Molecular determinants of alphavirus vector competence

对蚊媒虫媒病毒的传播和扩散知之甚少，部分原因是难以可靠地定位病媒和宿主进行采样。蚊子传播的甲病毒是世界各地各种人畜共患病的原因。然而，尚不清楚是什么原因导致某些地区在特定时间发生流行病，以及是什么触发了这些流行病的出现。在这种情况下，特别感兴趣的是蚊子物种对给定甲病毒的载体能力。媒介传播病毒的有效性取决于环境因素，主要是温度，但一般认为作为媒介的能力是由基因决定的。为了克服这些限制，虫媒病毒的遗传改变将允许适应蚊子载体，即感染中肠细胞或在蚊子内更快和更有效地复制。这种突变可能导致的严重后果，最近的基孔肯雅病毒（CHIKV）大流行令人印象深刻。CHIKV E1蛋白中的单个氨基酸交换导致白纹伊蚊中更好的传播率，导致仅在几年内非洲和亚洲就有数百万人感染病例。这项研究使用CHIKV作为模型系统，旨在定义决定载体能力的遗传元件，并允许病毒跨越载体物种甚至蚊子属。三种蚊子物种对CHIKV的易感性从高（白纹伊蚊）到弱（Ae.实验室中将使用从致倦库蚊（Culex vexans）到难治库蚊（Culex pipiens）。这种设置将使我们能够研究调节每种蚊子媒介物种感染性所需的病毒遗传变化。将使用三种独立的方法来强制和追踪相应的改变：（i）在体外将病毒在弱/非易感蚊子细胞中传代，（ii）通过使用非常高滴度的病毒溶液经口喂养并强制胸内接种，从而绕过中肠感染屏障，在体内将病毒在弱/非易感蚊子载体物种中传代，以及（iii）使用嵌合病毒。后者将通过将感染性CHIKV cDNA克隆内的片段与辛德毕斯病毒的相应区域交换来建立，辛德毕斯病毒是已知由Cx传播的另一种甲病毒。pipiens。最后，将使用CHIKV cDNA克隆在蚊子中进行具有在昆虫细胞中连续传代后获得的适应性突变和/或在蚊子中传代后获得的突变的标记拯救实验。这些研究将确定允许CHIKV打破物种屏障的遗传要求，从而允许这种虫媒病毒改变和/或扩展其载体宿主范围。

The transmission and spread of mosquito-borne arboviruses is poorly understood, in part because of the difficulty in reliably locating both vectors and hosts for sampling. Mosquito-borne alphaviruses are responsible for a variety of zoonotic illnesses throughout the world. However it is not known what causes epidemics to occur in some areas at particular times and what triggers their emergence. Of particular interest in this context is the vector competence of a mosquito species to a given alphavirus. How effective a vector can transmit a virus depends on environmental factors, mostly the temperature, but the general ability to serve as a vector is thought to be genetically defined. To overcome these restrictions, genetic alterations of an arbovirus will allow adaptation to the mosquito vector, i.e. infection of the midgut cells or faster and more efficient replication within the mosquito. The severe consequences such mutations can cause, was impressively demonstrated by the recent pandemic of Chikungunya virus (CHIKV). A single amino acid exchange in the CHIKV E1 protein resulted in better dissemination rates in Aedes albopictus leading to millions of human cases in Africa and Asia in only a few years. This study is using CHIKV as a model system, aiming to define genetic elements that determine vector competence and allow a virus to cross vector species or even mosquito genera. Three mosquito species differing in their susceptibility for CHIKV from high (Aedes albopictus) to weak (Ae. vexans) to refractory (Culex pipiens) will be used in the laboratory. This setup will allow us to investigate the genetic changes of the virus needed to modulate infectivity for each mosquito vector species. Three independent approaches will be used to force and trace the corresponding alteration(s): (i) passaging the virus in weak-/non-susceptible mosquito cells in vitro, (ii) passaging the virus in weak-/non-susceptible mosquito vector species in vivo via oral feeding using a very high titred virus solution and forced intrathoracic inoculation, thereby circumventing the midgut infection barrier, and (iii) using chimeric viruses. The latter will be established by exchanging fragments within an infectious CHIKV cDNA clone against the corresponding regions of Sindbis virus, another alphavirus known to be transmitted by Cx. pipiens. Finally, marker rescue experiments with adaptive mutations obtained after serial passage in the insect cells and/or mutations obtained after passage in the mosquitoes will be performed in the mosquitoes using the CHIKV cDNA clone. These studies will determine the genetic requirements that allow CHIKV to break the species barrier thereby allowing this arbovirus to change and/or extend its vector host range.