West Nile Virus Genetic Diversity is Maintained during Transmission by Culex pipiens quinquefasciatus Mosquitoes Doug E. Brackney, Kendra N. Pesko, Ivy K. Brown, Eleanor R. Deardorff, Jon Kawatachi, Gregory D. Ebel* Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America Abstract Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes. Citation: Brackney DE, Pesko KN, Brown IK, Deardorff ER, Kawatachi J, et al. (2011) West Nile Virus Genetic Diversity is Maintained during Transmission by Culex pipiens quinquefasciatus Mosquitoes. PLoS ONE 6(9): e24466. doi:10.1371/journal.pone.0024466 Editor: Patricia V. Aguilar, University of Texas Medical Branch, United States of America Received May 13, 2011; Accepted August 10, 2011; Published September 12, 2011 Copyright: 2011 Brackney et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the National Institute of Allergy and Infectious Disease, National Institutes of Health (NIH) under grant AI067380. DEB was supported by 5F32AI084432-02. ERD was supported by Institutional Research and Academic Career Development Awards via Award K12GM088021 from the National Institute of General Medical Sciences and the University of New Mexico Academic Science Education and Research Training fellowship. KP was supported by National Research Service Award Institutional Training Grant 5T32-AI07538-13. IKB and JK were supported by the University of New Mexico Initiative to Maximize Student Diversity, which is funded by the National Institute of General Medical Sciences, NIH under grant GM060201. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gebel@salud.unm.edu Introduction West Nile virus (WNV; Flaviviridae, Flavivirus) was introduced into North America in 1999 and has since spread across the continental United States and into Canada, Mexico, the Carribean, and South America [1]. Molecular epidemiologic studies of WNV in the US revealed that minor changes at the genomic level were associated with a dramatic shift in the genotypic composition of WNV circulating in North America [2–6]. Specifically, the introduced genotype, termed NY99, was displaced by a new variant, WN02. The WN02 genotype differs from NY99 by only a few nucleotide and/or amino acid changes, but is more efficiently transmitted by native Culex mosquitoes [5,7,8]. It was determined that the WN02 genotype requires a shorter extrinsic incubation period in mosquitoes (EIP, time from vector infection to transmission) thereby resulting in an increased vectorial capacity of local mosquitoes. Similarly, the emergence of Chikungunya virus (CHIKV; Togavir- idae, Alphavirus) seems to have been facilitated by analogous mutations that result in increased transmission efficiency by the vector Aedes albopictus [9,10]. Thus, relatively minor consensus genetic changes can significantly influence arbovirus transmission patterns and disease emergence. Determining the mechanistic underpinnings of genetic change in arboviruses is therefore critical to understanding their persistence and emergence. RNA viruses exist within hosts as a dynamic distribution of non- identical, but related viral variants [11–14]. High genetic diversity profoundly influences the population biology of RNA viruses, including WNV, polio, mumps and hepatitis C viruses [15–18]. In the case of WNV, high genetic diversity is associated with increased fitness in mosquitoes [19]. Population bottlenecks may reduce fitness by stochastically reducing the genetic diversity of the virus population. In vitro studies of vesicular stomatitis virus, an RNA virus, have demonstrated that repeated bottlenecks can lead to fitness loss through the action of Muller’s ratchet [20]. The extent to which mosquitoes impose such population bottlenecks on arthropod-borne viruses (arboviruses) is unclear. Analysis of WNV populations from naturally infected birds revealed that non- consensus, minority genotypes were shared among samples collected from multiple birds, suggesting that WNV populations may not be subject to bottlenecks during the natural transmission cycle [14]. Similarly, it was suggested that dengue virus type 1 (DENV1; Flaviviridae, Flavivirus) is not subject to widespread population bottlenecks during the natural transmission cycle because putatively defective genomes persist through complemen- tation, requiring frequent coinfection of cells in both mosquitoes and humans [21]. Similarly, a high frequency of coinfection of midgut cells has been reported for Venezuelan equine encephalitis virus (VEEV; Togaviridae, Alphavirus) in Aedes taeniorhynchus [22]. PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24466