GENETIC DETERMINANTS OF DENGUE VIRUS MOUSE NEUROVIRULENCE

在第一批登革热病毒株在非洲分离出来后不久， 登革1型或2型病毒的实验室连续脑内传代 (DEN1或DEN2）来减毒这些病毒 作为减毒活疫苗。 萨宾的研究显示， 在低传代水平时，这些病毒已失去大部分毒力。 此外，非致病性亲本的高度神经毒性突变体 DEN4株H241通过在小鼠脑内连续传代来选择。 DEN4小鼠适应突变体神经毒力的遗传基础是 通过比较含有三种基因的嵌合病毒， 亲本病毒或其神经毒力的结构蛋白基因 非神经毒性DEN4菌株背景序列的突变体 814669. 含有三种结构蛋白基因的嵌合体 小鼠神经毒力DEN4 H241在小鼠中具有高度神经毒力， 而含有其非- 小鼠适应亲本不是神经毒性的。 因此， DEN4突变体图中神经毒力的遗传位点 结构蛋白基因 亲本及其小鼠神经毒力突变株 发现它们的氨基酸差异只有5个。 结构蛋白 其中三个突变位于 (E)糖蛋白 其中两个氨基酸变化被鉴定为 DEN4小鼠神经毒力的重要决定因素：（i）单个 用Ile取代Thr434，消除了两个保守的 DEN4 E中的糖基化位点产生了一种病毒， 神经毒性的小鼠适应突变体;和（ii）Leu的Phe680 取代也产生了神经毒性病毒，但它的毒性较小， 比糖基化突变体更神经毒性。 亲本DEN1夏威夷 并对其小鼠神经毒力突变株进行了研究 绘制负责小鼠神经毒力的DEN1基因位点。 C-PreM-E结构蛋白区的13个氨基酸差异 被确认了身份 在13个变化中，7个位于E区，3个位于前M区， 3在C。

Shortly after the first strains of dengue virus were isolated in the laboratory, serial intracerebral passage of dengue type 1 or type 2 virus (DEN1 or DEN2) in mice was employed to attenuate these viruses for use as live attenuated vaccines in humans. Studies by Sabin revealed that at a low passage level these viruses had lost most of their virulence. In addition, a highly neurovirulent mutant of the non-pathogenic parental DEN4 strain H241 was selected by serial intracerebral passage in mice. The genetic basis for neurovirulence of the DEN4 mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes of the parental virus or its neurovirulent mutant on the background sequence of non-neurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes of mouse neurovirulent DEN4 H241 was highly neurovirulent in mice, whereas the chimera that contained the corresponding genes of its non- mouse adapted parent was not neurovirulent. Thus, some or all of the genetic loci for neurovirulence of the DEN4 mutant map within the structural protein genes. The parent and its mouse neurovirulent mutant were found to differ by only five amino acid differences in their structural proteins. Three of the mutations were located in the envelope (E) glycoprotein. Two of these amino acid changes were identified as important determinants of DEN4 mouse neurovirulence: (i) the single substitution of Ile for Thr434 which ablated one of the two conserved glycosylation sites in DEN4 E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant; and (ii) the Leu for Phe680 substitution also yielded a neurovirulent virus but it was less neurovirulent than the glycosylation mutant. The parental DEN1 Hawaii strain and its mouse neurovirulent mutant were also studied in an attempt to map the DEN1 genetic loci responsible for mouse neurovirulence. Thirteen amino acid differences in the C-PreM-E structural protein region were identified. Among the 13 changes 7 are located in E, 3 in PreM and 3 in C.