Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage phi6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837-7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the phi6 RdRP (phi6-siRNAs) was considerably more effective than single-site siRNAs. The phi6-siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.
