Byas, A. | bioRxiv Science

bioRxiv2023

Intracellular diversity of WNV within circulating avian peripheral blood mononuclear cells reveals host-dependent patterns of polyinfection

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Author SummaryWNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

microbiology↗

bioRxiv2017

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

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 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.\n\nAUTHOR SUMMARYIn general, mosquito-borne viruses like ZIKV must replicate in two very different host environments: an insect and a mammalian host. RNA viruses such as ZIKV must maintain genetic diversity in order to adapt to these changing conditions. During this transmission cycle, several barriers exist which can severely restrict viral genetic diversity, causing bottlenecks in the virus population. It is critical to understand these bottlenecks during virus transmission as this will provide important insights into the selective forces shaping arbovirus evolution within and between hots. Here, we employ a set of barcoded ZIKV constructs containing a degenerate stretch of nucleotides that can be tracked using next-generation sequencing. We found that the insertion site in the genome was an important determinant of the resulting diversity of the genetic barcode. We also found that bottlenecks varied between different mosquito populations and patterns of genetic diversity were distinct among individual mosquitoes within a single population, highlighting the randomness of virus dissemination in mosquitoes. Our study characterizes a new tool for tracking bottlenecks during virus transmission in vivo and highlights the importance of both viral and host factors on the maintenance of viral diversity.

molecular biology↗