兹卡病毒感染细胞培养系统和<em>体外</em>干扰素的预防作用
Summary
Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.
Abstract
寨卡病毒（ZIKV）是链接到胎儿发育异常，如小头畸形，眼缺陷，和受损生长一个新兴的病原体。 ZIKV是黄病毒科的RNA病毒。 ZIKV主要是由蚊子传播的，但也可以通过母体传播给胎儿的垂直传播和性接触。迄今为止，有可用的，以保护那些被病毒感染没有可靠的治疗或疫苗的选项。可重复的，有效的兹卡病毒感染的细胞培养系统的发展是研究ZIKV复制以及药物和疫苗发展的分子机制的关键。在这方面，描述的哺乳动物细胞为基础的体外寨卡病毒培养病毒的生产和生长分析系统的协议在这里报道。对一个细胞单层和斑块比色法检测病毒滴度形成斑块由寨卡病毒的详细介绍。病毒基因组复制动力学和双链RNA基因组replicatory中间体被确定。此培养平台利用屏幕对一小部分产生的干扰素α（IFN-α）的鉴定细胞因子的文库，IFN-β和IFN-γ作为寨卡病毒生长的有效抑制剂。总之， 在体外感染寨卡病毒培养体系和各种病毒学测定法证明了在本研究中，这也在研究界中进一步阐明病毒致病机制和病毒毒力的演变极大地受益的可能性。抗病毒的IFN-α还可以作为预防，暴露后预防性，和治疗的高危人群寨卡病毒感染，包括病毒感染的孕妇选项进行评估。
Introduction
兹卡病毒（ZIKV）是小头畸形及妊娠结局不佳有关1,4重要的人类病原体。 ZIKV属于集合医学相关的黄病毒可导致神经缺陷，如登革热，西尼罗河，和圣路易脑炎病毒。病毒传播的主要方式是通过蚊子载体埃及伊蚊 ，并且，此外，性传播也有报道5,6。 ZIKV已经成为一个主要的全球性健康问题，由于蚊子的地域扩张分布及其与出生缺陷密切相关。 ZIKV最早是在1947年从兹卡森林定点恒河猴隔离，乌干达和第一个人类病例报告，1952年7,8。个人认为感染了ZIKV存在轻度症状，如发热，皮疹，头痛，结膜炎和肌肉/关节疼痛。受感染的孕妇可以传输ZIKV发育中的胎儿1。 žIKV感染也被链接到吉兰巴雷综合征，外周神经自体免疫脱髓鞘病症9。
的寨卡病毒基因组由一个正感，单链RNA分子，其长度为约10.8千碱基的。基因组的结构被组织成5'NCR-C-的prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-3'NCR，与非编码区域（NCR）侧翼的蛋白质编码区6。一个单一的多聚（3419 AA）被翻译的是合作和翻译后切割成10小肽。无论是5'NCR和3'NCR RNA茎环结构在病毒基因组翻译和复制生效的重要组成部分。基因组的结构部件是由衣壳，膜和包膜蛋白。非结构蛋白是基因组复制的关键。
目前，寨卡病毒株被分成三个主要的基因型：西非，东非和亚洲6,10-13。有人提出，蔓延到西非和亚洲，后来进一步发展12东非血统。亚洲基因型负责在美洲当前疫情。寨卡病毒可以是在这两个蚊子和哺乳动物细胞中培养。初级真皮成纤维细胞，未成熟树突状细胞，皮层神经祖细胞，Vero细胞易受寨卡病毒感染10,14,15。 I型和II型干扰素已显示限制在皮肤成纤维细胞15 ZIKV增长。这项研究的目的是提供用于生产和亚洲基因型ZIKA病毒株PRVABC59的化验在哺乳动物细胞培养系统的逐步，详细的协议，并证明这种传染病文化体系作为药物开发平台的效用。此资源具有对兹卡病毒和神经学研究界进一步阐明我受益非浅的潜力病毒发病机理和病毒的毒性进化TS机制。
Protocol
Representative Results
Discussion
这里， 在体外培养寨卡病毒精简协议被提出。关键步骤包括，确定最佳的终点扩大病毒培养，测量滴度，并量化基因组复制被提供。寨卡病毒是一种人类病原体，因此，在处理感染性物质，生物安全程序必须严格遵守。阿猴肾细胞系，Vero细胞，用于展示各种病毒学检测。寨卡病毒复制动力学可以在各种组织和物种的细胞不同。可以如前面15所描述的使用的其他细胞系。寨卡病毒已?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Aaron Brault and Dr. Brandy Russell of the Centers for Disease Control and Prevention (CDC), USA for providing Zika viral strain PRVABC59. We thank Nicholas Ten of Yale University for copy-editing this manuscript. This work was supported by the Cedars-Sinai Medical Center Institutional Programmatic Research Award to V.A.
Materials
| Dulbecco’s modified Eagle’s medium (DMEM) | Sigma Life Science | D5796 | |
| HEPES | Life Technologies | 15630080 | |
| Glutamax | Life Technologies | 35050061 | |
| 2.5% Trypsin, 10X [-] Phenol Red | Corning | 25-054-C1 | |
| Trypan Blue Stain 0.4% | Life Technologies | T10282 | |
| Countess – Automated Cell Counter | ThermoFisher Scientific | C10227 | |
| Countess-cell counting chamber slides | ThermoFisher Scientific | C10283 | |
| Rneasy Mini Kit | Qiagen | 74104 | |
| Nanodrop 2000 | Thermo Scientific | Nanodrop 2000 | |
| mouse monoclonal anti-dsRNA antibody J2 | English & Scientific Consulting Kft. | 10010200 | |
| Goat anti-rabbit IgG Alexa Fluor 594 | Life Technologies | A11020 | |
| SUPERSCRIPT III RT | Life Technologies | 18080085 | |
| SYBR QPCR SUPERMIX W/ROX | Life Technologies | 11744500 | |
| QuantStudio12K Flex Real-Time PCR System | Thermo Fischer | 4471088 | |
| RNase-Free DNase | Promega | M6101 | |
| Vero Cell Line | ATCC | CCL-81 | |
| Zika viral strain PRVABC59 | Centers for Disease Control and Prevention (CDC) | ||
| IL-6 | Peprotech | 200-06 | |
| IL-1 alpha | Peprotech | 200-01A | |
| TNF-alpha | Peprotech | 300-01A | |
| Interferon alpha A | R & D Systems | 11100-1 | |
| Interferon beta | Peprotech | 300-02BC | |
| Interferon gamma | Peprotech | 300-02 | |
| Centrifuge 5415R | Eppendorf | 5415R | |
| Centrifuge 5810R | Eppendorf | 5810R | |
| Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI | Nikon | Visit Nikon for Request |
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