Location: Bee Research Laboratory
Project Number: 8042-21000-291-055-A
Project Type: Cooperative Agreement
Start Date: Feb 13, 2022
End Date: Sep 30, 2023
The goal of the CA is to improve the health of the European honey bee, Apis mellifera (hereafter, Am) by providing practical tools and resources to the U.S. beekeeping industry for combating the ectoparasitic mite, Varroa destructor (hereafter, Vd) and the harmful viruses they vector. The applied and basic research proposed in this project will, respectively, evaluate and expand strategies employed by beekeepers to control Vd mites; and design and test an innovative, cDNA clone-based virus vector that targets Vd mites. The project will focus on Deformed wing virus (DWV) and related viruses, specifically Varroa destructor virus-1 (VDV1) (together, DWV-like viruses), which are principal viruses affecting Am health worldwide. The accuracy of evaluations and tests, and the efficacy of designs, and control strategies, all depend on a refined model of Vd vectoring and transmission of DWV-like viruses, and the effects of different transmission routes on virus diversity and virulence in Am honey bees. Development of this model requires filling knowledge gaps related to the vectoring of DWV-like viruses by Vd and viral dynamics in both mites and bees. The project will both fill gaps in the model and guide our work for making more effective tools and accurate resources by addressing the following 3 objectives: 1. Use genetically tagged, cDNA-derived DWV type-A and Varroa destructor virus-1 (VDV1) to unravel key aspects of Vd-mediated vectoring and transmission of DWV-like viruses to honey bees; 2.Determine how changes in the occurrence of Vd-mediated transmission affect the population dynamics of DWV-like viruses in Am workers and colonies; and 3. Assess the utility of a novel DWV cDNA clone-based vectors for RNAi-based control of Vd mites.
1. Use genetically tagged, cDNA-derived DWV type-A and Varroa destructor virus-1 (VDV1) to unravel key aspects of Vd-mediated vectoring and transmission of DWV-like viruses to honey bees; The cDNA will modify the cDNA clone of the U.S. VDV1 with proven infectivity in honey bees, and those for U.S, VDV1-DWV recombinants (to be designed) will be modified by inserting the GFP-coding sequence into viral cDNA. The foreign (GFP) insert will be flanked by the sequences coding for the proteolytic cleavage peptides targeted by the DWV 3C protease. The full-length cDNA plasmid constructs will be linearized using unique restriction site located at the cDNA 3’ (PmeI for DWV-A and VDV1) and will be used as templates generate in vitro RNA transcripts using T7 RNA polymerase. Total RNA will be extracted from individual bees treated with RNAse free DNAse and subjected to purification using RNA purification columns. The purified DNAse treated RNA will be used to generate cDNA using reverse transcriptase, random (N6) primer will be used for total amplification, and negative strand specific tagged primers will be used for negative viral RNA strand detection. Replication of the mite-transmitted viruses in the pupae will be tested using DWV or VDV1-specific or GFP-tag-specific RT-qPCR for the number of genomic equivalents (GE) and the negative RNA strand. The clonal identity of DWV and VDV1 will be confirmed by sequencing PCR products or by restriction enzyme digestion of the RT-PCR products corresponding to the 5' untranslated regions of clone-derived viral RNA, which contain introduced restrictions sites. Selected total RNA extracts from honey bees will be used for RNA-seq. Analysis of Illumina HiSeq2500 reads (paired-end 150 nt, at least 10-20 million per library) will be performed. 2. Determine how changes in the occurrence of Vd-mediated transmission affect the population dynamics of DWV-like viruses in Am workers. Here, we will use laboratory experiments that test how restricted access to brood and different levels of Vd mites affect the population dynamics of DWV in honey bees and virulence of DWV strains in honey bees. In the first study, we will investigate how restricting access of Vd mites to pupae affects the population dynamics of DWV in Am workers exposed to low or high levels of highly Vd mites, and their subsequent infectivity to naïve pupae. Reproductive-stage Vd mites usually acquire high levels DWV-like viruses from host pupae. 3. Assess the utility of a novel DWV cDNA clone-based vectors for RNAi-based control of Vd mites. This will be the first ever attempt to test the suitability of novel cDNA clone-based virus vector designed to deliver dsRNA for controlling Vd mites (Fig.5). Delivery of dsRNA targeting Vd mites in bees was recently achieved in a by expressing Vd-targeting dsRNA in the cells of bacteria which colonizes honey bee guts (Leonard et al., 2020). We will construct cDNA DWV-A vectors with inserted genetic material that disrupts via RNAi key aspects of Vd mite physiology. The viruses and the genetic material will replicate in honey bee cells, which Vd mites consume.