裂谷热病毒:繁殖、定量和储存

Current Protocols in Microbiology Pub Date : 2019-09-19 DOI:10.1002/cpmc.92

MaRyka R. Smith, Erin E. Schirtzinger, William C. Wilson, A. Sally Davis

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引用次数: 16

摘要

裂谷热病毒是撒哈拉以南非洲和阿拉伯半岛流行的一种节肢动物传播的人畜共患疾病。裂谷热暴发在胎羊和新生羊中死亡率高达100%。在感染反刍动物和人类宿主后，裂谷热病毒感染有时会引起严重的肝炎和许多其他器官的病理。被包膜的病毒粒子包含一个主要为负义的三边单链RNA基因组，该基因组编码病毒在哺乳动物宿主和昆虫载体中复制所需的蛋白质。流行国家通常使用减毒的裂谷热病毒毒株对牲畜进行疫苗接种，但在非流行地区，没有商业许可的人类或牲畜疫苗。在实验室中，RVFV可以很容易地在体外使用细胞培养系统进行繁殖和操作。本文介绍了在我们的实验室中已被证明成功的裂谷热病毒研究中常规使用的技术。©2019 by John Wiley &基本方案1:裂谷热病毒在哺乳动物细胞中的繁殖基本方案2:裂谷热病毒通过空斑测定定量基本方案3:裂谷热病毒通过50%组织培养感染剂量(TCID50)测定定量基本方案4:裂谷热病毒通过形成病灶测定定量基本方案5:储存和消毒备用方案1:裂谷热病毒在MRC-5细胞中的繁殖备用方案2:裂谷热病毒在MRC-5细胞中的繁殖RVFV在蚊子衍生细胞中的传播替代方案3:使用荧光可视化的TCID50检测支持方案1:计算在选定的感染倍数下感染烧瓶所需的病毒量支持方案2:通过斑块测定或焦点形成测定计算病毒滴度支持方案3:通过Reed和muenchmethod计算TCID50滴度支持方案4:计算抗体体积用于焦点形成测定

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Rift Valley Fever Virus: Propagation, Quantification, and Storage

Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative-sense, single-stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non-endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells

Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay

Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID₅₀) assay

Basic Protocol 4: Quantification of Rift Valley fever virus by focus-forming assay

Basic Protocol 5: Storage and disinfection

Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC-5 cells

Alternate Protocol 2: Propagation of RVFV in mosquito-derived cells

Alternate Protocol 3: TCID₅₀ detection using fluorescence visualization

Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection

Support Protocol 2: Calculation of the virus titer by plaque assay or focus-forming assay

Support Protocol 3: Calculation of the TCID₅₀ titer by the method of Reed and Muench

Support Protocol 4: Calculation of the antibody volume for the focus-forming assay

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Current Protocols in Microbiology Immunology and Microbiology-Parasitology

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期刊介绍： Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.

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