Dynamic evolution in the key honey bee pathogen deformed wing virus: Novel insights into virulence and competition using reverse genetics

Abstract

The 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 honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and 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 coexisting DWV genotypes, thereby devising a reverse-genetics 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 coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.

The honeybee (Apis mellifera) developmental state shapes the genetic composition of the deformed wing virus-A quasispecies during serial transmission

“…Like others before us, we also could not distinguish DWV quasispecies from symptomatic and asymptomatic adult bees 24 and found the DWV quasispecies dynamics to be dominated by rapid selective processes 20,22,33 .…”

“…This is a natural foundation for the appearance of multiple strains adapted to different contexts. Since there is little difference between DWV-A and DWV-B in pathogenicity 47,49 , titres 20,33,47,49 or adult mortality 22 , the relative persistence of DWV-A, DWV-B and/or their recombinants in bee colonies may well be driven largely by pupal mortality and the effectiveness of social hygiene 50 , where the strains differ most.…”

“…Of course, these two alternatives are not mutually exclusive. It has been conclusively shown that DWV is directly responsible for the deformed wing symptoms 25,29,30 ; that these symptoms are directly related to both the amount of virus and the transmission route 14,22,24,25 , and that the different major DWV variants (and especially their recombinants) have different replicative capacities in both honeybees and V. destructor 20,21,[31][32][33] , as well as different relationships with honeybee pathology at individual bee 24,34 and colony level 20,21,31,35 . What is less well known is how the virus adapts genetically during these transitions and transmissions.…”

Co-incubation of dsRNA reduces proportion of viable spores of Ascosphaera apis, a honey bee fungal pathogen

“…Additionally, there is always an evolutionary arms race between combating the pathogen and escape of the pathogen from any selective pressure. For example, DWV appears to infect or replicate as a recombinant pool of genotypes, thus the current RNAi methods need to keep pace with the varying sequences of DWV (namely, researchers must be concerned that the chosen dsRNA sequence will lose efficacy as the complementary viral sequence changes, as already observed in bees (Ryabov et al, 2019) ). Therefore, a combination of intelligently-designed, diverse and synergistic honey bee disease strategies from apicultural management practices to scientific advancements, will eventually and undoubtedly lead to improved honey bee-dependent agriculture.…”

Cell Lines for Honey Bee Virus Research

“…A honey bee cell line would allow for in depth study of virus-host molecular interactions. This will be facilitated in particular by the establishment of infectious clones of honey bee viruses such as those of DWV [101,102], that allow for reverse genetic analysis of gene function. Mechanisms of virus binding and entry into the cell, replication, encapsidation and release from the cell along with host cell antiviral response could be delineated by use of a honey bee cell line.…”