Big Sioux River virus. A second novel dicistrovirus, designated Big Sioux River Virus (BSRV) after its place of discovery, is most similar to the Rhopalosiphum padi Virus (RhPV). Four contigs of size 1473, 861, 1164 and 1311 nt (GenBank JF423195-8) derived from the non-structural region, the IRES, and the capsid gene. BSRV shares low amino acid identity with RhPV; only 78% in the non-structural region and 69% in the capsid gene. This level of amino acid divergence is consistent with the taxonomic rank of a new species ( Figure S2 ). Twenty-eight incidences of BSRV were detected from 197 time-course samples by specific PCR with most individual colony detections occurring in samples collected from April to July 2009 in Mississippi and South Dakota. Incidence was low from October onwards ( Figure 3B ). Peak abundance was 7.64610 3 copies per 100 ng of RNA sample (approximately 3.8610 6 copies per bee) and occurred in August ( Figure 4 ). Of note, BSRV associated significantly with Nosema apis infections (p = 0.003, OR 6.0) and also with ALPV-Brookings (p = 0.014, odds ratio (OR) = 4.5).