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. 2023 Feb 10;10(2):140.
doi: 10.3390/vetsci10020140.

Molecular Detection and Phylogenetic Analysis of Deformed Wing Virus and Sacbrood Virus Isolated from Pollen

Ralitsa Balkanska  1 , Rositsa Shumkova  2 , Nedyalka Atsenova  3 , Delka Salkova  4 , Heliana Dundarova  3   5 , Georgi Radoslavov  3 , Peter Hristov  3
Affiliations

Molecular Detection and Phylogenetic Analysis of Deformed Wing Virus and Sacbrood Virus Isolated from Pollen

Ralitsa Balkanska et al. Vet Sci. .

Abstract

Among many pathogens and pests, honey bee viruses are known as one of the most common cause of diseases in honey bee colonies. In this study, we demonstrate that pollen grains and bee bread are potential sources of viral DNA. We extracted DNA from 3 types of pollen samples: directly provided by beekeepers (n = 12), purchased from trade markets (n = 5), and obtained from honeycombs (bee bread, n = 10). The extracted DNA was used for molecular detection (RT-PCR analysis) of six of the most widely distributed honey bee viruses: deformed wing virus, sacbrood virus, acute bee paralysis virus, black queen cell virus, Kashmir bee virus, Israeli acute paralysis virus, and chronic bee paralysis virus. We successfully managed to establish only the deformed wing virus (DWV) and the sacbrood virus (SBV), with different distribution frequencies depending on the territory of the country. The phylogenetic analyses of Bulgarian isolates were performed with the most similar sequences available in molecular databases from other countries. Phylogenies of Bulgarian viral strains demonstrated genetically heterogeneous populations of DWV and relatively homogenous populations of SBV. In conclusion, the results obtained from the current study have shown that pollen is a valuable source for molecular detection of honey bee pathogens. This allows epidemiological monitoring of honey bee diseases at a regional and a national level.

Keywords: RT-PCR; epidemiology; honey bee viruses; phylogeny; pollen.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Map showing the geographic localization and the positive and negative (DWV and SBV) pollen samples.
Figure 2
Figure 2
Frequency (%) of the positive (successfully amplified fragment) pollen samples (DWV and SBV) in different regions of the country. The analysis included a total of 27 pollen samples: directly provided by beekeepers (n = 12), purchased from the trade markets (n = 5), or obtained from honeycombs (bee bread, n = 10).
Figure 3
Figure 3
Phylogeny of deformed wing virus (DWV) isolates from Bulgaria and other countries. The unrooted phylogenetic tree was constructed based on the alignment of parts of nonstructural protein gene sequences of DWV isolates (382 bp) (nucleotide positions 8566–8960 bp according to RefSeq Acc. no. NC_004830) from Bulgaria using the neighbor-joining method under the maximum composite likelihood model. The indicated branching topology was evaluated by bootstrap resampling of the sequences 10,000 times, and nodes supported by bootstrap values > 50 are shown. Each isolate is indicated by the country of isolation and GenBank accession number. Bulgarian isolates identified by this study are shown in blue and red (the isolates from honey bees) [48].
Figure 4
Figure 4
Phylogeny of sacbrood virus (SBV) isolates from Bulgaria and other countries. The unrooted phylogenetic tree was constructed based on the alignment of the part of nonstructural protein gene sequences of SBV isolates (382 bp) (nucleotide position 7747–8172 bp according to RefSeq Acc. No. NC_002066) from Bulgaria using the neighbor-joining method under the maximum composite likelihood model. The indicated branching topology was evaluated by bootstrap resampling of the sequences 10,000 times, and nodes supported by bootstrap values > 50 are shown. Each isolate is indicated by the country of isolation and GenBank accession number. Bulgarian isolates identified by this study are shown in blue and red (the isolates from honey bees) [48].
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