Genetic identification and phylogenetic analysis of honeybee viruses from Serbia
Partial nucleotide sequences of four honeybee viruses (Acute bee paralysis virus, ABPV; Chronic bee paralysis virus, CBPV; Deformed wing virus, DWV; and Sacbrood virus; SBV) were amplified and determined from honeybee samples collected in Serbia. The sequences were identified by the Basic Local Alignment Tool (BLAST) search ( against the GenBank database. The genetic relationships between the Serbian honeybee virus sequences and related sequences deposited in the GenBank database were statistically analysed. After nucleotide alignments (ClustalW algorithm) sequences were subjected to phylogenetic analysis using Maximum Parsimony and Neighbor-Joining algorithms. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6 (Tamura, Stecher, Peterson, Filipski, and Kumar 2013) using default parameters. Inferred relationships are shown in phylograms:
The Serbian ABPV sequences (KL4, GenBank accession number KM001899, and KL5) have shown 97 to 93% nucleotide identities to ABPV sequences. Phylogenetic analysis of the Serbian sequences and nine further European ABPV sequences (Figure 1.) indicate that the Serbian sequences are forming a monophyletic cluster with full identity, and are relatively closely related to Hungarian ABPV sequences. ABPV sequences from Western and Northern Europe however form a separate cluster. The closer genetic relatedness between Serbian and Hungarian sequences could be explained by geographical distances, and the intensity of honeybee exchange and trade between beekeepers of the two countries. The results of the phylogenetic analysis clearly indicate that the Serbian ABPV is different form the Kashmir bee virus (KBV), which has been detected so far in the Western part of Europe.
Figure 1.Phylogenetic analysis of partial genome coding sections of 12 ABPVs.Phylogram of the Maximum Parsimony analysis. Viruses are indicated with GenBank accession numbers (if available) and country of origin. KBV was used as outgroup to root the tree. Numbers at nodes show percent values of bootstrap analysis of 1000 replicates. Bar on the left shows genetic distance.
A partial section of the first RNA segment of CBPV was determined in two Serbian bee samples (CBPV 1, GenBank accession number KM001900, and CBPV-3). The nucleotide sequences were 96 to 93% identical with CBPV sequences in the GenBank database. Phylogenetic analysis of the Serbian sequences and ten CBPV sequences (Figure 2.) show that the Serbian sequences are identical to each other and they exhibit a relatively long genetic distance from the other CBPVs. However, they are monophyletic with sequences of CBPVs from France, Belgium and Spain. Similar genetic distance is seen between a CBPV sequence from Switzerland and other European CBPV sequences; altghough the Swiss sequence is not monophyletic with the other European sequences. These findings may indicate that CBPV (which is taxonomically and genetically very different from the other three honeybee viruses analysed in this study), may have different epizootiological character, and hence, less intensively involved in the geographical spread of honeybeevirus strains. Alternatively, unique genetic properties (higher mutation rate or segment reassortment events) may explain the genetic seclusion of the Serbian CBPVs.
Figure 2.Phylogenetic analysis of partial genome coding sections of 12 CBPVs. Phylogram of the Maximum Parsimony analysis. Viruses are indicated with GenBank accession numbers (if available) and country of origin. FJ345326 was used as outgroup to root the tree. Numbers at nodes show percent values of bootstrap analysis of 1000 replicates. Bar on the left shows genetic distance.
Nine Serbian DWV sequences (Serbia D1, KM001902; Serbia D2, KM001903; Serbia D3, KM001904; Serbia D4, KM001905; Serbia D5;Serbia D6, KM001906; Serbia D7, KM001907;Serbia D8, KM001908; and Serbia D9, KM001909) were identified and analysed. BLAST search found 99 to 98% nucleotide identities with DWV sequences in the database. Maximum Likelihood analysis shows close genetic relationships between the Serbian sequences and other DWV sequences from Europe and Asia (Figure 3.). One DWV sequence from South America (JQ413340 from Chile) is relatively separated from the other DWVs. Two Serbian sequences (D4 and D5) are identical. The nine Serbian sequences are grouped in four clusters: D1, D4 and D5 are in one group; D2 and D6 form another group; D3 and D9 form the third group; D8 is clustered together with sequences from the United Kingdom, while D7 is in ancestral position. However, due to the relatively short sequences and high identity rates, this clustering is poorly supported by the bootstrap analysis. The close genetic distances between DWV support the idea of evolutionary recent world-wide distribution of DWV, potentially connected to the geographic expansion of its main vector, the parasitic mite Varroa destructor. The Serbian sequences are, however, clearly different from the Varroa destructor virus-1 (VDV), a closely related iflavirus, isolated from the mite. Phylogenetic analysis also supports that the “Kakugo virus”, which has been isolated from aggressive bees in Japan, is a variant of DWV. – diskusija.
Figure 3.Phylogenetic analysis of partial genome coding sections of 18DWVs. Phylogram of the Maximum Parsimony analysis. Viruses are indicated with GenBank accession numbers (if available) and country of origin. VDVs (AY251269 and JF440525) were used as outgroup to root the tree. Numbers at nodes show percent values of bootstrap analysis of 1000 replicates. Bar on the left shows genetic distance.
The partial nucleotide sequences of three Serbian SBVs (S1, KM001901; KL2 and KL25) were identified and analysed. They have shown 99 to 94% sequence identity rates with other SBVs in the database. The phylogenetic analysis revealed two main genetic lineages of the virus (Figure 4.). The three Serbian sequences were 100% identical with each other, and clustered together with SBV sequences from the continental Europe. However, the Serbian sequence is relatively distant from the other sequences in the cluster. Further analysis involving sequences from neighbouring countries could reveal, whether similar separation exists with SBV, as it was seen with ABPV. Two sequences from the United Kingdom and one from Russia shows a separated cluster from the other European sequences. A third branch of the lineage is represented by a South Korean sequence. The other genetic lineage contains Far-Eastern sequences. These viruses were mainly detected in Apiscerana, and are geographically and ecologically separated from the Apismellifera-derived viruses.
Figure 4.Phylogenetic analysis of partial genome coding sections of 20SBVs. Phylogram of the Maximum Parsimony analysis. Viruses are indicated with GenBank accession numbers (if available) and country of origin. Numbers at nodes show percent values of bootstrap analysis of 1000 replicates. Bar on the left shows genetic distance.
The genetic analyses of Serbian honeybee viruses confirmed their identity and revealed potential genetic relationships between representatives of the same virus species, detected in honeybees world-wide.
Reference:
Tamura K, Stecher G, Peterson D, Filipski A, and Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution:30 2725-2729.