Tissue-Resolved Virome Analysis of Natural Chronic Bee Paralysis Virus Outbreak in Apis mellifera Reveals Segment Stoichiometry Shifts and Divergent Covert Infections

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.