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MicroRNAs (miRNAs) are important gene regulatory molecules involved in a broad range of cellular activities. Although the existence and functions of miRNAs are clearly defined and well established in eukaryotes, this is not always the case for those of viral origin. Indeed, the existence of viral miRNAs is the subject of intense controversy, especially those of RNA viruses. Here, we characterized the miRNA transcriptome of cultured human liver cells infected or not with either of the two Ebola virus (EBOV) variants: Mayinga or Makona; or with Reston virus (RESTV). Bioinformatic analyses revealed the presence of two EBOV-encoded miRNAs, miR-MAY-251 and miR-MAK-403, originating from the EBOV Mayinga and Makona variants, respectively. From the miRDB database, miR-MAY-251 and miR-MAK-403 displayed on average more than 700 potential human host target candidates, 25% of which had a confidence score higher than 80%. By RT-qPCR and dual luciferase assays, we assessed the potential regulatory effect of these two EBOV miRNAs on selected host mRNA targets. Further analysis of Panther pathways unveiled that these two EBOV miRNAs, in addition to general regulatory functions, can potentially target genes involved in the hemorrhagic phenotype, regulation of viral replication and modulation of host immune defense. [ABSTRACT FROM AUTHOR]