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Clinical and Diagnostic Laboratory Immunology, January 2001, p. 93-104, Vol. 8, No. 1
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.1.93-104.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Sacbrood Virus of the Honeybee (Apis mellifera): Rapid Identification and Phylogenetic Analysis Using Reverse Transcription-PCR

Elvira Grabensteiner,1 Wolfgang Ritter,2 Michael J. Carter,3 Sean Davison,4 Hermann Pechhacker,5 Jolanta Kolodziejek,1 Otto Boecking,6 Irmgard Derakhshifar,7 Rudolf Moosbeckhofer,7 Elisabeth Licek,8 and Norbert Nowotny1,*

Institute of Virology1 and Institute for Fish and Bee Diseases, University of Veterinary Sciences, A-1210 Vienna8, and Institute for Apiculture, Federal Office and Research Centre of Agriculture, A-1226 Vienna,7 Austria; Department of Bee Pathology, Institute of Animal Health, D-79108 Freiburg, Germany2; School of Biological Sciences, University of Surrey, Guilford, Surrey GU2 7XH, United Kingdom3; Department of Microbiology, University of the Western Cape, 7535 Bellville, South Africa4; Institute for Apiculture, Federal Office and Research Centre of Agriculture, A-3293 Lunz, Austria5; and Institute for Apiculture, University of Bonn, D-53127 Bonn, Germany6

Received 13 July 2000/Returned for modification 17 October 2000/Accepted 27 October 2000

Sacbrood virus (SBV) infects larvae of the honeybee (Apis mellifera), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.

* Corresponding author. Mailing address: Institute of Virology, University of Veterinary Sciences, Veterinärplatz 1, A-1210 Vienna, Austria. Phone: 43 1 25077 2304. Fax: 43 1 25077 2390. E-mail: Norbert.Nowotny{at}vu-wien.ac.at.

Clinical and Diagnostic Laboratory Immunology, January 2001, p. 93-104, Vol. 8, No. 1
1071-412X/01/$04.00+0   DOI: 10.1128/CDLI.8.1.93-104.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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