CC BY
0TRACKING THE VIRAL THREAT TO UZBEKISTAN'S HONEY
BEES IN SPRING 2024
1Minhyeok Kwon, 2Sobirov Sokhibkhon Shomurhon Ugli, 3Abdurakhimov Abdulaziz Abdujalil Ugli, 4Sun-Ho Kwon, 5Kaxramanov Boymaxmat Abdiazizovich, 6Chuleui Jung,
7Eui-Joon Kil
1Graduate Student, Andong National University 2'3Student, Tashkent State Agrarian University 4Graduate Student, Andong National University 5Tashkent State Agrarian University, Professor 6Andong National University, Professor 7Andong National University, Associate Professor https://doi.org/10.5281/zenodo.11428830
Abstract. The health of honey bees, a cornerstone of the beekeeping industry, is under threat from various viruses that can cause symptoms ranging from wing deformities to paralysis, thereby reducing productivity. This study focuses on the detection and analysis of seven major honey bee viruses in Uzbekistan, a critical region on the Silk Road that could play a significant role in the transmission of pathogens due to its strategic location and active exchange with neighboring countries. Utilizing multiplex one-step RT-PCR, we conducted diagnostics on samples collected from 20 sites across four regions in Uzbekistan during the spring of2024. Our findings reveal the presence of black queen cell virus in all samples and deformed wing virus in nearly all, with regional variations in virus occurrence. Notably, chronic bee paralysis virus and sacbrood virus were detected in specific regions, indicating potential localized spread. Sequence analysis showed high identity with previous isolates from Uzbekistan, suggesting regional transmission patterns. These results highlight the urgent need for comprehensive research on honey bee viruses in Uzbekistan and underscore the importance of monitoring these pathogens to safeguard the apiculture industry and biodiversity.
Key words: detection, honey bee, RT-PCR, Uzbekistan, virus.
In the beekeeping industry, the health of honey bees is a critical factor, and viruses are a significant pathogen impacting their well-being (Gisder and Genersch, 2015). Infected honey bees often exhibit symptoms such as wing deformities and paralysis, which directly reduce their productivity (de Miranda et al., 2010; de Miranda and Genersch, 2010; Genersch and Aubert, 2010). However, in some cases, honey bees show no external signs of infection, leading to the detection of multiple viral infections in seemingly healthy colonies upon diagnosis (Amiri et al., 2020). Reports on honey bee viruses are increasingly being confirmed with the revolutionary advancements in diagnostic technologies (Beaurepaire et al., 2020). As with other viruses, molecular diagnostic methods based on nucleic acid amplification, such as (RT-)PCR, and serological diagnostic methods based on the interaction between viral proteins and their corresponding antibodies, are primarily used (Cassedy et al., 2021; Cuc et al., 2021). Virome analysis based on Next-Generation Sequencing (NGS) technology has made it easier to identify the presence of various known and novel viruses within individual honey bees, leading to the continual reporting of novel viruses such as the Lake Sinai virus (Kwon et al., 2023; Runckel et al., 2011). Furthermore, the ease of virus detection has facilitated the observation of international movement patterns of various viruses, confirming that these pathogens are not confined to specific regions but are emerging across continents. The spread of these viruses is presumed to be driven
by the movement of infected honey bee colonies or by vectors such as mites that can transmit several key viruses (McMenamin and Genersch, 2015).
Situated at the heart of the Silk Road, bridging Asia and Europe, Uzbekistan could serve as a pivotal intermediary in the transmission of pathogens. Additionally, the active exchange with Russia and Asian countries further amplifies the potential for virus mediation. Despite this significance, research on honey bee pathogens in Uzbekistan has been scant. The significance of researching honey bee virus patterns in Uzbekistan is not only vital for the country's apiculture industry but also for understanding the spread of viruses in neighboring nations and beyond, into Europe and Asia.
We conducted diagnostics for seven major species of viruses [acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV) and sacbrood virus (SBV)] in honey bee (Apis mellifera) samples collected in the spring of 2024 from 20 sites of four regions in Uzbekistan: Samarqand, Jizzax, Navoiy, and Qashqadaryo (Table 1).
Table 1. Sample information
No. Collection (label) site Number repetitions of Circumstance Coordinate
1 Agricultural area 40°05'03.0"N 67°48'26.0"E
2 Agricultural area 40°04'51.0"N 67°53'26.0"E
3 Jizzax (Ji) 5 Urban area 40°08'26.0"N 67°47'46.0"E
4 Urban area 40°08'47.0"N 67°45'16.0"E
5 Urban area 40°08'43.0"N 67°45'21.0"E
6 Urban area 40°06'36.2"N 65°20'57.3"E
7 8 Navoiy (Na) 4 Urban area Agricultural area 40°07'42.0"N 65°22'13.9"E 40°01'48.8"N 64°48'16.3"E
9 Urban area 40°04'51.4"N 65°22'08.9"E
10 Urban area 39°37'43.0"N 67°02'32.0"E
11 Urban area 39°37'44.0"N 67°02'32.0"E
12 Samarqand (Sa) 5 Urban area 39°37'45.0"N 67°02'32.0"E
13 Agricultural area 39°28'34.0"N 67°02'24.0"E
14 Agricultural area 39°25'28.0"N 67°02'20.0"E
15 Agricultural area 38°57'18.0"N 66°45'10.0"E
16 Agricultural area 38°57'34.0"N 66°45'19.0"E
17 18 Qashqadaryo (Ka) 6 Agricultural area Agricultural area 38°59'55.0"N 66°38'54.0"E 38°59'55.0"N 66°38'38.0"E
19 Agricultural area 38°59'31.0"N 66°39'12.0"E
20 Agricultural area 38°59'15.0"N 66°39'13.0"E
Pooling was performed by randomly selecting two honey bees from each of the five hives sampled from each farm, resulting in a total of ten bees per pooling group, followed by the extraction of total RNA using the TRIzol® protocol. The quality of the extracted RNA was measured with a NanoPhotometer® NP80 (IMPLEN, Munich, Germany). Virus diagnostics from the prepared RNA were carried out via multiplex one-step RT-PCR, utilizing SuPrimeScript
RT-PCR Premix (2X) (GENETBIO, Daejeon, South Korea), employing eight specific primers for seven viruses as introduced in Table 2. During multiplex RT-PCR, the eight primers were grouped into three based on the length of the amplification products, and the results were verified through agarose gel electrophoresis.
Table 2. Primer sets used in this study
Virus Sequence (5' ^ 3') Tm (°C) Size (bp)
ABPV TGAGAACACCTGTAATGTGGGT GGCAGGTATAGTATTTCGGAACG 56 1271
BQCV AAATGCCAATGTGGACCAAA GATAGGGCTGCTATCCACCG 56 370
CBPV RNA1 CCTCCCGTCATGATTTCCCC TGCATTGTCATTGCTGGAAC 56 974
CBPV RNA2 CGTTATCTCAGGTCCGGCAA GAGCAAAGACGCTGAGGAGA 56 612
DWV CGGTGCGACTGAAACTTCTA CATACGTTCTTGCTCCAGCG 56 610
IAPV ATTCCTGTGTCGGAGCAGTG CAAAGTATCCTCAAGTTGTGGG 56 870
KBV GATGAACGTCGACCATTGA TGTGGGTGGCTATGAGTCA 56 414
SBV GGAGGCCTGGGAAAAGAGTG TTCCAACTGCACCACAGGTT 56 535
Amplification products identified were subjected to Sanger sequencing (Macrogen, Seoul, South Korea) for base sequence verification, and the outcomes were analyzed using NCBI BLAST and CLC genomics workbench (version 24.0.1).
According to the RT-PCR results, ABPV, IAPV, and KBV were not detected in samples from any region, whereas BQCV was found in all samples. DWV was also detected in almost all samples, except for two. CBPV was only identified in the Jizzax and Navoiy regions, and SBV was found exclusively at two sites in the Navoiy region (Table 3). Although further analysis on more samples is required, these results indicate regional variations in the occurrence of viruses.
Table 3. Results of RT-PCR diagnostics for seven virus species at 20 sites secured in this study
Virus Jizzax Navoiy Samarqand Qashqadaryo
1 2 3 4 5 1 2 3 4 1 2 3 4 5 1 2 3 4 5 6
ABPV N N N N N N N N N N N N N N N N N N N N
BQCV D D D D D D D D D D D D D D D D D D D D
CBPV RNA1 D D D D N D D D D N N N N N N N N N N N
CBPV RNA2 D D D D N D D D D N N N N N N N N N N N
DWV D D D D D D D D D D D D D N D D N D D D
IAPV N N N N N N N N N N N N N N N N N N N N
KBV N N N N N N N N N N N N N N N N N N N N
SBV N N N N N N D D N N N N N N N N N N N N
* 'D' indicates a positive result, and 'N' indicates a negative result for each virus
Although only partial sequences were analyzed, when the PCR amplification products of each virus were examined through Sanger sequencing, it was confirmed that they showed the highest identity with the sequences identified by our team in Uzbekistan in 2022. DWV exhibited a 98% identity with the isolates from Yanghikurgan (OR912391.1) and Galvasay (OR912375.1), and BQCV showed around 98% similarity with the Yanghikurgan isolates (OR912388.1 and OR912392.1) (Table 4). CBPV, which had not been detected by this research team previously, was identified for the first time in Uzbekistan; for RNA1, sample 1 showed 94.24% similarity with an isolate collected from honey bees in Chongqing, China, in 2019 (MZ821995.1), and sample 2 was 96.04% similar to an isolate separated in Slovenia in 2010 (KY937971.1). RNA2 was 98% similar to an isolate separated from honey bees in Austria in 2018 (MK637523.1). While there may be some differences when analyzing the complete genome sequence, the regional characteristics bridging European and Asian countries were partially reflected in the sequence similarities.
Table 4. BLAST results for the amplicons in this study
Sampl Max Tota l Scor e Quer y Cove r E value Per.
Virus e Name Accession Isolate Scor e Ident
Ji2 OR912375. 1 Galvasay (U1) 1051 1051 99% 0 98.33 %
OR912391. 1 Yanghikurgan (U4) 1046 1046 99% 0 98.16 %
KY909333. 1 Vespa_crabro_DWV_PI_20 16 1024 1024 99% 0 97.50 %
OR361537. 1 DWV-A-OR-OCT2018-EVR24 1024 1024 99% 0 97.50 %
MH267696. 1 DWV_MS 1013 1013 99% 0 97.16 %
DWV
Sa2 OR912391. 1 Yanghikurgan (U4) 985 985 100% 0 98.06 %
OR912375. 1 Galvasay (U1) 985 985 100% 0 98.06 %
KY909333. 1 Vespa_crabro_DWV_PI_20 16 957 957 100% 0 97.18 %
OR361537. 1 DWV-A-OR-OCT2018-EVR24 957 957 100% 0 97.18 %
OR496436. 1 Bonghwa 952 952 100% 0 97.00 %
Na1 OR912392. 1 Yanghikurgan (U4) 536 536 99% 1.00E -147 98.05 %
BQC OR912388. 1 Yanghikurgan (U3) 531 531 99% 6.00E -146 97.73 %
V OR496415. 1 Uiseong 508 508 99% 3.00E -139 96.43 %
MZ821800. 1 BQCV_No1_Acc003-BJ2019 508 508 99% 3.00E -139 96.43 %
Qa1
Na1
CBP V
RNA 1
Na2
Na1
CBP V
RNA
2
Na2
MZ821804. 1
BQCV_No5_Acc019-SD2017
505 505 99%
4.00E -138
96.10
%
OR912392. 1
OR912388. 1
KY741959. 1
OR496405. 1
OR496415. 1
Yanghikurgan (U4)
Yanghikurgan (U3)
GS1
Andong
Uiseong
224 224 100%
224 224 100%
207 207 100%
207 207 100%
206 206 99%
4.00E
-54
4.00E
-54
4.00E
-49
4.00E
-49
1.00E
-48
MZ821995. 1
MZ821969. 1
MZ821963. 1
MZ821979. 1
OK491520. 1
CBPV_No22_Am066-CQ2019
CBPV_No9_Am010-GS2018
CBPV_No6_AM-AH-2017-
3
CBPV_No14_Am025-HLJ2017
known 11
1221 1221 99% 0
1218 1218 99% 0
1201 1201 99% 0
1199 1199 99% 0
1199 1199 99% 0
KY937971. 1
ON648749. 1
OR582946. 1
OR582954. 1
OR582951. 1
M92/2010
CBPV RNA1 376/2020
FR20HM439
FR20PA427.1
FR19HM425.0'
1439 1439 99% 0
1428 1428 99% 0
1406 1406 99% 0
1400 1400 99% 0
1397 1397 99% 0
MK637523. 1
KY937972. 1
ON648750. 1
ON648752. 1
MZ821976. 1
AUT-17 segment RNA2, complete sequence
M92/2010
CBPV RNA2 376/2020
CBPV RNA2 341/2019
CBPV_No12_Am020-HB2019
1066 1066 99% 0
1055 1055 99% 0
1055 1055 99% 0
1050 1050 99% 0
1005 1005 99% 0
MK637523. 1
KY937972. 1
ON648750. 1
AUT-17
M92/2010
CBPV RNA2 376/2020
1022 1022 100% 0
1011 1011 100% 0
1011 1011 100% 0
98.44
%
98.44
%
96.09
%
96.09
%
96.06
%
94.24
%
94.12
%
93.74
%
93.73
%
93.74
%
96.04
%
95.81
%
95.36
%
95.25
%
95.02
%
98.83
%
98.49
%
98.49
%
98.33
%
96.99
%
98.61
%
98.27
%
98.27
%
°N648752. CBPV RNA2 341/2019 1005 1005 100% 0 98 09 1 %
MZ821976. CBpV_No 12_Am020- 944 944 100% 0 96.19
1 HB2019 %
The recent findings underscore the significance of research on honey bee pathogens, which has been inadequately conducted in Uzbekistan and neighboring countries, despite the importance of the apiculture industry. Building upon these results, further research—including virome analysis to obtain the full-length genome nucleotide sequences of the viruses identified in this study, and to check for the emergence of novel or mutant strains—could be a valuable asset in tracking the spread of honey bee viruses.
Acknowledgments. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (No. NRF-2022K1A3A9A05036394).
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