Kawano H, Rostapshov V, Rosen L, Lai C J
Molecular Viral Biology Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1993 Nov;67(11):6567-75. doi: 10.1128/JVI.67.11.6567-6575.1993.
Mouse-adapted dengue type 4 virus (DEN4) strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes from mouse neurovirulent DEN4 strain H241 proved to be highly neurovirulent in mice, whereas the chimera that contained the corresponding genes from its non-mouse-adapted parent was not neurovirulent. This finding indicates that most of the genetic loci for the neurovirulence of the DEN4 mutant lie within the structural protein genes. A comparison of the amino acid sequences of the parent and its mouse neurovirulent mutant proteins revealed that there were only five amino acid differences in the structural protein region, and three of these were located in the envelope (E) glycoprotein. Analysis of chimeras which contained one or two of the variant amino acids of the mutant E sequence substituting for the corresponding sequence of the parental virus identified two of these amino acid changes as important determinants of mouse neurovirulence. First, the single substitution of Ile for Thr-155 which ablated one of the two conserved glycosylation sites in parental E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant. Thus, the loss of an E glycosylation site appears to play a role in DEN4 neurovirulence. Second, the substitution of Leu for Phe-401 also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. These findings indicate that at least two of the genetic loci responsible for DEN4 mouse neurovirulence map within the structural protein genes.
鼠适应株登革4型病毒（DEN4）H241株对小鼠具有高度神经毒力，而其未适应小鼠的亲代病毒则很少具有神经毒力。通过比较同型嵌合病毒来研究适应小鼠的突变体神经毒力的遗传基础，这些嵌合病毒在非神经毒力的DEN4 814669株背景序列中含有来自亲代病毒或神经毒力突变体的三个结构蛋白基因。含有来自小鼠神经毒力DEN4 H241株的三个结构蛋白基因的嵌合体在小鼠中被证明具有高度神经毒力，而含有来自其未适应小鼠亲代的相应基因的嵌合体则没有神经毒力。这一发现表明，DEN4突变体神经毒力的大多数遗传位点位于结构蛋白基因内。亲代及其小鼠神经毒力突变体蛋白的氨基酸序列比较显示，结构蛋白区域仅有五个氨基酸差异，其中三个位于包膜（E）糖蛋白中。对含有突变体E序列中的一个或两个变异氨基酸替代亲代病毒相应序列的嵌合体分析确定，其中两个氨基酸变化是小鼠神经毒力的重要决定因素。首先，将亲代E中两个保守糖基化位点之一的苏氨酸-155替换为异亮氨酸，产生的病毒几乎与适应小鼠的突变体一样具有神经毒力。因此，E糖基化位点缺失似乎在DEN4神经毒力中起作用。其次，将苯丙氨酸-401替换为亮氨酸也产生了一种神经毒力病毒，但它的神经毒力低于糖基化突变体。这些发现表明，负责DEN4小鼠神经毒力的遗传位点中至少有两个位于结构蛋白基因内。
