Reaction conditions for polymerase chain reaction (PCR) amplifications of select samples were performed under the following conditions: 5 mL of 1:10 dilution of PCR-amplified DNA and 10 pmol of each forward and reverse primers were amplified with Taq polymerase with the following cycling conditions: 95uC for 5 min; 95uC for 30 s, 50-60uC for 30 s, 72uC for 1 min, 35 cycles; final elongation 72uC for 7 min, hold at 4uC. Select samples were Sanger sequenced directly from ExoI and SAP treated PCR product or from colony PCR of TOPO cloned (Invitrogen) gel-extracted bands. Bands produced by PCR assays for known honey bee pathogens were sequenced until each molecular weight product was unambiguously associated with either a true positive or non-target amplification of the honey bee genome or microbiome. All PCR results for the four novel viruses were confirmed by Sanger sequencing.