EVOLUTIONARY CONSEQUENCES OF DENGUE VIRUS EMERGENCE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。亚细亚登革热病毒在非洲和亚洲森林树冠上的一个未知水库中传播，不会导致人类登革热，登革热是由地方性登革热病毒引起的，登革热病毒只在人类和叮咬人的蚊子之间传播。由于尚未确定防止新登革热病毒的屏障，因此无法评估新登革热病毒株出现的风险。解释人类体内不存在森林病毒的三种假设包括：1.森林病毒比地方性病毒更不适合人类；2.森林病毒比地方性病毒更不适合叮咬人的蚊子；3.森林病毒和地方性病毒同样适合人类和叮咬人的蚊子，但它们占据着不同的空间生态位(森林树冠和森林地面)。为了区分这些假说，我的实验室比较了两种二型登革热病毒和八种流行的二型登革热病毒的全基因组序列，并在组成登革热病毒基因组的十个基因和两个非翻译区中的每一个确定了两组之间保守的遗传差异。接下来，将在人类肝脏(HH-7)细胞和蚊子(C6/36)细胞中测量四种二型登革热病毒和四种地方性二型登革热病毒的复制动力学和复制幅度，这代表了适应性的一个组成部分。如果假设1或2是正确的，那么在人类或蚊子细胞中，雅尔瓦尔病毒的复制水平应该分别显著低于地方性病毒，而两组之间的差异将支持假设3。显示低复制的雅尔瓦尔病毒将在该细胞类型上连续传代，以允许适应，然后对其进行测序，以确定可能与人类感染适应相关的候选突变。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sylvatic dengue viruses circulate in an unknown reservoir in the forest canopies of Africa and Asia and do not contribute to human dengue disease, which is caused by endemic dengue viruses that circulate only between humans and human-biting mosquitoes. Because the barriers that prevent sylvatic viruses have not been identified, the risk that sylvatic viruses pose for emergence of new dengue strains cannot be assessed. Three hypotheses to explain the absence of sylvatic viruses in humans include: 1. Sylvatic viruses are less fit in humans than endemic viruses 2. Sylvatic viruses are less fit in human-biting mosquitoes than endemic viruses 3. Sylvatic viruses and endemic viruses are equally fit in both humans and human-biting mosquitoes, but occupy separate spatial niches (the forest canopy versus the forest floor). To distinguish among these hypotheses, my lab has compared the whole genome sequence of two sylvatic dengue type 2 viruses with eight endemic dengue type 2 viruses and identified conserved genetic differences between the two groups in each of the ten genes and two untranslated regions that compose the dengue virus genome. Next the kinetics and magnitude of replication, which represents one component of fitness, of a panel of four sylvatic dengue type 2 viruses and four endemic dengue type 2 viruses will be measured in human liver (HuH-7) cells and mosquito (C6/36) cells. If hypotheses 1 or 2 is correct then sylvatic viruses should show significantly lower levels of replication than endemic viruses in human or mosquito cells, respectively, whereas a lack of difference between the two groups would support hypothesis 3. Sylvatic viruses showing low replication will be serially passaged on that cell type to allow for adaptation and then sequenced to identify candidate mutations that could be associated with adaptations for human infection.