Tandem repeat sequence of duck circovirus serves as downstream sequence element to regulate viral gene expression.

Duck circovirus (DuCV) has a small, single-stranded circular DNA genome of approximately 1.99 kb. Through a genome sequence analysis using the dottup program, we found that a quadruple tandem repeat sequence (QTR) in the intergenic region between the rep and cap genes of the DuCV genome, but not in other circoviruses. The QTR was also substantially different and evolutionarily conserved in the genotype 1 and 2 DuCV strains. Furthermore, a luciferase reporter assay demonstrated that QTR functioned as a downstream sequence element (DSE) of polyadenylation signals to enhance mRNA stability, which was dependent on four copies but not the QTR direction. Cap and Rep expression derived by subgenomic constructs also revealed a critical role of QTR in regulating viral gene expression. Finally, a reverse genetic study of a DuCV-based minicircle DNA technique found that a deletion of QTR induced a significant deficiency in viral genes transcription and replication. Our findings were the first to report that QTR only exists in the DuCV genome and serves as a novel molecular marker of DuCV genotyping, and has revealed its crucial biological function in regulating viral gene expression.