The use of RNA-dependent RNA polymerase for the taxonomic assignment of Picorna-like viruses (order Picornavirales) infecting Apis mellifera L. populations

Background: Single- stranded RNA viruses, infectious to the European honeybee, Apis mellifera L. are known to reside at low levels in colonies, with typically no apparent signs of infection observed in the honeybees. Reverse transcription- PCR ( RT- PCR) of regions of the RNA- dependent RNA polymerase ( RdRp) is often used to diagnose their presence in apiaries and also to classify the type of virus detected. Results: Analysis of RdRp conserved domains was undertaken on members of the newly defined order, the Picornavirales; focusing in particular on the amino acid residues and motifs known to be conserved. Consensus sequences were compiled using partial and complete honeybee virus sequences published to date. Certain members within the iflaviruses, deformed wing virus ( DWV), Kakugo virus ( KV) and Varroa destructor virus ( VDV); and the dicistroviruses, acute bee paralysis virus ( ABPV), Israeli paralysis virus ( IAPV) and Kashmir bee virus ( KBV), shared greater than 98% and 92% homology across the RdRp conserved domains, respectively. Conclusion: RdRp was validated as a suitable taxonomic marker for the assignment of members of the order Picornavirales, with the potential for use independent of other genetic or phenotypic markers. Despite the current use of the RdRp as a genetic marker for the detection of specific honeybee viruses, we provide overwhelming evidence that care should be taken with the primer set design. We demonstrated that DWV, VDV and KV, or ABPV, IAPV and KBV, respectively are all recent descendents or variants of each other, meaning caution should be applied when assigning presence or absence to any of these viruses when using current RdRp primer sets. Moreover, it is more likely that some primer sets ( regardless of what gene is used) are too specific and thus are underestimating the diversity of honeybee viruses.