MicroRNAs (miRNAs) are a conserved class of small non-coding RNA genes with 19 to 25 nucleotides in length that are found in all higher eukaryotes as well as some DNA viruses. MiRNAs have been elucidated to exert important regulatory functions in many biological and pathological processes, by imperfect base pairing to the 3’ untranslated region (UTR) of target mRNAs, leading to translational repression or mRNA degradation. In the recent years, the rapid development of next generation sequencing (NGS) technologies has tremendously enhanced the identification of novel miRNA genes as well as the profiling of miRNA expression levels. In this study, NGS method has been applied to two different gammaherpesviruses (γ-herpesviruses) associated models: Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) in human and murine gammaherpesvirus 68 (MHV-68)-infected cell lines from

mouse. As a result, two novel miRNA precursors from EBV and six novel miRNA precursors from MHV-68 have been successfully identified and characterized. In addition, the completion of MHV-68 miRNA set has revealed a unique viral tRNA (vtRNA)-miRNA-miRNA structure in MHV-68 genome. Furthermore, expression levels of the viral miRNAs suggest a distinct pattern during different stages of the viral life

cycle. On the other hand, the profiling of cellular miRNAs also defined a number of miRNAs that have been dysregulated in EBV-positive NPC tissues compared to healthy control tissues, and in MHV-68-infected compared to non-infected NIH 3T3 murine fibroblasts. Among them, miR-15 and miR-16 were upregulated upon EBV or MHV-68 infection. The tumor suppressor gene BRCA1 has been revealed to be the repressed target protein of miR-15/16 in both models, implying an interesting role of miRNAs in the pathogenesis of γ-herpesviruses. Meanwhile, to facilitate the analysis of NGS data, an automated software was designed and developed. Additional information gained during the analysis processes revealed the possible mis-annotations existing in the miRNA registry database, suggesting that the definition and characterization of novel miRNA genes has to be performed with much more caution.