Synergic and antagonistic effect of small hairpin RNAs targeting the NS gene of the influenza A virus in cells and mice

• 10 shRNAs targeting the NS gene of the IAV were tested individually or in mixtures 16 h after infection.
• shRNA targeting the NS1 transcript decreased the virus titre in vitro and in vivo.
• A synergistic effect of the shRNAs was observed.
• NS1 mRNA was significantly reduced in lungs treated with shRNAs.
• The levels of RIG-1, IFN-α, IFN-β and IFN-γ mRNAs in lungs were not altered.

In the present study, we demonstrate the effect of individual and mixtures of shRNAs targeting the NS gene to treat an established infection of influenza A virus (IAV). We prepared 10 shRNAs targeting the NS gene of the IAV, and these shRNAs were tested individually or in mixtures 16 h after infection. Our results revealed: (i) shRNA targeting the NS1 transcript decreased the virus titre up to 21% (P < 0.01), (ii) shRNA targeting NEP transcript did not influence the replication of IAV in the infected cells; (iii) a mixture of shRNAs targeting the NS1 transcript was less effective than the individual shRNAs and decreased the virus titre up to 42% in vitro; (iv) a mixture of individually inactive shRNAs targeting the NEP transcript significantly inhibited the replication of IAV in vitro; (v) the activities of the individual shRNAs in vivo predominantly corresponded to their activities in vitro; (vi) a synergistic effect of the shRNAs was observed in vivo; and (vii) a shRNA targeting the region common to both the NS1 and NEP transcripts, shNS593, exhibited the strongest inhibition and reduced the virus titre up to 16.4% in vitro, prolonged the survival of the mice by three days and abolished the protective effect of other shRNAs in vivo. shRNAs inhibited influenza virus infection in a gene-specific manner. NS1 mRNA was significantly reduced in lungs treated with shRNAs and the levels of RIG-1, IFN-α, IFN-β and IFN-γ mRNAs shRNAs were not altered.