Effects of Arboviral Population Diversity on Transmission

描述(由申请人提供)：这项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，而是以基因组上带有突变的变异体的“群”的形式存在。虫媒病毒(节肢动物传播的病毒)还没有表现出变异的特征，这种病毒需要脊椎动物宿主和媒介(如蚊子或扁虱)的感染。在感染蚊子的过程中，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。我假设，在感染蚊子媒介后，变异量减少，导致虫媒病毒的进化速度比只有一个宿主的病毒慢。进化的速度对于了解病毒的生态和出现是很重要的。委内瑞拉马脑炎病毒(VEEV)和两种天然蚊子媒介将被用来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之所以引起人们的兴趣，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，没有获得许可的疫苗或治疗方法。该病毒在墨西哥传播，并在美国的马类和人类中引发疫情。病毒多样性将通过3个特定目标进行调查。首先，将与已确定的流行病媒介库蚊的瓶颈图谱进行比较，以确定流行媒介狭纹夜蛾的瓶颈图谱。其次，VEEV IE和IC亚型的宿主内变异情况将在蚊媒带喙库蚊和带喙伊蚊以及适当的啮齿动物宿主感染后确定，作为传播周期的一部分。将使用一种既定的方案来调查解剖蚊子瓶颈对病毒多样性的影响，以启动与自然发生的传播周期相似的实验室传播周期。 将使用下一代测序技术确定多样性水平，以确定在整个传播周期中存在的少数群体。这种多样性的减少可能会由于随机效应而导致适应度的降低。将通过实验传输周期来测试这种分集降低所引起的漂移水平。第三，当通过基因操作提高聚合酶的保真度时，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，在病毒存活过程中，突变的病毒在复制过程中表现出高保真，将用于实验感染。预计病毒存活率将会下降。该项目的结果将加深我们对蚊媒中虫媒病毒种群遗传动态的了解，并确定自然选择和遗传漂移对新虫媒病毒的持续和出现的影响程度。这些结果将转化为其他新出现的虫媒病毒，如登革热、西尼罗河和基孔肯雅病毒，并为产生有效的虫媒病毒出现模型提供数据。

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.