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在传播和进化中的重要性。病毒不是以无性繁殖群体的形式存在，而是以基因组突变的变异“群”的形式存在。虫媒病毒（节肢动物传播的病毒）的变异尚未被表征，这种病毒需要感染脊椎动物宿主和媒介（如蚊子或蜱虫）。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，从而对病毒施加了瓶颈。据推测，在感染蚊子载体后，变异的数量减少，导致虫媒病毒的进化速度比单一宿主的病毒慢。进化的速度对于理解病毒生态和出现是很重要的。将使用委内瑞拉马脑炎病毒（VEEV）和两种天然蚊子媒介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，目前还没有获得许可的疫苗或治疗方法。该病毒在墨西哥传播，并在美国引起了马科动物和人类的爆发。病毒多样性将通过三个具体目标进行调查。首先，将与已经确定的地方性病媒窄体库蚊的瓶颈情况进行比较，确定流行病媒窄体棘孔虫的瓶颈情况。其次，根据蚊媒带喙伊蚊和带喙伊蚊与适当的啮齿动物宿主的感染情况，以及作为传播周期的一部分，将确定VEEV亚型IE和IC的宿主内变异概况。将使用已建立的方案来启动一个接近自然发生的实验室传播周期，研究蚊子解剖瓶颈对病毒多样性的影响。

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.