Evaluation of single and dual siRNAs targeting rabies virus glycoprotein and nucleoprotein genes for inhibition of virus multiplication in vitro

Abstract

Small interfering RNAs (siRNAs) targeting rabies virus (RV) glycoprotein (G) and nucleoprotein (N) genes were evaluated as antiviral agents against rabies virus in vitro in BHK-21 cells. To select effective siRNAs targeting RV-G, a plasmid-based transient co-transfection approach was used. In this, siRNAs were expressed as short hairpin RNAs (shRNAs), and their ability to inhibit RV-G gene expression was evaluated in cells transfected with a plasmid expressing RV-G. The nine different siRNAs designed to target RV-G exhibited varying degrees of knockdown of RV-G gene expression. One siRNA (si-G7) with considerable effect in knockdown of RV-G expression also demonstrated significant inhibition of RV multiplication in BHK-21 cells after in vitro challenge with the RV Pasteur virus-11 (PV-11) strain. A decrease in the number of fluorescent foci in siRNA-treated cells and a reduction (86.8 %) in the release of RV into infected cell culture supernatant indicated the anti-rabies potential of siRNA. Similarly, treatment with one siRNA targeting RV-N resulted in a decrease in the number of fluorescent foci and a reduction (85.9 %) in the release of RV. As a dual gene silencing approach where siRNAs targeting RV-G and RV-N genes were expressed from single construct, the anti-rabies-virus effect was observed as an 87.4 % reduction in the release of RV. These results demonstrate that siRNAs targeting RV-G and N, both in single and dual form, have potential as antiviral agent against rabies.

A review on current status of antiviral siRNA

“…Rabies virus (RV) is a member of family Rhabdoviridae and causes fatal zoonotic disease in both humans and animals. It is responsible for 50 000 to 55 000 human deaths annually in Asia and Africa . Brandao et al used siRNAs against rabies virus nucleoprotein mRNA, which decreased virus titers in BHK‐21 cells .…”

“…Brandao et al used siRNAs against rabies virus nucleoprotein mRNA, which decreased virus titers in BHK‐21 cells . Other workers developed siRNAs targeting rabies virus glycoprotein and nucleoprotein showing remarkable knockdown effects and significant inhibition of RV multiplication and release …”

Rabies Control and Treatment: From Prophylaxis to Strategies with Curative Potential

“…Initially, plasmids encoding siRNA, or another form of RNAi that target specific RABV genes, were constructed and tested for anti-rabies efficacy [124,125,126]. Although the plasmid-based approaches achieved some degree of anti-rabies effect, the low efficiency of siRNA delivery hindered the substantial protection against RV through anti-rabies RNAi approach.…”

Small Non-coding RNAs Associated with Viral Infectious Diseases of Veterinary Importance: Potential Clinical Applications

“…In mice, inoculation of both siRNA vectors resulted in a 50% protection against a subsequent lethal RV injection. siRNAs simultaneously targeting the glycoprotein G and N genes led to an 87% reduction in viral release, demonstrating that siRNAs directed against different targets may act synergistically and increase efficacy of siRNA-based interventions against RV ( 85 ). Taken together, the above results do suggest that use of siRNAs constitutes a promising approach to interventions against RV.…”