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.

Genetic Drift during Systemic Arbovirus Infection of Mosquito Vectors Leads to Decreased Relative Fitness during Host Switching

“…In addition, these data, combined with previously reported data on intrahost WNV populations in various avian species (Grubaugh et al, 2015), demonstrate that purifying selection is weaker in mosquitoes compared to birds.…”

“…new genotype linked to a shorter extrinsic incubation period in local mosquitoes), promoting its spread throughout the Americas (Moudy et al, 2007). Several studies have assessed how different host types impact WNV population structure, and have shown that WNV populations are more diverse in mosquitoes compared to birds (Grubaugh et al, 2015; Jerzak et al, 2005; Jerzak et al, 2007). In mosquitoes, purifying selection is weak and virus diversification is driven by the action of RNA interference (RNAi), which creates an intracellular milieu that favors rare genotypes (Brackney et al, 2009; Brackney et al, 2015).…”

“…WNV evolutionary dynamics have been shown to vary in response to environmental conditions (Bertolotti et al, 2008). Indeed, different avian hosts of WNV have distinct impacts on virus population structure and fitness (Grubaugh et al, 2015). The impacts of different mosquito species on WNV population biology and fitness, however, have not been directly addressed.…”

Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling

“…True variants are identified as those with frequencies exceeding the expected error rate according to some predetermined statistical test. Despite being employed in many NGSbased studies of viral diversity (3,4,12,35,36), few of these algorithms have been benchmarked using defined viral populations. To our knowledge, none have been tested under conditions that mimic those found in patient-derived samples.…”

Isolation of a Novel Insect-Specific Flavivirus fromCuliseta melanurain the Northeastern United States

“…Total RNA (15 lL) from virus-infected C6/36 cell culture supernatants (virus isolate 502-13) were prepared for whole genome sequencing, as previously described (Grubaugh et al 2015). In brief, the samples were treated with DNase, amplified using the Ovation RNA-Seq System V2 (NuGEN, San Carlos, CA), sheared using the Covaris S2 Focusedultrasonicator (Covaris, Woburn, MA), and prepared using the Ovation Ultralow Library Kit (NuGEN, San Carlos, CA).…”