Molecular diagnosis of Kashmir bee virus infection

SUMMARYMolecular protocols were applied to the diagnosis of Kashmir bee virus (KBV) infection in the honey bee, Apis mellifera. Procedures based on either serology (chemiluminescent Western blotting) or nucleic acid amplification (RT-PCR, reverse transcription-polymerase chain reaction) proved to be very effective, although it was possible in some circumstances to detect viral infection simply by Coomassie blue staining of SDS-PAGE gels. Under laboratory conditions, KBV infections were readily distinguished from those induced by acute paralysis virus, a closely related isolate, by using monospecific antisera raised against the putative picornavirus-like VP4 polypeptide. Since only a very small amount of material was required for either Western blotting or RT-PCR, a single bee could in theory be examined for the presence of a variety of different pathogens. PCR primers based on sequences from the viral RNA polymerase gene amplified a 417 bp amplicon from KBV-infected, but not healthy, pupae. The same primers generated an amplicon of identical size from preparations of both acute paralysis virus and black queen cell virus, suggesting possible contamination with KBV.