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Abstract：
Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.
摘要：
尽管有高效的黄热病病毒（YFV）疫苗，黄热病的爆发经常发生在非洲和南美洲，死亡率很高，强调迫切需要抗病毒药物来管理未来的疫情。为了支持针对YFV的抗病毒药物的发现和开发，我们表征了一组针对三种YFV结构蛋白和五种非结构蛋白的兔多克隆抗体，并证明了这些抗体试剂与病毒RNA代谢标记的结合，双链RNA染色和膜浮选测定是研究YFV多蛋白加工的强大工具，复制复合物的形成，病毒RNA合成和高通量抗病毒药物的发现。具体来说,病毒多蛋白的蛋白水解过程可以通过蛋白质印迹分析来分析。免疫荧光染色和膜浮选测定可以揭示NS5蛋白的主要核定位以及细胞内病毒非结构蛋白分布与YFVRNA复制灶之间的关系。以抗YFVNS4B蛋白的抗体为例，细胞内蛋白质和高含量成像试验已被开发用于高通量发现抗病毒药物.YFVNS4B靶向抗病毒剂BDAA和NS5RNA依赖性RNA聚合酶抑制剂（Sofosbuvir）的协同抗病毒作用也通过高含量成像测定得到证明。显然,本文建立的基于抗体的测定不仅有助于发现和开发针对YFV的抗病毒剂，而且还提供了有价值的工具来剖析抗病毒剂抑制YFV复制的分子机制。
