title: Inhibition of Respiratory Syncytial Virus Replication by Simultaneous Targeting of mRNA and Genomic RNA Using Dual-Targeting siRNAs.
abstract: We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0.01 was reduced more than MOI 0.1; (iii) reduction in N transcript was greater than F; and (iv) finally, in combination pre-treatment with two shRNAs, the reduction was not significant as compared to single shRNA transfection. shRNAs also inhibited the production of RSV progeny as shown by viral load in infected HEp-2 cells. (i) Virus load reduction was greater at MOI 0.01 than 0.1 and (ii) significant load reduction was not seen with combination shRNA pre-treatment. The antiviral potency was also confirmed by plaque assay and western blot analysis. Our results provided further evidence that RNAi could be a powerful treatment option against respiratory viruses.
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran.
authors.forename: Somayeh Shatizadeh
authors.initials: SS
authors.lastname: Malekshahi
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran.
authors.forename: Vahid
authors.initials: V
authors.lastname: Salimi
authors.affiliation: Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Arefian
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran.
authors.forename: Ghazal
authors.initials: G
authors.lastname: Fatemi-Nasab
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran.
authors.forename: Sarvin
authors.initials: S
authors.lastname: Adjaminejad-Fard
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran.
authors.forename: Jila
authors.initials: J
authors.lastname: Yavarian
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran. mokhtari@sina.tums.ac.ir.
authors.forename: Talat
authors.initials: T
authors.lastname: Mokhtari-Azad
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors*; D012367:RNA, Viral / Q000037:antagonists & inhibitors*; D012136:Respiratory Syncytial Viruses / Q000187:drug effects / Q000502:physiology*; D019562:Viral Load / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D012333:RNA, Messenger; D012367:RNA, Viral
keywords: Dual-targeting siRNA; Fusion; Genomic RNA; Nucleoprotein; RSV; mRNA