利用反向遗传学技术发现寨卡病毒的关键神经毒力位点

The outbreak and prevalence of Zika virus (ZIKV) has become a public health emergency since 2016. As a classical neurotropic virus, ZIKV infection in human can lead to severe neurological complications, such as microcephaly in fetus and Guillain-Barré syndrome in adults, whereas the pathological mechanisms behind these diseases remains unclear. Recently, we have successfully isolated several ZIKV strains from the Pacific islands and the Americas, and established the neurovirulence animal models in fetal and neonatal mice. In this project, we aim to develop the neurovirulence model in non-human primate (NHP) model, and investigate the replication dynamics of ZIKV in the central nervous systems as well as the neuropathological damage in ZIKV-infected brains by using the fetus, neonatal mice and NHP model systematically. Then, the differences in the neurovirulence and other important biological phenotypes of various ZIKV strains belonging to different evolutionary clades will be compared comprehensively with the established models. Furthermore, the potential determinants of ZIKV neurovirulence will be predicted based on sequence comparison and phylogenetics analysis. Using reverse genetics approaches, mutations corresponding to the potential neurovirulence determinants will be introduced into the infectious clone of ZIKV strain FSS13025 and GZ01, respectively, and a series of virus mutants will be generated and their neurovirulence will be tested using different animal models, and the corresponding critical determinants of viral neurovirulence will be identified. At last, by a combination of multiple biochemical and virological technologies, the molecular mechanism how the determinants influence viral neurovirulence and microcephaly will be revealed. Our project will reveal a panel of novel neurovirulence determinants and the relevant attenuation mechanisms, which would be important clues for the understandings of the connections between the evolution of ZIKV neurovirulence, ZIKV prevalence and the emergence of microcephaly. Our project will also provide reliable animal models for the investigation of ZIKV pathogenicity and the safety evaluation of ZIKV vaccine candidates.

寨卡病毒是一种典型的嗜神经病毒，感染后可导致小头畸形严重神经系统疾病，但其致病机制尚不十分清楚。去年寨卡疫情暴发以来，我们成功分离获得了多株不同来源的寨卡病毒，利用建立的胎鼠和乳鼠动物模型发现当前流行株的神经毒力远强于早期分离株。本研究将进一步建立基于恒河猴的神经毒力模型，明确寨卡病毒在颅内的复制动力学及其导致神经系统损伤的特征；系统比较不同进化地位的寨卡病毒分离株在不同动物模型神经毒力的差异，结合生物信息学预测潜在的关键毒力位点；进一步通过反向遗传学技术构建一系列病毒突变体，发现和鉴定决定病毒神经毒力的关键位点；综合运用多种技术手阐明相应关键位点影响病毒神经毒力的作用机制。本项目可为寨卡病毒致病机制研究以及疫苗安全性评价提供可靠模型，同时有望发现全新的毒力位点与减毒靶标，对于深刻理解寨卡病毒的暴发流行与毒力进化具有重要的科学意义。

2015年南美暴发的寨卡疫情被WHO宣布为国际突发的公共卫生事件。此次疫情伴随出现大量的新生儿小头畸形病例，提示寨卡病毒可能具有更强的侵染神经系统的能力，解析其独特的致病机制成为亟需解决的关键科学问题。本项目重点围绕寨卡病毒的神经毒力特征开展研究，旨在建立包括胎鼠、乳鼠、成鼠在内的系统的神经毒力评价模型，在此基础上结合系统进化和序列比对结果，预测与寨卡病毒神经毒力相关的关键位点，进一步利用反向遗传学技术等手段，构建获得了携带相应突变的重组病毒株，通过与亲本病毒进行体外和体内生物学特性比较，鉴定出影响寨卡病毒神经嗜性和致病性的关键位点，并结合生化和病毒学多种技术手段，对相应关键位点影响病毒神经毒力的分子机制进行了深入探讨。本项目取得了如下三方面的重要发现：1）发现了一系列位于病毒结构蛋白和非结构蛋白中影响神经毒力（K101R）、复制效力（N8）和垂直传播能力（M2634V）的关键氨基酸位点，初步明确了上述位点发挥相应生物功能的作用机制；2）鉴定获得了寨卡病毒基因组3’非编码区中影响病毒复制的关键功能元件（DB12和RCS3），初步阐明其分别通过改变RNA结构的紧密度和sfRNA的产生来影响病毒RNA的复制；3）发现了寨卡病毒3’非编码区的关键神经毒力元件Musashi1结合基序，并成功实现了利用该元件调控病毒神经毒力。上述研究结果对于深刻理解寨卡病毒暴发流行和毒力进化具有重要的科学意义，发现的全新毒力位点有望成为疫苗设计的减毒靶标。