于了解与ZIKV相关的中枢神经系统(CNS)相关疾病,以及快速测试抗病毒药物和疫苗对ZIKV的疗效的必要性。正是在这种背景下,我们开发了使用ZIKV特定焦点形成测定(FFA)对外围和CNS中ZIKV定子进行快速分析的方法。
小型动物模型对于了解疾病进展和早期评估疫苗、治疗和抗病毒药物非常重要。我们已经建立了研究阿尔博病毒病的小动物模型,利用各种小鼠菌株来模拟人类感染,并保护病毒病原体8,9,10,11, 12,13,14,15,16,17,18,19,20 21,22.利用这个以往的经验,我们开始修改用于评估WNV和登革热病毒的技术,这是一种相关的黄病毒,用于评估两个外围器官的ZIKV丁打剂以及CNS21、2324.这些方法比其他测定的优点是:1) 它们结合收集外周和CNS器官进行分析的能力;2) 该方法适用于流式细胞测定,用于测量先天和适应性免疫反应,以及同一器官中同一动物的病毒定子;3)收获技术适用于组织学分析;4)ZIKV FFA是一种快速的高通量方法,用于病毒性皮底分析;5)这些方法可用于评估感染大多数病原体的动物器官中的病毒性定子25。
Protocol
本研究的所有程序均符合圣路易斯大学动物护理和使用委员会制定的指导方针。SLU 获得国际实验室动物护理评估与认证协会 (AAALAC) 的完全认可。 1. 器官隔离 注:病毒在室温 (RT) 下不稳定,因此必须仔细规划一次收获的动物数量,以保存病毒性定位剂。 根据所需的表型,使用所选剂量和路线感染小鼠。对于此方案,感染8-10周大的雄性?…
Representative Results
使用上述协议评估ZIKV针,通过皮下(SC)注射到脚垫上,小鼠感染了ZIKV(PRVABC59)。 在这里,给ZIKV的1 x 105 FFU给8-12周大的Ifnar1-/-小鼠SC不是致命的,但病毒可以在外围和CNS中复制。此剂量和途径用于研究宿主病原体免疫反应和致病性。给ZIKV的1 x 105 FFU注射8-12周大的Ifnar1-/-小鼠静脉注射(IV)是80至100%的致命性,动物在病毒注射后8至14天之间?…
Discussion
ZIKV感染可引起神经系统疾病,因此目前动物模型研究的发病机制、免疫反应和疫苗和抗病毒药物的保护功效需要专注于中枢神经系统内的病毒控制。关注中枢神经系统疾病的挑战之一是,它往往以研究周围感染为代价。这里提出的器官分离方法侧重于需要快速评估在外围和CNS的ZIKV感染,以便评估中枢介ZIKV相关疾病,并建立抗病毒药物临床前测试模型,治疗和疫苗。该技术的另一个好处是,它还允许高度?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Pinto博士的资金来自圣路易斯大学医学院的种子赠款和圣路易斯大学医学院的启动基金。Brien博士的资助来自NIH NIAID的K22AI104794早期调查员奖以及圣路易斯大学学院的种子补助金。对于所有受资助的个人,资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有作用。
Materials
| 1-bromo-3-chloropropane (BCP) | MRC gene | BP151 | |
| 10cc syringe | Thermo Fisher Scientific | BD 309642 | |
| 18G needle | Thermo Fisher Scientific | 22-557-145 | |
| 1cc TB syringe | Thermo Fisher Scientific | 14-823-16H | |
| 20cc syringe | Thermo Fisher Scientific | 05-561-66 | |
| 24 tube beadmill | Thermo Fisher Scientific | 15 340 163 | |
| 3.2 mm stainless steel beads | Thermo Fisher Scientific | NC9084634 | |
| 37C Tissue Culture incubator | Nuair | 5800 | |
| 4G2 antibody | in house | ||
| 96 well flat bottom plates | Midsci | TP92696 | |
| 96well round bottom plates | Midsci | TP92697 | |
| Basix 1.5ml eppendorf tubes | Thermo Fisher Scientific | 02-682-002 | |
| Concentrated Germicidal Bleach | Staples | 30966CT | |
| CTL S6 Analyzer | CTL | CTL S6 Universal Analyzer | |
| curved cutting scissors | Fine Science Tools | 14061-11 | |
| Dulbecco’s Modified Eagle’s Medium – high glucose With 4500 mg/L glucose | MilliporeSigma | D5671 | |
| Ethanol (molecular biology-grade) | MilliporeSigma | e7023 | |
| Fetal Bovine Serum | MilliporeSigma | F0926-500ML | |
| Forceps | Fine Science Tools | 11036-20 | |
| Glacial acetic acid | MilliporeSigma | 537020 | |
| Goat anti-mouse HRP-labeled antibody | MilliporeSigma | 8924 | |
| HEPES 1 M | MilliporeSigma | H3537-100ML | |
| Isopropanol (molecular biology-grade) | MilliporeSigma | I9516 | |
| Ketamine/Xylazine cocktail | Comparative Medicine | ||
| L-glutamine | MilliporeSigma | g7513 | |
| Magmax RNA purification kit | Thermo Fisher Scientific | AM1830 | |
| Methylcellulose | MilliporeSigma | M0512 | |
| Microcentrifuge | Ependorf | 5424R | |
| MiniCollect 0.5ml EDTA tubes | Bio-one | 450480 | |
| o-ring tubes | Thermo Fisher Scientific | 21-403-195 | |
| one step q RT-PCR mix | Thermo Fisher Scientific | 4392938 | |
| Paraformaldehyde | Thermo Fisher Scientific | EMS- 15713-S | |
| Phosphate Buffered Saline | MilliporeSigma | d8537-500ml | |
| Proline multichannel pipettes | Sartorius | 72230/72240 | |
| Proline single channel pipettes | Sartorius | 728230 | |
| RNAse free water | Thermo Fisher Scientific | 10-977-023 | |
| RNAzol BD | MRC gene | RB192 | |
| Rocking Platform | Thermo Fisher Scientific | 11-676-333 | |
| RPMI 1640 | Fisher | MT10040CV | |
| Saponin | MilliporeSigma | s7900 | |
| spoon/spatula | Fine Science Tools | 10090-17 | |
| straight cutting scissors | Fine Science Tools | 14060-11 | |
| Triton X-100 | MilliporeSigma | t8787 | |
| True Blue Substrate | VWR | 95059-168 | |
| Trypsin | MilliporeSigma | T3924-100ML |
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Cite This Article
Brien, J. D., Hassert, M., Stone, E. T., Geerling, E., Cruz-Orengo, L., Pinto, A. K. Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse. J. Vis. Exp. (150), e59632, doi:10.3791/59632 (2019).