Experimental Evolution of an RNA Virus in Wild Birds: Evidence for Host-Dependent Impacts on Population Structure and Competitive Fitness

Abstract

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Author Summary

Viruses are constantly emerging into new areas and pose significant challenges to public health. Chikungunya and West Nile viruses (WNV), both mosquito-borne RNA viruses, are quintessential examples of how increased globalization has facilitated the expansion of viruses into new territories. Rapid evolution of both of these agents has contributed to their rapid spread and health burden. Thus, characterizing how selection shapes zoonotic RNA viruses in their natural hosts is important to understand their emergence. As an ecological generalist able to infect hundreds of bird species, WNV is an excellent tool to study how different animal hosts can differentially drive virus evolution. We examined the genetic composition and fitness of WNV produced during replication in wild-caught American crows, house sparrows and American robins, species that range in mortality following WNV infection (crows the highest, robins the lowest). We demonstrate host-dependent effects on WNV population structure and fitness. Our study provides insights on how different virus-animal interactions can influence the success of a virus in the next host and ultimately the success of virus emergence into new host systems.