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Using Barcoded Zika Virus to Assess virus Population Structure in vitro and in Aedes aegypti Mosquitoes

By James Weger-Lucarelli, Selene M Garcia, Claudia Rückert, Alex Byas, Shelby L. O’Connor, Matthew Aliota, Thomas C Friedrich, David H O'Connor, Gregory D Ebel

Posted 21 Nov 2017
bioRxiv DOI: 10.1101/222984 (published DOI: 10.1016/j.virol.2018.06.004)

Arboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) replicate in both mammalian and insect hosts where they encounter a variety of distinct host defenses. To overcome these pressures, arboviruses exist as diverse populations of distinct genomes. However, transmission between hosts and replication within hosts can involve genetic bottlenecks, during which population size and viral diversity may be significantly reduced, potentially resulting in large fitness losses. Understanding the points at which bottlenecks exist during arbovirus transmission is critical to identifying targets for preventing transmission. To study these bottleneck effects, we constructed 4 barcoded ZIKV clones - 2 with an 8-base-pair degenerate insertion in the 3 prime UTR and 2 with 8 or 9 degenerate synonymous changes in the coding sequence, theoretically containing thousands of variants each. We passaged these viruses 3 times each in 2 mammalian and 2 mosquito cell lines and characterized selection of the barcode populations using deep sequencing. Additionally, the viruses were used to feed three recently field-caught populations of Aedes aegypti mosquitoes to assess bottlenecks in a natural host. The barcoded viruses replicated well in multiple cell lines in vitro and in vivo in mosquitoes and could be characterized using next-generation sequencing. The stochastic nature of mosquito transmission was clearly shown by tracking individual barcodes in Ae. aegypti mosquitoes. Barcoded viruses provide an efficient method to examine bottlenecks during virus infection.

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