Haoran Qin

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.

Chronic bee paralysis virus (CBPV) is a bipartite positive-sense RNA virus that causes significant colony losses in honey bees, yet its tissue-level infection dynamics remain poorly characterized. We performed tissue-resolved virome analysis of two naturally infected Apis mellifera apiaries during a CBPV outbreak in Shandong, China. The apiaries shared a queen source and overwintering site but were separated by over 80 km at the time of sampling and developed contrasting disease patterns. Symptomatic foragers showing characteristic clinical signs of CBPV infection, including trembling, flightlessness, crawling, and hairless dark cuticle, and asymptomatic foragers were collected. Head, thorax, and midgut tissues were then dissected and used for RNA sequencing, yielding 36 RNA-seq libraries. In symptomatic bees, CBPV dominated the virome, with viral loads highest in heads and lowest in midguts. The RNA1/RNA2 copy ratio revealed a conserved tissue-specific gradient, shifting from RNA2 predominance in heads and thoraces to RNA1 enrichment in midguts (p = 0.024 for each apiary). This directional imbalance was validated by multiple controls and conserved across two genetically distinguishable CBPV populations, implicating the host tissue environment as the primary driver. In asymptomatic bees, the two apiaries harbored markedly different covert viromes, with CBPV dominant in the heads and thoraces of one apiary and near-exclusive Lake Sinai virus dominance in the other. These findings reveal that CBPV segment stoichiometry is modulated in a tissue-dependent manner in vivo and that viral genetic composition and covert virome composition can diverge notably between epidemiologically connected apiaries, providing additional insight into the infection biology of CBPV under natural conditions.