Investigating Virus-Host Interactions in Cultured Primary Honey Bee Cells.

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.

Metatranscriptome Analysis of Sympatric Bee Species Identifies Bee Virus Variants and a New Virus, Andrena-Associated Bee Virus-1.

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.

The Honey Bee Gene Bee Antiviral Protein-1 Is a Taxonomically Restricted Antiviral Immune Gene.

Insects have evolved a wide range of strategies to combat invading pathogens, including viruses. Genes that encode proteins involved in immune responses often evolve under positive selection due to their co-evolution with pathogens. Insect antiviral defense includes the RNA interference (RNAi) mechanism, which is triggered by recognition of non-self, virally produced, double-stranded RNAs. Indeed, insect RNAi genes (e.g., dicer and argonaute-2) are under high selective pressure. Honey bees (Apis mellifera) are eusocial insects that respond to viral infections via both sequence specific RNAi and a non-sequence specific dsRNA triggered pathway, which is less well-characterized. A transcriptome-level study of virus-infected and/or dsRNA-treated honey bees revealed increased expression of a novel antiviral gene, GenBank: MF116383, and in vivo experiments confirmed its antiviral function. Due to in silico annotation and sequence similarity, MF116383 was originally annotated as a probable cyclin-dependent serine/threonine-protein kinase. In this study, we confirmed that MF116383 limits virus infection, and carried out further bioinformatic and phylogenetic analyses to better characterize this important gene-which we renamed bee antiviral protein-1 (bap1). Phylogenetic analysis revealed that bap1 is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach) and that the majority of bap1 amino acids are evolving under neutral selection. This is in-line with the results from structural prediction tools that indicate Bap1 is a highly disordered protein, which likely has relaxed structural constraints. Assessment of honey bee gene expression using a weighted gene correlation network analysis revealed that bap1 expression was highly correlated with several immune genes-most notably argonaute-2. The coexpression of bap1 and argonaute-2 was confirmed in an independent dataset that accounted for the effect of virus abundance. Together, these data demonstrate that bap1 is a taxonomically restricted, rapidly evolving antiviral immune gene. Future work will determine the role of bap1 in limiting replication of other viruses and examine the signal cascade responsible for regulating the expression of bap1 and other honey bee antiviral defense genes, including coexpressed ago-2, and determine whether the virus limiting function of bap1 acts in parallel or in tandem with RNAi.

Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health.

Honey bees (Apis mellifera) are important pollinators of plants, including those that produce nut, fruit, and vegetable crops. Therefore, high annual losses of managed honey bee colonies in the United States and many other countries threaten global agriculture. Honey bee colony deaths have been associated with multiple abiotic and biotic factors, including pathogens, but the impact of virus infections on honey bee colony population size and survival are not well understood. To further investigate seasonal patterns of pathogen presence and abundance and the impact of viruses on honey bee colony health, commercially managed colonies involved in the 2016 California almond pollination event were monitored for one year. At each sample date, colony health and pathogen burden were assessed. Data from this 50-colony cohort study illustrate the dynamic nature of honey bee colony health and the temporal patterns of virus infection. Black queen cell virus, deformed wing virus, sacbrood virus, and the Lake Sinai viruses were the most readily detected viruses in honey bee samples obtained throughout the year. Analyses of virus prevalence and abundance revealed pathogen-specific trends including the overall increase in deformed wing virus abundance from summer to fall, while the levels of Lake Sinai virus 2 (LSV2) decreased over the same time period. Though virus prevalence and abundance varied in individual colonies, analyses of the overall trends reveal correlation with sample date. Total virus abundance increased from November 2015 (post-honey harvest) to the end of the almond pollination event in March 2016, which coincides with spring increase in colony population size. Peak total virus abundance occurred in late fall (August and October 2016), which correlated with the time period when the majority of colonies died. Honey bee colonies with larger populations harbored less LSV2 than weaker colonies with smaller populations, suggesting an inverse relationship between colony health and LSV2 abundance. Together, data from this and other longitudinal studies at the colony level are forming a better understanding of the impact of viruses on honey bee colony losses.

The Heat Shock Response in the Western Honey Bee (Apis mellifera) is Antiviral.

Honey bees (Apismellifera) 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.

Honey Bee and Bumble Bee Antiviral Defense.

Bees are important plant pollinators in both natural and agricultural ecosystems. Managed and wild bees have experienced high average annual colony losses, population declines, and local extinctions in many geographic regions. Multiple factors, including virus infections, impact bee health and longevity. The majority of bee-infecting viruses are positive-sense single-stranded RNA viruses. Bee-infecting viruses often cause asymptomatic infections but may also cause paralysis, deformity or death. The severity of infection is governed by bee host immune responses and influenced by additional biotic and abiotic factors. Herein, we highlight studies that have contributed to the current understanding of antiviral defense in bees, including the Western honey bee (Apis mellifera), the Eastern honey bee (Apis cerana) and bumble bee species (Bombus spp.). Bee antiviral defense mechanisms include RNA interference (RNAi), endocytosis, melanization, encapsulation, autophagy and conserved immune pathways including Jak/STAT (Janus kinase/signal transducer and activator of transcription), JNK (c-Jun N-terminal kinase), MAPK (mitogen-activated protein kinases) and the NF-κB mediated Toll and Imd (immune deficiency) pathways. Studies in Dipteran insects, including the model organism Drosophila melanogaster and pathogen-transmitting mosquitos, provide the framework for understanding bee antiviral defense. However, there are notable differences such as the more prominent role of a non-sequence specific, dsRNA-triggered, virus limiting response in honey bees and bumble bees. This virus-limiting response in bees is akin to pathways in a range of organisms including other invertebrates (i.e., oysters, shrimp and sand flies), as well as the mammalian interferon response. Current and future research aimed at elucidating bee antiviral defense mechanisms may lead to development of strategies that mitigate bee losses, while expanding our understanding of insect antiviral defense and the potential evolutionary relationship between sociality and immune function.

Recently identified bee viruses and their impact on bee pollinators.

Bees are agriculturally and ecologically important plant pollinators. Recent high annual losses of honey bee colonies, and reduced populations of native and wild bees in some geographic locations, may impact the availability of affordable food crops and the diversity and abundance of native and wild plant species. Multiple factors including viral infections affect pollinator health. The majority of well-characterized bee viruses are picorna-like RNA viruses, which may be maintained as covert infections or cause symptomatic infections or death. Next generation sequencing technologies have been utilized to identify additional bee-infecting viruses including the Lake Sinai viruses and Rhabdoviruses. In addition, sequence data is instrumental for defining specific viral strains and characterizing associated pathogenicity, such as the recent characterization of Deformed wing virus master variants (DWV-A, DWV-B, and DWV-C) and their impact on bee health.

Abiotic and biotic factors affecting the replication and pathogenicity of bee viruses.

Bees are important pollinators of plants in both agricultural and non-agricultural landscapes. Recent losses of both managed and wild bee species have negative impacts on crop production and ecosystem diversity. Therefore, in order to mitigate bee losses, it is important to identify the factors most responsible. Multiple factors including pathogens, agrochemical exposure, lack of quality forage, and reduced habitat affect bee health. Pathogen prevalence is one factor that has been associated with colony losses. Numerous pathogens infect bees including fungi, protists, bacteria, and viruses, the majority of which are RNA viruses including several that infect multiple bee species. RNA viruses readily infect bees, yet there is limited understanding of their impacts on bee health, particularly in the context of other stressors. Herein we review the influence environmental factors have on the replication and pathogenicity of bee viruses and identify research areas that require further investigation.

Honey bee colony losses and associated viruses.

Recent large-scale colony losses among managed Western honey bees (Apis mellifera) have alarmed researchers and apiculturists alike. Here, the existing correlative evidence provided by monitoring studies is reviewed which (i) identified members of the deformed wing virus and acute bee paralysis virus clades as lethal pathogens for entire colonies, and (ii) identified novel viruses whose impact on honey bee health remains elusive. Also discussed in this review is related evidence obtained via controlled experimental infection assays and RNAi approaches underscoring the damage inflicted by some of these viruses on individuals and colonies. The relevance of the ectoparasitic mite Varroa destructor acting as mechanical and biological virus vector for the enhanced virulence of certain viruses or mite selected virus strains is carefully considered.