AbstractThe impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States (US) honey bee colonies. Phylogenetic analysis showed that the divergent US DWV genotypes are of monophyletic origin, which were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to co-existing DWV genotypes, thereby devising a reverse genetic system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to co-exist. Mutational and recombination events were observed across clone progeny providing new insights into the forces that drive and constrain virus diversification. Accordingly, herein we propose a new model of Varroa-induced DWV dynamics whereby an initial selective sweep is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. This selection likely reflects the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.Author SummaryVirulence of Deformed wing virus (DWV), a major pathogen of honey bees, showed a sharp and significant increase following the introduction of its vector, the mite Varroa destructor. Varroa vectoring resulted in genetic changes of DWV, including reduction of DWV diversity to nearly clonal levels in the UK and Hawaii. Contrary to the previous reports, we discovered that virulent DWV populations circulating across the Varroa-infested United States included many divergent genotypes generated following a strong bottleneck event. We designed a series of the full-length infectious cDNA clones that captured the diversity of a typical virulent DWV population from a declining Varroa-infested colony, effectively establishing first reverse genetic system for an invertebrate RNA virus quasispecies, in order to investigate interactions between the virus genotypes. We demonstrated that individual co-existing DWV genotypes and diverse natural DWV populations replicated equally well indicating that complementation between isolates was not required to enable DWV replication to high levels. Also, no obvious competitive exclusion was detected between genotypes in mixed infections suggesting DWV genotypes are adapted to co-exist to maintain overall population diversity. We suggest that introduction of Varroa resulted in an initial selective sweep of DWV diversity which was followed by DWV diversification driven by selection for new genotypes capable of evading host defenses, specifically antiviral RNA interference.
Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants
