Location: Bee Research Laboratory
Project Number: 8042-21000-291-034-I
Project Type: Interagency Reimbursable Agreement
Start Date: Oct 1, 2020
End Date: Dec 31, 2024
Objective:
The goal of this three-year project is to improve the health of the European honey bee Apis mellifera (hereafter, Am) by providing practical tools and resources to the US 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 (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 general aim of the three-year project is to contribute innovative tools and resources for advancing research of honey bee viruses and for advising beekeepers of best management practices for combating Vd mites and the harmful viruses they vector. However, before effective tools and accurate resources are developed, the model of Vd vectoring of DWV-like viruses must first be refined. 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.
Approach:
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 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.
For the Vd mite passage assay, phoretic Vd mites are passage from infected pupae to naïve pupae and then tested for stable infections, determined using RT-qPCR at >109 GE. For infectivity assay, mites will be maintained on naïve Am pupae for 48 hr. to transmit the viruses.
All colonies are housed in 10-frame hive boxes and are monitored for general health and mite levels. Colonies as sources of naïve pupae and adult bees for experiments are treated with Apivar (amitraz). Colonies as sources for harvesting mites are kept at a separate apiary that is dedicated as a ‘mite farm’. All colonies are fed 50% sugar water as needed (natural pollen is abundant all season).
Total RNA will be extracted from individual bees using Trizol, treated with RNAse free DNAse and subjected to purification using RNeasy RNA purification columns. The purified DNAse RNA treated will be to generate cDNA using SuperscriptIII 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, which will be carried out in University of Maryland, Baltimore Institute for Genome Sciences. Analysis of Illumina HiSeq2500 reads (paired-end 150 nt, at least 10-20 million per library) will be performed.
