Molecular Detection and Phylogenetic Relationships of Honey Bee-Associated Viruses in Bee Products

Simple Summary One of the major threats to the health status of honey bee colonies is presented by honey bee-associated viruses. Therefore, the detection and identification of viruses in bee colonies is an important step in the fight against them. In this study, we used a novel and non-invasive approach-molecular analysis of environmental RNA as a tool for detection of six of the most widespread viruses in honey bee colonies in bee products (pollen, bee bread, and royal jelly) from different regions in Bulgaria. The obtained results showed successful detection and identification of the Deformed wing virus, the Acute bee paralysis virus, the Black queen cell virus, and the Israeli acute paralysis virus in all the investigated bee products. Phylogenetic analysis of the detected Bulgarian isolates was implemented to reveal the phylogenetic relationships with the highly similar worldwide strains available in the GenBank database. Ultimately, honey bee products represent a valuable source of eRNA, which allows the monitoring of virus infections in bee colonies at local, regional, and even national levels.Abstract In the last few years, the isolation and amplification of DNA or RNA from the environment (eDNA/eRNA) has proven to be an alternative and non-invasive approach for molecular identification of pathogens and pests in beekeeping. We have recently demonstrated that bee pollen and bee bread represent suitable biological material for the molecular identification of viral RNA. In the present study, we extracted total RNA from different bee products (pollen, n = 25; bee bread, n = 17; and royal jelly, n = 15). All the samples were tested for the presence of six of the most common honey bee-associated viruses-Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Chronic bee paralysis virus (CBPV), Sacbrood virus (SBV), Kashmir bee virus (KBV), and Black queen cell virus (BQCV)-using a reverse transcription polymerase chain reaction (RT-PCR). We successfully detected six records of DWV (10.5%, 6/57), four of ABPV (7.0%, 4/57), three of Israeli acute paralysis virus (IAPV) (5.3%, 3/57), and two of BQCV (3.5%, 2/57). Using ABPV primers, we also successfully detected the presence of IAPV. The obtained viral sequences were analyzed for phylogenetic relationships with the highly similar sequences (megablast) available in the GenBank database. The Bulgarian DWV isolates revealed a high homology level with strains from Syria and Turkey. Moreover, we successfully detected a DWV strain B for the first time in Bulgaria. In contrast to DWV, the ABPV isolates formed a separate clade in the phylogenetic tree. BQCV was closely grouped with Russian isolates, while Bulgarian IAPV formed its own clade and included a strain from China. In conclusion, the present study demonstrated that eRNA can be successfully used for molecular detection of honey bee-associated viruses in bee products. The method can assist the monitoring of the health status of honey bee colonies at the local, regional, and even national levels.