MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions.DNA viruses encode miRNAs that regulate the viral life cycle.However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery.Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV;Hu/China/H2/1982), which is a typical cytoplasmic RNA virus.Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p.Both of the novel virally encoded miRNAs were clearly detected in infected cells.Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent.Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells.Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS).Genomic location analysis revealed novel miRNAs locatedin the coding region of the viral genome.Overall,our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection.This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus.These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do.These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derivedmiRNAs.—Shi, J., Sun, J., Wang, B., Wu, M.,Zhang,J., Duan, Z., Wang, H., Hu, N., Hu, Y.NovelmicroRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.FASEB J.28,000-000 (2014).