Detection of Honeybee Viruses in Northern Thailand
DetectionDetection ofof HoneybeeHoneybee VirusesViruses inin NorthernNorthern ThailandThailand
PanuwanPanuwan ChantawannakulChantawannakul andand SirikarnSirikarn SanpaSanpa
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand *[email protected] A bee tree (Bombax sp.)of A. dorsata
Modern beekeeping Sampling sites Six honey bee viruses were surveyed using the RT-PCR and real-time PCR technique to obtain an overview of bee viruses which are spreading through the apiaries in our region. • Acute bee paralysis virus (ABPV) • Black queen cell virus (BQCV) • Chronic bee paralysis virus (CBPV) • Deformed wing virus (DWV) • Kashmir bee virus (KBV) • Sacbrood virus (SBV) Sample collection
Bees
Alcohol 70% RNA extraction PrimersPrimers usedused forfor PCRPCR diagnosisdiagnosis
Virus Forward primer Reverse primer Length reference (bp) ABPV TGAGAACACCTGTA ACCAGAGGGTTGAC 452 Tentcheva et ATGTGG TGTGTG al., 2004b BQCV GGACGAAAGGAAG ACTAGGAAGAGACT 424 Tentcheva et CCTAAAC TGCACC al., 2004b CBPV AGTTGTCATGGTTA TCTAATCTTAGCAC 455 Ribiére et al., ACAGGATACGAG GAAAGCCGAG 2002 DWV TTTGCAAGATGCTG GTCGTGCAGCTCGA 395 Tentcheva et TATGTGG TAGGAT al., 2004a KBV GATGAACGTCGACC TGTGGGTTGGCTAT 393 Stoltz et al., TATTGA GAGTCA 1995 SBV GGATGAAAGGAAAT CCACTAGGTGATCC 426 Tentcheva et TACCAG ACACT al., 2004b PCRPCR reactionreaction •• InnitialInnitial denaturationdenaturation 9595 °°CC 22 min
•• DenatureDenature 9595 °°CC 3030 s •• AnnealingAnnealing 5656 °°CC 4545 s 4040 cycles •• ExtensionExtension 7272 °°CC 1 1 min
•• FinalFinal extensionextension 7272 °°CC 10 10 min for KBV, annealing temperature was 47 °°CC PercentagesPercentages ofof apiariesapiaries infectedinfected withwith honeybeehoneybee viruses.viruses.
Lamphun province (9) Lampang province (9)
100 100 80 80 60 adult 60 adult 40 pupae 40 pupae 20 20 %of apiaries % of apiaries %of 0 0 ABPV BQCV CBPV DWV KBV SBV ABPV BQCV CBPV DWV KBV SBV virus virus
Chiang Rai province (5) Phayao province (8)
100 100 80 80 60 adult 60 adult 40 pupae 40 pupae 20 20 % of apiaries %of % of apiaries of % 0 0 ABPV BQCV CBPV DWV KBV SBV ABPV BQCV CBPV DWV KBV SBV virus virus Nan province (6) Phrae province (5)
100 100 80 80 60 adult 60 adult 40 pupae 40 pupae 20 20 % of apiaries %of % of apiaries of % 0 0 ABPV BQCV CBPV DWV KBV SBV ABPV BQCV CBPV DWV KBV SBV virus virus
Chiang Mai province (4)
100 80 60 adult 40 pupae 20 % of apiaries %of 0 ABPV BQCV CBPV DWV KBV SBV virus 63 70 60 46 adult 50 33 % 40 pupae 30 20 20 11 10 4 2 4 0 DWV ABPV SBV KBV
virus
Detection of ABPV, DWV, KBV and SBV in honeybee from 46 Thai apiaries. FrequenciesFrequencies ofof simultaneoussimultaneous virusvirus infectionsinfections inin honeybeehoneybee samples.samples.
No. of Type of infection No. of Percentage Origin viruses sample 0 8 17 1LPU, 3LPA, 1PY, 2PH, 1CM 1 ABPV 7 15 6PY, 1CM DWV 15 33 8LPU, 2LPA, 2NAN, 3PH 2 ABPV,DWV 8 17 1CR, 1PY, 4NAN, 2CM 3 ABPV, DWV, KBV 1 2 1LPA ABPV, DWV, SBV 5 11 1LPA, 4CR
4 ABPV, DWV, KBV, SBV 2 4 2LPA
LPU, Lamphun; LPA, Lampang; CR, Chiang Rai; PY, Phayao; NAN, Nan; PH, Phrae; CM, Chiang Mai. 4% 13% 17%
free from virus 1 virus 2 viruses 17% 3 viruses 4 viruses
49%
Lampang
ABPV, DWV, KBV, SBV Virus Accession Homologous % Homology no. region
Acute bee paralysis EF219380 RNA polymerase 97 virus gene
Deformed wing AJ489744 gene for 94 virus polyprotein
Kashmir bee virus AF197908 RNA polymerase 96 gene
Sacbrood virus AF092924 polyprotein 91 Phylogenetic tree illustrating the genetic relationship among SBV strains generated by the neighbor-joining program. Apis cerana bee
• Fewer viruses found
– Black Queen Cell virus (BQCV)
– Deformed Wing Virus (DWV) Real time PCR (Apis cerana)
PCR Amp/Cycle Chart BQCV
DWV TropilaelapsTropilaelaps mitemite
PPrimersrimers design Internal positive control (Tropilaelaps mites) Nuclear ribosomal (ITS1-5.8S-ITS2) gene sequences Tropilaelaps clareae EF025474 Tropilaelaps koenigerum EF025475 Tropilaelaps mercedesae EF025471 Tropilaelaps thaii EF025477 TropilaelapsTropilaelaps mitemite
IPC
DWV TropilaelapsTropilaelaps mitemite Sample number Mean Ct values (±SD) DWV Internal control (ITS) T1 32.48 (±0.6222) 19.50
T2 32.63 (±0.0565) 24.77 (±0.1272)
T4 30.94 (±0.3818) 20.94
T5 38.22 22.65 (±0.3111)
T6 33.36 (±0.1262) 23.00 (±0.1343) Conclusions
• The most prevalent virus in adult populations during our survey was DWV, which occurred in all seven provinces and in 33% of all samples, followed by ABPV (20%), SBV (4%) and KBV (2%). • Similarly in pupae, DWV was found in 63% of all apiaries; ABPV, 46%; SBV, 11%, and KBV, 4%.
• In some sites of the Lampang province, more than one virus could be detected; DWV, ABPV, SBV and KBV. • Twenty of 46 apiaries (43%) were infested with either V. destructor or T. clarae and 15 of 46 (33%) were infested with both mites.
• Fewer viruses found in cerana bees . • T. clarae carried DWV . ThankThank youyou
• Thailand Research Fund and The Commission on Higher Education
• Department of Biology, Faculty of Science, Chiang Mai University
• All students in Bee protection center, Chiang Mai University
• Beekeepers and Northern Beekeeper Association of Thailand
• National Bee Unit, Central Science Laboratory, United Kingdom