Objective To investigate the effect of Spodoptera frugiperda Sf9 cell exosomal microRNA (miRNA) on proliferation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV).

Method Exosomes were purified using discontinuous sucrose mass fraction gradient ultracentrifugation. Then the purified exosome samples were observed under transmission electron microscopy, and conducted particle diameter analysis using nanoparticle tracking analysis. By sRNA hight throughput sequencing analysis, exosomally differentially expressed miRNAs were identified and biological pathways corresponding to their potential target genes were predicted. Cellular experiments such as mimic transfection and virus infection, as well as qPCR were performed to verify the differential expression of exosomal miRNAs, and detect the effect of the differentially expressed miRNA of sfr-miR-1a-3p on the proliferation of AcMNPV.

Result Exosomes from Sf9 cells were successfully purified, and AcMNPV infection effectively stimulated exosome secretion from Sf9 cells. After 72 h of AcMNPV infection in Sf9 cells, a total of 11 differentially expressed miRNAs were identified in exosomal miRNAs by comparing with the Rfam database, in which eight miRNAs’ transcription levels showed consistent trends with the sequencing results. Potential target genes were mainly enriched in the cAMP signaling pathway, PI3K-Akt signaling pathway, cancer pathway, and human papilloma virus signaling pathway, suggesting that differentially expressed exosomal miRNAs may participate in insect innate immune responses. Overexpressing sfr-miR-1a-3p significantly promote the expression level of the AcMNPV gene vp39.

Conclusion This study conducts sRNA sequencing to screen differentially expressed miRNAs in AcMNPV-infected Sf9 cell exosomes, demonstrating that AcMNPV promotes infection by enhancing exosome secretion and affecting surrounding uninfected cells via miRNA sfr-miR-1a-3p transfer. This research provides an important theoretical support for understanding the mechanism by which insect exosomal miRNAs regulate virus proliferation.