Honey bees (Apis mellifera) are an agriculturally important pollinator species that live in easily managed social groups (i.e., colonies). Unfortunately, annual losses of honey bee colonies in many parts of the world have reached unsustainable levels. Multiple abiotic and biotic stressors, including viruses, are associated with individual honey bee and colony mortality. Honey bees have evolved several antiviral defense mechanisms including conserved immune pathways (e.g., Toll, Imd, JAK/STAT) and dsRNA-triggered responses including RNA interference and a non-sequence specific dsRNA-mediated response. In addition, transcriptome analyses of virus-infected honey bees implicate an antiviral role of stress response pathways, including the heat shock response. Herein, we demonstrate that the heat shock response is antiviral in honey bees. Specifically, heat-shocked honey bees (i.e., 42 °C for 4 h) had reduced levels of the model virus, Sindbis-GFP, compared with bees maintained at a constant temperature. Virus-infection and/or heat shock resulted in differential expression of six heat shock protein encoding genes and three immune genes, many of which are positively correlated. The heat shock protein encoding and immune gene transcriptional responses observed in virus-infected bees were not completely recapitulated by administration of double stranded RNA (dsRNA), a virus-associated molecular pattern, indicating that additional virus–host interactions are involved in triggering antiviral stress response pathways.

Bees are important plant pollinators in agricultural and natural ecosystems. High average annual losses of honey bee (Apis mellifera) colonies in some parts of the world, and regional population declines of some mining bee species (Andrena spp.), are attributed to multiple factors including habitat loss, lack of quality forage, insecticide exposure, and pathogens, including viruses. While research has primarily focused on viruses in honey bees, many of these viruses have a broad host range. It is therefore important to apply a community level approach in studying the epidemiology of bee viruses. We utilized high-throughput sequencing to evaluate viral diversity and viral sharing in sympatric, co-foraging bees in the context of habitat type. Variants of four common viruses (i.e., black queen cell virus, deformed wing virus, Lake Sinai virus 2, and Lake Sinai virus NE) were identified in honey bee and mining bee samples, and the high degree of nucleotide identity in the virus consensus sequences obtained from both taxa indicates virus sharing. We discovered a unique bipartite + ssRNA Tombo-like virus, Andrena-associated bee virus-1 (AnBV-1). AnBV-1 infects mining bees, honey bees, and primary honey bee pupal cells maintained in culture. AnBV-1 prevalence and abundance was greater in mining bees than in honey bees. Statistical modeling that examined the roles of ecological factors, including floral diversity and abundance, indicated that AnBV-1 infection prevalence in honey bees was greater in habitats with low floral diversity and abundance, and that interspecific virus transmission is strongly modulated by the floral community in the habitat. These results suggest that land management strategies that aim to enhance floral diversity and abundance may reduce AnBV-1 spread between co-foraging bees.

Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap.

Honey bee colony losses are influenced by multiple abiotic and biotic factors, including viruses. To investigate the effects of RNA viruses on honey bees, we infected bees with a model virus (Sindbis-GFP) in the presence or absence of double-stranded RNA (dsRNA). In honey bees, dsRNA is the substrate for sequence-specific RNA interference (RNAi)-mediated antiviral defense and is a trigger of sequence-independent\antiviral responses. Transcriptome sequencing identified more than 200 differentially expressed genes, including genes in the RNAi, Toll, Imd, JAK-STAT, and heat shock response pathways, and many uncharacterized genes. To confirm the virus limiting role of two genes (i.e., dicer and mf116383) in honey bees, we utilized RNAi to reduce their expression in vivo and determined that the virus abundance increased. To evaluate the role of the heat shock stress response in antiviral defense, bees were heat stressed post-virus infection and the virus abundance and gene expression were assessed. Heat-stressed bees had reduced virus levels and a greater expression of several heat shock protein encoding genes (hsps) compared to the controls. To determine if these genes are universally associated with antiviral defense, bees were infected with another model virus, Flock House virus (FHV), or deformed wing virus and the gene expression was assessed. The expression of dicer was greater in bees infected with either FHV or Sindbis-GFP compared to the mock-infected bees, but not in the deformed wing virus-infected bees. To further investigate honey bee antiviral defense mechanisms and elucidate the function of key genes (dicer, ago-2, mf116383, and hsps) at the cellular level, primary honey bee larval hemocytes were transfected with dsRNA or infected with the Lake Sinai virus 2 (LSV2). These studies indicate that mf116383 and hsps mediate dsRNA detection and that MF116383 is involved in limiting LSV2 infection. Together, these results further our understanding of honey bee antiviral defense, particularly dsRNA-mediated antiviral responses, at both the individual bee and cellular levels.