The Prevalence of Seven Crucial Honeybee Viruses Using Multiplex RT-PCR and Their Phylogenetic Analysis

Turkish Journal of Veterinary and Animal Sciences Turk J Vet Anim Sci (2021) 45: 44-55 http://journals.tubitak.gov.tr/veterinary/ © TÜBİTAK Research Article doi:10.3906/vet-2004-139 The prevalence of seven crucial honeybee viruses using multiplex RT-PCR and their phylogenetic analysis 1, 2 Abdurrahman Anıl ÇAĞIRGAN *, Zafer YAZICI 1 Department of Virology, İzmir/Bornova Veterinary Control Institute, İzmir, Turkey 2 Department of Virology, School of Veterinary Medicine, Ondokuz Mayis University, Samsun, Turkey Received: 26.04.2020 Accepted/Published Online: 26.12.2020 Final Version: 23.02.2021 Abstract: Honey bee viruses may be of severe adverse effect on bee colonies. Till now, more than twenty honey bee viruses have been identified. The aim of this study was to investigate the prevalence of seven honey bee viruses, namely Israeli acute paralysis virus (IAPV), deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV) using a multiplex reverse transcription polymerase chain reaction (mRT-PCR). A total of 111 apiaries were randomly selected and adult bees and larvae samples were obtained from seemingly healthy colonies located at Aegean region of west Turkey. The presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), acute bee paralysis virus (ABPV), and sacbrood virus (SBV) was detected, while Israeli acute paralysis virus (IAPV) or Kashmir bee virus (KBV) couldn’t be detected in studied colonies. The results showed the virus with the highest prevalence was DWV followed by BQCV, ABPV, SBV, and CBPV. PCR products for DWV, BQCV, ABPV, SBV, CBPV were sequenced and compared to the Genbank database. The Turkish strains demonstrated heterogeneous populations of DWV, BQCV, and relatively homogenous populations of ABPV, CBPV, and SBV. Based on these results, SBV and CBPV had formed different branch and genotypes when compared to previous studies. In conclusion, this study provides information about the distinctive honey bee viruses for future researches in Turkey, which holds the largest number of managed colonies in Europe. Key words: Apis mellifera, deformed wing virus, black quenn cell virus, sacbrood virus, chronic bee paralysis virus, acute bee paralysis virus 1. Introduction virus (IAPV), deformed wing virus (DWV), sacbrood Turkey has tremendous beekeeping potential with 7.4 virus (SBV), acute bee paralysis virus (ABPV), black queen million colonies ranking just after China and India. cell virus (BQCV), Kashmir bee virus (KBV), and chronic Twenty-five percent of these colonies are located in the bee paralysis virus (CBPV) are thought to be primarily Aegean region, which has an important place in the world responsible for the majority of severe disease in apiaries of beekeeping [1]. around the globe [8,9]. IAPV, KBV, and ABPV belong Until now, approximately twenty-four different viruses to the genus Aparavirus, BQCV belongs to the genus affecting Apis genus have been identified [2]; this number Cripavirus, and both genera belong to the Dicistroviridae changes continuously with the identification of new family. SBV and DWV belong to the genus Iflavirus of the viruses such as Apis Rhabdovirus-1, Apis Rhabdovirus-2 Iflaviridae family, while CBPV is not classified yet [10,11]. [3], and Varroa orthomyxovirus-1 [4]. It is known that a Characteristic symptoms of DWV include shrunken large part of honey bee viruses is of single-stranded RNA, wings that make flight impossible, decreased body size, and while filamentous virus (AmFV) and Apis iridescent virus discoloration in adult bees [12–14]. SBV can infect either (AIV) have DNA genome. Apart from cronic bee paralysis larvae or adult bee, but it causes larval death [14]. ABPV virus that has an isometric shape, most of the other viruses leads to typical symptoms in brood with discoloured larvae have indistinguishable using particle morphology [5-7]. and paralysed adult bees [15]. BQCV causes mortality In general bee viruses belong to one of these two in queen larvae and pupae [16]. IAPV leads to rapidly families known as Iflaviridae and Dicistroviridae. They are progressing paralysis, trembling, inability to fly, darkening nonenveloped, single-stranded, and positive-sense RNA and loss of hair from the thorax and abdomen [7,17]. In viruses [6]. Seven viruses namely Israeli acute paralysis experimental infections, KBV is extremely lethal to adults * Correspondence: [email protected] 44 This work is licensed under a Creative Commons Attribution 4.0 International License. ÇAĞIRGAN and YAZICI / Turk J Vet Anim Sci and larvae without clearly defined disease symptoms The cycling conditions consisted of one cycle at 50 °C [16]. Characteristic symptoms of CBPV are paralysis and for 30 min for reverse transcription followed by initial trembling, hairless and shiny, sometimes black individuals denaturation at 95 °C for 7 min, followed by denaturation crawling at the hive entrance [5,18]. Although many bee at 95 °C 20 s, annealing at 58 °C for 20 s, extension at 72 viruses can cause fatal disease at the individual or colony °C for 1 min, for 45 cycles, with a final extension at 72 level, they usually persist and spread harmlessly at low °C for 10 min (Techne TC-412, United Kingdom). The titres within and between bee populations. Difficult results were analyzed using 2.5% agarose gel and staining identification, unknown disease progress, the diagnosis, with ethidium bromide in TAE buffer at 80 V for 80 min and management of disease progress of viral diseases cause (Thermo, USA), the photographs was taken by Vilber difficulties comparing to other bee pathogens [7]. Lourmat, France. Many bee viruses are common throughout the world. The PCR products were sequenced in Forward Also, bee viruses have been the subject of different studies and Reverse by Microsynt (Balgach, Switzerland). conducted in Turkey [19–23]. Turkey is located in both Nucleotide sequence results were assembled and edited Europe and Asia, and it is situated at the crossroads of using DNADynamo DNA sequence analysis software. the Balkans, the Middle East, and the Mediterranean. The consensus nucleotide sequences that were obtained Therefore, it is an important country in terms of the were verified using the basic local alignment search bilateral transmission of viruses in the area. Hence, in this tool (BLAST) at the National Central for Biotechnology study, we investigated seven honey bee viruses in local Information (NCBI) [26]. colonies in Turkey. In addition, a phylogenetic analysis was 2.3. Phylogenetic analysis performed to provide information about the geographic In order to determine phylogenetic relationships among distribution of different virus strains. strains of DWV, SBV, ABPV, BQCV, and CBPV viruses in Turkey, the following sequences were first identified: 2. Materials and methods 484 nt sequences encoding a helicase gene of DWV, 317 nt sequences encoding a polyprotein protein of SBV, 719 2.1. Collection of bee samples and isolation of total RNA nt sequences encoding a capsid protein of ABPV, 383 nt The samples were randomly collected at a 95% confidence sequences encoding a capsid protein of BQCV and 1293 interval and 3% expected prevalence from apiaries in nt sequences encoding a putative RNA-dependent RNA seven Aegean provinces (Aydın, Denizli, İzmir, Kütahya, polymerase (RdRP) of CBPV. The sequencing primers Manisa, Muğla, Uşak) using the multistep sampling are provided in Table. The other sequences, which had method [24] (Figure 1). A total of 111 apiaries, including been reported before, were obtained from GenBank. The 30 adult bees and 30 larvae from each apiary, were selected multiple sequence alignments of the data were performed in this method. A pool consisting of 30 adult bees and 30 using the ClustalW algorithm using MEGA 6 software. larvae from each apiary was created and homogenized Then, the best DNA/Protein models were identified to use with 9 mL Eagle Minimum Essential Medium (EMEM) for phylogeny. Phylogenetic trees were constructed using (Sigma–Aldrich Corp., Dorset, United Kingdom) and MEGA 6 software [32] with the maximum likelihood (ML) then centrifuged at 3500 rpm and 4 °C for 30 min. The and neighbor-joining (NJ) methods with the Tamura-3 supernatant was stored at –80°C until analyzed. parameter. The best substitution models were selected Samples were collected between March 2017 and for each tree, a bootstrap value of 1000 replicates for all August 2017. There were no overt clinical symptoms in methods. any of the collected bee samples. The presence of bacterial, Amino acid distances were calculated using the parasitic, and fungal diseases was not checked. Jones–Taylor–Thornton method with gamma distribution For RNA extraction, 200 µL of supernatant was used. (JTT+G), and a bootstrap value of 1000 replicates [32,33]. Total RNA extraction was carried out using High Pure Similarity and identity of amino acid and nucleotide were Viral RNA Kit (Roche, Germany) in accordance with the determined using an online programme (imed.med.ucm. manufacturer’s instructions. es/Tools/sias.html). 2.2. mRT-PCR amplification and nucleotide sequencing The multiplex PCR method developed by Cagirgan and 3. Results Yazici [25] was used in this study. The final optimum 3.1. Virus detection reaction mixture consisted of 18.5 µL water, 10 µL reaction For adult bees from 111 apiaries sampled, 22 were positive buffer (Roche), 7 µL forward primer (0.4 µM each primers), for DWV. In this study, DWV was the most frequently 7 µL reverse primer (0.4 µM each primers), 1.5 µL MgCl2 detected viral agent in the apiaries, occurring in 19.8% of (2 mM), 5 U/µL Taq polymerase (Roche) and 5 µL RNA those tested. BQCV was detected in 20 apiaries, and it was (samples), in a total volume of 50 µL. the second most prevalent viral agent with a prevalence 45 ÇAĞIRGAN and YAZICI / Turk J Vet Anim Sci Figure 1.

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