Effects of Arboviral Population Diversity on Transmission

描述（由申请人提供）：本研究计划着眼于病毒多样性对传播和进化的重要性。病毒不是以克隆种群的形式存在，而是以基因组沿着发生突变的变异体“成群”的形式存在。虫媒病毒（节肢动物传播的病毒）的变异尚未得到表征，虫媒病毒需要感染脊椎动物宿主和载体（如蚊子或蜱）。在蚊子的感染过程中，有几个解剖屏障减少了病毒颗粒的数量，因此对病毒施加了瓶颈。假设在蚊子载体感染后，变异量减少，导致虫媒病毒与具有单一宿主的病毒相比进化速率降低。进化的速度对于理解病毒生态学和出现是很重要的。委内瑞拉马脑炎病毒（VEEV）和两种天然蚊子载体将用于模拟野生型传播中存在的病毒变异，以及蚊子感染对病毒多样性的影响。VEEV之所以令人感兴趣，是因为它既是一种重要的自然出现的病毒，也是一种潜在的生物武器，没有许可的疫苗或治疗方法。该病毒在墨西哥传播，并在美国的马和人类中引起疫情。将使用3个特定目标调查病毒多样性。首先，将确定流行病媒介Psorophora confinnis的瓶颈概况与已经确定的地方病媒介带尾库蚊的瓶颈概况进行比较。其次，将确定蚊子载体C感染后VEEV亚型IE和IC的宿主内变异概况。带喙伊蚊（Aedes taeniorhynchus）和适当的啮齿类宿主，单独地和作为传播周期的一部分。蚊子解剖学瓶颈对病毒多样性的影响将使用既定的协议进行调查，以启动一个接近自然发生的实验室传播周期。 将使用下一代测序技术确定多样性水平，以确定整个传输周期中存在的少数群体。由于随机效应，多样性的这种减少可能导致适应度的降低。将通过一个实验传输周期来测试由这种多样性降低引起的B漂移水平。第三，当通过遗传操作增加聚合酶保真度时，将检查病毒完成传播周期的能力的变化。据推测，病毒变异有助于 因此，突变为在复制中表现出高保真度的病毒将用于实验感染。预计病毒存活率将降低。该项目的结果将增强我们对蚊子媒介中发生的虫媒病毒种群遗传动力学的理解，并确定自然选择和遗传漂变影响新虫媒病毒持续存在和出现的程度。这些结果将转化为其他新兴的虫媒病毒，如登革热，西尼罗河和基孔肯雅病毒，并提供数据以生成虫媒病毒出现的有效模型。

DESCRIPTION (provided by applicant): This research proposal looks at the importance of viral diversity on transmission and evolution. Viruses exist not as clonal populations but as 'swarms' of variants with mutations along the genome. Variation has not been characterised in arboviruses (arthropod-borne viruses), which require infection of a vertebrate host and a vector (such as a mosquito or tick). During infection of the mosquito there are several anatomical barriers that reduce the number of viral particles, thus a bottleneck is imposed on the virus. It i hypothesised that, upon infection of the mosquito vector, the amount of variation decreases, leading to a decreased rate of evolution in the arboviruses as compared to viruses with a single host. The rate of evolution is important to understand viral ecology and emergence. Venezuelan equine encephalitis virus (VEEV) and two natural mosquito vectors will be used to model viral variation present in wild-type transmission, and the impact that mosquito infection has on viral diversity. VEEV is of interest because it is both an important, naturally emerging virus and a potential bioweapon with no licensed vaccine or therapy. The virus circulates in Mexico and has caused outbreaks in equids and humans in the U.S. Viral diversity will be investigated using 3 specific aims. First, the bottleneck profile of the epidemic vector Psorophora confinnis will be determined compared to the bottleneck profile already determined for the enzootic vector Culex taeniopus. Secondly, the profile of intra-host variation of VEEV subtypes IE and IC will be determined upon infection of the mosquito vectors C. taeniopus and Aedes taeniorhynchus and an appropriate rodent host, individually and as part of a transmission cycle. The effect of the anatomical mosquito bottlenecks on viral diversity will be investigated using an established protocol to initiate a laboratory transmission cycle that approximates to that occurring naturally. The levels of diversity will be determined using Next Generation Sequencing technology to determine the minority populations present throughout the transmission cycle. This decrease in diversity may lead to a reduction in fitness due to stochastic effects. The level of drift caused b this decrease in diversity will be tested via an experimental transmission cycle. Thirdly, the change in the ability of the virus to complete a transmission cycle when the polymerase fidelity is increased by genetic manipulation will be examined. It is hypothesized that viral variation aids in viral survival therefore viruses mutated to exhibit high-fidelity in their replication will be ued in experimental infections. It is expected that viral survival will decrease. The results of this projct will enhance our understanding of arboviral population genetic dynamics occurring in the mosquito vector as well as determine the degree to which natural selection and genetic drift influence the persistence and emergence of new arboviruses. The results will translate to other emerging arboviruses such as dengue, West Nile and chikungunya, as well as provide the data to generate effective models of arbovirus emergence.