Low-Frequency iSNVs Reveal Cryptic CBPV Transmission Between Epidemiologically Linked Apiaries

Chronic bee paralysis virus (CBPV) is a serious pathogen of honey bees and has expanded across major beekeeping regions in recent years. Understanding viral connectivity between apiaries is therefore essential for disease control. However, consensus-based phylogenetic analysis has limited power to resolve epidemiological links between closely managed colonies. To address this, we applied virome sequencing and intra-host single-nucleotide variant (iSNV) profiling to investigate shared variation and possible viral exchange between two apiaries across two consecutive years. One apiary was translocated ~80 km away each spring and returned each winter to within ~1 km of a stationary apiary. This seasonal translocation mirrors the long-distance movements and winter aggregations common in commercial beekeeping operations in China and the United States, providing a natural microcosm of the migratory practices that drive large-scale viral connectivity. Parallel 16S rRNA gene profiling of the midgut microbiota revealed no detectable association between bacterial community composition and CBPV infection status. iSNV analysis identified extensive shared low-frequency variants between the two apiaries across both years, including formerly private variants that became detectable in both sites after winter co-location. In contrast, consensus phylogenies placed two co-located apiaries in separate clades, suggesting that standard surveillance may underestimate cryptic viral connectivity. We cannot rule out the possibility that the observed shared iSNVs reflect shared ancestry, earlier contact, or common external sources. Furthermore, the direction of viral exchange cannot be determined from observational data with limited replication and uneven sequencing depth. These results show that finer-scale iSNV profiling highlights patterns of viral connectivity in managed bee systems, providing a model for high-resolution surveillance of RNA viruses in mobile managed host populations where conventional methods underestimate connectivity, though validation in controlled experimental settings is needed to establish causal transmission pathways.