MOLECULAR BIOLOGY OF DENGUE AND OTHER FLAVIVIRUSES

有超过60种血清学相关的虫媒黄病毒， 包括重要的人类病原体，如蚊子传播的黄色 发热病毒、日本脑炎病毒和登革热病毒，以及 蜱传脑炎病毒 其中，四种登革热病毒 血清型（DEN 1 -4）在人类发病率方面是最重要的， 据估计，每年有数百万人参与其中。为此 因此，世界卫生组织高度重视 登革热病毒疫苗的研制。因为它们的重要性 在人类疾病中，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， 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。 DEN 4 RNA基因组，大小约为1.1万个核苷酸（nt）， 编码一种长的多蛋白，切割后产生10种病毒蛋白 包括三种病毒体结构蛋白，即，衣壳 前体膜蛋白（PreM）和包膜蛋白（C） 蛋白（E）和七种非结构蛋白（NS）。DEN 4基因组 含有5'非编码（NC）序列和3' NC序列， 据预测，它会形成一个稳定的二级结构， 在病毒复制过程中发挥作用。经过不懈努力，我们 成功构建了一个全长DEN 4 cDNA克隆， 当转染到易感细胞中时具有感染性的转录物 在文化中。 引入了一系列长度不等的缺失 插入DEN 4 cDNA克隆的5 ′或3 ′ NC序列区;大多数的 缺失突变体被证明是可行的。我们发现大多数可行的3' 缺失突变体产生的噬斑在猿猴LLC-MK 2上出现较晚， 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，类似于 到5' NC缺失突变体构建较早，也复制较少 并且在LLC-MK2细胞中获得比野生型更低的滴度 型病毒。回收和鉴定含有 在3' NC区域或5' NC区域中的缺失，并且是限制性的 在猴LLC-MK2细胞中复制，使我们能够准备一份菜单， 待考虑用于活体的候选减毒突变 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.