Abstract
BACKGROUND: Honey bees (Apis mellifera) are essential pollinators that support global food production and economic stability. Their health and survival are threatened by diminishing floral resources, pesticide exposure, parasitic mites, and microbial and viral diseases. Among these stressors, viral infections are particularly challenging, often exacerbated by the parasitic mite Varroa destructor, a competent vector of multiple honey bee viruses. Understanding the mechanisms underlying honey bee-virus interactions is critical for mitigating the negative impact of infections on colony health. One understudied aspect is the role of microRNAs (miRNAs) in viral pathogenesis and antiviral defense. miRNAs are short, non-coding RNAs produced by both hosts and pathogens that act as post-transcriptional regulators of gene expression and can influence host-pathogen dynamics during infection. In this study, we used a honey bee-derived cell line to test the hypothesis that viral infection perturbs honey bee- and viral-encoded miRNA expression. METHODS: Small RNA libraries from honey bee AmE-711 cells subjected to one of four treatments: media change only (uninfected), heat-killed Acute bee paralysis virus (ABPV), the viral mimic Poly(I:C), or infectious ABPV, were prepared using an Illumina Revvity NextFlex V4 Small RNA kit and sequenced in single-read 51-base mode on the Illumina NextSeq 2000 platform using the NextSeq 2000 P3 Reagents kit. Sequencing data were analyzed using miRDeep2 and sRNAtoolbox to identify differentially expressed (DE) miRNAs, which were subsequently validated by RT-qPCR assay. RESULTS: Sequencing yielded > 3.6 x 10(8) raw reads that were assigned to 12 small RNA libraries, from which 481 unique miRNAs were identified. Moreover, 15 miRNAs were DE in ABPV-infected cells compared to uninfected cells: miR-2b-5p, miR-33-5p, miR-133-3p, miR-6001-3p, miR-996-3p, miR-965-3p, miR-125-5p, miR-13b-3p, miR-79-3p, miR-971-3p, miR-277-3p, miR-92c-5p, miR-6065-3p, miR-965-5p, and miR-3786-5p. We highlight some of the DE miRNAs identified in ABPV-infected cells that show regulatory effects in other systems in response to infection. CONCLUSION: This study identified miRNAs differentially expressed in ABPV-infected cells, suggesting roles in either antiviral defense or promoting viral pathogenesis through suppression of host immune responses. These results provide a foundation for functional studies using honey bee cell lines to clarify the cellular mechanisms governing honey bee-virus interactions.