Dengue virus (DV) contains a single-stranded, positive-sense RNA genome, and the 3' non-coding regions (NCRs) have been demonstrated to play crucial roles in vital RNA replication and translation initiation. In this study, we report the presence of a species of small sub-genomic RNA (sgRNA) derived from the 3' NCR in DV-infected cell cultures and animal tissues. Further experiments demonstrate that these sgRNAs range from 390 to 430 nt in length and that they accumulate together with viral genomic RNA during replication. Sequence analysis and secondary structure prediction indicate that the stem-loop structures within the 3' NCR are responsible for the production of DV sgRNAs. Unexpectedly, a unique DV isolate produced three species of sgRNAs, compared to only one in the other isolates. Collectively, the structural and biochemical properties of DV sgRNAs warrant further study of their biological functions during the viral life cycle and pathogenesis. (C) 2009 Elsevier Inc. All rights reserved.