To evaluate
antiviral potential of adenoviral vector-delivered
small interfering RNA (
siRNA) against
rabies, recombinant, replication-defective adenoviral vectors (rAdV) encoding siRNAs targeting rabies virus (RV) polymerase (L) and
nucleoprotein (N) genes were developed. The siRNAs were delivered as small hairpin RNAs (shRNAs) through these vectors. Treatment of BHK-21 cells with rAdV expressing
siRNA targeting L gene (rAdV-L) and N gene (rAdV-N) (100 MOI) and their subsequent
infection with RV (0.001 MOI, RV PV-11), reduced RV fluorescent foci by 48.2% (mean±SEM; 48.17±0.6540, N=6) and 41.8% (mean±SEM; 41.83±0.3073, N=6), respectively, with respect to that of BHK-21 cells treated with rAdV expressing negative control
siRNA (rAdV-Neg) indicating inhibition of multiplication of RV in BHK-21 cells in response to adenoviral vector mediated
siRNA delivery. Also, the similar treatment of BHK-21 cells with rAdV-L and rAdV-N and similar subsequent
infection of them with RV resulted in reduction in RV
mRNA transcript levels for their respective targets (RV L gene for rAdV-L and N gene for rAdV-N).
mRNA transcript level for RV L gene was reduced by 17.88-fold (mean±SEM; 17.88±0.06638, N=6) in cells treated with rAdV-L and that for RV N gene was reduced by 5.7-fold (mean±SEM; 5.7±0.04472, N=6), in cells treated with rAdV-N, in comparison with that in cells treated with rAdV-Neg, as analyzed by using real-time PCR. These in vitro studies showed that between these two, adenoviral vector mediated delivery of
siRNA targeting RV L gene was comparatively more effective in inhibiting RV multiplication in BHK-21 cells than that of
siRNA targeting RV N gene (p<0.0001). Localized treatment (
intramuscular injection in masseter muscle) of mice with 10(7) plaque forming units of either rAdV-L or rAdV-N and subsequent lethal RV
infection (15-20LD(50) of CVS-11) at the same site, through the same route, although resulted in 50% protection (3 out of 6 mice survived) against lethal
rabies, the survival patterns for groups of mice treated with either rAdV-L or rAdV-N and that treated with rAdV-Neg did not differ significantly (p=0.5234). These results indicated that adenoviral vector mediated
siRNA delivery, in vitro in BHK-21 cells inhibited RV multiplication in vitro in BHK-21 cells;
siRNA targeting RV L gene used in this study was comparatively more efficient in doing this than that targeting RV N gene used in this study; in vivo in mice inhibited RV multiplication in mice and imparted partial protection against lethal
rabies and so it may have a potential to be used as an alternative
antiviral approach against
rabies, although further study is required to establish its efficacy for this purpose.