RNA interference (RNAi)-based sequence-specific gene silencing is applied to 
identify gene function and also possesses great potential for inhibiting virus 
replication both in animals and plants. Small interfering RNA (siRNA) molecules 
are the inducers of gene silencing in the RNAi pathway but may also display 
immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 
nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex 
when introduced into the cells, while longer siRNA molecules are first processed 
by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have 
applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality 
double-stranded RNA molecules that are specific for the UL29 gene of herpes 
simplex virus (HSV). The 653 nt long double-stranded RNA molecules were 
converted to siRNA and DsiRNA pools using Dicer enzymes originating from human 
or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in 
length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA 
targeting the UL29 were used as references. The impact of these siRNAs on cell 
viability, inflammatory responses, gene silencing, and anti-HSV activity were 
assayed in cells derived from human nervous system and skin. Both pools and the 
canonical single-site siRNAs displayed substantial antiviral activity resulting 
in four orders of magnitude reduction in virus titer. Notably, the pool of 
DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, 
whereas the immunostimulation effect was in relation to the length with the 
single-site siRNAs. Our results also propose differences in the processivity of 
the two Dicers.