A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.
A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.
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DOI：
10.1371/journal.ppat.1004290
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发表时间：
2014-07
期刊：
影响因子：
6.7
通讯作者：
Lee YM
中科院分区：
文献类型：
--
作者：
Yun SI;Song BH;Kim JK;Yun GN;Lee EY;Li L;Kuhn RJ;Rossmann MG;Morrey JD;Lee YM
日本脑炎病毒（JEV）是一种蚊媒黄病毒，可导致人类致命的神经系统疾病，是具有公共卫生意义的最重要的新兴病原体之一。 JEV 代表乙脑血清群，其中还包括西尼罗河病毒、墨累河谷脑炎病毒和圣路易斯脑炎病毒。在该血清群中，乙脑病毒是一种可通过疫苗预防的病原体，但其神经毒力的分子基础仍不清楚。在这里，我们构建了最广泛使用的乙脑减毒活疫苗 SA14-14-2 的感染性 cDNA，并从 cDNA 中拯救出分子克隆病毒 SA14-14-2MCV，该病毒表现出与其亲本 SA14-14-2 相同的体外生长特性和体内减毒表型。为了阐明神经毒力的分子机制，我们通过在小鼠中连续脑内传代，从SA14-14-2MCV（LD50，>1.5×105 PFU）中选择了三种独立的高神经毒力变体（LD50，<1.5 PFU）。完整的基因组序列比较揭示了总共八个点突变，其中常见的单个 G1708→A 取代将病毒 E 糖蛋白第 244 位的 Gly 替换为 Glu。使用我们的感染性 SA14-14-2 cDNA 技术，我们证明 E-244 处的这种单一的 Gly 到 Glu 变化足以赋予小鼠致命的神经毒力，包括病毒传播和中枢神经系统组织炎症的快速发展。 E-244 的全面定点诱变与基于同源的结构建模相结合，证明了 E-244 在 E 二聚化结构域的 ij 发夹内对 JEV 神经毒力具有新的重要调节作用。在小鼠和人类神经元细胞中，我们进一步表明，E-244 突变改变了 JEV 的体外感染性，与体内神经毒力水平直接相关，但对病毒 RNA 复制没有显着影响。我们的结果为开发针对乙脑病毒和可能的其他脑炎黄病毒的新型治疗和预防策略迈出了关键的一步。一组引起人类致命性脑炎的蚊媒黄病毒是所有新出现的具有全球意义的人类病原体中最重要的一种。该组包括日本脑炎 (JE)、西尼罗河病毒、圣路易斯脑炎病毒和墨累谷脑炎病毒。在这项工作中，我们通过构建包含全长 SA14-14-2 cDNA 的感染性细菌人工染色体，开发了 SA14-14-2（一种在乙脑流行地区最常用的乙脑活疫苗）的反向遗传学系统。利用这种感染性SA14-14-2 cDNA，结合JEV感染的小鼠模型，我们鉴定了一种关键的病毒神经毒力因子，即与病毒E糖蛋白融合环相邻的ij发夹中的保守单氨基酸，它调节病毒对体内中枢神经系统神经元以及体外小鼠和人类神经元细胞的感染性。因此，我们的研究结果阐明了乙脑病毒和其他密切相关的脑炎黄病毒引起的神经毒力的分子基础，这是了解其神经发病机制的重要一步。从临床角度来看，病毒神经毒力因子及其作用的发现将直接应用于设计一类新型广谱抗病毒药物，以治疗和预防乙脑病毒和其他分类学相关的神经亲性黄病毒感染。
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses. A group of mosquito-borne flaviviruses that cause fatal encephalitis in humans is among the most important of all emerging human pathogens of global significance. This group includes Japanese encephalitis (JE), West Nile, St. Louis encephalitis, and Murray Valley encephalitis viruses. In this work, we have developed a reverse genetics system for SA14-14-2, a live JE vaccine that is most commonly used in JE-endemic areas, by constructing an infectious bacterial artificial chromosome that contains the full-length SA14-14-2 cDNA. Using this infectious SA14-14-2 cDNA, combined with a mouse model for JEV infection, we have identified a key viral neurovirulence factor, a conserved single amino acid in the ij hairpin adjacent to the fusion loop of the viral E glycoprotein, which regulates viral infectivity into neurons within the central nervous system in vivo and neuronal cells of mouse and human in vitro. Thus, our findings elucidate the molecular basis of the neurovirulence caused by JEV and other closely related encephalitic flaviviruses, a major step in understanding their neuropathogenesis. From a clinical perspective, the discovery of the viral neurovirulence factor and its role will have direct application to the design of a novel class of broad-spectrum antivirals to treat and prevent infection of JEV and other taxonomically related neurotropic flaviviruses.
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影响因子：
3.7
作者：
Chen, LK;Lin, YL;Chin, C
通讯作者：
Chin, C
影响因子：
5.4
作者：
Arroyo, J;Guirakhoo, F;Chambers, TJ
通讯作者：
Chambers, TJ
影响因子：
11.8
作者：
Gubler, Duane J.
通讯作者：
Gubler, Duane J.
DOI：
10.1016/j.str.2004.04.024
发表时间：
2004-07
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Dokland T;Walsh M;Mackenzie JM;Khromykh AA;Ee KH;Wang S
通讯作者：
Wang S
影响因子：
11.4
作者：
Bressanelli, S;Stiasny, K;Rey, FA
通讯作者：
Rey, FA