Erzincan İlinde Bal Arısı (Apis mellifera L.) Kolonilerinde Tespit Edilen Deforme Kanat Virüslerünün Moleküler Karakterizasyonu

Abstract

Iflaviridae ailesine ait olan deforme kanat virüsü (DWV), tüm dünyada bal arısı kolonilerinde ciddi maddi kayıplara neden olan yıkıcı bir virüstür. 2020'de (Kasım) Erzincan ilinden toplanan virüs şüphesi olan bal arısı örnekleri, DWV enfeksiyonuna karşı moleküler testlerle tarandı. Test edilen 12 örneğin 6'sında muhtemel virüs varlığını gösteren yaklaşık 700 bp DNA bandı elde edildi. Virüsün varlığını gösteren bantlardan rastgele seçilen iki amplifiye edilmiş DNA bandı pGEM-T Easy vektörüne klonlanmış ve elektriksel olarak kompetan E. coli bakterisine aktarılmıştır. Klonlamadan elde edilen rekombinant plazmitler, E. coli'den saflaştırılmış ve yeni nesil dizileme (NGS) ile dizilenmiştir. DWV'nin kısmi nükleotid dizilerinin 711 nükleotid içerdiği tespit edilmiş ve bu diziler MW962981 ve MW962982 erişim numarası ile NCBI GenBank'a kaydedilmiştir. Her iki izolatın en yüksek dizi benzerliği, nükleotid düzeyinde %98.03 ile %98.17 arasında değişmiştir. Ayrıca, Erzincan DWV dizilerinin filogenetik ilişkileri, GenBank'ta kayıtlı farklı ekolojik bölgelerden 15 benzersiz dizi kullanılarak ortaya çıkarılmıştır. Filogenetik ağaç, DWV Erzincan izolatlarının İngiltere (HM067438), İspanya (MK262743) ve İsveç (MN746311) izolatları ile kümelendiğini göstermiştir. Literatür bilgilerimize göre, bu çalışma ile DWV (Deforme kanat virüsü) Türkiye'nin Erzincan ili bal arılarında ilk kez rapor edilmiştir.

Keywords

• Deforme kanat virüsü
• bal arısı
• RT-PCR
• klonlama

Molecular characterization of Deformed wing viruses identified in honeybee (Apis mellifera L.) colonies in Erzincan province of Turkey

Abstract

Deformed wing virus (DWV), which belongs to the Iflaviridae family, is a devastating virus causing severe financial losses in honey bee colonies all over the world. In 2020 (November), virus-suspected honey bee specimens collected from Erzincan province were screened by molecular assays against DWV infection. In 6 of 12 samples, an expected approximately 700 bp DNA bands were achieved, indicating probably virus-positive. Two randomly selected amplified DNA band suggesting the presence of the virus was further cloned into a pGEM-T Easy vector and electrically transformed into E. coli competent bacteria. Recombinant plasmids obtained from cloning were purified from E. coli and sequenced by next-generation sequencing (NGS). The partial nucleotide sequences of DWV were ascertained to contain 711 nucleotides and these sequences were recorded in the NCBI GenBank with MW962981 and MW962982 accession number. The highest sequence similarity of both isolates ranged from 98.03% to 98.17%, at the nucleotide level. Furthermore, phylogenetic relationships of Erzincan DWV sequences were revealed using 15 unique sequences from different ecological regions registered in the GenBank. Phylogenetic tree displayed that DWV Erzincan isolates are clustered with UK (HM067438), Spain (MK262743), and Sweden (MN746311) isolates. According to our best knowledge, DWV has been reported for the first time in the honey bees from Erzincan province of Turkey by this study.

Keywords

• Deformed wing virus
• honey bee
• RT-PCR
• cloning

References

1. Abd-El-Samie, EM., Basuny, NK., & Seyam, H. (2021). Molecular characterization of viruses found in honeybee (Apis mellifera) colonies infested with Varroa destructor and Nosema cerana in Egypt. Molecular and Cellular Probes. 57, 101731.
2. Bailey, L., & Ball, BV. (1991). Honey bee pathology. Academic Press; London, United Kingdom.
3. Barroso-Arévalo, S., Fernández-Carrión, E., Goyache, J., Molero, F., Puerta, F., & Sánchez-Vizcaíno, J.M. (2019). High Load of Deformed Wing Virus and Varroa destructor Infestation Are Related to Weakness of Honey Bee Colonies in Southern Spain. Frontiers in Microbiology, 10, 1331.
4. Berenyi, O., Bakonyi, T., Derakhshıfar, I., Koglberger, H., & Nowotny, N. (2006). Occurrence of six honey bee viruses in diseased Austrian apiaries. Applied and Environmental Microbiology 72, 2414-2420.
5. Bingöl, M., & Erkan, C. (2014). Van İli Arı Hastalıkları ve Zararlılarının Belirlenmesine Yönelik Bir Araştırma Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi. 5(2), 14-20.
6. Cagirgan, A.A., Yildirim, Y., & Usta, A. (2020). Phylogenetic analysis of deformed wing virus, black queen cell virus and acute bee paralysis viruses in Turkish honeybee colonies. Medical. Veterinary. 76 (8), 480-484.
7. Cagirgan, A.A., & Yazici, Z. (2021). The prevalence of seven crucial honeybee viruses using multiplex RT-PCR and their phylogenetic analysis. Turkish Journal of Veterinary and Animal Sciences 45(1), 44-55.
8. Chen, Y.P., Pettis, J.S., Collins, A., & Feldlaufer, M.F. (2006). Prevalence and transmission of honey bee viruses. Applied Environmental Microbiology, 72, 606–611.

9. Chen, Y.P., Smith, I.B., Collins, A.M., Pettis, J.S., & Feldlaufer, M.F. (2004). Detection of Deformed wing virus infection in honey bees, Apis mellifera L., in the United States. American Bee Journal, 144, 557-559.
10. Cirkovic, D., Stevanovic, J., Glavinic, U., Aleksic, N., Djuric, S., Aleksic, J., & Stanimirovic, Z. (2018). Honey bee viruses in Serbian colonies of different strength. Peer J. DOI: 10.7717/peerj.5887.
11. Cox-Foster. D.L., Conlan, S., Holme,s E.C., Palacios, G., Evans, J.D., Moran, N.A., Quan, P.L., Briese, T., Hornig, M., Geiser, D.M., Martinson, V., VanEngelsdorp, D., Kalkstein, A.L., Drysdale, A., Hui, J., Zhai, J.H., Cui, L.W., Hutchison, S.K., Simons, J.F., Egholm, M., Pettis, J.S., & Lipkin, W.I. (2007). A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318, 283-287 DOI: 10.1126/science.1146498
12. Doğanay, A., & Girişgin, A.O. (2017). Bal arisi yetiştiriciliği ürünleri hastaliklari. In: Doganay A, Aydin L (editors). 1st ed. Bursa: Dora Basın Yayın Dağıtım; 21-146.
13. Fievet, J., Tentcheva, D., Gauthier, L., De Miranda, J., Cousserans, F., Colin, M. E., & Bergoin, M. (2006). Localization of deformed wing virus infection in queen and drone Apis mellifera L. Virology Journal, 3, 16.
14. Foissac, L., Gentit, P., Svanetia-Dumas, L., Dulucq, M. J., & Candresse, T. (2001). Polyvalent detection of fruit tree tricho, capillo, and foveaviruses by nested RT-PCR using degenerated and inosine-containing primers (PDO RT-PCR). Acta Horticulturae, 550, 37-43.
15. Francis, R.M., Nielsen, S.L., & Kryge, P. (2013). Varroa-Virus interaction in collapsing honey bee colonies. PLoS ONE, 8:e57540. DOI: 10.1371/ journal.pone.0057540
16. Genersch, E., & Aubert, M. (2010). Emerging and re-emerging viruses of the honey bee (Apis mellifera L.). Vet. Res. 41:54 DOI: 10.1051/vetres/2010027
17. Genersch, E., Von Der Ohe, W., Kaatz, H., Schroeder, A., Otten, C., Buchler, R., Berg, S., Ritter, W., Muhlen, W., Gisder, S., Meixner, M., Liebig, G., & Rosenkranz, P. (2010). The German bee monitoring project: A long term study to understand periodically high winter losses of honey bee colonies. Apidologie, 41, 332-352, DOI: 10.1051/apido/2010014.
18. Gülmez, Y., Bursalı, A., & Tekin, Ş. (2009). First molecular detection and characterization of deformed wing virus (DWV) in honeybees (Apis mellifera L.) and mite (Varroa destructor) in Turkey. African Journal of Biotechnology. 8 (16), 3698-3702.
19. Hung, K-LJ., Kingston, J.M., Albrecht, M., Holway, D.A., & Kohn, J.R. (2018). The worldwide importance of honey bees as pollinators in natural habitats. Proceedings of the Royal Society B: Biological Sciences, 285, 20172140.
20. Kalayci, G., Cagirgan, A.A., Kaplan, M., Pekmez, K., Beyazit, A., Ozkan, B., Yesiloz, B., & Arslan, F. (2020). The role of viral and parasitic pathogens affected by colony losses in Turkish apiaries.Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 26 (5), 671-677.
21. Karapınar, Z., Oğuz, B., Dinçer, E., & Öztürk, C. (2018). Phylogenetic analysis of black queen cell virus and deformed wing virus in honeybee colonies infected by mites in Van, Eastern Turkey. Medicine Veterinary. 74 (7), 460-465.
22. Lanzi, G., De Miranda, J.R., Boniotti, M.B., Cameron, C.E., Lavazza, A., Capucci, L., Camazine, S. M., & Rossi, C. (2006). Molecular and biological characterization of deformed wing virus of honey bees (Apis mellifera L.). Journal of Virology, 80, 4998–5009.
23. Locke, B., Semberg, E., Forsgren, E., & de Miranda, J.R. (2017). Persistence of subclinical deformed wing virus infections in honeybees following Varroa mite removal and a bee population turnover. PLoS ONE. 12 (7), e0180910.
24. Maramorosch, K., & Shatkin, A. (2007). Honey bee viruses. Advances in Virus Research. Academic Press. 33-80.
25. Martin, S.J., Ball, B.V., & Carreck, N.L. (2013). The role of deformed wing virus in the initial collapse of varroa infested honey bee colonies in the UK. Journal of Apicultural Research, 52, 251–258.
26. Muz, D., Muz, & M.N. (2009). Survey of the occurrence of Deformed Wing Virus and multiple parasites of queens (Apis mellifera L.) in apiaries with collapsed colonies in Hatay, Turkey. Journal of Apicultural Research, 48(3), 204–208.
27. Muz, D., Muz, & M.N. (2017). Tekirdağ’da “Koloni Kaybı Sendromu” Benzeri Kayıp Görülen Arılıklarda Bazı Patojenlerinin Araştırılması. Kocatepe Veterinary Journal, 10(1), 21-28.
28. Muz, M.N., & Muz, D. (2008). Occurrence and genetic analysis of DWV (Deformed wing virus) and ABPV (Acute bee paralysis virus) and Varroa destructor-1 virus (VD-1) in Turkey. Proceedings of the 2nd International Forum of Apimondia-Apiquality, Villa Mondragone, Rome, Italy. pp. 94.
29. Posada-Florez, F., Childers, A.K., Heerman, M.C., Egekwu, N.I., Cook, S.C., Chen, Y., Evans, J.D., & Ryabov, E.V. (2019). Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner. Scientific Reports. 9. 12445. 10.1038/s41598-019-47447-3.
30. Reyes-Quintana, M., Espinosa-Montaño, L.G., Prieto-Merlos, D., Koleoglu, G., Petukhova, T., Correa-Benítez, A., & Guzman-Novoa, E. (2019). Impact of Varroa destructor and deformed wing virus on emergence, cellular immunity, wing integrity and survivorship of Africanized honey bees in Mexico. Journal Invertebrate Pathology, 164, 43-48.
31. Roberts, J.M.K., Anderson, D.L., & Durr, P.A. (2017). Absence of deformed wing virus and Varroa destructor in Australia provides unique perspectives on honeybee viral landscapes and colony losses. Scientific Reports, 7, 6925.
32. Rodríguez, M., Vargas, M., Antúnez, K., Gerding, M., Castro FO., & Zapata, N. (2014). Prevalence and phylogenetic analysis of honey bee viruses in the Biobío Region of Chile and their association with other honey bee pathogens. Chilean journal of agricultural research, 74(2), 170-177.
33. Rüstemoğlu, M. (2019). Uluslararası Arıcılık Araştırmaları ve Sürdürülebilir Kırsal Kalkınma Stratejileri Kongresi. 11-13 Ekim 2019. Bingöl, Türkiye.
34. Rüstemoğlu, M. (2020). Isolation and Characterization of Two Important Honeybee-Killing Virus Species, Deformed Wing Virus (DWV) and Black Queen Cell Virus (Bqcv) From Messor Concolorants (Hymenoptera: Formicidae). Applied Ecology and Environmental Research, 18, 7407-7415. 10.15666/aeer/1805_74077415.
35. Rüstemoğlu, M., & Sipahioğlu, H.M. (2016) : Occurrence and molecular characterization of acute bee paralysis virus (ABPV) in honeybee (Apis mellifera) colonies in Hakkari province. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi, 26, 174-182.
36. Rüstemoğlu, M., & Sipahioğlu, H.M. (2019). Occurrence and prevalence of six honey bee viruses in Hakkari (Turkey) and their genomic divergence. Munis Entomology & Zoology, 14 (2), 574-583
37. Ryabov, E.V., Fannon, J.M., Moore, J.D., Wood, G.R., & Evans, D.J. (2016). The Iflaviruses Sacbrood virus and Deformed wing virus evoke different transcriptional responses in the honeybee which may facilitate their horizontal or vertical transmission. Peer J. 4: e1591.
38. Schöning, C., Gisder, S., Geiselhardt, S., Kretschmann, I., Bienefeld, K., Hilker, M., & Genersch, E. (2012). Evidence for damage-dependent hygienic behaviour towards Varroa destructor parasitised brood in the western honey bee, Apis mellifera. Journal of Experimental Biology, 215, 264–271.
39. Shumkova, R., Neov, B., Sirakova, D., Georgieva, A., Gadjev, D., Teofanova, D., Radoslavov, G., Bouga, M., & Hristov, P. (2018). Molecular detection and phylogenetic assessment of six honeybee viruses in Apis mellifera L. colonies in Bulgaria. PeerJ. , 6: e5077.
40. Tentcheva, D., Gauthier, L., Jouve, S., Canabady-Rochelle, L., Dainat, B., Cousserants, F., Colin, M. E., Ball, B. V., & Bergoin, M. (2004). Polymerase chain reaction detection of deformed wing virus (DWV) in Apis mellifera and Varroa destructor. Apidologie, 35, 431– 439.
41. Tutkun, E., & Boşgelmez, A., (2003). Balarısı zararlıları ve hastalıkları teşhis ve tedavi yöntemleri. Bizim Büro Basımevi, Selanik Caddesi 18/11, Ankara.
42. Yue, C., Schroder, M., Gisder, S., & Genersch, E. (2007). Vertical-transmission routes for deformed wing virus of honeybees (Apis mellifera). Journal of General Virology, 88, 2329-2336.