Molecular determinants of alphavirus vector competence

人们对蚊媒虫媒病毒的传播和传播知之甚少，部分原因是很难可靠地定位病媒和宿主进行采样。蚊媒甲型病毒是世界各地各种人畜共患病的罪魁祸首。然而，目前尚不清楚是什么原因导致某些地区在特定时间发生疫情，以及是什么触发了疫情的出现。在这方面特别令人感兴趣的是蚊子物种对给定甲型病毒的媒介能力。媒介传播病毒的有效程度取决于环境因素，主要是温度，但作为媒介的一般能力被认为是由基因决定的。为了克服这些限制，虫媒病毒的基因改变将使其适应蚊子媒介，即感染中肠细胞或在蚊子体内更快和更有效地复制。最近的基孔肯雅病毒(CHIKV)大流行令人印象深刻地证明了这种突变可能造成的严重后果。CHIKV E1蛋白中的单一氨基酸交换导致了更好的白纹伊蚊传播率，导致在短短几年内在非洲和亚洲发生了数百万人感染病例。这项研究使用CHIKV作为模型系统，旨在定义决定媒介能力的遗传因素，并允许病毒跨越媒介物种甚至蚊子属。三种蚊子对CHIKV的敏感性从高(白纹伊蚊)到弱(Ae.Vexans)到耐火材料(淡色库蚊)将在实验室使用。这一设置将使我们能够调查调节每个蚊子媒介物种的传染性所需的病毒的基因变化。将使用三种独立的方法来强制和追踪相应的改变(S)：(I)在体外将病毒在弱/不敏感蚊子细胞中传递，(Ii)通过口服极高滴度的病毒溶液并强制胸腔接种，在体内通过弱/不敏感蚊媒物种传递病毒，从而绕过中肠感染屏障，以及(Iii)使用嵌合病毒。后者将通过交换具有感染性的CHIKV cDNA克隆中的片段来对抗辛德比斯病毒的相应区域来建立，辛德比斯病毒是另一种已知由CX传播的甲型病毒。皮皮恩斯。最后，利用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，将使用CHIKV cDNA克隆在蚊子中进行标记挽救实验。这些研究将确定允许CHIKV打破物种屏障从而允许这种虫媒病毒改变和/或扩大其媒介宿主范围的遗传要求。

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.