. Identification of possible new viruses in Bumble bees by next-generation sequencing

The past decade has witnessed an increasing interest in studying bumble bee (Bombus spp.) populations due to their role in agricultural food production and natural ecosystems. Some studies showed that some diseases have been transmitted from commercially reared bumble bees to wild populations and led to noticeable population reduction in some species. Other studies demonstrated that RNA viruses can be transmitted to bumble bees by sharing diverse foraging sites with honey bees (Apis mellifera L.). A recent study showed that bumble bee infection in the wild does not occur by spillover from honey bees. High throughput sequencing (HTS) technologies can provide comprehensive insights into the bee virome, leading to a better understanding of the bee-pathogen interactions, including the viral diseases and their impacts. In the present study, we used HTS technologies to characterize the RNA virome from a pool of five forage bumble bees collected from garden spaces. Bumble bees were homogenized in TRIzol reagent (Thermo Fisher), followed by isopropanol precipitation. The total RNAs were purified by RNeasy Plant Mini Kit (QIAGEN) and treatment with DNAse (Promega). PolyA enrichment of total RNAs, Illumina TruSeq RNA library construction, followed by 2 × 100 NovaSeq sequencing were outsourced to Macrogen Inc. (Seoul, Korea). The obtained reads (36,919,326 reads with 44.531% GC contents) were checked for their quality by FastQC and assembled using metaSPAdes version 3.9.0 with the "only-assembler" parameter and multiple kmers (-k, 71, 81, and 91). A total of 55831 contigs were assembled and blasted against the NCBI viral sequences using Blast+ package (v. 2.9.0; Blastx with E-value < 1e-10). Bioinformatic analyses showed that 4.8% (2663 contigs) were of non-host origin, out of which 31 contigs showed significant similarity to viral sequences. The results demonstrated the occurrence of a number of viruses belonging to different genera, such as Partitiviridae, Secoviridae, Riboviridae, Hepeviridae, Dicistroviridae and other belonging to unclassified Riboviria realm. Most of the viruses found are common in invertebrates; others are common on vertebrates and/or plants. Furthermore, further investigations by RT-PCR confirmed the presence of some viruses infecting honey bees, like Black queen cell virus (Triatovirus) and also, the presence of Mayfield virus 1-like (unclassified Riboviria). The use of HTS technologies allowed discovering viruses that may pose threats to the bumble bees worldwide.