Abstract: Objective To evaluate the anti-viral effects of a plasmid expressing an inverted-repeat RNA targeting dengue virus
type-2 (DENV-2) pre-membrane (prM) gene. Method Suckling mice were inoculated with live DENV-2 in the brain. The total
RNA was extracted from the brain tissue of the infected mice, and the prM gene fragments were amplified by RT-PCR and
then subcloned into Xho I/EcoR I of the pcDNA3.1(+) plasmid in antisense orientation to construct the plasmid pcDNA-asprM.
DENV-2 prM sequences were also subcloned into pMD18-T-vector in sense orientation to construct the plasmid pMD18-TprM.
pcDNA-irRNA was constructed by inserting in sense orientation the prM fragment isolated from pMD18-T-prM into the
Nhe I/Kpn I of pcDNA-asprM. The plasmid pcDNA-irRNA was transfected into BHK-21 cells and the anti-viral effects were
analyzed by semi-quantitative PCR and real-time PCR. Results Transfection with the plasmid pcDNA-irRNA caused a
reduction of NS3 mRNA expression level by 28% in BHK-21 cells following a 96-h challenge with DENV-2 as compared to the
cells without plasmid transfection (positive control). The viral copies in pcDNA-irRNA-transfected cells was 1.44-fold lower
than those in the positive control cells following a 72-h virus challenge, and the mRNA expression levels of NS1 were also
significantly lower in the transfected cells at 96 h after viral challenge (P<0.05) as shown by real-time quantitative PCR.
Conclusion The inverted-repeat RNA for DENV-2 prM gene silencing can suppress DENV-2 replication in BHK-21 cells,
which provides a basis for developing dengue virus gene vaccine.