GENETIC LOCI RESPONSIBLE FOR GROWTH RESTRICTION OF MOUSE-ADAPTED DENGUE VIRUSES

登革1型或2型病毒在小鼠脑内连续传代 前面显示了为小鼠神经毒性突变体进行选择，这也 事实证明，它对人类来说是弱化的。以类似的方式，一种神经毒性 用连续脑内传代法筛选DEN4突变体(菌株H241) 在老鼠身上。DEN4 H241神经毒株(N)的复制效率低于 在猴LLC-MK2细胞中表达其DEN4-H_241亲本(P)。一种非典型DEN4 含DEN4 H241N C-Prem-E结构蛋白基因的嵌合体 对LLC-MK2细胞的生长也有明显的抑制作用。分析 DEN4 H241N及其衍生的C-Prem-E嵌合体病毒粒子蛋白的研究 在蚊子C6/36细胞中，M的产生很少。那里 有大量证据表明未成熟的含有Prem的黄病毒病毒粒子 比成熟的含M病毒的传染性更低。因为这个原因， 我们构建了一个嵌合体面板，其中包含一个或多个 突变的C-Prem-E序列的变异氨基酸取代 相应的亲本病毒序列。这些嵌合体是 分析以确定这些突变中是否有任何一个是导致 低效的Prem卵裂。包含两个替换的突变体 Prem，即Lys165-Glu和Val188-Ala，有或没有Thr81-Ile C中的取代，表现出减少的Prem裂解。Prem的缩减 还观察到含有多个突变的嵌合体的切割 在E中，即Thr434-Ile、Ser435-Pro和Phe680-Leu，但例外 含有Thr434-Ile和Ser435-Pro的嵌合体。高效的Prem 病毒需要切割才能实现高水平的复制 猴LLC-MK2细胞。同样，减少的遗传基础 小鼠传代MD-1疫苗株在日本的复制效率 LLC-MK2细胞与其亲本DEN1夏威夷株相比 调查过了。E中独立的三个代换的组合 导致猴细胞复制受到限制。单款Val207-Ala Prem的替代也是限制的独立责任 在LLC-MK2细胞中复制MD-1或其衍生的嵌合体。这 Prem突变位于M的N端下游， 它是由宿主细胞呋喃酶从Prem上切割出来的。Val207-Ala 突变不会影响蚊子细胞中Prem的切割，但这 突变可能会限制猴细胞中Prem的切割，因此， 对限产负责。

Serial intracerebral passage of dengue type 1 or type 2 virus in mice was shown previously to select for mouse neurovirulent mutants which also proved to be attenuated for humans. In a similar manner a neurovirulent mutant of DEN4 (strain H241) was selected by serial intracerebral passage in mice. DEN4 H241 neurovirulent (N) replicated less efficiently than its DEN4 H241 parent (P) in simian LLC-MK2 cells. An intratypic DEN4 chimera containing the C-PreM-E structural protein genes from DEN4 H241N also exhibited marked restriction of growth in LLC-MK2 cells. Analysis of virion proteins of DEN4 H241N or its derived C-PreM-E chimera grown in mosquito C6/36 cells indicated that very little M was produced. There is considerable evidence that immature PreM-containing flavivirus virions are less infectious than the mature M-containing virus. For this reason, we constructed a panel of chimeras that contained one or more of the variant amino acids of the mutant C-PreM-E sequence substituting for the corresponding sequence of the parental virus. These chimeras were analyzed to determine if any of these mutations was responsible for inefficient PreM cleavage. Mutants which contained two substitutions in PreM, i. e., Lys165-Glu and Val188-Ala, with or without the Thr81-Ile substitution in C, exhibited reduced PreM cleavage. Reduction of PreM cleavage was also observed for chimeras which contain multiple mutations in E, i.e., Thr434-Ile, Ser435-Pro, and Phe680-Leu, with the exception of the chimera containing Thr434-Ile and Ser435-Pro. Efficient PreM cleavage was required for virus to achieve high level of replication in simian LLC-MK2 cells. Similarly, the genetic basis for the reduced replicative efficiency of the mouse-passaged MD-1 vaccine strain in LLC-MK2 cells compared to its parental DEN1 Hawaii strain was investigated. A combination of three substitutions in E independently caused restriction of replication in simian cells. A single Val207-Ala substitution in PreM was also independently responsible for restriction of replication of MD-1 or its derived chimera in LLC-MK2 cells. This PreM mutation is located immediately downstream of the N-terminus of M, which is cleaved from PreM by the host cell furin enzyme. The Val207-Ala mutation did not affect PreM cleavage in mosquito cells, but this mutation might possibly restrict PreM cleavage in simian cells and thus, be responsible for growth restriction.