Generation of a Single-Cycle Replicable Rift Valley Fever Vaccine.
Generation of a Single-Cycle Replicable Rift Valley Fever Vaccine.
复制标题
单周期可复制裂谷热疫苗的产生。
DOI：
10.1007/978-1-4939-3387-7_9
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Makino,Shinji
中科院分区：
文献类型：
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作者：
Murakami,Shin;Terasaki,Kaori;Makino,Shinji
裂谷热病毒（白蛉病毒属，布尼亚病毒科）是一种虫媒病毒，可在撒哈拉以南非洲国家的人类和牲畜中引起严重疾病。该病毒携带三边、单链和负义RNA基因组，称为L、M和S RNA。通过对动物和人类进行有效的疫苗接种，可以预防裂谷热病毒的传播。虽然RVFV的MP-12毒株是一种候选减毒活疫苗，但MP-12在幼鼠和免疫缺陷小鼠中表现出神经侵袭性和神经毒力。因此，对于某些个体，特别是免疫功能低下的个体，MP-12的使用值得关注。为了提高MP-12的安全性，我们已经生成了一个单循环、可复制的MP-12 (scMP-12)，它携带L RNA， S RNA编码绿色荧光蛋白代替病毒非结构蛋白NSs， M RNA编码缺乏内质网检索信号和膜融合功能缺陷的突变包膜蛋白。scMP-12在稳定表达病毒包膜蛋白的Vero细胞系Vero- g中进行高效扩增，而在naïve细胞中进行单周期复制，颅内接种后在哺乳小鼠中完全没有神经毒力。单次给小鼠接种scMP-12疫苗可产生保护性免疫。因此，scMP-12代表了一种新的、有希望的裂谷热候选疫苗。在这里，我们描述了scMP-12的生成方案，通过使用反向遗传系统，建立Vero-G细胞，并在Vero-G细胞中滴定scMP-12。
Rift Valley fever virus (RVFV)(genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. The virus carries a tripartite, single-stranded, and negative-sense RNA genome, designated as L, M, and S RNAs. RVFV spread can be prevented by the effective vaccination of animals and humans. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, MP-12 showed neuroinvasiveness and neurovirulence in young mice and immunodeficiency mice. Hence, there is a concern for the use of MP-12 to certain individuals, especially those that are immunocompromised. To improve MP-12 safety, we have generated a single-cycle, replicable MP-12 (scMP-12), which carries L RNA, S RNA encoding green fluorescent protein in place of a viral nonstructural protein NSs, and an M RNA encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function. The scMP-12 undergoes efficient amplification in the Vero-G cell line, which is a Vero cell line stably expressing viral envelope proteins, while it undergoes single-cycle replication in naïve cells and completely lacks neurovirulence in suckling mice after intracranial inoculation. A single-dose vaccination of mice with scMP-12 confers protective immunity. Thus, scMP-12 represents a new, promising RVF vaccine candidate. Here we describe protocols for scMP-12 generation by using a reverse genetics system, establishment of Vero-G cells, and titration of scMP-12 in Vero-G cells.