MOLECULAR BIOLOGY OF DENGUE AND OTHER FLAVIVIRUSES

有60多种血清学上相关的昆虫传播的黄病毒， 包括重要的人类病原体，如蚊媒黄斑病菌 发热病毒、乙型脑炎病毒、登革热病毒和 蜱传播的脑炎病毒。其中，四种登革热病毒 在人类发病率方面，血清型(DEN1-4)最重要 据估计，每年涉及数百万人。为了这个 原因是，世界卫生组织已高度重视 开发登革热病毒疫苗。因为它们的重要性 在人类疾病方面，我们研究了登革热的分子生物学。 病毒，最初侧重于基因组组织和机制 基因表达和病毒复制。有人认为这是一种 信息可能会让我们更好地了解登革热 病毒的发病机制，并最终设计分子策略 预防登革热病毒病。 DEN4 RNA基因组，大小约为11000个核苷酸(NT)， 编码一种长多聚蛋白，它被切割成10种病毒蛋白 这包括三种病毒粒子结构蛋白，即衣壳 蛋白(C)、前体膜蛋白(PREM)和包膜 蛋白质(E)和七种非结构蛋白(NS)。DEN4基因组 包含5‘非编码(NC)序列和3’NC序列，每个序列 它被预测会形成一个稳定的二级结构 在病毒复制过程中发挥作用。经过相当大的努力，我们 成功构建了能产生RNA的DEN4全长cDNA克隆 将转录本导入敏感细胞时具有感染性 在文化上。引入了一系列长度不等的删除 插入到DEN4基因克隆的5‘或3’NC序列区域； 事实证明，缺失突变体是可行的。我们展示了最可行的3‘ 缺失突变体产生了在猿猴LLC-MK2上出现较晚的斑块 细胞或在蚊子C6/36细胞上表现出小斑块形态 与野生型病毒相比。大多数3‘NC缺失突变体，相似 早些时候构建的5‘Nc缺失突变体，复制的也更少 并在LLC-MK2细胞中获得比野生株更低的效价 输入病毒。具有活力的突变体的回收和鉴定 3‘nc区或5’nc区中的缺失 在猴LLC-MK2细胞中的复制，允许我们准备一个菜单 考虑在活体中使用的候选衰减突变 登革热病毒疫苗。

There are more than 60 serologically related insect-borne flaviviruses, including important human pathogens, such as the mosquito-borne yellow fever virus, Japanese encephalitis virus, and dengue viruses and the tick-borne encephalitis viruses. Among them, the four dengue virus serotypes (DEN1-4) are most important in terms of human morbidity which is estimated to involve millions of individuals every year. For this reason, the World Health Organization has assigned a high priority to the development of a dengue virus vaccine. Because of their importance in human disease, we investigated the molecular biology of the dengue viruses, focusing initially on genome organization and the mechanisms of gene expression and viral replication. It was thought that this information might allow us to gain a better understanding of dengue virus pathogenesis and eventually, to design molecular strategies for the prevention of dengue virus disease. DEN4 RNA genome, approximately 11 thousand nucleotides (nt) in size, codes for a long polyprotein that is cleaved to yield 10 viral proteins that include the three virion structural proteins, i.e., the capsid protein (C), the precursor membrane protein (PreM), and the envelope protein (E) and seven nonstructural proteins (NS). The DEN4 genome contains a 5' non-coding (NC) sequence and a 3' NC sequence, each of which is predicted to form a stable secondary structure that is thought to play a role during viral replication. After considerable effort, we successfully constructed a full-length DEN4 cDNA clone that yielded RNA transcripts that were infectious when transfected into susceptible cells in culture. A series of deletions varying in length were introduced into the 5' or 3' NC sequence region of the DEN4 cDNA clone; most of the deletion mutants proved to be viable. We showed that most viable 3' deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared to the wild type virus. Most 3' NC deletion mutants, similar to 5' NC deletion mutants constructed earlier, also replicated less efficiently and attained a lower titer in LLC-MK2 cells than the wild type virus. Recovery and identification of viable mutants that contained a deletion in the 3' NC region or 5'NC region and that were restricted in replication in simian LLC-MK2 cells, allowed us to prepare a menu of candidate attenuating mutations to be considered for use in a live dengue virus vaccine.