Introduction
Honey bees are crucial pollinators that play a vital role in sustaining global agricultural production. However, massive colony losses in honey bee populations have been reported worldwide (Highfield et al., Reference Highfield, El Nagar, Mackinder, Noël, Hall, Martin and Schroeder2009; McMenamin and Genersch, Reference McMenamin and Genersch2015). Viral infections were recognised as the main cause of colony collapse disorder (Martin et al., Reference Martin, Ball and Carreck2013; McMenamin and Genersch, Reference McMenamin and Genersch2015). Mostly viruses cause in-apparent infections in honey bee colonies. However, clinical symptoms can be observed when colonies are stressed (Chen and Siede, Reference Chen and Siede2007). The presence of Varroa destructor (V. destructor) in bee colonies significantly affects colony stress and increases the incidence of colony losses due to viral infections (Martin et al., Reference Martin, Ball and Carreck2013; McMenamin and Genersch, Reference McMenamin and Genersch2015).
Deformed wing virus (DWV) is reported to be the most common viral agent in honey bee colonies infested with Varroa mites (Yue and Genersch, Reference Yue and Genersch2005; Schroeder and Martin, Reference Schroeder and Martin2012; Chen et al., Reference Chen, Wang, Jia, Feng, Hu, Chen and Zheng2021). DWV has a positive, single-strand RNA genome, and it belongs to the Iflavirus genus of the Iflaviridae family (Ongus et al., Reference Ongus, Peters, Bonmatin, Bengsch, Vlak and van Oers2004; Lanzi et al., Reference Lanzi, de Miranda, Boniotti, Cameron, Lavazza, Capucci, Camazine and Rossi2006). The viral genome encodes four major structural proteins (VP1, VP2, VP3 and VP4) and four non-structural proteins (an RNA helicase, a genome-linked viral protein, a 3C-like cysteine protease and an RNA-dependent RNA polymerase) (Škubník et al., Reference Škubník, Nováček, Füzik, Přidal, Paxton and Plevka2017). DWV is classified into four main genetic variants: DWV-A, DWV-B (Varroa destructor virus-1 (VDV-1)), DWV-C and DWV-D (de Miranda et al., Reference De Miranda, Brettell, Chejanovsky, Childers, Dalmon, Deboutte, de Graaf, Doublet, Gebremedhn, Genersch, Gisder, Granberg, Haddad, Kaden, Manley, Matthijnssens, Meeus, Migdadi, Milbrath, Mondet, Remnant, Roberts, Ryabov, Sela, Smagghe, Somanathan, Wilfert, Wright, Martin and Ball2022). Recent studies have reported that recombination events frequently occurred between DWV-A and DWV-B variants (Moore et al., Reference Moore, Jironkin, Chandler, Burroughs, Evans and Ryabov2011; Ryabov et al., Reference Ryabov, Childers, Chen, Madella, Nessa, van Engelsdorp and Evans2017). DWV can cause subclinical infection in honey bees. However, wing deformity, paralysis and reduced body size can be seen in infected bees (Schroeder and Martin, Reference Schroeder and Martin2012). VDV-1 causes wing deformities and shortened life expectancy in infected adult bees (Benaets et al., Reference Benaets, Van Geystelen, Cardoen, De Smet, de Graaf, Schoofs, Larmuseau, Brettell, Martin and Wenseleers2017; Brettell et al., Reference Brettell, Mordecai, Schroeder, Jones, da Silva, Vicente-Rubiano and Martin2017).
Beekeeping is important for both ecological and economic reasons in Türkiye. Türkiye has around 8.1 million bee colonies and ranks second position on the list of the world's largest honey producers (TEPGE, 2021). However, honey production per colony is considerably lower than those of countries such as China, Brazil, the USA and Canada (FAOSTAT, 2021). The reasons why beekeeping has not yet reached its full potential in Türkiye remain unclear, as there are no comprehensive regular disease monitoring programmes. However, bee diseases are expected to play a significant role in honeybee losses in Türkiye as they have contributed to significant honey bee losses worldwide (Oğuz et al., Reference Oğuz, Karapınar, Dinçer and Değer2017; Karapınar et al., Reference Karapınar, Oğuz, Dinçer and Öztürk2018; Muz and Muz, Reference Muz and Muz2018). The presence of DWV in honey bees has been reported in Türkiye (Muz and Muz, Reference Muz and Muz2017; Karapınar et al., Reference Karapınar, Oğuz, Dinçer and Öztürk2018; Mayack and Hakanoğlu, Reference Mayack and Hakanoğlu2022). Yet, there is limited information about the genetic variants of DWV circulating in Türkiye. Monitoring and determining the prevalence of viral infections in honey bees are important to ensure the continuity of beekeeping. Therefore, in this study, it was aimed to investigate the prevalence and genetic diversity of DWV in honey bees and to determine the role of V. destructor in the transmission of genetic variants of DWV.
Materials and methods
Honey bee (Apis mellifera) sampling
This study was conducted from March 2022 to April 2023 in the Hatay Province (36°12′12.1′N, 36°09′31.4′E) (fig. 1). Hatay Province has 15 districts. However, five districts (Antakya, Arsuz, Dörtyol, Kırıkhan and Samandağ) were randomly selected for sampling due to budget limits. The selected five districts had 593 apiaries (54.0% of the apiaries in the Hatay Province) (TUIK, 2021). In this study, a two-stage sampling was used to select the apiaries and bees in the apiaries for the detection of DWV strains that were circulating in bees and Varroa mites. First, the method specified by Thrusfield (Reference Thrusfield2007) was used to determine the number of apiaries required for sampling. It was determined that 62 apiaries were required with a 90% confidence level, 10% acceptable error and 50% expected apiaries level DWV prevalence. The list of apiaries in the selected districts was obtained from the Hatay Province Beekeepers Association. From the list, 62 apiaries were randomly selected using the randomise tool in Microsoft Excel software (Microsoft Corporation, USA), and contacted with apiaries owners. The contacted apiary owners agreed to participate in this study. The method described by Pirk et al. (Reference Pirk, de Miranda, Kramer, Murray, Nazzi, Shutler, van der Steen and van Dooremalen2013) was used to determine the minimum number of bees to be examined within each colony, and it was determined that 25 bees should be sampled in each colony with 95% probability. Adult bee samples were obtained from four randomly selected colonies in each apiary according to the signs of the diseases. The swollen abdomen, wing deformations, discoloration, shrinking body, crawling on the ground and flightlessness were observed in the sampled bees. The collected bees were placed into dry ice and transported to the laboratory where they were kept frozen at −80°C until analysis.
Figure 1. The location of the study area. The red colour represents the location where the study took place.
V. destructor determination in honey bee samples
The presence of V. destructor in adult honey bee samples was examined morphologically using a stereo microscope (Motic, Wetzlar, Germany) with 30× magnification (OIE, 2021). A total of 100 adult bees from each apiary were tested. The apiaries which had infestation rates ≥3 mites per 100 adult bees were considered as Varroa infested (Jack and Ellis, Reference Jack and Ellis2021). Detected Varroa mites were separated by considering the apiaries where they were detected, and pools of Varroa mites were created using sterile phosphate buffer saline (PBS).
Nucleic acid extraction
The frozen bees from each colony in each apiary were pooled and were considered as one single sample. Furthermore, V. destructor from each colony in each apiary were also pooled. The samples were homogenised in sterile falcon tubes (50 ml) containing sterile PBS by using the TissueRuptor (Qiagen, Hilden, Germany). The falcon tubes then were centrifuged for 30 min at 5000 rpm at 4°C, and 50 μl of supernatant sample was taken for RNA extraction. Nucleic acid extraction was performed using a commercial kit (High Pure Viral Nucleic Acid Kit, Roche, Germany) following the manufacturer's instructions. The obtained extracts were stored at −80°C until analysis.
DWV-A and VDV-1 screening by real-time RT-PCR
For the detection of DWV-A and VDV-1, separate real-time RT-PCR assays were performed using VP3 gene region-specific probes and primers described by Schurr et al. (Reference Schurr, Tison, Militano, Cheviron, Sircoulomb, Riviere, Chabert, Thiery and Dubois2019) with a commercial master mix kit (AgPath-ID One-Step RT-PCR, ThermoFisher Scientific, MA, USA). For DWV-A amplification, 25 μl reaction mix containing primers and probe (350 nM, F: 5′-GCGGCTAAGATTGTAAATTG-3′, R: 5′-GTGACTAGCATAACCATGATTA-3′, and probe 100 nM (6-Fam) 5′-CCTTGACCAGTAGACACAGCATC-3′ (Tamra), targeting the region between nucleotides 4258 and 4329 of the DWV-A genome) and 5 μl of extracted RNA was prepared, while 25 μl reaction mix containing primers and probe (1200 nM, F: 5′-GGTCTGAAGCGAAAATAG-3′ and R: 5′-CTAGCATATCCATGATTATAAAC-3′, and probe 400 nM (6-Fam) 5′-CCTTGTCCA GTAGATACAGCATCACA-3 (Tamra), targeting the region between nucleotides 4218 and 4290 of the VDV-1 genome) and 5 μl of extracted RNA was prepared for VDV-1 amplification. The cycling conditions were: reverse transcription at 45°C for 10 min, followed by initial PCR activation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 1 min at 60°C. Positive DWV RNA obtained from the Department of Parasitology (Veterinary Faculty, Hatay Mustafa Kemal University, Antakya, Türkiye) was used as positive control, whereas nuclease-free water was used as negative control in all analyses. The cycle threshold value (Ct) equal or lower than 35 was considered as positive (De Miranda et al., Reference De Miranda, Bailey, Ball, Blanchard, Budge, Chejanovsky, Chen, Gauthier, Genersch, de Graaf, Ribière, Ryabov, Smet and van der Steen2013).
RT-PCR amplification, sequence and phylogenetic analyses
The samples found positive by real-time RT-PCR assay (cut-off for positivity was Ct value ≤ 35) were subjected to one-step RT-PCR using primers (F: 5′-TGACTTAACGGCTGAAATGAATCA-3′ and R: 5′-TTCATTTCCTCCACTAAGCGCTGATT-3′, targeting an 866-bp long sequence spanning nucleotide positions 5386–6252 of DWV genome) (fig. 2) described by Wang et al. (Reference Wang, Xie, Shreeve, Ma, Pallett, King and Possee2013). The reaction mix was prepared in a final volume of 50 μl, containing primers (400 nM) and 5 μl of extracted RNA, with a commercial master mix kit (MegaFi Pro One-Step RT-PCR, ABM, Canada). The amplification conditions were as follows: 15 min at 60°C, 30 s at 98°C, followed by 45 cycles with 20 s at 95°C, 20 s at 55°C, 30 s at 72°C and a final extension cycle 10 min at 72°C. PCR products were separated by gel electrophoresis in 1.5% agarose gel containing GelRed (Biotium, USA). PCR products of 866 bp were purified from gel using a commercial purification kit (HibriGen Gel Extraction Kit, Kocaeli, Türkiye), and sequenced in reverse and forward directions by BM Laboratory (Ankara, Türkiye). Nucleotide sequences were analysed using the Bioedit software (version 7.0.5.3), and compared with sequences obtained from the GenBank. Sequences from the GenBank were selected based on the criteria of the information available regarding the genetic variants, the location and the year of isolation. The phylogenetic tree of polyprotein gene was constructed by the maximum likelihood method with Kimura's two-parameter substitution model using MEGA software (version 11.0). The lowest BIC score in MEGA software was used to determine the best-fitting nucleotide substitution model for the alignment of datasets (Fei et al., Reference Fei, Guo, Fan, Wang, Wu, Li and Ma2019).
Figure 2. Schematic of the DWV genome showing the sequenced region (at positions 5386–6252) in this study denoted by black triangles. Structural genes are represented by blue while non-structural genes are represented by orange.
Nucleotide sequence accession numbers
The nucleotide sequences of the VDV-1 isolates detected in this study are available in the GenBank database under accession numbers OQ849766 and OQ849767.
Recombination analysis
Recombination analysis of the field isolates detected in this study was performed using RDP4 software (Martin et al., Reference Martin, Murrell, Golden, Khoosal and Muhire2015). The following algorithms were used in the RDP4 analysis: (1) RDP with a window size of 40, (2) Geneconv (default settings), (3) Bootscan with a window size of 300, step size = 20, bootstrap = 100 and Jin and Nei model selected for the Neighbour-Joining tree, (4) χ2 using variable sites per window = 60, (5) Chimaera using variable sites per window = 60, (6) Siscan with a window size of 300 and (7) 3SEQ (default settings). The presence of recombination event was considered when the P value of at least three algorithms was below 0.05 and the recombination consensus score was between 0.4 and 0.6 (Fei et al., Reference Fei, Guo, Fan, Wang, Wu, Li and Ma2019). Furthermore, the presence of recombination was confirmed by the genetic algorithm for recombination detection (GARD) analysis available in the Datamonkey web interface (Delport et al., Reference Delport, Poon, Frost and Kosakovsky Pond2010).
Results
V. destructor frequencies in apiaries
V. destructor was detected in 46 (74.2%) of the 62 apiaries, whereas the number of apiaries that achieved ≥3% of infestation with V. destructor was 17 (27.4%) (table 1).
Table 1. The prevalence and district distribution patterns of V. destructor mite, DWV-A and VDV-1 in Hatay Province
Detection of DWV-A and VDV-1
DWV-A specific viral RNA was not detected, whereas VDV-1 specific RNA was detected in four (6.5%) of the 62 apiaries. Each VDV-1 positive apiary was in the Antakya (n = 2), Arsuz (n = 1) and Kırıkhan (n = 1) districts in the Hatay Province. However, DWV-A and VDV-1 specific RNA was not detected in V. destructor pools. In this study, positive samples showed Ct values ranging between 18.51 and 34.48.
By using a RT-PCR method, two strong amplicon bands of about 860 bp were obtained from two of the four samples that were found positive by VDV-1 real-time RT-PCR. This situation can be explained by the low viral load in other real-time RT-PCR positive samples (Ct value > 30).
Nucleotide and amino acid sequence analyses of VDV-1 isolates
Analysis of the polyprotein gene revealed that the nucleotide homology between the isolates detected in this study was 97.8% when compared with each other, and 91.2–98.4% when compared with DWV-A and VDV-1 isolates from other countries, the lowest with a Chinese DWV isolate (MF770715) and the highest with United Kingdom VDV-1-DWV recombinants (HM067437, HM162354 and HM162360).
The amino acid homology between the isolates detected in this study was 99.4%, whereas homology with the previously characterised DWV-A and VDV-1 isolates ranged between 97.9 and 100.0%.
In this study, a total of 38 nucleotide substitutions were detected in field isolates when polyprotein gene region of the field isolates was compared with the Netherlands VDV-1 (NC_006494), a reference isolate. Six of the 38 nucleotide substitutions have not been reported in other strains included in this study (table 2). Furthermore, 26 of the 38 nucleotide substitutions were non-synonymous substitutions. Three of the 26 amino acid substitutions have not been previously reported in other strains included in this study (table 3).
Table 2. Nucleotide changes in the helicase coding region of the field isolates detected in this study compared to Netherlands VDV-1
a VDV1-DWV recombinant strains; Pos. nt, nucleotide position in the polyprotein gene.
*Mutations found exclusively in the field isolates detected in this study and never reported in other strains included in this study.
Table 3. Amino acid changes in the helicase coding region of the field isolates detected in this study compared to Netherlands VDV-1
a VDV1-DWV recombinant strains; Pos. aa, amino acid position in the helicase gene.
* Mutations found exclusively in the field isolates detected in this study and never reported in other strains included in this study.
Phylogenetic relationships of the VDV-1 isolates
The phylogenetic tree based on the polyprotein gene revealed that VDV-1 isolates detected in this study were clustered separately from the previously detected DWV-A and VDV-1 isolates (fig. 3). Furthermore, the field isolates detected in this study formed separate branches from the VDV-1-DWV recombinants that were previously detected in the United Kingdom.
Figure 3. Phylogenetic tree of the VDV-1 polyprotein gene, only values above 50% were reported. The sequences obtained in this study are marked with a round black dot.
Recombination breakpoints
Recombination analyses indicated that field isolates, TUR/Hatay/Antakya A3 and TUR/Hatay/Kirikhan K1, were determined as recombinants at the breakpoint in the positions 5923–6244 nt and 5908–6202 nt, respectively, with high level of confidence (table 4). The identified breakpoints were located in the helicase-coding region of the genome. The potential parental isolates of TUR/Hatay/Antakya A3 were Netherlands VDV-1 (AY251269) and New Zealand DWV (MN538208), whereas potential parental isolates of TUR/Hatay/Kirikhan K1 isolate were United Kingdom VDV-1 (KC786222) and Chile DWV A1 (JQ413340) (fig. 4). Two field isolates, TUR/Hatay/Antakya A3 and TUR/Hatay/Kirikhan K1, were also identified as recombinant by GARD analysis (RHS, LHS P-values < 0.05).
Table 4. Possible recombination events in VDV-1 isolates described by RDP4
R, RDP; G, Geneconv; B, Bootscan; M, MaxChi; C, Chimaera; S, Siscan; T, 3Seq; NS, not significant.
Figure 4. Phylogenetic analyses based solely on the recombination site using UPGMA in RDP4. (A) TUR/Hatay/Antakya A3 and (B) TUR/Hatay/Kirikhan K1. The colour code used is explained in the figure.
Discussion
DWV is one of the most common honey bee viruses in the world (Gülmez et al., Reference Gülmez, Bursalı and Tekin2009; Kevill et al., Reference Kevill, Highfield, Mordecai, Martin and Schroeder2017; Fei et al., Reference Fei, Guo, Fan, Wang, Wu, Li and Ma2019; Brasesco et al., Reference Brasesco, Quintana, Di Gerónimo, Genchi García, Sguazza, Bravi, Fargnoli, Reynaldi, Eguaras and Maggi2021; de Miranda et al., Reference De Miranda, Brettell, Chejanovsky, Childers, Dalmon, Deboutte, de Graaf, Doublet, Gebremedhn, Genersch, Gisder, Granberg, Haddad, Kaden, Manley, Matthijnssens, Meeus, Migdadi, Milbrath, Mondet, Remnant, Roberts, Ryabov, Sela, Smagghe, Somanathan, Wilfert, Wright, Martin and Ball2022). V. destructor plays an important role in the transmission of DWV among honey bees (Piou et al., Reference Piou, Schurr, Dubois and Vétillard2022). DWV mostly persists in bee colonies and causes asymptomatic infections. Asymptomatic infections can remain latent and spread, resulting in large-scale colony losses, since colonies are rarely sampled and investigated for infections (Highfield et al., Reference Highfield, El Nagar, Mackinder, Noël, Hall, Martin and Schroeder2009). Therefore, this study focused on the prevalence of DWV variants and the role of V. destructor in the transmission of DWV variants in Türkiye.
In the present study, considering the V. destructor infestation rate of 3% as relevant, out of the 62 apiaries examined, 17 (27.4%) were positive. This rate is lower than the results of the previous studies that reported the rate of V. destructor in apiaries ranging between 35.0 and 90.0% in Türkiye (Cakmak et al., Reference Cakmak, Aydin, Gulegen and Wells2003a, Reference Cakmak, Aydin and Gulegen2003b; Gülmez et al., Reference Gülmez, Bursalı and Tekin2009; Gümüşova et al., Reference Gümüşova, Albayrak, Kurt and Yazici2010). These differences in V. destructor rates in different regions of Türkiye could be explained by the climatic conditions, sampling time and use of drugs against Varroa mites (Cobey, Reference Cobey2001). Furthermore, observed lower detection rate can be attributed to the small number of adult bees per colony for the Varroa mite laboratory examination and diagnostic methods used in this study. According to the apiary owners' report, all sampled apiaries have been using drugs, containing amitraz, against Varroa for a long time, and last Varroa treatments were conducted 1–2 months before sampling. However, V. destructor mite was detected in these apiaries. This situation may be explained by the ability of mites to develop drug resistance. It has been reported that amitraz resistance can develop in Varroa mites due to long-term drug use (El Agrebi et al., Reference El Agrebi, Traynor, Wilmart, Tosi, Leinartz, Danneels, de Graaf and Saegerman2020).
DWV can infect both larvae and adult forms of honey bee, and is very common all over the world (Chen et al., Reference Chen, Pettis, Collins and Feldlaufer2006; Martin and Brettell, Reference Martin and Brettell2019). Reported DWV rates in honey bees were 18.6% in Spain (Antúnez et al., Reference Antúnez, Anido, Garrido-Bailón, Botías, Zunino, Martínez-Salvador, Martín-Hernández and Higes2012), 20.3% in Brazil (Teixeira et al., Reference Teixeira, Chen, Message, Pettis and Evans2008), 57.3% in Denmark (Nielsen et al., Reference Nielsen, Nicolaisen and Kryger2008), 87.0% in Greece (Bacandritsos et al., Reference Bacandritsos, Granato, Budge, Papanastasiou, Roinioti, Caldon, Falcaro, Gallina and Mutinelli2010), 91.0% in Austria (Berényi et al., Reference Berényi, Bakonyi, Derakhshifar, Köglberger and Nowotny2006) and 97.0% in France (Tentcheva et al., Reference Tentcheva, Gauthier, Zappulla, Dainat, Cousserans, Colin and Bergoin2004). Previous studies conducted in different regions of Türkiye reported that the rate of DWV in apiaries ranged between 25.2 and 74.2% (Kalaycı et al., Reference Kalaycı, Cagırgan, Kaplan, Pekmez, Beyazıt, Ozkan, Yesiloz and Arslan2020; Cagırgan and Yazici, Reference Cagırgan and Yazici2021; Usta and Yildirim, Reference Usta and Yildirim2022). However, there is no information about the genetic variants of DWV that were detected in Türkiye. DWV-A was not detected in this study. The possible explanations for the difference could be due to the level of beekeepers’ knowledge and awareness of DWV, the conditions of care and feeding, types of beekeeping (migratory or stationary) and sampled bees (larvae or adult bees). In this study, only adult bees were investigated for the presence of DWV. Furthermore, the reason for not detecting DWV-A in this study may be related with the sampling method. In this study, apiaries were sampled randomly and bees were randomly collected from each apiary. However, in most of the previous studies with high prevalence, samples were collected from apiaries with signs of disease.
VDV-1 is one of the most significant infectious agents causing colony losses (Ryabov et al., Reference Ryabov, Childers, Chen, Madella, Nessa, van Engelsdorp and Evans2017). In this study, VDV-1 was detected in four (6.5%) of 62 apiaries. In this study, VDV-1 detection rate is lower than the results of previous studies. A previous study conducted in the United States revealed that detection rate of VDV-1 was 66.0% (Ryabov et al., Reference Ryabov, Childers, Chen, Madella, Nessa, van Engelsdorp and Evans2017). Another study conducted in Argentina reported that the rate of VDV-1 was 47.0% (Brasesco et al., Reference Brasesco, Quintana, Di Gerónimo, Genchi García, Sguazza, Bravi, Fargnoli, Reynaldi, Eguaras and Maggi2021). The possible explanations for the difference could be due to the types of beekeeping, the conditions of care and feeding and level of beekeepers’ knowledge and awareness. Furthermore, results of the study suggest that detection of low rate of V. destructor in the study area may contribute to the low rate of VDV-1 in sampled apiaries when considering the role of Varroa mites in the transmission of VDV-1 (Ongus et al., Reference Ongus, Roode, Pleij, Vlak and van Oers2006).
In this study, VDV-1 was detected in apiaries where V. destructor was detected, whereas DWV-A was not detected. This result is consistent with the results of previous studies that reported VDV-1 is the most common variant in honey bee colonies (Kevill et al., Reference Kevill, Stainton, Schroeder and Martin2021; Piou et al., Reference Piou, Schurr, Dubois and Vétillard2022). This situation could be explained by the fact that VDV-1 multiplies more efficiently and with higher viral load in the V. destructor than DWV-A (Gisder and Genersch, Reference Gisder and Genersch2021; Piou et al., Reference Piou, Schurr, Dubois and Vétillard2022).
Comparison of TUR/Hatay/Kirikhan K1 and TUR/Hatay/Antakya A3 sequences showed that they had 97.8 and 99.4% nucleotide and amino acids homology, respectively. Although the homology of the two isolates was relatively high, they do not belong to the same strain (fig. 3). Furthermore, analysis of the polyprotein gene region showed that three amino acid substitutions detected in filed isolates had not been previously reported in other isolates (table 3). One of these mutations (A1870T (hydrophobic to hydrophilic)) was detected in the TUR/Hatay/Kirikhan K1 isolate, and two mutations (P1888S (hydrophobic to hydrophilic) and C1910R (hydrophilic to hydrophilic)) were detected in the TUR/Hatay/Antakya A3 isolate. Further studies are needed to investigate the functional roles of these amino acid changes in viral replication and virulence.
The phylogenetic tree based on polyprotein gene showed that TUR/Hatay/Kirikhan K1 and TUR/Hatay/Antakya A3 isolates had a closer relationship with VDV-1-DWV recombinants which were detected in the United Kingdom than other DWV and VDV-1 isolates (fig. 3). This situation could be explained by recombination between different genetic variants due to factors such as queen bee imports from different countries. Recombination is a strategy for viruses to adapt to new environmental conditions and hosts. Also, recombination is a mechanism that can facilitate the transmission of viruses from Varroa mites to bees (Mordecai et al., Reference Mordecai, Wilfert, Martin, Jones and Schroeder2016). Recombination events between DWV-A and VDV-1 viruses have been reported in the United Kingdom and the United States (Moore et al., Reference Moore, Jironkin, Chandler, Burroughs, Evans and Ryabov2011; Ryabov et al., Reference Ryabov, Childers, Chen, Madella, Nessa, van Engelsdorp and Evans2017). Furthermore, Zioni et al. (Reference Zioni, Soroker and Chejanovsky2011) have reported recombinant events in Israel, a flight distance of approximately 380 km from Hatay Province, where this research was carried out. A recombination event between DWV-A and VDV-1 has not been previously reported in Türkiye. To the best our knowledge, this is the first report of VDV1-DWV recombinants in honey bees in Türkiye. It has been reported that VDV-1-DWV recombinants detected in the United Kingdom were associated with mite transmission (Moore et al., Reference Moore, Jironkin, Chandler, Burroughs, Evans and Ryabov2011). In this study, V. destructor was also detected in apiaries where VDV-1-DWV recombinants were determined.
In this study, recombination points were detected in the helicase gene. This result is consistent with the results of previous studies that reported helicase gene region contains recombinant points (Moore et al., Reference Moore, Jironkin, Chandler, Burroughs, Evans and Ryabov2011; Dalmon et al., Reference Dalmon, Desbiez, Coulon, Thomasson, Le Conte, Alaux, Vallon and Moury2017). A high degree of similarity in parental sequences can encourage recombination events (Kirkegaard and Baltimore, Reference Kirkegaard and Baltimore1986). It has been reported that helicase coding region has the highest identity among virus variants (Dalmon et al., Reference Dalmon, Desbiez, Coulon, Thomasson, Le Conte, Alaux, Vallon and Moury2017). Therefore, the helicase gene region can be considered as recombination ‘hot spots’, and associated with the emergence of new virus variants. In this study, only partial genome sequences of the helicase gene were used to identify potential recombination events. The findings of the study are limited by the lack of whole genome sequencing of field isolates. Further studies are needed to determine other recombinants and recombination breakpoints among the circulating DWV-A and VDV-1 isolates in Türkiye.
Conclusions
Türkiye has a significant geographical position that connects Asia and Europe, and ranks first position on the list of the Europe's honey producers. Therefore, it is very important to understand the prevalence of honey bee viruses in Türkiye. This study underlines the presence of VDV-1-DWV recombinants in honey bees in Türkiye. VDV-1-DWV recombinants detected in this study showed a number of specific nucleotide and deduced amino acid motifs that are not observed in other DWV and VDV-1 isolates. VDV-1-DWV recombinants have been reported to be highly virulent in the United Kingdom (Ryabov et al., Reference Ryabov, Wood, Fannon, Moore, Bull, Chandler, Mead, Burroughs and Evans2014). The occurrence of recombination events may threaten beekeeping by contributing to high levels of genetic diversity and increased host susceptibility. Therefore, further studies are necessary to determine the impact of VDV-1-DWV recombinants and their virological and antigenic properties.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Author contributions
A. Z. and M. Y. collected samples. A. Z. and I. E. isolated the RNA. M. Ş. performed the molecular analyses. M. Ş. wrote the original draft. All authors read and approved the final manuscript.
Financial support
This study has been supported by Necmettin Erbakan University Scientific Research Projects Coordination Unit under project number 221229001.
Competing interests
None.
Ethical standards
This study was approved by the Experimental Animal Ethics Committee of Hatay Mustafa Kemal University (No. 2021/02-02).
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