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Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.