Dietemann, Vincent

Hu, Fu-Liang

Neumann, Peter

Yañez, Orlando

Zheng, Huo-Qing

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A scientific note on Israeli acute paralysis virus infectionof Eastern honeybee Apis cerana and vespine predatorVespa velutinaOrlando YAÑEZ1, Huo-Qing ZHENG2, Fu-Liang HU2, Peter NEUMANN1,3,Vincent DIETEMANN1,41Swiss Bee Research Centre, Federal Department of Economic Affairs EVD, Research Station AgroscopeLiebefeld-Posieux ALP, Schwarzenburgstrasse 161, CH-3003 Bern, Switzerland2College of Animal Sciences, Zhejiang University, Hangzhou 310058, China3Department of Zoology and Entomology, Rhodes University, Grahamstown 6140, South Africa4Social Insect Research Group, Zoology and Entomology Department, University of Pretoria, Private Bag X20Hatfield 0028, Pretoria, South AfricaReceived 17 August 2011 – Revised 20 January 2012 – Accepted 6 February 2012Apis mellifera / Apis cerana / Vespa velutina / IAPV / virus / honeybeeThe Israeli acute paralysis virus (IAPV; Maori etal. 2007; de Miranda et al. 2010) is widespread inWestern honeybees (Apis mellifera) for whom itcan be a marker of colony losses (Cox-Foster et al.2007). It has also been found in other Hymenoptera(e.g., Vespula vulgaris; Singh et al. 2010), but theability of the virus to replicate in such alternativehosts still has to be confirmed, as has been shownrecently for the ectoparasite Varroa destructor (DiPrisco et al. 2011). We investigated whether IAPVcan be found in Western and Eastern (Apis cerana)honeybees and in their hornet predator Vespavelutina in China and whether this virus is able toreplicate in these organisms. Positive-strand RNAvirus replication requires the production ofnegative-strand RNA replicative intermediates.Detection of negative-strand RNA of IAPV inA. cerana and V. velutina would strongly supporttheir function as alternative hosts of this virus.Eastern honeybee workers (N=180) were sam-pled from six healthy colonies in Jinhua, ZhejiangProvince, China. Western honeybee workers (N=90) were obtained from three healthy colonies atZhejiang University in Hangzhou subject topredation by V. velutina wasps, which werecaught in flight (N=10). Samples were storedimmediately at −20°C until RNA extraction.RNAwas extracted from 30 pooled honeybeesper colony of each species and from the wholebodies of five V. velutina hornets. Extractions weredone with the total RNA Kit II (E.Z.N.A™)following the manufacturer's recommendations.The same kit was used for the separate extractionof the head, thorax and abdomen of the remainingfive V. velutina hornets in order to locate in whichbody parts these viruses occurred. The extractedRNA was eluted in 60 μL of RNAse-free water.Reverse transcription was performed using M-MLV RT Thermoscript® RT-PCR (Invitrogen)with 9 μL of extracted RNA using randomhexamer oligonucleotides as primers in 20 μL ofreaction final volume, following the manufacturerrecommendations. Tenfold dilutions of the result-ing cDNA were used for the PCR assays. IAPVdetection was performed using MyTaq™ reagentsin 50 μL solutions containing 2 μL templatecDNA, 10 μL of 5X reaction buffer, 2 μL of eachforward and reverse primers (10 μM) and 0.25 μLof Taq polymerase (1.25 U). The primers usedare described in Di Prisco et al. (2011) (sense: 5′-Corresponding author: V. Dietemann,vincent.dietemann@alp.admin.chManuscript editor: Peter RosenkranzApidologie (2012) 43:587–589* INRA, DIB and Springer-Verlag, France, 2012DOI: 10.1007/s13592-012-0128-yGCGGAGAATATAAGGCTCAG-3′; antisense:5′-CTTGCAAGATAAGAAAGGGGG-3′ gener-ating a PCR product of 586 bp). The PCR assaysconsisted of 2 min incubation at 95°C and 35cycles of 20 s at 95 °C for denaturation, 20 s at57°C for annealing, 30 s at 72°C extension andthen a final step of 7 min at 72°C. The specificityof individual PCR bands was confirmed bysequencing. Moreover, we analysed the samplesfor Kashmir bee virus (KBV) and acute beeparalysis virus (ABPV) (primers described inShen et al. 2005 and Gauthier et al. 2007,respectively) in order to verify the occurrenceof related viruses (de Miranda et al. 2010).For the detection of IAPV-negative-strandRNA, reverse transcription was performed usingM-MLV RT Thermoscript® RT-PCR (Invitrogen)at 55°C for 50 min followed by 70°C for 10 minfor inactivation in 20-μL volume. The taggedprimer (5′-agcctgcgcacgtggGCGGAGAATATAAGGCTCAG-3′; Di Prisco et al. 2011) wasused for the reverse transcription. The resultingcDNAs were purified for the excess of primers(Boncristiani et al. 2009) using the Kit Nucleo-Spin Extract II and eluted in 30 μL elutionbuffer. PCR was performed using MyTaq™reagents. The amplifications were performedunder the same conditions as for the diagnosticPCR assays using the Tag oligonucleotide (5′-AGCCTGCGCACCGTGG-3′; Yue and Genersch2005) as forward primer. PCR reactions ofpurified samples without Tag primer were in-cluded as negative control.IAPV was detected in all three A. melliferacolonies and in all ten V. velutina hornets, but inonly one A. cerana colony out of the six sampled(Figure 1). The sequences were compared withthat in accession number EF219380 (Maori et al.2007) confirming the identity of IAPV. KBV andABPV were not detected in our samples. IAPV-negative-strand RNA was not detected inA. cerana, but could be found in A. mellifera andin nine out of the ten V. velutina individuals,including their heads, thoraces and abdomens.Our data show that, in China, both Western andEastern honeybees as well as the hornet predator V.velutina are carriers of IAPV, the latter probablydue to foodborne contamination via infected honey-bee prey. The virus was rarely found in A. cerana,which could be due to the absence of IAPV innearby A. mellifera colonies that may have acted asreservoirs (Singh et al. 2010). The detection ofIAPV-negative-strand replicative intermediates inV. velutina strongly suggests that the virus repli-cates in the wasp. Given the presence of negative-strand RNA in the heads and thoraces of thepredator, it is unlikely that these replicative inter-mediates are of honeybee origin. If this were thecase, replicative intermediates from the preywould only be expected in the abdomen of thehornet.We detected evidence of IAPVreplication inA. mellifera, but not in A. cerana. The virus maynot be able to replicate in A. cerana although thesebees were carriers. We consider this unlikely giventhe closer phylogenetic proximity between the twohoneybee species than between A. mellifera andV. velutina or varroa in which evidence of replica-tion was found (this study; Di Prisco et al. 2011).Figure 1. IAPV detection and negative-strand detection. Lanes 1 and 12: size scales; lanes 2 to 4: detection ofIAPV in representative individuals of A. mellifera (Am), A. cerana (Ac) and V. velutina (Vv); lanes 5 to 8:detection of negative-strand RNA of IAPV in a representative individual of V. velutina (Vv) as well as heads (H),thoraces (T) and abdomens (A) of a pool of five V. velutina individuals. No negative-strand RNA of IAPV wasdetected in A. cerana (lane 9). NC PCR negative control (lane 10); PNC purification negative control (lane 11).588 O. Yañez et al.The detection of IAPV in A. cerana andV. velutina, together with that of various otherhoneybee viruses in predatory or non-predatoryHymenoptera (Anderson 1991; Celle et al. 2008;Dainat et al. 2009; Genersch et al. 2006; Singh etal. 2010), suggests that honeybee viruses canexpand to multiple non-Apis species. With thedetection of signs of replication of IAPVin a wasp,our results indicate that a non-Apis Hymenopteracould not only be a carrier (Singh et al. 2010), butalso an additional host for this honeybee virus. Ourresults and those of others suggest that the numberof pathogens shared between social insects andtheir associated arthropods has been considerablyunderestimated. Their role could be relevant forthe health of honeybees and other insects given thepotential increase of pathogen virulence followinghost shifts (Woolhouse et al. 2001).ACKNOWLEDGEMENTSWe wish to thank Xiao-Ling Su and Jin-Lian Zhangfor their help in the field, Qi-Yun Hua for logisticalassistance, Zhang-Gen Yu for access to his apiary forsampling and Benjamin Dainat for laboratory support.Financial support was provided to OY and PN by theCOST-funded project VIVA and toVDby the Qian-TangRiver expert programme. The funding agencies were notinvolved in study design; in the collection, analysis andinterpretation of data; in the writing of the report; and inthe decision to submit the paper for publication.Note scientifique sur l’infection par le virus israéliende la paralysie aiguë (IAPV) de l’abeille orientaleApis cerana et de son prédateur Vespa velutinaEine wissenschaftliche Notiz zur Infektion der östlichenHonigbiene Apis cerana und der predatorischenWespe Vespa velutina mit dem israelischen aktutenParalyse Virus (IAPV)REFERENCESAnderson, D.L. (1991) Kashmir bee virus—a relativelyharmless virus of honey bee colonies. Am. Bee J.131, 767–770Boncristiani, H.F., Di Prisco, G., Pettis, J.S., Hamilton, M.,Chen, Y.P. (2009)Molecular approaches to the analysisof deformed wing virus replication and pathogenesis inthe honey bee, Apis mellifera. Virol. J. 6, 221Celle, O., Blanchard, P., Olivier, V., Schurr, F., Cougoule,N., Faucon, J.P., Ribière, M. 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(2011) Varroa destructor is aneffective vector of Israeli acute paralysis virus inhoney bees, Apis mellifera. J. Gen. Virol. 92, 151–155Gauthier, L., Tentcheva, D., Tournaire, M., Dainat, B.,Cousserans, F., Colin, M.E., Bergoin, M. (2007)Viral load estimation in asymptomatic honey beecolonies using the quantitative RT-PCR technique.Apidologie 38, 426–435Genersch, E., Yue, C., Fries, I., de Miranda, J.R. (2006)Detection of Deformed wing virus, a honey bee viralpathogen, in bumble bees (Bombus terrestris andBombus pascuorum) with wing deformities. J.Invertebr. Pathol. 91, 61–63Maori, E., Lavi, S., Mozes-Koch, R., Gantman, Y.,Peretz, Y., Edelbaum, O., Tanne, E., Sela, I. (2007)Isolation and characterization of Israeli acute paral-ysis virus, a dicistrovirus affecting honeybees inIsrael: evidence for diversity due to intra- and inter-species recombination. J. Gen. Virol. 88, 3428–3438Shen, M., Cui, L., Ostiguy, N., Cox-Foster, D. 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A scientific note on Israeli acute paralysis virus infection of Eastern honeybee Apis cerana and vespine predator Vespa velutina

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A scientific note on Israeli acute paralysis virus infection of Eastern honeybee Apis cerana and vespine predator Vespa velutina

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