title: Expression of a single siRNA against a conserved region of NP gene strongly inhibits in vitro replication of different Influenza A virus strains of avian and swine origin.
abstract: Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals.
authors.affiliation: Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Cremona, Italy; Laboratorio Colture Cellulari, Reparto Substrati Cellulari e Immunologia Cellulare, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Stoppani
authors.affiliation: Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Cremona, Italy.
authors.forename: Ivan
authors.initials: I
authors.lastname: Bassi
authors.affiliation: Laboratorio Colture Cellulari, Reparto Substrati Cellulari e Immunologia Cellulare, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Dotti
authors.affiliation: Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Cremona, Italy; UOS/IRGB/CNR, Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Lizier
authors.affiliation: Laboratorio Colture Cellulari, Reparto Substrati Cellulari e Immunologia Cellulare, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy.
authors.forename: Maura
authors.initials: M
authors.lastname: Ferrari
authors.affiliation: Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Cremona, Italy. Electronic address: franco.lucchini@unicatt.it.
authors.forename: Franco
authors.initials: F
authors.lastname: Lucchini
mesh_terms: D000818:Animals; D001717:Birds; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D061985:Madin Darby Canine Kidney Cells; D019590:Nucleocapsid Proteins; D009976:Orthomyxoviridae Infections / Q000662:veterinary / Q000821:virology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000032:analysis; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013552:Swine; D014758:Viral Core Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects*
chemical_list: C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D034741:RNA, Small Interfering; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins
keywords: Influenza; Nucleoprotein; Orthomyxoviridae; RNA interference; siRNA