Positive sense RNA viruses, like LSV 1 and 2, utilize a negative strand template to produce viral genome copies, therefore detection of the negative-strand intermediate is indicative of an actively replicating infectious virus [39, 64, 65] . We used negativestrand specific RT-PCR to detect the replicative forms of both LSV1 and LSV2 ( Figure S5 ). cDNA synthesis reactions were performed using tagged negative strand-specific LSV1 and 2 primers followed by exonuclease I digestion of excess unincorporated RT-primers [65] (Materials and Methods and Table S2 ). PCR amplification using a tag-specific forward primer and LSVspecific reverse primers confirmed the presence of the replicative forms of both LSV1 and LSV2 in the July RNA sample ( Figure  S5 ). Together, this data and the abundance of LSV1 and 2, compared to other significant honey bee viruses, suggests that LSV1 and LSV2 are novel honey bee viruses that may play significant roles in colony health.