Toward Control of Rift Valley Fever Virus Replication

裂谷热病毒(RVFV)是白喉病毒属的一种典型病毒，属于布尼亚病毒科， 属于NIAID A类病原体清单和CDC潜在生物恐怖主义制剂清单。裂痕 谷热是撒哈拉以南非洲的一种地方性疾病，可通过蚊子传播的爆炸性疾病出现。 流行病，杀死成群的羊和牛，造成巨大的经济损失；人类正在 同时感染，导致出血热、脑炎和视网膜血管炎。许多不同的 蚊子，包括几种原产于北美的蚊子，是RVFV流行的合格媒介 变速箱。RVFV引入北美可能会引起普遍的恐慌 人口减少，对牲畜的影响可能会对经济产生毁灭性的影响。 尽管针对病毒包膜蛋白的体液免疫可引起保护性免疫，但 目前还没有适合大规模人类疫苗接种计划的RVFV疫苗。本申请的目的是 通过修改MP-12减毒株来开发安全、活的RVFV疫苗。利用反向遗传学和 在小鼠模型中，我们将首先研究RVFV毒力减弱的遗传基础，通过检测RVFV在 野生型RVFV囊膜蛋白中一种特定氨基酸的毒力。MP-12展品 幼鼠的神经侵袭性，我们将调查这种表型是否是特定病毒的结果 在感染过程中不断积累的突变。此外，我们将产生MP-12变种，这种变种不太可能 以产生神经侵入性突变体。基于这样的假设，增加分子的正净电荷 MP-12的包膜蛋白使病毒毒力进一步减弱，我们将产生一系列MP-12 通过在包膜蛋白中引入正电荷氨基酸的变化，我们将测试它们的 诱导小鼠中和抗体的能力及其毒力。如果这些病毒表现出 毒力降低，免疫原性差，另一组携带人密码子的突变病毒进行了优化 将产生包膜蛋白基因，并测试其毒力、免疫原性和保护效果 免疫Wt RVFV攻击小鼠。密码子优化也将作为一种减少 蚊子的传染性。该项目的最终产品将是第二代MP-12疫苗， 安全性更高，适合最终产品开发和人体测试。衰减策略将 也适用于其他新兴的布尼亚病毒疫苗以及其他RNA病毒的开发。

Rift Valley fever virus (RVFV) is a prototypical virus of the genus Phlebovirus, in the family Bunyaviridae, and belongs to the NIAID Category A list pathogens and the CDC list of potential bioterrorism agents. Rift Valley fever is an endemic disease of sub-Saharan Africa that can emerge in explosive mosquito-borne epidemics, decimating herds of sheep and cattle and resulting in enormous economic losses; humans are infected concurrently, resulting in hemorrhagic fever, encephalitis, and retinal vasculitis. Many different mosquitoes, including several native to North America, are competent vectors for RVFV epidemic transmission. The introduction of RVFV into North America would likely cause panic in the general population, and the effects on livestock could have a devastating economic impact. Although humoral immunity against the viral envelope proteins elicits protective immunity, there is currently no RVFV vaccine suitable for massive human vaccination programs. The present application aims to develop safe, live RVFV vaccines by modifying the attenuated MP-12 strain. Using reverse genetics and the mouse model, we will first study the genetic basis of RVFV virulence attenuation by examining the role in viral virulence of a specific amino acid in the envelope protein of wild-type RVFV. MP-12 exhibits neuroinvasiveness in young mice, and we will investigate whether this phenotype is the result of specific viral mutations accumulating during infection. Furthermore, we will generate MP-12 variants that will be less likely to produce neuroinvasive mutants. Based on the hypothesis that increasing the positive net charge of envelope proteins of MP-12 causes further attenuation of viral virulence, we will generate a series of MP-12 mutants by introducing positive charge amino acid changes into the envelope proteins, and we will test their competence for eliciting neutralizing antibodies and their virulence in mice. If these viruses demonstrate a reduced virulence and poor immunogenicity, another set of mutant viruses carrying human-codon optimized envelope protein genes will be generated and tested for virulence, immunogenicity and efficacy in protecting immunized mice from wt RVFV challenge. Codon optimization will also be tested as a method to reduce mosquito infectivity. The final product of this project will be a second generation MP-12 vaccine with increased safety, suitable for final product development and human testing. The attenuation strategies will also be applicable to other emerging bunyaviral vaccine development as well as other RNA viruses.