Molecular Epidemiology of Common Pathogenic Viruses Infected Varroa Mites in Iranian Apiaries

1. Introduction 
Honey bee (Apis mellifera), a vital important factor in the agricultural industry, is pollinator for a wide range of crops [1]. However, over the past few decades, honey bee health has declined globally because of the spread of numerous parasites and pathogens, including bacteria and viruses, a problem facilitated by the international trade in honey bees [2]. Among parasitic agents, Varroa destructor is an external parasite of honey bees which effects on the apiculture industry [3].
There are six major common pathogenic viruses which are of particular importance, including acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), black queen cell virus (BQCV), deformed wing virus (DWV), Sacbrood virus (SBV), and Kashmir virus (KBV) [4]. These honey bee viruses can transmit between bees in a colony both horizontally and vertically. Horizontal transmission occurs through Varroa mites, interaction between adult bees, and feeding of pupae by nurse bees, and vertical transmission occurs through drones and queens [5]. In colonies with heavy infested with Varroa mites, the detection of high viral loads in sick or dead bees suggests that Varroa mite infestation may stimulate the virus to replicate and induce disease from a state of asymptomatic infection to symptomatic infection. Varroa mites can activate ABPV and play a role in causing observable inoculation of bees by reducing bee defenses and directly injecting the virus into the hemolymph. Also, virus replication can be caused by other factors. Some studies have shown that ABPV was detected in honeybees in apiaries while ABPV-positive Varroa mites were not diagnosed. This proposes that varroa mites are not the only factor causative to the spread of ABPV infection [5]. SBV infection has been detected in large numbers of adult bees in apiaries infected with Varroa mites. It has been reported the high levels of virus in Varroa mites, especially in the mouthparts and digestive tract. Also, the positive relationship between the occurrence of SBV in Varroa mite samples and SBV in adult bee samples was detected [5]. 
It has been shown that KBV can transmit from KBV-infected mites to healthy pupae and also mites in colonies without any KBV infection [6]. Varroa mites are responsible for the spread of DWV to all tissues of the honey bee by feeding on the hemolymph and reactivating viral infections. When DWV is transmitted by Varroa mites, this virus can have devastating effects on honey bees by causing morphological deformities and even death [7].
Considering the role of Varroa mites in transmitting viral diseases to honey bees, it seems it is necessary to investigate the prevalence of viral infections in Varroa mites. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) method was used to identify common honey bee pathogenic viruses in Varroa mites.

2. Materials and Methods
2.1. Data collection and sample preparation
Sampling was accomplished randomly based on the number of apiaries in Iran by province and prevalence rate of 40% (reported by Iran Veterinary Organization). Using Cochran’s formula (Equation 1), population size was estimated [8]:

1. N = z2[pq]/d2
where, d or error was equal 0.06, p was equal 0.4 and q was equal 0.6 and z or the normal variable was equal 1.96 with a confidence factor of 95%. Thus, N≈260. So, the number of 274 apiaries from 31 provinces of Iran were considered, whereas for each apiary, a number of hives were randomly selected regardless of the clinical symptoms of the disease. Then, a number of bees were collected from each comb in the sterile containers (overall more than 50 adult bees for each apiary). All collected adult bee samples from each apiary were pooled and were sent to the lab using a cold chain for further analysis. 
To count and separate mites in each sample, 200–250 bees were immersed in ethanol and shaken for 10 minutes. Then, using a mesh (2–3 mm pores), the bees and mites were separated, counted, and the percentage determined. For each province, all the isolated mites were mixed together and crushed well in a sterile porcelain mortar. After that, 200 µL of DEPC water was added and homogenized. Afterward, the homogenized solution was transferred into a sterilized 1.5 mL tube and centrifuged at 20,000 g for 1 min. The supernatant was collected for RNA extraction and stored at -20 °C until use [9].

2.2. RNA extraction 
RNA extraction for Varroa mite samples was performed using QIA amp -Viral RNA Mini kit and cDNA synthesis was performed using kit (Biotech, Germany) according to the manufacturer’s instructions in a 0.2 mLtube and a final volume of 20 µL. Primers introduced by Berényi et al. [10] were used. PCR reaction was performed using (1.5 mM mgcl2) Taq 2× Master mix (Ampliqon, Denmark). The molecular weight of the PCR product was examined on a 1.2% agarose gel containing safe stain DNA.

2.3. Purification and DNA sequencing
PCR products were purified using High Pure PCR Product Purification Kit [Roche, Germany] according to the manufacture´s instruction [11]. For sequencing, two replicates of purified PCR products of DWV isolated from Varroa mite samples were selected and sequenced by Microsynth (Switzerland).

2.4. Statistical analyses and sequence analysis
To estimate the number of apiaries for sampling, population size was calculated using Cochran’s formula [8]. Then, results of molecular process were evaluated by descriptive statistics. Phylogenetic tree of sequences of DWV isolated from Varroa mite samples were assembled by MEGAX 4.0 program using the neighbor-joining method [12] and bootstrap support was estimated based on 1000 replicates. 
To ensure the results, given the high prevalence of DWV among mite samples, 2 samples of PCR products from DWV positive cases that had strong bands were selected and sent to Microscience (Switzerland) for sequencing. Sequencing was performed using the forward primer of each sample. The obtained sequences were blasted to determine the similarity with other sequences available in the NCBI database and the homology was examined. Sequence comparison and information on genetic parameters such as the number of mutations, nucleotide diversity, and the number of sites where similar substitutions occurred, as well as construction of a phylogenetic tree, were conducted using CLC Sequence Viewer software (v. 6.8.2.)

3. Results 
Figure 1 shows the results of PCR products of DWV, KBV and SBV infection which were isolated from Varroa mites in different provinces.

As presented, out of 274 samples in all provinces, 122(44.52%) apiaries were positive for Varroa mite which belonged to 26 provinces of the country. In these 26 provinces, viral infection was observed in 24 provinces which 22(84.61%), 4(15.38%) and 12(46.15) provinces were positive for DWV, KBV and SBV, respectively, and no infection was observed for ABPV, BQCV and CBPV. In four provinces, viral infection for DWV, KBV and SBV was observed, simultaneously. In six provinces, DWV and SBV was observed, simultaneously. Table 1 shows the number of varroa mite positive samples and positive studied viruses in infected varroa mite samples in each province.

As mentioned earlier, we only sequenced DWV isolated from V. destructor mites isolated from bee samples. The identified nucleotide differences between strains and phylogenetic tree for this virus are shown in Figures 2 and 3.

The phylogenetic tree shows three clades. DWV isolate obtained from the V. destructor mite in our study had a minimal genetic distance with the isolate obtained from the bee sample reported by Torkaman et al. [13] in the same study, as well as to the Iranian isolate registered in the NCBI database (Hamedan-MW566438) with an isolate from China and Iraq, indicating the existence of a common ancestor among these strains. The isolates from Belgium and France are in the same clade, and the sequence from Australia is an out-group. Phylogenetic analysis of DWV in Varroa mites showed a substitution mutation (G → A) at position 2499 in comparison with bee samples in the same study [13].

4. Discussion 
Recent findings support the key role of Varroa mites in the spread of infected viruses of honeybees. Honey bee viral infestation occurs consistently in honey bee populations in spite of the absence of clinical signs suggesting the prevalence of the colony diseases may be caused by environmental factors which cause the activation of virus replication in honey bees [5]. The V. destructor mite is broadly recognized as the most destructive risk to the colonies of honey bee on a global scale. Varroa mite infestation in honey bee colonies is linked to viral infections, and then, leads to a decline in bee populations and accelerated colony losses [14]. Research has demonstrated the association between Varroa mites and viruses, emphasizing the efficiency of Varroa mites in transmitting viruses among honey bees and colonies. Therefore, Varroa mites control can reduce viral infections in honey bee colonies [14]. This pest is an external parasite of honey bees, which can be important in the transmission of viral diseases. Given the vital role of V. destructor in the transmission of honey bee viruses, it is important to identify the viruses that replicate within the mites [15].
When a honey bee colony is infected by Varroa mites, the risk of colony collapse increases due to the weakening caused by the Varroa mites’ and continuous feeding on the hemolymph of developing larvae, pupae and adult bees. In fact, it has been shown that Varroa can be an active carrier of about 19 honey bee viruses. Therefore, this parasite can increase viral titers and, by overcoming the mechanical and immune defenses of the honey bee, by reducing nutrients and blood cells in the hemolymph, can effectively transmit viruses by direct injection into the hemolymph [3]. Regarding the growth of the Varroa mite population which is influenced by condition of the colony, the number of mites can increase between 12 and 800 times. Hence, Varroa mite can be a huge problem in apiculture industry [3].
Our results showed the prevalence of V. destructor mites in Iranian apiaries which was about 44.52%. Of the collected samples from all provinces, V. destructor mites were found in 26 provinces, whereas viral infection was found in isolated mites only in 24 provinces. Of these, 84.61%, 15.38% and 46.15% were positive for DWV, KBV, and SBV, respectively, and no infection was found for ABPV, BQCV, and CBPV. Torkaman et al. [13] also examined the prevalence of viral infection in honey bee samples collected as the same study in terms of infection with the six mentioned viruses. Their results showed that out of 274 honey bee samples, 7.66% ABPV, 7.66% BQCV, 4.01% CBPV, 90.15% DWV, 11.13% KBV and 33.21% SBV samples were positive. While, DWV, SBV and KBV viruses showed a higher prevalence than other viruses [13]. They also showed ABPV, BQCV, and CBPV were less prevalent than other studied viruses, which further highlights the role of Varroa mites in the transmission of viral diseases. The results of our study were consistent with those of Torkaman et al. [13], where both showing the highest infection rates for DWV and SBV. Varroa mites from 22 out of 26 provinces (84.61%) were infected with DWV, which, given the high infection rate of the studied apiaries with this virus (90.15%) [13]. It has been reported that the most common and virulent viruses affecting the health of A. mellifera in the world are DWV and BQCV [16]. Findings of present study also signifies DWV has a high prevalence in the V. destructor mite population as well as adult bees. Although viruses can be detected in mites, yet viral infection is also observed in mite-free colonies [17]. Given that free Varroa mites colonies also showed infection with some viruses, other transmission routes should be noted such as contaminated food (pollen, honey, etc.) and queens and male honey bees [13]. 
In previous study Torkaman et al. [13] showed that the most infestation was related to DWV and SBV, respectively, which is consistent with the high prevalence of infection of the V. destructor studied with these viruses. Shen et al. [18] showed that under natural and artificial infestations with Varroa mites, honey bee pupae contained significantly higher levels of KBV and DWV and also KBV structural proteins than pupae free from Varroa mites. Linear regression analysis showed a positive correlation between the number of Varroa mites introduced and the levels of viral RNAs. Their research suggests that infection by Varroa mites suppresses the immunity of honey bees and leads to the activation of a persistent and latent viral infection [18].
In Brazil, by examining six pathogenic viruses (DWV-BQCV-SBV-IAPV-ABPV-CBPV), Brzoskowski Chagas et al. [19] showed that cases of simultaneous infection with ABPV, BQCV, and DWV in both states. While, in all beekeeping with positive virus samples, the mite V. destructor was also reported, indicating the transmission of these viruses through Varroa mites [19]. Tentcheva et al. [5], examined six pathogenic viruses DWV-BQCV-SBV-KBV-ABPV-CBPV in adult bees, pupae and V. destructor samples from apparently healthy honey bee colonies in France using PCR technique. In their studied Varroa samples, four viruses were identified; DWV (100%), SBV (45%), ABPV (36%) and KBV (5%) of apiaries [5].
In Hungary, Bakonyi et al. [20] detected ABPV in Varroa mites in apiaries suffering from colony collapse disorder (CCD), while more than 87% of the collapsed apiaries showed ABPV infection in bee samples. In our study, ABPV infection was not observed in mite samples and infection of the total apiary rate with this virus was less than 8%, indicating Varroa mite infection along with viral diseases plays an effective role in viral infection of honey bee colonies [20].
Studies by Chen et al. showed that KBV can be transmitted from KBV-infected mites to healthy pupae and mites in a KBV-free colony [6]. Shen et al. identified KBV and SBV in honey bee samples and also, for the first time, in V. destructor mites. The simultaneous identification of viruses in V. destructor and honey bees emphasizes the importance of mites in the transmission of various honey bee viruses [18]. Furthermore, it was shown that DWV is not able to replicate in all mites. Interestingly, viral replication in mites is associated with DWV infection in honeybees, which suggests that symptoms are more likely to occur if the virus is transmitted by mites [21]. In another study conducted by Minoo et al. [22] rates of DWV and ABPV detection in Varroa mites were 100% and 77%, respectively, while, in Tropilaelaps mites, they detected 100% DWV, 57% ABPV, 30% CBPV and 23% VDV-1. Arzumanyan et al. reported infection of DWV in all Varroa mites [23]. The presence of DWV, SBV, KBV, IAPV, BQCV, and CBPV in Varroa mites collected from Korean apiaries has been reported by Moon et al. (2024) [24]. They showed the highest prevalence of viral infection belonged to DWV (87%), followed by IAPV (65.2%), SBV (17.4%), and less than 10% belonged to other viruses. In the present study, the prevalence of DWV was higher than SBV and KBV in Varroa mite samples, which was consistent with the above mentioned study. These results indicate that the V. destructor plays an effective role in the propagation and transmission of viral diseases in honey bees. This means that one of the reasons for the low prevalence of the pathogenic viruses including ABPV, BQCV and CBPV in honey bee samples [13] can be due to the non-infectiousness of the Varroa mite samples. Of course, it has been showed that there is no relationship between the spread of CBPV and the V. destructor mite [25].
Phylogenetic tree of the studied DWV showed the low genetic distance between the sequence obtained from the V. destructor and honeybee samples, which indicates the importance of the Varroa mite in the transmission of this virus among honeybees. The high viral replication in mites increases the probability of the mutation. Assuming that only some DWV mutations are capable of causing wing paralysis in developing honeybees, the possibility of such a mutation is possible with a high transcription rate in Varroa mites [7]. Our results showed a substitution mutation (G → A) at position 2499 in phylogenetic analysis of DWV in Varroa mites.
In the last decade, KBV has been recognized as the most important virus among several viruses related with CCD in apiaries infected with the V. destructor mite [10]. Reports show that in the beekeeping industry, control of Varroa mites and viruses is a vital factor in preserving the health of honey bees. So, beekeepers should take preventive methods to prevent the spread of bee diseases. Given the lack of treatment for viral diseases, management strategies may be the only way to control viral diseases [5]. Also, given the importance of the V. destructor mite in transmitting viral diseases, more attention should be paid to controlling this pest. Consequently, regular inspection of colonies by trained personal is essential to control or reduce viral infections. Recent research has suggested the use of thermal interventions, particularly hyperthermia, as a suitable method for controlling Varroa mites. These studies have also explained the effect of heat stress in decreasing viral infections among infected bees. Hyperthermia treatment may be effective in treating viral infections by decreasing Varroa mite infestation or by inducing the production of heat shock proteins, which have potential antiviral properties [26]. 

5. Conclusion
Our results showed the importance of the V. destructor mite in transmitting viral diseases in honey bee. The findings propose serious control of V. destructor, which is an active vector of honey bee viruses (especially DWV). should be placed on the program of regulatory organizations. These measures should be incorporated into the regulatory and apiculture management programs.

Acknowledgements
The authors would like to show their gratitude to Hossein Modirrousta for his help. Also, authors thank Iran Veterinary Organization for helping samples collection. 

Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.
Funding
This work was supported by Razi Vaccine and serum Research Institute, Karaj, Iran (Grant No.: 01-18-18-019-01019).

Authors' contributions
Conceptualization, study design, administrative, technical, and material support, review and editing: all authors; Data acquisition: Maryam Torkaman, Mojtaba Moharrami, and Masoumeh Bagheri; Analysis and data interpretation: Maryam Torkaman and Masoumeh Bagheri; Statistical analysis: Maryam Torkaman and Masoumeh Bagheri. Writing the original draft: Maryam Torkaman and Masoumeh Bagheri. 

Conflict of interest
The authors declared no conflict of interest. 

Data availability
The data that support the findings of this study are available on request from the corresponding author.

References

1. Calderone NW. Insect pollinated crops, insect pollinators and US agriculture: Trend analysis of aggregate data for the period 1992-2009. Plos One. 2012; 7(5):e37235 [DOI:10.1371/journal.pone.0037235] [PMID]
2. Neumann P, Carreck N. Honey bee colony losses. J Api Res. 2010; 49(1):1-6. [Link]
3. Whitehouse M, Yousuf F, Sainsbury J, Rangel J, Goodwin M. Innovations in Varroa mite management. Curr Opin Insect Sci. 2025; 68:101343. [DOI:10.1016/j.cois.2025.101343] [PMID]
4. Chen Y, Siede R. Honey Bee Viruses. Adv Virus Res. 2007; 70:33-80. [DOI:10.1016/S0065-3527(07)70002-7] [PMID]
5. Tentcheva D, Gauthier L, Zappulla N, Dainat B, Cousserans F, Colin ME, et al. Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl Environ Microbiol. 2004; 70(12):7185-91. [DOI:10.1128/AEM.70.12.7185-7191.2004] [PMID]
6. Chen Y, Zhao Y, Hammond J, Hsu HT, Evans J, Feldlaufer M. Multiple virus infections in the honey bee and genome divergence of honey bee viruses. J Invertebr Pathol. 2004; 87(2-3):84-93. [DOI:10.1016/j.jip.2004.07.005] [PMID]
7. Gisder S, Aumeier P, Genersc E. Deformed wing virus: Replication and viral load in mites (Varroa destructor). J Gen Virol. 2009; 90(2):463-7. [DOI:10.1099/vir.0.005579-0] [PMID]
8. Cochran WG. Sampling techniques (3rd ed.). New York: John Wiley & Sons. 1991. [Link]
9. Schäfer MO. Varroosis of honey bees (infestation of honey bees with Varroa spp.). In: Manual of diagnostic tests and vaccines for terrestrial animals. Paris: World Organisation for Animal Health; 2021. [Link]
10. Berényi O, Bakonyi T, Derakhshifar I, Köglberger H, Nowotny N. Occurrence of six honeybee’s viruses in diseased Austrian apiaries. Appl Environ Microbiol. 2006; 72(4):2414-20. [DOI:10.1128/AEM.72.4.2414-2420.2006] [PMID]
11. High pure PCR product purification kit. Basel: Roche; 2018. [Link]
12. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24(8):1596-9. [DOI:10.1093/molbev/msm092] [PMID]
13. Torkaman M, Moharrami M, Pourtaghi H, Nikbakht Brujeni G, Bagheri M. Epidemiological study and phylogenetic analysis of common pathogenic viruses of honeybee in Apiaries of Iran. Arch Razi Inst. 2025; 80(4):869-78. [DOI:10.66224/ARI.80.4.869]
14. Giacobino A, Molineri AI, Pacini A, Fondevila N, Pietronave H, Rodríguez G, et al. Varroa destructor and viruses association in honey bee colonies under different climatic conditions. Environ Microbiol Rep. 2016; 8(3):407-12. [DOI:10.1111/1758-2229.12410] [PMID]
15. Damayo JE, McKee RC, Buchmann G, Norton AM, Ashe A, Remnant EJ. Virus replication in the honey bee parasite, Varroa destructor. J Virol. 2023; 97(12):e0114923. [DOI:10.1128/jvi.01149-23] [PMID]
16. Zhang X, He SY, Evans JD, Pettis JS, Yin GF, Chen YP. New evidence that deformed wing virus and black queen cell virus are multi-host pathogens. J Invert Pathol. 2012; 109(1):156-9. [DOI:10.1016/j.jip.2011.09.010] [PMID]
17. Brettell, LE, Riegler M, O’brien, C, Cook JM. Occurrence of honey bee-associated pathogens in Varroa-free pollinator communities. J Invertebr Pathol. 2020; 171:107344.[DOI:10.1016/j.jip.2020.107344] [PMID]
18. Shen M, Cui L, Ostiguy N, Cox-Foster D. Intricate transmission routes and interactions between picorna-like viruses (Kashmir bee virus and sacbrood virus) with the honeybee host and the parasitic varroa mite. J Gen Virol. 2005; 86(8):2281-9. [DOI:10.1099/vir.0.80824-0] [PMID]
19. Brzoskowski Chagas D, Liz Monteiro F, da Silva Barcelos L, Iuri Frühauf M, Botton NY, Ribeiro LC, et al. Detection of honey bee viruses in apiaries in Southern Brazil through two standardized multiplex RT-PCR. J Apic Res. 2023; 62(5):1207-14. [DOI:10.1080/00218839.2022.2106089]
20. Bakonyi T, Farkas R, Szendröi A, Dobos-Kovács M, Rusvai M. Detection of acute bee paralysis virus by RT-PCR in honey bee and Varroa destructor field samples: Rapid screening of representative Hungarian apiaries. Apidologie. 2002; 33(1):63-74. [DOI:10.1051/apido:2001004]
21. Yue C, Genersch E. RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor). J Gen Virol. 2005; 86(12):3419-24. [DOI:10.1099/vir.0.81401-0] [PMID]
22. MinOo H, Kanjanaprachoat P, Suppasat T, Wongsiri S. Honey bee virus detection on Tropilaelaps and Varroa mites in Chiang Mai Thailand. J Apic Res. 2018; 33(2):77- 81. [DOI:10.17519/apiculture.2018.06.33.2.77]
23. Arzumanyan H, Avagyan H, Voskanyan H, Simonyan L, Simonyan J, Semirjyan Z, and Karalyan Z. First molecular detection of the presence of honey bee viruses in insects, Varroa destructor mites, and pollinated plants in an isolated region of Armenia. Vet World. 2023; 16(5):1029-34. [DOI:10.14202/vetworld.2023.1029-1034] [PMID]
24. Moon K, Cho S, Lee J, Lee SH, Seong KM, Kim YH. Molecular detection and phylogenetic analysis of six bee viruses from Varroa destructor in Korea. J Api Res. 2025; 64(4):1105-16. [DOI:10.1080/00218839.2024.2411769]
25. Ribiere M, Olivier V, Blanchard P. Chronic bee paralysis: A disease and a virus like no other? J Invertebr Pathol. 2010; 103(Suppl 1):120-31. [DOI:10.1016/j.jip.2009.06.013] [PMID]
26. Xu X, Zhou S, Huang J, Geng F, Zhu X, Abou-Shaara HF. Influence of hyperthermia treatment on varroa infestation, viral infections, and honey bee health in beehives. Insects. 2025; 16(2):168. [DOI:10.3390/insects16020168] [PMID]