Cross-Protective Effect of Antisense Oligonucleotide Developed Against the Common 3′ NCR of Influenza A Virus Genome

Abstract

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5' and 3' ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3' NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.

High antiviral effect of TiO2·PL–DNA nanocomposites targeted to conservative regions of (−)RNA and (+)RNA of influenza A virus in cell culture

“…the noncoding region located on the 3’-end of (+)cRNA after the stop codon (1544–1564), the nearby coding region close to the stop codon (1498–1516), the region including the AUG codon (34–54), and the 5’-noncoding region (2–22), where the nucleotide numbering is given for (+)cRNA. The short 5’- and 3’-terminal nontranslated regions (NTRs) are of considerable interest because all eight RNA segments of various IAV subtypes are known to contain conserved sequences of 13 or 12 nucleotides at their 5’ and 3’ ends ( 5’ AGUAGAAACAAGG 3’ and 5’ CCUGCUUUUGCU 3’ , respectively) [20–21]. …”

“…The coding region close to the stop codon was often used as the target for the action of siRNA [9,12,15–16]. The other regions of the NP gene were also studied as targets [8,11–12 21]. It can be concluded from these literature data that the most susceptible regions for the action of NA-based drugs are the first two regions mentioned above.…”

“…The DNA 4 morpholino oligonucleotide displayed very low activity in these experiments. It was shown [21] that a 12-mer oligonucleotide directed to the 3’-end of (−)RNA of all IAV segments and delivered into cells with lipofectamine inhibited the IAV replication by 60% (2.5-fold) at a MOI of 0.1 and 5 nM concentration.…”

Treatment of Influenza: Prospects of Post-Transcriptional Gene Silencing Through Synthetic siRNAs

“…This implied that a single antisense oligonucleotide could provide protection against more than one strain of influenza A virus. 90 Another in vivo study carried out by Tompkins et al revealed that influenza-specific siRNA treatments directed against the NP and PA genes were broadly effective and protected animals against lethal challenge with highly pathogenic avian influenza A viruses of the H5 and H7 subtypes. 91 Ge and co-workers also reported that siRNAs targeting the NP and PA gene segments (NP-1496 and PA-2087, respectively) provided a broadly effective inhibition of H1N1 influenza virus.…”

Small RNAs targeting the 5′ end of the viral polymerase gene segments specifically interfere with influenza type A virus replication

“…The recent dissection of the RNA processing and control has opened up the frontiers to innovative concept of antiviral therapy based on nucleic acid RNA interference (DeVincenzo, 2012;Spurgers et al, 2008) cleotides as potential antivirals have already been used by different research groups for many pathogenic respiratory viruses (Spurgers et al, 2008;DeVincenzo, 2012), including the influenza virus (Wong et al, 2010). In this context, antivirals based on antisense oligonucleotides, small RNA interfering molecules and microRNAs (miRNA) targeting different sequences of influenza virus genome have been reported (Li et al, 2011;Kumar et al, 2013;Ge et al, 2006;Kwok et al, 2009;Abe et al, 2001;Duan et al, 2008). Influenza virus genomic segments encoding for the viral polymerase subunits (PA, PB1 and PB2), have been described to play a pivotal role in the hierarchy of segment interactions,during the packaging of viral genome into the newly assembled particles (Palese and Shaw, 2007;Hutchinson et al, 2010).…”